OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR ... · May 2004 Volume 25 Number 2 OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC. A. B. C. D. Page 26 Page 23 1 2

May 2004Volume 25Number 2

OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC.

A. B.

C. D.Page26

Page 23

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Streptomycescoelicolor

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M I C R O B I O L O G Y A U S T R A L I A • M A Y 2 0 0 4 1

Vertical Transmission 2

First Words 3

In Focus 4

Genomes full of promise 4

Streptomyces and beyond 8

Taxonomy as a roadmap for search and discovery 13

Under the Microscope 16

Functional genomics of Streptomyces coelicolor 16

Streptomyces viewed from the inside:

the application of proteomics to a model streptomycete 17

Contributions of methylenomycin to the

genetics of antibiotic production 19

Superhosts for polyketide drug production 21

Streptomyces coelicolor in an oxygen-limited

liquid environment: adapt and escape 22

Streptomycetes and anaerobic stress survival 26

Alkaliphilic streptomycetes as a source of

novel secondary metabolites 27

Biodiscovery programme conducted at the

Gause Institute, Moscow, Russia 30

Exploiting and expanding actinomycete

diversity for antibiotic discovery 32

Horizontal gene transfer within streptomycetes 34

Uniqueness of the ‘Smart State’s’ microbial diversity:

from an actinomycete collection to biodiscovery at the

University of the Sunshine Coast 36

Emerging Microbiologists 40

ASM Affairs 43

What’s On 48

2 M I C R O B I O L O G Y A U S T R A L I A • M A Y 2 0 0 4

Vertical Transmission

The Australian Societyfor Microbiology Inc.

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EDITORDr Penny Bishop

EDITORIAL BOARDProf Mary BartonDr Chris Burke

Prof Peter ColoeAssoc Prof David EllisDr Tony Della Porta

Dr Ruth FoxwellDr Ailsa Hocking

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Prof Duncan Veal

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E-mail: [email protected]

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© 2004 The Australian Society forMicrobiology Inc. All rights reserved.

No part of this publication may bereproduced or copied in any form or by anymeans without the written permission of the

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ISSN 1324-4272

The opinions expressed in articles,letters and advertisements in

Microbiology Australia are not necessarilythose of the Australian Society for

Microbiology or the Editorial Board.

The March issue of Microbiology Australia

contained important information about

the various awards and prizes available to

ASM members. The closing date for most

of these is 1 June 2004. I would like to

encourage members to either apply for

or to nominate a colleague for one of

these awards – for full details see

www.theasm.com.au.

The Annual Scientific Meeting and

Exhibition offers an exciting programme

covering all aspects of microbiology and

registrations are now well advanced.

Early bird registrations close on 30 June

2004 so don’t be late. Abstract

submissions should be made online and

are due by 14 May 2004, so you may still

have a chance to get one in. The

workshops on antibiotic resistance,

biochemical ID, mycology and

parasitology will be of major interest to

many laboratories. All details regarding

the Sydney meeting are on the website

www.asm2004.org.au.

While on websites, the ASM site is

currently undergoing a major revision.

This site will now be managed by the

National Office and pages and/or links will

be made available to all branches and

SIGs. The National Office is working very

hard and the staff are doing a great job;

we have had a very successful

membership drive, the finances are now

back in the black and the conference

organisation is going really well. Well

done Chris, Janette, Meg and Lena.

I have just finished a national lecture tour

organised by ASM and sponsored by

Novartis – it was great to be able to visit all

the branches. I see an opportunity for

ASM to repeat this exercise with other

Australian speakers. It would be ideal if

we could set up a national speaker

programme using Australian expertise to

cover areas of interest to the members.

A special thank you to Tom Riley who has

recently stepped down from both the

Editorial Board and as coordinator of our

International Visitor Programme. Tom

has unselfishly served the ASM in these

roles for at least 15 years and has made a

major contribution to the society. John

MacKenzie is also standing down as a long

term member of the Editorial Board and I

also wish to acknowledge his worthwhile

contributions. Mary Barton will take over

the International Visitor Programme and I

also wish to welcome Mary Barton and

Bill Rawlinson to the Editorial Board of

Microbiology Australia.

Finally, I would also like to congratulate

the Editorial Board, especially Ailsa

Hocking (Chair) and Penny Bishop

(Editor) of Microbiology Australia for

also doing a great job. I, like many

members, have really enjoyed reading MA

and the quality and variety of the articles

have been excellent.

David EllisPresident ASM


Cover illustration: Scanning electron micrograph (x20,000) of Streptomyces sp.producer of antimycin complex showing aerial mycelium with straight chains ofspores. Courtesy D.I. Kurtböke and R. Locci, Department of Plant Pathology,University of Milan.


First Words

Dr Ipek KurtbökeConvener

13th International Symposiumon the Biology of Actinomycetes

Melbourne, VIC

The 13th International Symposium on the

Biology of Actinomycetes was held at the

Melbourne Convention Centre from 1-5

December 2003. The Conference

welcomed 355 participants from over 35

countries, with the excellent support of

the Government of Victoria. In particular,

Dr Amanda Caples, Director of

Biotechnology for the State Government

of Victoria was very supportive

throughout the event. The Honorable

Matt Viney, Parliamentary Secretary of the

Victorian Government for the Innovation

and Industry, Department of Industry

Innovation and Regional Development,

formally opened the Conference.

This was the first ISBA Meeting in the

southern hemisphere and provided

colleagues from the northern hemisphere

with opportunities to encounter the work

of the Actinomycetologists in this part of

the world. It has also paved the way

towards the establishment of the

Australian-New Zealand Actinomycete

Group (ANZAG).

The impressive programme included the

‘hot topics’ in the field, including

genomics, proteomics and bioinformatics,

as well as the application of the

Actinomycetes in biotechnology,

biodiscovery and biobusiness. It also

included the clinically important

Actinomycetes, the molecular aspects of

antibiotic resistance as well as alternative

therapies. The use of Actinomycetes in

environmental biotechnologies such as

bioremediation was also discussed.

ISBA’13 was followed by a workshop

entitled Commercial use of microbial

diversity organised by the World

Federation of Culture Collections (WFCC).

This timely event coincided with both the

Australian Federal and State Governments’

support initiatives to map Australian

biodiversity (including microorganisms)

and explore their potential biotechnological

applications.

Throughout the Conference and the post-

conference workshop, recent advances in

the natural products screening

programmes as well as eco-taxonomical

aspects of Actinomycete natural products

were the subject of discussions. This is a

topic of great importance in the Oceania

region, which contains unique microbial

diversity in both its marine and terrestrial

environments. The event, therefore, was

the start of an exciting opportunity in

Oceania both to make important

contributions to the field of drug

discovery through the acquisition and

screening of this biota and to attract

international partners.

ISBA Conferences have been in existence

since 1968 and live on traditions. It has a

strong, passionate bond that endures;

this is typical among researchers working

in the same scientific field. The

International Actinomycete Group

contains many eminent scientists

including Sir Prof David Hopwood, Prof

Actinomycetes, the millennium bugs: in Melbourne for the 13th International

Symposium on the Biology of ActinomycetesJulian Davies, Prof Stanley Cohen, ProfAlan Bull and Prof Michael Goodfellow.The traditions they have set up havecontinued over the years.

At this Conference, past achievementswere honoured and awards, in thememory of those who have contributed tothe field but not with us any longer, werepresented for the best post-graduatestudents. These were the Prof Tom CrossAward in Ecology, the Prof MarionMordarski Award in Biotechnology, theProf Yurii V Dudnik Award in AntibioticProduction, the Prof Bruna Petrolini Awardin Plant Pathology and the Dr GeorgeLeudemann Award in Biodiscovery. DrLeudemann’s family travelled from theUSA to present the award – this was one ofthe most touching moments of theConference. Dr Leudemann, whodiscovered the antibiotic Gentamicin, wasalso an expert on the ecology of the genusGeodermatophilus.

Melbourne, the biotechnology capital ofAustralia, provided an excellent meetingplace for all these eminent scientists.With strong local government support forscience, technology and research, and alarge biotechnology workforce, it madean ideal venue for the first southernhemisphere encounter – it has beenanother ISBA which will be rememberedfor long years to come.

The conference was proudly sponsored by:

• The University of the Sunshine Coast, Australia

• GRDC, Australia

• HAL Ltd., Australia

• The State Government of Victoria

• World Federation of Culture Collections

(WFCC)

• ESKITIS INSTITUTE (formerly Astra Zeneca

R&D-Griffith University), Australia

• Vicuron Pharmaceuticals, Italy

• Aventis Pharma, Germany

• Roche Molecular System, USA

• Syngenta, Switzerland


In Focus

Since the first bacterial genome was

sequenced in 1995, over 100 others have

been completed. They include many

pathogens, as well as other bacteria

chosen for their special interest, whether

academic or applied. The resulting

knowledge is revolutionising our

understanding of the bacterial world.

Streptomyces is a genus of soil-dwelling

bacteria with two unusual attributes – a

complex developmental cycle and the

ability to produce many of the antibiotics

applied in medicine, as well as important

drugs with other applications, such as

anti-cancer and anti-parasitic agents, and

immunosuppressants for use in tissue

transplantation. Over the past 2 years,

the complete genome sequences of two

representative streptomycetes have been

published 1, 2. The inventories of genes

deduced from the sequences are

throwing a powerful light on the

strategies used by the streptomycetes to

compete in the soil habitat, as well as

providing a huge potential toolbox for

making novel antibiotics by genetic

engineering.

Streptomyces genomescontain many genes

The genomes of such well-known

bacteria as Escherichia coli, the

workhorse of molecular genetics over the

past 50 years, and Bacillus subtilis, a

much-studied model for bacterial spore

formation, are circular DNA molecules

just over 4000 base pairs long, enough for

about 4000 genes. In contrast, the

Streptomyces chromosome is a linear

molecule of about twice the size, with

double the number of genes.

The Streptomyces colony is much more

complex than that of the other two

bacteria. Instead of consisting of a mass

of separate rod-shaped cells like those of

E. coli and B. subtilis, the Streptomyces

colony is a mould-like system of

interconnected, branching hyphae that

Genomes full of promise

first colonises the substrate as a so-called

vegetative mycelium and then, when the

food source is exhausted, gives rise to a

sporulating aerial growth (Figure 1).

Such a developmental programme might

be expected to require a large number of

genes to implement, and many have

indeed been characterised, but this does

not explain why the Streptomyces

chromosome has thousands more genes

than a Bacillus, which also needs to

programme developmental events, in its

case the production of heat-resistant

endospores inside the rod-shaped

mother cells. Most of the ‘extra’ genes

seem to play other roles in adapting

Streptomyces to live in the stress-rich

environment of the soil.

Soils contain a huge variety of potential

food sources, ranging from simple sugars

and inorganic sources of nitrogen to hard-

to-digest polymeric carbohydrates like

cellulose and chitin (derived from the

skeletons or walls of dead plants, insects

and fungi), and complex nitrogen sources

such as proteins. Moreover, the food

sources vary from time to time and from

place to place, so the Streptomyces

genome encodes many suites of enzymes

that can be called into play to deal with

the different food sources as they are

encountered.

Soils have many other variables too, such

as temperature, pressure, pH, and the

availability of oxygen and water, as well as

the presence of other organisms that may

represent competition to be met or

potential colleagues with which to

establish a symbiosis. The genome is full

of genes whose products would meet

these opportunities and threats, ensuring

that the organism can thrive under a

much wider set of conditions than most

other bacteria, which have instead

evolved to be supremely well adapted to a

limited set of habitats.

With such an arsenal of genes, many of

which are needed only under specific

circumstances, it is no surprise that the

genome is also provided with an

unprecedented number – for a bacterium

– of regulatory genes to switch on

different sub-sets of genes in response to

specific signals: one eighth of all the

genes fall into this category, twice the

proportion found in genomes half the

size.

The Streptomyceschromosome is linear

Why is the Streptomyces genome linear,

as in eukaryotes, rather than being

circular like those of most bacteria? The

answer is not obvious, especially because

linearity brings with it the need for a

special replication strategy to avoid the

loss of coding sequences from the ends of

the chromosome in each round of

replication (a consequence of the fact

that all DNA synthesis can only start with

an RNA primer that is removed once the

synthesis gets under way; if this is at the

end of a molecule, a gap is left in the

daughter strand). Eukaryotes overcome

this problem using their complex

telomeres, which constantly renew lost

Sir David HopwoodDepartment of Molecular Microbiology

John Innes CentreNorwich, NR4 7UH, UKTel: (44) 1603 450000Fax: (44) 1603 450778

E-mail:[email protected]


In Focus

end sequences. Instead, streptomycetes

have evolved a unique system to patch

the gaps, using primer proteins

permanently bound to the free ends.

The question about linearity becomes

even more intriguing when we find that

Streptomyces chromosomes occasionally

mutate to a circular form by fusion of the

ends, and they continue to replicate

perfectly well 3. Linearity almost certainly

represents an earlier state found in the

ancestors of modern streptomycetes, and

indeed in present-day actinomycetes with

smaller genomes and narrower ecological

niches, such as the mycobacteria that

cause tuberculosis and leprosy 4, 5.

Perhaps a clue to chromosome linearity is

the finding that the genomes of the two

sequenced streptomycetes show a

biphasic structure 1, 2, with a central core

containing unconditionally essential genes

such as those for cell division, central

metabolism, DNA replication, transcription

and translation, and arms representing

nearly half the genome and packed with

genes that would be adaptive under

various sets of conditions (Figure 2).

Comparing the two Streptomyces

genomes, the arms differ more strongly

than the core in gene content, telling us

that the arms are probably evolving at a

faster rate by acquiring genes through

horizontal transfer from other

microorganisms, often on transposons.

The arms can also exchange their ends

with those of linear, transmissible

plasmids, providing a potential route to

such horizontal transfer. This

recombination process depends on

genome linearity.

Engineering novel antibioticsWhat are the consequences of Streptomyces

genome sequencing for antibiotic discovery

and development? Over the past 10 years, a

new field of biotechnology has grown from

a glint in the eye to one that has produced

drug candidates in Phase I clinical trials.

This is ‘combinatorial biosynthesis’ of

‘unnatural natural products’. It stems from

genetic studies of the biosynthesis of two

chemical classes of antibiotics, the

polyketides and the non-ribosomally

synthesised peptides. The former includes

blockbuster antibacterials like the

tetracyclines and erythromycin, as well as

anti-tumour drugs such as adriamycin and

the important anti-parasitic agent

avermectin. The peptides include the most

important immunosuppressants.

Genetic studies have revealed that both

classes of compounds are made on giant

enzymatic assembly lines that determine

the complex product structure by a linear

arrangement of catalytic sites acting in

succession on the molecule as it travels

along the assembly line 6. Such

programming of the chemistry – by the

nature, number and arrangement of the

catalytic sites – is encoded in the

Streptomyces genome and is readily

amenable to manipulation by genetic

engineering in a combinatorial fashion to

generate compounds that are ‘natural’

because they are made in microorganisms

but ‘unnatural’ because they are not

found in nature. Kosan Biosciences Inc, a

leader in this field based in Hayward

California, calls this approach “doing

chemistry by genetics”.

Current examples of this technology

involve gene clusters for already known

Figure 1. Scanning electron micrographs of a Streptomyces colony. Clockwise fromtop left: edge of a colony showing vegetative hyphae colonising thesubstrate; mature vegetative mycelium with aerial branches beginning todevelop; an immature chain of spores in an aerial hypha; a mature chainof spores [photographs by Kim Findlay, John Innes Centre].


In Focus

metabolites, but one of the excitements

of whole genome sequencing is that it has

revealed far more clusters of biosynthetic

genes for structurally complex chemicals

than had even been suspected, never

mind proven. The genome sequence of

Streptomyces coelicolor, the most

studied laboratory model for the genus,

revealed two dozen clusters of such

genes, while that of Streptomyces

avermitilis, the industrial producer of

avermectin, showed more than 30 1, 2.

Most of these clusters are in the arm

regions, emphasising the conditionally

adaptive nature of their products in the

soil environment.

Nearly all the clusters are different

between the two streptomycetes, telling

us that sequencing more Streptomyces

genomes will reveal an enormous number

of such gene clusters. The toolbox of

potential spare parts for combinatorial

biosynthesis is evidently huge.

Whole genome sequencing of antibiotic

producing streptomycetes has another

potential application. Many unnatural

natural products are made by engineered

strains at very low levels. This arises from

a variety of causes, such as poor substrate

availability, siphoning off of substrates by

competing pathways, low tolerance of the

novel compound by the engineered

producer, and a plethora of regulatory

influences.

An exciting goal is to engineer a ‘super-

host’ that made more of the desired

product by addressing each of these

problems in the light of the genome

sequence. For example, a missing

pathway for a novel substrate could be

introduced from another microorganism,

potential transport proteins for product

export could be added, and genes for

competing pathways could be identified

in the host genome and deleted. Thus,

while yield optimisation will continue to

be in part empirical and involve

traditional strain improvement by random

mutagenesis and screening, because of

the sheer complexity of the regulatory

circuits involved, rational steps will

increasingly become possible as we come

to understand more and more about the

genetic endowment of streptomycetes by

the application of the new techniques of

functional genomics.

References1. Bentley SD, Chater KF, Cerdeño-Tarraga A-M et

al. Complete genome sequence of the model

actinomycete Streptomyces coelicolor A3(2).

Nature 2001; 417:141-147.

2. Ikeda H, Ishikawa J, Hanamoto A et al. Complete

genome sequence and comparative analysis of the

industrial microorganism Streptomyces avermitilis.

Nature New Biology 2003; 21:526-531.

3. Lin YS, Kieser HM, Hopwood DA & Chen CW. The

chromosomal DNA of Streptomyces lividans is

linear. Molecular Microbiology 1993; 10:923-933.

4. Cole ST, Brosch R, Parkhill J et al. Deciphering the

biology of Mycobacterium tuberculosis from the

genome sequence. Nature 1998; 393:537-544.

5. Cole ST, Eiglmeier K, Parkhill J et al. Massive

gene decay in the leprosy bacillus. Nature 2001;

409:1007-1011.

6. Donadio S, Staver MJ, McAlpine JB, Swanson SJ &

Katz J. Modular organization of the genes

required for complex polyketide biosynthesis.

Science 1991; 252:675-679.

Figure 2. Genetic content of the Streptomyces coelicolor genome. The core of thechromosome is in dark blue and the arms in light blue. Ori denotes theorigin of chromosome replication and the blue circles at the ends of thechromosome are protein molecules responsible for priming the specialDNA synthesis that ensures complete replication of the linearchromosome. The outer two multi-coloured circles show the predictedgenes on the two DNA strands as coloured bars; note that the genedensity is just as high in the arms as in the core. The next, incomplete,circle includes a selection of essential genes, for cell division, DNAreplication, transcription, translation and amino acid biosynthesis; notetheir location in the core region of the chromosome. For a fullexplanation of the figure, see reference 1 [reprinted by permission fromNature, ©(2002) Macmillan Publishers Ltd].

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Streptomycescoelicolor

8,667,507bp

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Before prescribing, please review Approved Product Information. Full Approved Product Information isavailable on request from Pfizer. VFEND (voriconazole). Indications: invasive aspergillosis, serious Candida infections (including C.krusei), including oesophageal and systemic Candida infections (hepatosplenic candidiasis, disseminated candidiasis, candidaemia), seriousfungal infections caused by Scedosporium spp and Fusarium spp, other serious fungal infections in patients intolerant of, or refractory to othertherapy. Contraindications: hypersensitivity to voriconazole or excipients, coadministration with terfenadine, astemizole, cisapride, pimozide,quinidine, rifampicin, carbamazepine, phenobarbital, ergot alkaloids (ergotamine, dihydroergotamine), sirolimus. Precautions: hypersensitivity to other azoles, *exercise cautionin patients with potentially proarrhythmic conditions (eg. cardiomyopathy and electrolyte disturbances) as QT prolongation has been reported rarely, flushing and nausea duringinfusion; if severe consider cessation, monitor for hepatotoxicity, monitor for renal toxicity particularly in combination with nephrotoxic medications, exfoliative cutaneous reactionsrare, safety and effectiveness not established in children <2 years, avoid concomitant phenytoin or rifabutin unless benefit outweighs risk of toxicity due to these agents, monitorlevels of concomitant cyclosporine or tacrolimus, galactose intolerance, Lapp lactase deficiency or glucose-galactose malabsorption, pregnancy Category B3; ensure effectivecontraception in women of child-bearing potential lactation, driving or operating machinery. Adverse Reactions: most commonly reported were visual disturbances, fever, rash,vomiting, nausea, diarrhoea, headache, peripheral oedema and abdominal pain. Dosage and Administration: IV 6 mg/kg q12 hours (for first 24 hours) then 3–4 mg/kg q12hours or oral 200–400 mg q12 hours (for the first 24 hours) then 100–200 mg bd depending on indication and body weight. Please refer to Approved Product Information for completed dosing schedule. Pfizer Pty Ltd, ABN 50 008 422 348, 38–42 Wharf Road, West Ryde, NSW 2114. * Please note changes in Product Information.

References. 1. VFEND Approved Product Information. 2. Herbrecht R et al. N Engl J Med 2002; 347:408–415. ® Registered Trademark Pfizer Inc. www.pfizer.com.au 02/04 AP94064 PFXVF5331

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PBS Information: This product is not listed on the PBS.

Designed for


In Focus

In terms of commercial value, the

streptomycetes rank high among the

bacteria, having been the principal source

of numerous and valuable therapeutic

agents for more than half a century.

The current world market for antibiotics

is upwards of $US 25 million/year, of

which a significant proportion are the

products of bacterial fermentation,

mainly from Streptomyces species. In

terms of the industrial exploitation of

microbial products in general, only the

yeasts are more important, having

provided humans with sustenance and

pleasure for thousands of years.

The development of streptomycetes as

sources of antibiotics and as industrial

microorganisms was spearheaded by

Selman Waksman who was the first to

identify their extraordinary capacity to

make antibiotics 1. Incidentally, this

discovery also led to the modern

pharmaceutical industry, as we know it

now.

It has been estimated that the

streptomycetes alone may produce

upwards of 250,000 different biologically

active molecules of different classes 2. The

complex biosynthetic pathways of many of

these molecules have been elucidated, the

gene clusters cloned, and a number have

been expressed in heterologous hosts for

more detailed studies. There must remain

many more small molecule products to be

isolated since analyses of the completed

genome sequences of several

Streptomyces species reveal the presence

of some 25 different small molecule

biosynthetic pathways, in each genome!

Like most biosynthetic pathways, the

evolutionary origins of the gene clusters

involved in small molecule syntheses are

little understood. Interestingly, there is

recent evidence that some of the pathway

genes were acquired by lateral gene

transfer involving bacterial, fungal and

plant sources 3.

In more recent years, the search for small

molecules with pharmacologic activity

has been extended to other bacterial

genera, as the discovery of new and useful

molecules from the streptomycetes has

become more and more infrequent. One

problem might be that many of the

biosynthetic pathways are cryptic and

conditions for their expression have not

been found.

Are there any specific phenotypic

characteristics that might define certain

classes of bacteria as good producers of

small molecules? Might it be possible to

identify entirely new bacterial genera

useful in this sense?

In fact (perhaps not surprisingly), a very

large number of bacterial species are able

to produce biologically-active small

molecules, many of which have

demonstrated antibiotic activity. These

include Gram-negative bacteria such as

myxobacteria, pseudomonads and even

Escherichia coli. Among the Gram-

positives, Bacillus sp. are prolific

Streptomycetes and beyond

producers of antibiotics and several of

these molecules are still on the market.

Perhaps the most distinctive

characteristics of bacteria producing a

great diversity of small molecules are

large genomes (often 8Mb or larger), with

a high G+C content (>70%), complex

cell development, and a cosmopolitan

distribution as befits saprophytic

organisms with the capacity to metabolise

many different substrates 4 (Figure 1).

However, if the search for organisms

producing novel and useful chemicals is

to be successfully continued, it will be

necessary that the quest for producing

organisms be expanded to include new

families of bacteria, both cultivatable and

non-cultivatable. As a result of work by

several leading scientists in the field,

there have been major advances in the

studies of bacterial phylogeny, and the

identification of novel and unsuspected

taxonomic groups lends credence to the

probability that this approach will help in

the identification of new classes of

antibiotic-producing bacteria. Thus the

streptomycetes are only the tip of the

iceberg of small molecule discovery.

This becomes obvious when we realise

that the Streptomycetaceae are but one of

the members of the order

Actinomycetales, of which there may be at

least 40 known families and well over 100

different genera (and if the estimates of

the number of non-cultivatable bacteria

are correct, there will be many more!).

Many have large genomes with DNA of

high G+C content and have already been

shown to produce small molecules with

useful biological activities. However, we

can expand this chemical and biological

horn-of-plenty even further; since the

order Actinomycetales is but one of the

groups within the class Actinobacteria, a

recently characterised and growing family

that has been assigned its own branch of

the prokaryotic tree. Many of the

Prof Julian DaviesDept. of Microbiology and Immunology

University of British ColumbiaVancouver, BC, CanadaTel: (1) 604 822 5856Fax: (1) 604 822 6041

E-mail: [email protected] Cubist Pharmaceuticals

Lexington, Mass, USA


In Focus

Actinobacteria have been identified only

from 16S rRNA sequences obtained from

environmental DNA and it can be

assumed that there are a large number of

unidentified members of this class. We do

not know how many of the Actinobacteria

have the genetic capacity to produce

small molecules but they may be a

veritable cornucopia of therapeutically

active compounds.

An obvious question to be asked is: “What

are the roles of so many compounds of

diverse chemical structures in the biology

of their producing organisms and the

communities that produce them?”

The full extent of the microbial

population of the biosphere is very poorly

appreciated. Microbes define the limits of

the conditions of life in terms of

temperature (<0–>1400), pH (1–12.8),

pressure and chemical parameters. In

fact, the growth of bacteria defines the

limits of all forms of life, since there is no

other form of life without bacteria.

Why have so few of the microbial

population been identified? This is due

mostly to the lack of the right techniques

to isolate and identify bacteria, especially

from more complex environments. Some

habitats, such as soils, are rich in

microbial content, whereas others, such

as marine environments and those

presenting extremes of chemical and

physical stress, appear to have

significantly lower microbial diversity.

The past 20-30 years have seen great

advances in the cataloguing of bacterial

populations in selected situations by the

use of molecular tools such as DNA

isolation, cloning and sequencing. New

approaches using metagenomic analysis

offer great promise, especially for non-

expressed pathways, but there are many

technical challenges that need to be

overcome before these approaches can

be applied generally. The results so far

are encouraging but, in reality, only serve

to emphasise the enormity of the

problem of identifying the components of

the microbiosphere 5.

One community property that has excited

interest in the past few years is the intense

cooperativity of microbial populations.

This has been realised for some time in the

sense that the survival of all life forms is

absolutely dependent of bacterial

interactions and, in another sense, the

nature of host-pathogen relationships.

This cooperativity is omnipresent in

environmental communities and it has

been proposed that microbial species

always exist in consortia 6, even though a

limited number of strains can be grown in

isolation under laboratory conditions.

However, true microbial life is not

represented on Petri plates! There must

be many biochemical signalling or

communication processes that maintain

microbial communities in stable forms

(even when subjected to fluctuation due

to the pressures of environmental

change). It can be assumed that most, if

not all, microbial interactions have a

chemical basis. Thus the chemical

ecology of microbial populations is a very

important field of study.

Figure 1. Genome size, G+C content and small molecules [modified from L Shimkets].

Genome Size, G+C Content and Small Molecules(modified from L. Shimkets)

Mycoplasma

Chlamydia

Neisseria

Staphylococci

Mycobacteria

Enterobacteria

Pseudomonads

Actinomycetes

52

37

66

50

67

75

Increasing Genome Size and %G+C

SPECIALISTS GENERALISTS

Seco

ndar

yM

etab

olite

Prod

uctio

n

42

40

PRODUCERS OF SMALL MOLECULES

Producers of QS Autoinducers

71Myxobacteria

Cyanobacteria60+


In Focus

There has been considerable interest in

the phenomenon of quorum-sensing in

Gram-positive and Gram-negative

pathogens, in which a variety of simple

organic pheromones are used to sense

population density and regulate metabolic

functions (in the microbe and the host).

As mentioned earlier, most bacterial

producers of biologically-active small

molecules must exist within cosmopolitan

communities which are likely subject to

many fluctuations; a variety of chemical

signals are therefore needed to regulate

metabolic functions in microbes under

these conditions. Both intercellular

signalling and contact-dependent

regulation must be investigated.

Recent studies identifying a range of

activities of antibiotics and other small

molecules demonstrate that they possess

two separate classes of biological activities

depending on whether they are tested at

low (sub-inhibitory) or high (inhibitory)

concentrations 7. The former are

characterised by the modulation (up

regulation or repression) of some 5% of

the cellular transcripts, while the latter

show strong repression of most

transcripts with the exception of a small,

specific number of functions. This is a

clear example of chemical hormesis

which is defined as low dose stimulation

followed by higher dose inhibition of a

biological function 8. In this respect, it is

worth noting that most well known

antibiotics, when used therapeutically at

optimum concentrations, are static and

not cidal agents.

Studies of this type have been performed

with different bacterial species with

compounds that have distinct

macromolecular targets (transcription,

translation, cell-wall metabolism). Thus it

appears that several different forms of

cell-regulatory processes operate in

environmental communities; small

molecules (produced at low

concentrations) act on other members of

the microbial community through

intracellular macromolecular targets to

modulate their metabolic activity and so

influence the stability of the community.

At the present time, the pharmaceutical

industry is suffering from a ‘pipeline-

drought’ due to the failure of the

combinatorial chemistry/high-throughput

screening approaches that were so

heavily promoted (at great cost) over the

past 10 years. Concurrently, the pace of

discovery of naturally-occurring

compounds slowed considerably, largely

due to the low rate of return in terms of

novel active compounds. This is in spite

of the fact that natural products represent

an astronomical number of compounds

that, by definition, must be biologically

active!

The fault obviously does not lie in the

sources but in the creativity of the

searchers; the traditional screening

approaches for active molecules must be

replaced. It may be that the newly

recognised hormetic activities of

naturally-occurring small molecules and

improved methods for the isolation and

characterisation of microbes from

different environments, in combination

with imaginative use of phylogenetic

relationships, will provide new and

successful approaches to the discovery of

antibiotics and other pharmaceutically-

active molecules. One thing is certain,

the drugs are there for us to find.

AcknowledgementI would like to thank Dick Baltz for his

helpful comments

References1. Waksman SA & Woodruff HB. Bacteriostatic and

bactericidal substances produced by soilactinomyces. Proc. Soc. Exp. Biol. Med. 1940;45:609-614.

2. Watbe MG, Tickoo R, Jog MM & Bohle BD. Howmany antibiotics are produced by the genusStreptomyces? Arch. Microbiol. 2001; 176:386-390.

3. Bode HB & Muller R. Possibility of bacterialrecruitment of plant genes associated with thebiosynthesis of secondary metabolites. PlantPhysiology 2003; 132:1153-1161.

4. Bentley SD et al. Complete genome sequence ofthe model actinomycete Streptomyces coelicolorA3(2). Nature 2002; 417:141-147.

5. Curtis TB, Sloan WT, & Scannell JW. Estimatingprokaryotic diversity and its limits. Proc. Natl.Acad. Sci. USA 2002; 99:10494-10499.

6. Buckley MR. Microbial communities:advantages of multicellular cooperation. Am.Acad. Microbiol Colloquium, Washington, DC,2003. Available at http://www.asmusa.org

7. Goh EB, Yim G, Tsui W, McClure J, Surette MG &Davies J. Transcriptional modulation of bacterialgene expression by subinhibitory concentrationsof antibiotics. Proc. Natl. Acad. Sci. USA 2002;99: 17025-17030.

8. Conolly RB & Lutz WK. Nonmonotonic dose-response relationships: mechanistic basis, kineticmodeling and implications for risk assessment.Toxicol. Sci. 2004; 77:151-157.

When it comes to treating Candida,

Diflucan really can. Diflucan has

proven efficacy†1–4 in systemic Candida

infections and is well-tolerated.†1–4 And

with oral and IV formulations, Diflucan

effectively treats Candida in any setting.

Oral / IV

fluconazole/Pfizer

*

†Compared to amphotericin B. Before prescribing, please refer to Abridged Product Information in this publication. FullApproved Product Information is available on request from Pfizer. PBS dispensed price: 50 mg(28) = $177.42; 100 mg (28) = $328.62; 200mg (28) = $624.50; 200 mg (IV) = $196.46. Pfizer Australia Pty Ltd, ABN 50 008 422 348, 38–42 Wharf Road, WestRyde, NSW 2114. References. 1. Anaissie EJ et al. Am J Med 1996; 101:170–176. 2. Anaissie EJ et al. Clin Infect Dis 1996; 23:964–972. 3. Malik IA

et al. Am J Med 1998; 105:478–483. 4. Winston DJ et al. Am J Med 2000; 108:282–289. *Trademark Pfizer Inc. www.pfizer.com.au 02/04 AP35055 PFXDI5348

PBS Information: Authority required. Refer to PBS Schedule for full authority requirement information.

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In Focus

The ‘golden age’ of antibiotic therapy 1 isthreatened by new, existing, emergingand re-emerging pathogens 2. Since thefirst penicillin-resistant Staphylococcusaureus was noted in 1951, a half-centuryof successful anti-infective therapy hasbeen built on the discovery of newantibiotics and their second, third andfourth generation derivatives.

Historically, natural products have animpressive track record, but have provedcomplex at every level of the drugdiscovery, development and productionprocess. Once rediscovery of knownmetabolites exceeded the discovery ofnovel products, major efforts in drugdiscovery were directed away fromnatural product screening 3.

The lure of simple molecules and rationaldesign was irresistible, but these strategies– “driven by chemistry, guided bypharmacology” – have not beendemonstrably successful 4. A massive,parallel, combinatorial chemistry andscreening experiment over billions of yearsin the biosphere has generated complexmolecules distinctly different from thoseproduced by chemical synthesis 5.

Search and discovery for new naturalproduct drugs is so difficult becausemicrobial diversity is so large;biogeography and ecology mean it isheterogeneously distributed overenvironments, space and time, and thechemical diversity sought is dispersedwithin this biological diversity. Molecularecology has revealed the extent of ourignorance of prokaryotic diversity, butculture methods can isolate thisadditional diversity 6. So, dereplication ofbiological diversity is important inselecting the input to new highthroughput, target-based and hightechnology screens.

Taxonomy has three roles inbioprospecting 7:

• Enabling the classification, and thenthe detection and identification oforganisms.

• Predicting metabolic potential.

• De-replicating isolates for screening.

In this view, a general purposehierarchical classification ofmicroorganisms is both possible andpredictive. The opposing view is that aspecial purpose, artificial classification isneeded to detect and identify diversity forbioprospecting. Poor identification oforganisms and partial identification oftheir metabolic potential leave a confusedand incongruent pattern which supportsthis latter view. The challenge is tounderstand how evolutionary andenvironmental forces have shaped thedistribution of bioactive natural productsacross biological diversity, and theimplications for search and discoverystrategies 8.

TaxonomyLateral gene transfer challenges theconstruction of a general purposehierarchical classification, but this remainsthe current taxonomic model. Despite thelimitations of 16S rRNA sequence data fordiscriminating species 9 and in resolvingthe relative branching of deep-rootedphylogenetic lineages 10, it has beenadopted as the framework for prokaryotictaxonomy in Bergey’s Manual 11. The lack

of congruence of some protein gene

phylogenies with 16S rRNA phylogenies

has been attributed to lateral gene transfer,

though whole genome data does find

congruent trees, which largely support the

complexity hypothesis 12. The major

evolutionary lineages are well-defined,

though their deep relationships are

ambiguous, but a consensus of congruent

trees is emerging from core genes – giving

a phylogenetic signal and a Darwinian tree

representing evolutionary relatedness

back to the roots of evolution 10, 13.

In Escherichia coli 755 of 4,288 open

reading frames (ORFs) may have been

acquired since it diverged from Salmonella

enterica ~100 million years (Myr) ago 14.

Anomalous base-composition patterns can

be ‘reverse ameliorated’ by applying

specific rates of mutation/substitution

determined for E. coli 15, until they match

an extant bacterial group, to estimate the

age of transferred genes. In E. coli, the

average age was estimated at 6.7 Myr and

most recently acquired DNA was insertion

sequence elements, fragments of

prophages and remnants of transfer

mechanisms, which are rapidly lost. Once

these are excluded, the average age of the

remaining genes is still only 14.4 Myr. At

this rate, E. coli should have ~1600Kb of

acquired DNA since diverging from S.

enterica, <275Kb of potentially beneficial

genes is present – all recently acquired.

So, these genes are short term

acquisitions. Nevertheless, all the

phenotypic characters separating E. coli

from S. enterica arise from this DNA, not

the estimated 22Kb of mutational change.

Lateral gene transfer is important to

speciation, but may not be so destructive

of the phylogenetic signal – a conclusion

supported by the fact that, even for

evolutionary traits involving well-

established examples of lateral gene

transfer, the gene trees are congruent with

the 16S phylogeny for most organisms 8.

Taxonomy as a roadmap for search and discovery

Dr Alan WardSchool of Biology

University of Newcastle upon TyneNewcastle NE1 7RU, UKTel: (44)191 222 7709Fax: (44)191 222 5228


Prof Michael GoodfellowSchool of Biology

University of Newcastle upon TyneNewcastle NE1 7RU, UKTel: (44)191 222 7706



In Focus

Predicting metabolicpotential

Actinomycetes produce two thirds ofnatural product drugs, withstreptomycetes responsible for 80% ofthese. It seems clear that particulartaxonomic groups, despite theconfounding effect of screening effort, areprolific natural product producers. Wholegenome sequencing supports this – the ‘S.coelicolor’ A3(2)16 and S. avermitilis 17

genomes, both contain >20 naturalproduct gene clusters, far more than othergenomes, e.g. Bacillus subtilis with three,four in Pseudomonas aeruginosa, two inRalstonia solanacearum and none inmost genomes 16.

This means that it is predictable thatmyxobacteria should be a target group toexplore for novel bioactive naturalproducts 18; ‘rare’ genera in theactinomycetes are potential producers ofnovel bioactive secondary metabolites 19;and selective isolation of Amycolatopsis isa target for search and discovery 20. Thesegroups are difficult to isolate, but novelmetabolites from those isolates indicatethey will be prolific producers.

But a strategy of random sampling, genuslevel dereplication and screening only forcommercial significance, meancarbapenems 21 (1978) and monobactams 22

(1981) were the last two major scaffoldsgiving clinically successful antibiotics.Rational search and discovery will needmuch more effort to characterise andmap bio- and chemical diversity.

With more than 5,000 natural productsfrom streptomycetes and more than 48natural products from Streptomyceshygroscopicus 18, predicting metabolicpotential requires a fine level oftaxonomic discrimination if we are to usetaxonomy as a roadmap to discover novelmetabolites. The distribution ofundiscovered bioactive natural productsacross microbial diversity, and theoptimum search and discovery strategiesto exploit them depend upon themechanisms of evolution of organismsand genes. If lateral gene transfer is soextensive that microbial and chemicaldiversity are uncoupled and chemical

diversity, more or less, randomly

distributed, then understanding

biological diversity will be ineffective as a

roadmap to metabolic potential.

But, if environment exerts a selective

pressure which overlays lateral gene

transfer upon vertical gene inheritance,

and the rates of evolutionary change and

biogeographical dispersal are not too

rapid, then exploiting the predictive

power of taxonomy will depend on our

understanding of biological diversity. The

distribution of genes within that diversity,

and the ecology and biogeography of the

organisms, will guide search and

discovery strategies.

Antibiotic resistance genes, in antibiotic

gene clusters, are subjected to strong

selective pressure and extensively

transferred. The acquisition of resistance

genes has been documented in

actinomycetes – e.g. streptomycin

resistance but, also, sometimes,

biosynthetic genes 23. Streptomycin

biosynthesis seems distributed across

many streptomycetes, suggesting lateral

gene transfer plays a significant role in the

distribution of natural product

biosynthesis across biodiversity.

In contrast in 12 reference strains

received as Streptomyces hygroscopicus

or S. violaceusniger 8, five fell outside the

S. violaceusniger 16S rRNA clade –

(S. auranticolor NRRL 8097T,

S. phaeoluteichromogenes NRRL B-5799T,

S. phaeogriseichromogenes NRRL 2834T,

S. phaeoluteigriseus NRRL 5182T and S.

sparsogenes NRRL 2940T). Three

additional species were added to the S.

violaceusniger clade, (S. albiflaviniger and

S. griseiniger and S. geldanamycinus).

The metabolic profiles of the well

characterised members of the S.

violaceusniger clade were determined,

they all show the same pattern of HPLC-

detected secondary metabolites

consisting of geldanamycin, eliaophylin,

nigericin and a characteristic polyene, a

set of metabolites consistent with those

previously found in S. hygroscopicus

strains 24, 25.

DereplicationWoese 26 argues the “radical insight” ofPace 27 to use rRNA for characterisation ofprokaryotes in the environment, “freedmicrobiologists to explore the microbialworld in its entirety” and “made irrelevantwhether organisms existed in pre-culture”. Molecular ecology hasundoubtedly revealed the extent of ourignorance 28 – it has still not provided thatability to “detect and identify organisms”and “define ecological niches inorganismal terms” with which Woese 26

distinguished the classical ecology ofhigher organisms from the “pseudo-ecology” possible for microbiologists.

It is significant that an estimate of>150,000 bioactive metabolites still to bediscovered from Streptomyces 3 is difficultto evaluate. It is clear that brute forcescreening will only be successful by dintof ever decreasing chance. Rationalsearch and discovery strategies needmuch more effort to characterise andmap bio- and chemical diversity – butscreening combinatorial numbers ofsimple molecules or clone librariesgenerated from environmental meta-genomes of unknown size, will notrequire less effort.

Systematics, including taxonomy, like therest of biology, is currently in the throesof a technological revolution.Developments in high throughputsequencing are providing unprecedentedamounts of data. High throughputtechniques for phenotypiccharacterisation, whole genome andmulti-locus sequencing, micro-arrays andsubtractive hybridisation will define theunit of diversity for prokaryotes 9 providerapid and accurate methods fordereplication and identification, and buildup a roadmap for search and discoverystrategies by providing that ability to“detect and identify organisms” and“define ecological niches” in terms of theorganisms present.

References1. Demain AL & Elander RP. The ß-lactam

antibiotics: past, present and future. Antonievan Leuwenhoek 1999; 75:5-19.

2. Cohen ML. Changing patterns of infectiousdisease. Nature 200; 406:762-767.


In Focus

3. Watve MG, Tickoo R, Jog MM & Bhole BD. Howmany antibiotics are produced by the genusStreptomyces? Arch. Microbiol. 2001; 176:386-390.

4. Drews J. Drug discovery: a historicalperspective. Science 2000; 287:1960-1964.

5. Henkel T, Brunne RM, Muller H & Reichel F.Statistical investigation into the structuralcomplementarity of natural products andsynthetic compounds. Angewandte Chemie-International Edition 1999; 38:643-647.

6. Sait M, Hugenholtz P & Janssen PH. Cultivationof globally distributed soil bacteria fromphylogenetic lineages previously only detectedin cultivation-independent surveys. Environ.Microbiol. 2002; 4:654-666.

7. Bull AT, Ward AC & Goodfellow M. Search anddiscovery strategies for biotechnology: theparadigm shift. Microbiol. Mol. Biol. Reviews2000; 64:573-606.

8. Ward AC & Goodfellow M. In: MicrobialDiversity and Bioprospecting. ASM Press,Washington, 2004, p.288-313.

9. Stackebrandt E, Frederiksen W, Garrity GM et al.Report of the ad hoc committee for the re-evaluation of the species definition inbacteriology. Int. J. Syst. Evol. Microbiol. 2002;52:1043-1047.

10. Gribaldo S & Philippe H. Ancient phylogeneticrelationships. Theoret. Pop. Biol. 2002; 61:391-408.

11. Ludwick W & Klenk H-P. In: Bergey’s Manual ofSystematic Bacteriology (2nd ed). Springer, NewYork, 2001, p.49-65.

12. Jain R, Rivera MC & Lake JA. Horizontal genetransfer among genomes: the complexityhypothesis. Proc. Nat. Acad. Sci. USA 1999;96:3801-3806.

13. Brochier C, Bapteste E, Moreira D & Philippe H.2002. Eubacterial phylogeny based ontranslational apparatus proteins. Trends Genet.2002; 18:1-5.

14. Lawrence JG & Ochman H. Moleculararchaeology of the Escherichia coli genome.Proc. Nat. Acad. Sci. USA 1998; 95:9413-9417.

15. Lawrence JG & Ochman H. Amelioration ofbacterial genomes: rates of change andexchange. J. Mol. Evol. 1997; 44:383-397.

16. Bentley SD, Chater KF, Cerdenõ-Tárraga AM et al.Complete genome sequence of modelactinomycete Streptomyces coelicolor A3(2).Nature 2002; 417:141-147

17. Ömura S, Ikeda H, Ishikawa J et al. Genomesequence of an industrial microorganismStreptomyces avermitilis: deducing the ability ofproducing secondary metabolites. Proc. Nat.Acad. Sci. USA 2001; 98:12215-12220

18. Strohl WR. In: Microbial Diversity andBioprospecting. ASM Press, Washington, 2004,p.336-355.

19. Lazzarini A, Cavaletti G, Toppo G & Marinelli F.Rare genera of actinomycetes as potentialproducers of new antibiotics. Antonie vanLeeuwenhoek 2001; 79:399-405.

20. Tan GYA, Ward AC & Goodfellow M. Selectiveisolation of Amycolatopsis strains fromenvironmental samples using antimicrobial

Fluconazole is a triazole antifungal agent.

INDICATIONS: 1. Cryptococcal meningitis in patients unable to tolerate amphotericin B. 2. Maintenance therapy to prevent relapse of cryptococcal meningitis in patients with AIDS.3. Oropharyngeal and oesophageal candidiasis in AIDS and other immunosuppressed patients.4.Secondary prophylaxis of oropharyngeal candidiasis in patients with HIV infection. 5. Seriouslife-threatening Candida infections in patients unable to tolerate amphotericin B. 6. Vaginalcandidiasis, when topical therapy has failed.

CONTRAINDICATIONS: Sensitivity to fluconazole, to related azole compounds or excipients.Concomitant use with cisapride or terfenadine.

PRECAUTIONS: PREGNANCY (Category D); lactation (has been found in breast milk atconcentrations similar to plasma, hence its use in nursing mothers is not recommended);immunocompromised patients who develop rashes; allow for salt content and volume of theinfusion solution; patients who develop abnormal liver function tests should be monitored for thedevelopment of more severe hepatic injury and Diflucan should be discontinued if clinical signsand symptoms consistent with liver disease develop that may be attributable to fluconazole.

**Some azoles, including fluconazole, have been associated with prolongation of the QTinterval on the electrocardiogram. During post-marketing surveillance, there have been very rarecases of QT prolongation and torsade de pointes in patients taking fluconazole. These reportsincluded seriously ill patients with multiple confounding risk factors, such as structural heartdisease, electrolyte abnormalities and concomitant medications that may have been contributory.Fluconazole should be administered with caution to patients with these potentially proarrhythmicconditions.

Drug Interactions: Oral contraceptives; warfarin; sulphonylureas; hydrochlorothiazide;phenytoin; theophylline; astemizole; cyclosporin; rifabutin; rifampicin; tacrolimus; zidovudine;short acting benzodiazepines.

ADVERSE REACTIONS: Headache; nausea; vomiting; abdominal pain; diarrhoea; skin rash;acne; mild transient elevations in hepatic transaminases; clinical hepatitis; cholestasis; fulminanthepatic failure; anaphylaxis; rare cases of leukopenia and thrombocytopenia (causal relationshipnot established); **QT prolongation, torsade de pointes.

DOSAGE & ADMINISTRATION: Normally administered orally; if not possible, by intravenousinfusion (not exceeding 200 mg/hour). Base daily dose on the infecting organism and thepatient’s response to therapy. Continue until clinical evidence or laboratory tests indicate thatactive fungal infection has subsided. Patients with AIDS and cryptococcal meningitis or recurrentoropharyngeal candidiasis often require maintenance therapy to prevent relapse. Diflucan IV hasbeen used safely for up to 14 days. Diflucan intravenous infusion is compatible with Ringer’ssolution; Normal saline. Avoid mixing with any other drug prior to infusion. Adults: Cryptococcalmeningitis: 400 mg on day 1, then 200–400 mg daily. Continue 10–12 weeks after CSFbecomes culture negative. Patients not responding to treatment for up to 60 days are unlikely torespond to Diflucan. Prevention of relapse of cryptococcal meningitis: 100–200 mg daily.Oropharyngeal candidiasis: 100 mg on day 1, then 50 mg daily for 2–3 weeks. Oesophagealcandidiasis: 200 mg on day 1, then 100 mg daily for 2–3 weeks and in severe cases for 2weeks following resolution of symptoms. Secondary prophylaxis against oropharyngealcandidiasis: 150 mg as a single dose once weekly. Serious and life-threatening candidalinfections: 400 mg on day 1, then 200–400 mg daily for at least 4 weeks and for at least 2 weeks following resolution of symptoms. Vaginal candidiasis when topical therapy has failed:150 mg as a single oral dose. Children: Mucosal candidiasis: 3 mg/kg daily. A loading dose of6 mg/kg may be used on day 1. Systemic candidiasis and cryptococcal infection: 6–12 mg/kgdaily. Impaired renal function in adults and children: reduce dose in accordance with theguidelines given for adults. Children below 4 weeks of age: Neonates excrete fluconazoleslowly. Weeks 0–2: same mg/kg dosing as in older children at 72-hour intervals. Weeks 2–4:same dose every 48 hours.

PRESENTATION: Hard Gelatin Capsules: 50 mg, 100 mg, 200 mg – packs of 28; 150 mg –packs of 1. Powder for Oral Suspension: 35 mL bottle containing 50 mg/5 mL of orangeflavoured suspension when reconstituted. Solution for Injection: 2 mg/mL in sodium chloridesolution; 50 mL and 100 mL vials.

Pfizer Pty Ltd (ABN 500 8422 348) 38–42 Wharf Road, West Ryde, NSW 2114. Full Product Information: TGA approved 30 October 1997, Date of last amendment 18 August 2003.(**Please note changes in Product Information at the last amendment). Abridged PI prepared 18 September 2003. *Trademark Pfizer Inc.www.pfizer.com.au 02/04 PFXDI5378 AP35057

Oral / IV

*

(fluconazole/Pfizer)ABRIDGED PRODUCT INFORMATION

agents. In: Abstracts of the 148th Meeting of theSociety for General Microbiology. Heriot-WattUniversity, Edinburgh, U.K. 2001, p.69.

21. Albers-Schonberg G, Arison BH, Hensens OD etal. Structure and absolute configuration ofthienamycin. J. Am. Chem. Soc. 1978; 100:6491–6499.

22. Sykes RB, Cimarusti CM, Bonner DP et al.Monocyclic-lactam antibiotics produced bybacteria. Nature 1981; 291:489–491.

23. Egan S, Wiener P, Kallifidas D & Wellington EMH.Phylogeny of Streptomyces species and evidencefor horizontal transfer of entire and partialantibiotic gene clusters. Antonie vanLeeuwenhoek 2001; 79:127-133.

24. Allen IW & Ritchie DA. Cloning and analysis ofDNA-sequences from Streptomyces hygroscopicusencoding geldanamycin biosynthesis. Mol. Gen.Genet. 1994; 243:593-599.

25. Fang AQ, Wong GK & Demain AL. Enhancementof the antifungal activity of rapamycin by thecoproduced elaiophylin and nigericin. J.Antibiot. 2000; 53:158-162.

26. Woese CR. In: Biodiversity of Microbial Life:Foundation of Earth’s Biosphere. John Wiley &Sons, Inc., New York, 2002, p.xvi-xxxii.

27. Stahl DA, Lane DJ, Olsen GT & Pace NR.Characterisation of a Yellowstone hot springmicrobial community by 5S rRNA sequences.Appl. Environ. Microbiol. 1985; 45:1379-1384.

28. Whitman WB, Coleman DC & Wiese WT.Prokaryotes: the unseen majority. Proc. Nat.Acad. Sci. USA 1998; 95:6578-6583.


Under the Microscope

Following the decision, in the late 1990s,

to sequence the genome of Streptomyces

coelicolor A3(2), the Biotechnology and

Biological Sciences Research Council

(BBSRC) of the UK committed substantial

funding for a coordinated functional

genomics programme aimed at building a

detailed understanding of this model

actinomycete species. For current

information on the S. coelicolor genome

and genome-related tools, see: http://jic-

bioinfo.bbsrc.ac.uk/S.coelicolor/.

The programme involves a number of UK

centres, covering the following post-

genomic technologies:

• DNA microarray development,

provision and training: Colin Smith

(University of Surrey, formerly at

UMIST, Manchester).

• Proteomics: Keith Chater (John Innes

Centre, Norwich).

• Systematic gene disruption: Keith

Chater and Tobias Kieser (JIC) and

Paul Dyson (University of Swansea).

• Bioinformatics: Andy Brass (University

of Manchester); Douglas Kell (UMIST,

formerly at University of Aberystwyth);

Chater (JIC); Smith (Surrey).

These technologies are now well

established in the respective laboratories

and are starting to provide new biological

insights. Whole genome DNA

microarrays have been produced for gene

expression profiling and ‘genomotyping’,

more than 1,000 protein spots have been

identified, more than 1,000 genes have

been knocked out by ‘PCR targeting’ and

in vitro transposition technologies, and

genome, transcriptome and proteome

databases are actively under development.

We have established a BBSRC-supported

resource centre at the University of

Surrey (UniS) for providing Streptomyces

DNA microarrays and associated training

(http://www.surrey.ac.uk/sbms/Fgenomic)

(e-mail, [email protected]). We

have focussed largely on producing PCR-

based DNA microarrays, although we

have recently completed successful side-

by-side trials with long oligonucleotide

arrays; a complete ‘long oligo’ set has

been produced in collaboration with

MWG Biotech, and are now being used to

produce arrays at UniS (Figure 1).

The PCR-generated microarrays have

been tested with commercially important

streptomycetes such as S. clavuligerus

and found to readily detect their

respective orthologous genes. This

opens the way for exploiting the arrays

more broadly in commercial and

taxonomic research programmes.

A great advantage of having the in-house

capacity to produce spotted arrays is that

they can be customised. For example,

gene probes representing antibiotic gene

clusters from other species can be

designed and spotted alongside the ‘core’

S. coelicolor genes, allowing the parallel

measurement of species-specific gene

expression.

For generating PCR products, we

designed an automated primer selection

programme. Similarity searches on each

Functional genomics of Streptomyces coelicolor

Prof Colin P SmithSchool of Biomedical and

Molecular SciencesUniversity of Surrey

Guildford, Surrey GU2 7XH, UKTel: (44) 1483 68 6937Fax: (44) 1483 30 0374


Figure 1. A Streptomyces coelicolor whole genome DNA microarray produced bythe UniS Functional Genomics Laboratory. The cDNA is labelled with Cy3(green) and the genomic DNA is labelled with Cy5 (red). Each spot isapprox 150 microns in diameter.



candidate probe predicted potential

cross-hybridisations and allowed

selection of ‘unique’ PCR products; these

were then generated by a two-stage

procedure, firstly using gene-specific

primers with universal tags and,

subsequently, using universal primers,

one of them being 5’-amidated. Arrays

comprising the majority of open reading

frames have been produced and

protocols for RNA isolation, cDNA

synthesis and hybridisation have been

optimised.

We are now exploiting DNA microarrays

to study global patterns of gene

expression in Streptomyces and are

particularly focussing on investigating

patterns of gene expression in time-

course experiments to investigate

changes that correlate with

developmental and metabolic transitions.

Our analysis has concentrated mainly on

‘surface-grown’ cultures and has revealed

dramatic changes in gene expression at

the ‘decision’ phase prior to the onset of

aerial mycelium and secondary

metabolite formation. Several transiently

induced novel genes have already been

identified that are likely to play roles in

the regulation of development and

antibiotic production.

In parallel, the proteomics effort has

identified (by 2-D PAGE and subsequent

MALDI-MS) more than 1,000 protein

spots (e.g. see http://qbab.aber.ac.uk/s_

coeli/referencegel/ for information), and a

significant number of unusual post-

translational modifications have been

revealed.

Streptomyces coelicolor A3(2) has

become the model system for this genus

of antibiotic-producing bacteria, thanks to

the life-time commitment of Sir David

Hopwood to its genetic analysis 1. The

determination of its complete genome

sequence 2 has made it an even more

valuable model, opening up many

analytical possibilities that emerge

directly from the sequence, and also

various functional genomics approaches.

One particularly exciting prospect is the

high throughput application of MALDI-

ToF mass spectrometry to the analysis of

the overall protein content of cell or

culture extracts. In such a proteomics

approach, proteins separated by multiple

fractionations, most typically by

isoelectric properties and size in classical

2D gel electrophoresis, are digested with

a protease (typically trypsin) before mass

spectrometric analysis. Usually, the

resulting mass fingerprint reveals about

half of the fragments predicted for any

one protein, with a mass accuracy

sufficient to give unambiguous

identification of the cognate gene.

Proteomic analysis of S. ceolicolor

extends the range of global gene

expression analysis beyond the

Streptomyces viewed from the inside: the application of proteomics

to a model streptomycete

Dr Andy HeskethProf Keith Chater

Department of Molecular MicrobiologyJohn Innes Centre

Norwich Research Park, Colney, NorwichNR4 7UH, UK

Tel: (44) 1603 450000Fax: (44) 1603 450778

E-mail: [email protected]@bbsrc.ac.uk

Figure 1. Comparison of 2D gel separations reveals that the abundance of geneproducts from SCO7511, one of three genes for glyceraldehyde-3-phosphate dehydrogenase, is increased by mutation in bldA, whilesynthesis from another, SCO1947, is not significantly affected. The thirdhomologue was not detected.

Parent (early stationary)bldA (early stationary)

SCO1947SCO7511

SCO1947 SCO7511mwt

pI pI



transcription level provided by microarray

analysis, and allows post-transcriptional

regulatory events to be observed and

characterised. For the first time,

processes such as antibiotic production

are being glimpsed from the point of view

of the gene product and how

modification may affect its function.

An initial proteomic survey of liquid-

grown mycelium identified about 10% of

the theoretical proteome 3. Many

hypothetical proteins of unknown

function were observed (they are

therefore no longer hypothetical!), as

were enzymes belonging to secondary

metabolite pathways. The technique is

sensitive enough to detect at least some

regulatory proteins, including 11 of the 82

response regulators of two-component

systems, making these important proteins

amenable to a proteomic approach.

However, certain groups of proteins,

notably integral membrane proteins

including many regulatory kinases, were

absent from the mapping data and appear

to be intractable to 2D gel based

proteomics.

Overall, protein synthesis from about 50%

of the genome could be detected under

the conditions employed (although not

all detectable proteins are abundant

enough to easily allow identification). An

average of 1.2 protein spots per gene

indicated the frequent occurrence of

post-translational modifications. Of 88

examples that were closely scrutinised,

modifications of 10 proteins were

successfully characterised from their

peptide mass fingerprint data.

The S. coelicolor genome contains 20

gene clusters that encode enzymes

characteristic of secondary metabolism,

including three for production of

antibiotics 2. Secondary metabolism

typically begins as cultures enter

stationary phase, and understanding the

underlying regulatory events will facilitate

development of rational approaches for

improving the efficiency of antibiotic

fermentations.

Holt et al 4. used 2D gel electrophoresis to

follow changes in protein synthesis

associated with production of the

antibiotic bialaphos in Streptomyces

hygroscopicus but, because of the

absence of a genome sequence, proteins

of interest could not readily be identified 4.

In S. coelicolor, we have identified

proteins from eight of the 20 secondary

metabolite clusters using proteomics,

including three for novel compounds 3.

Interestingly, some of these proteins also

showed evidence of post-translational

modifications, suggesting that secondary

metabolism is significantly regulated at

the post-transcriptional level. They

included six of the 14 proteins identified

from the cluster responsible for

production of the aromatic polyketide

antibiotic actinorhodin 5.

This may have significant implications for

efforts to produce novel hybrid antibiotic

structures from PKS clusters by

expression in vitro, in heterologous

hosts, or under heterologous promoters.

Clearly, analysis of the regulation of

antibiotic production at the

transcriptome level alone will not provide

a complete picture.

The bldA gene, which has pleiotropic

effects on antibiotic production 6, encodes

the only tRNA that can efficiently translate

mRNA containing the rare leucine codon

UUA, which occurs in 145 S. coelicolor

genes. Thus, in a bldA mutant, TTA-

containing genes, (including the redZ and

actII-ORF4 genes encoding pathway-

specific regulatory proteins for the

undecylprodigiosin and actinorhodin

antibiotic pathways respectively), can be

transcribed, but not usually translated,

into protein. A further 13 regulatory

genes contain a TTA codon.

In order to reveal the global

consequences of bldA mutation we,

together with the group of CP Smith,

have studied changes in both the

proteome and transcriptome during

growth in liquid culture. Most proteome

differences between the bldA mutant and

the parent strain are in proteins which

change in abundance during growth of

the parent from mid-exponential to

stationary phase, including gene products

from six secondary metabolite clusters

(Figure 1).

Only one difference corresponding to the

complete absence of a protein from a

TTA-containing ORF in bldA was

observed. Some differences can be

attributed to previously established links

with TTA-containing regulatory genes,

while several appear to be the result of

polar effects on protein synthesis from

genes immediately downstream of TTA-

containing ORFs. However, most of the

changes detected between the

proteomes are probably indirect effects of

the absence of the 15 regulators encoded

by TTA-containing genes. Continuing

analysis of the proteome and

transcriptome data is expected to

produce an extensive molecular

phenotype of the bldA mutation.

References1. Hopwood DA. Forty years of genetics with

Streptomyces: from in vivo through in vitro to insilico. Microbiology 1999; 145:2183-2202.


3. Hesketh A, Chandra G, Shaw A, Rowland J, KellDB, Bibb M & Chater K. Primary and secondarymetabolism, and post-translational proteinmodifications, as portrayed by proteomicanalysis of Streptomyces coelicolor. Mol.Microbiol. 2002; 46(4):917-932

4. Holt TG, Chang C, Laurent-Winter C, Murakami T,Garrels JI, Davies JE & Thompson CJ. Globalchanges in gene expression related to antibioticsynthesis in Streptomyces hygroscopicus. Mol.Microbiol. 1992; 6:969-80.

5. Hesketh A & Chater KF. Evidence fromproteomics that some of the enzymes ofactinorhodin biosynthesis have more than oneform and may occupy distinctive cellularlocations. J. Ind. Microbiol Biotechnol. 2003;30:523-529.

6. Lawlor EJ, Baylis HA & Chater KF. Pleiotropicmorphological and antibiotic deficiencies resultfrom mutations in a gene encoding a tRNA-likeproduct in Streptomyces coelicolor A3(2). Genes& Development 1987; 1:1305-1310.



In the early 1970s, shortly after David

Hopwood established his Streptomyces

coelicolor group at the John Innes

Institute in Norwich, UK, Alan Vivian

showed that a non-chromosomal genetic

element, SCP1 caused production of, and

resistance to, a diffusible inhibitory

substance 1.

At the same time, in Japan, Haneishi and

colleagues had identified an antibiotic

produced by Streptomyces violaceoruber

SANK 95570 (a close relative of S.

coelicolor), as the epoxycyclopentanone

antibiotic methylenomycin (Figure 1) 2.

Fred Wright and Ralph Kirby subsequently

confirmed that SCP1 did indeed specify

methylenomycin production in S.

coelicolor 3, 4. Kirby also isolated a

number of methylenomycin deficient

(mmy) mutants, certain pairwise

combinations of which could co-

synthesise the antibiotic when grown

near one another. Among the mutants,

‘secretors’ apparently synthesised an

intermediate convertible to

methylenomycin by a second group

(‘converters’), while members of a third

group failed to co-synthesise with each

other or any other mmy mutant 5.

These first ever genetic studies of

antibiotic biosynthesis in Streptomyces,

the pre-eminent natural source of

antibiotics, led to the suspicion that

antibiotic production in these organisms

might generally be plasmid-determined,

but in fact that turned out to be a

misleading exception rather than the rule.

It would be many years before another set

of antibiotic pathway genes would be

found on a plasmid, and in the meantime

numerous sets had been found in various

chromosomal locations.

In the 1980s, the methylenomycin genes

played a significant part in the

development of cloning systems for

Streptomyces. In Stanley Cohen’s lab in

Stanford, Mervyn Bibb carried out the

(equal) first successful cloning in

Streptomyces when he isolated the

methylenomycin resistance gene mmr 6.

Bob Neal later showed this to be the first

example of an integral membrane-located

transporter conferring antibiotic self-

resistance 7. A couple of years later, parts

of the mmy gene cluster were the first

genes to be isolated by the novel

technique of mutational cloning. This

work led to the initial demonstration by

Celia Bruton of what has become a

paradigm, the clustering of genes for

antibiotic production (some in large

operons) with genes for resistance and

pathway-specific regulation 8, 9 (Figure 2).

Revelations from sequencing the methylenomycin

biosynthetic gene cluster:a complex regulatory cascade

Sequencing of most of the mmy cluster

by Celia Bruton and Nigel Hartley in 1997

provided an enormous advance in our

thinking about the pathway and its

regulation. Spurred on by the availability

of this database, Greg Challis proposed a

tentative pathway for the biosynthesis of

methylenomycin 10. An unexpected

feature of the sequence was the presence

of a homologue (mmfL) of afsA, the

biosynthetic gene for the famous γ-

butyrolactone signalling molecule A-

factor in Streptomyces griseus,

accompanied by not one, but two genes

for putative γ-butyrolactone receptors

(mmyR and mmfR, homologues of arpA

of S. griseus). The presence of these

genes suggested that a γ-butyrolactone-

related signalling molecule was involved

in regulating methylenomycin

production. We now know that Kirby’s

original secretor mutants are indeed

secretors of this molecule (now called M-

factor), and are deleted for the

methylenomycin biosynthetic pathway

genes, the converse being true of the

converter mutants.

The sequence of mmfL revealed the

presence of a TTA codon, the rarest of six

leucine codons in Streptomyces DNA

(which is very GC-rich). The gene for the

cognate tRNA, bldA, can be deleted

without affecting growth, but such

mutants are developmentally defective

and lose the ability to make most

antibiotics, presumably due to the

inability to efficiently translate TTA-

containing genes involved in these

processes. A bldA mutant made no M-

factor, and it has been unambiguously

Contributions of methylenomycin to thegenetics of antibiotic production

Dr Sean O’Rourke

Prof Keith ChaterDepartment of Molecular Microbiology

John Innes CentreNorwich Research Park, Colney, Norwich

NR4 7UH, UKTel: (44) 1603 450000Fax: (44) 1603 450778

E-mail: [email protected]’[email protected]

Figure 1. Structure of Methylenomycin A.

O

OCO2H



shown that this TTA codon is responsible.

However, making M-factor production

independent of bldA does not restore

methylenomycin production. Further

inspection of the cluster revealed another

TTA-containing gene (mmyB) with a likely

regulatory character, which needs to be

translated in order for methylenomycin to

be made.

Thus, regulation of methylenomycin

production consists of two consecutive

regulatory systems, one for production of

the likely γ-butyrolactone M-factor, the

other for the methylenomycin

biosynthetic genes, with the product of

the first system being necessary for

activation of the second system, but both

systems also being dependent on the

pleiotropically acting unlinked host gene

bldA.

The methylenomycin system, though

largely ignored by natural products

specialists, has contributed many

significant new concepts to the genetics

of antibiotic production over a period of a

third of a century. We expect that its

further study will continue to reveal yet

more regulatory complexities, and

contribute to a greater understanding of

secondary metabolism.

References1. Vivian A. Genetic control of fertility in

Streptomyces coelicolor A3 (2): plasmid

involvement in the interconversion of UF and IF

strains. J. Gen. Micro 1971; 69:353-364.

2. Haneishi T, Kithara N, Takiguchi Y, Arai M &

Sugawara S. New antibiotics, methylenomycins A

and B. Producing organism, fermentation and

isolation, biological activities and physical and

chemical properties. J. Antibiot 1974; 27:386-392.

3. Kirby R, Wright LF & Hopwood DA. Plasmid-

determined antibiotic synthesis and resistance in

Streptomyces coelicolor. Nature 1975; 254:265-

267.

4. Wright LF & Hopwood DA. Identification of the

antibiotic determined by the SCP1 plasmid of

Streptomyces coelicolor A3 (2). J. Gen. Micro

1976; 95:96-106.

5. Kirby R & Hopwood DA. Genetic determination

of methylenomycin synthesis by the SCP1

plasmid of Streptomyces coelicolor. J. Gen.

Micro 1977; 98:239-252.

6. Bibb MJ, Schottel JL & Cohen SN. A DNA cloning

system for interspecies gene transfer in

mmyR

mmfPmmfH

mmfLmmfR

mmyTmmyO

mmyGmmyJ

mmrmmyK

mmyP

mmyA

mmyCmmyX

mmyD mmyEmmyQ

mmyBmmyY

mmyF orf1orf2

orf3orf4

BiosynthesisResistance

M-factor system Biosynthesis Regulatory

TTATTA

antibiotic-producing Streptomyces. Nature

1980; 284:526-31.

7. Neal RJ & Chater KF. Nucleotide sequence

analysis revels similarities between proteins

determining methylenomycin A resistance in

Streptomyces and tetracycline resistance in

eubacteria. Gene 1987; 58:229-241.

8. Chater KF & Bruton CJ. Mutational cloning and

the isolation of antibiotic production genes.

Gene 1983; 26:67-78.

9. Chater KF & Bruton CJ. Resistance, regulatory

and production genes for the antibiotic

methylenomycin are clustered. EMBO Journal

1985; 4:1893-1897.

10. Challis GL & Chater KF. Incorporation of [U-

13C] glycerol defines plausible early steps in the

biosynthesis of methylenomycin A in

Streptomyces coelicolor A3 (2). Chem. Commun

2001; 10:935-936.

Figure 2. Methylenomycin gene cluster (mmy).

Applications for the followingASM Awards are closing soon!

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For further information visit the ASM websitewww.theasm.com.au



Polyketides are a rich source of

therapeutic agents used in human

medicine, including antibiotics,

antifungals, immunosuppressants and

anticancer agents. Sometimes the natural

producer of these polyketides can be

difficult, or impossible, to cultivate. More

often, the titre of the desired polyketide is

very low.

Rather than focus on development of

many individual polyketide-producing

organisms, scientists at Kosan Biosciences

have developed three Streptomyces spp.

as generic hosts for expression of

polyketide synthase (PKS) genes that

have been cloned from the original

producers. The three hosts are,

Streptomyces coelicolor, S. fradiae and

Saccharopolyspora erythraea.

These three streptomycetes underwent

conventional strain improvement and

fermentation development to enhance

the production of their endogenous

polyketides; then the endogenous PKS

genes were deleted to create ‘clean

hosts’; finally, genetic methods were

developed so that large PKS genes cloned

from other sources could be introduced

and expressed in these hosts by

fermentation.

Heterologous expression of PKS genes in

these ‘super-hosts’ offers several advantages

over conventional improvements to

separate strains:

• Genes from difficult or unculturable

organisms can be expressed.

• The hosts have already been

optimised for poyketide production,

and often produce more of the

desired polyketide than the original

producer of that polyketide.

• Fermentation methods do not have to

be developed from scratch.

• Polyketides that could interfere with

purification have been deleted from

these hosts.

• They are genetically tractable, and a

robust set of expression vectors have

been developed for these strains.

Kosan uses these strains to support its

efforts to develop polyketide drugs with

new or improved pharmacological

properties. For example, genes from

different antibiotic-producing organisms

were combined in the S. fradiae

‘Superhost’ to produce a hybrid

polyketide antibiotic that had not

previously been found in nature, as

shown in Figure 1. In this case, the

original source of each polyketide

antibiotic produced less than 0.1g/L of

their respective polyketides, whereas the

hybrid polyketide was produced at 1.3g/L

in the S. fradiae host.

Superhosts for polyketide drug production

Dr Peter RevillKosan Biosciences Inc3832 Bay Center Place

Hayward, CA 94545, USATel: (1) 510 732 8400Fax: (1) 510 732 8401


OO

O

O

OH

O

O

O

O

OO

HO O O

O

HON

OH

OH

OO

O

O OH

O

O

O O

O

HON

OH

OH

ON

O

OH

O

O

O

OO

OHO

OO

OO

HO

Chalcomycin from Streptomyces bikiniensis

Spiramycin from Streptomyces ambofaciens

OS.ACP

O

O

HO

methoxymalonate from Streptomyces hygroscopicus

O

O

O

OH

O

OO

OO

O

HO O O

O

HO N OH

OH

Tylosin from Streptomyces fradiae

Novel hybrid antibiotic from the Streptomyces fradiae “Superhost”

Figure 1. Novel hybrid antibiotic from the Streptomyces fradiae ‘Superhost’.



IntroductionStreptomycetes are mycelial soil bacteria

that undergo a complex developmental

cycle on solid media. Spores germinate

and form a branched, vegetative

mycelium.

Several signals trigger the formation of

hydrophobic aerial hyphae that

differentiate further into reproductive

chains of spores. Differentiation is

accompanied by the production of

secondary metabolites, e.g. antibiotics.

We have studied the development of

Streptomyces coelicolor in standing liquid

cultures. The media in these cultures

show steep oxygen gradients 1 which are

similar to those found in flooded soils.

For example, nutrient rich media were

already anoxic 1-2mm below the surface.

How does S. coelicolor cope with this?

Recent research indicates that this

bacterium adapts and escapes.

Despite the oxygen limitation in the

standing liquid cultures, S. coelicolor

readily colonised the medium (Figure 1),

implying the presence of an active

anaerobic metabolism 1. Growth of S.

coelicolor under anaerobic conditions

had not been reported before. Yet,

various genes within the genome are

predicted to be involved in low oxygen

stress, nitrate and nitrite respiration, and

fermentation (http://www.sanger.ac.uk/

Projects/S_coelicolor/). This indicates

that this bacterium is fully equipped to

grow under these conditions.

Hyphae in the aqueous anaerobic

environment not only grow freely in the

medium but also attach to and grow over

the hydrophobic surface of the well

(Figure 1) 1, 2. Attachment of hyphae was

reduced in strains in which the rdlA and

rdlB genes had been deleted. These

genes encode for homologous secreted

proteins called rodlins. Rodlins are only

produced by hyphae in contact with a

hydrophobic environment such as a

hydrophobic solid or the air and form (or

are part of) a rodlet-decorated outer cell

wall layer (Figure 2).

The finding that the rdl genes are not

only expressed under aerobic conditions,

but also in oxygen-limited conditions,

suggests that regulation of these genes

and possibly other developmentally

regulated genes as well, is not signalled

through oxygen levels.

After a period of submerged growth,

hyphae in the liquid standing culture

migrated to the air interface (Figure 1) 1.

How hyphae move to this interface is

currently being studied. Possibly,

buoyancy of S. coelicolor is provided by

the formation of gas vesicles encoded for

by two gas vesicle gene clusters that are

contained in the genome. Hyphae in

shaken liquid cultures do not float when

these cultures are no longer shaken

which indicates that shear forces may

have a negative effect on flotation.

The observed decrease in oxygen tension

and nutrient limitation in standing liquid

cultures could form additional triggers for

becoming buoyant. At the air interface

floating colonies were formed that

produced sporulating aerial hyphae

(Figure 1) similar to those on solid agar

media. Interestingly, the floating colonies

were fixed at the air interface by a rigid

light reflecting film. However, this film

does not seem to be involved in enabling

hyphae to escape the water to grow into

the air. This was concluded from a recent

study showing that chaplins

(hydrophobic cell surface proteins

involved in aerial mycelium formation)

fulfil this function.

A strain in which six out of eight chaplins

genes were deleted was strongly affected

in its formation of aerial hyphae 3, 4.

However, the light reflecting film was still

formed 3. Aerial growth on solid medium

could be restored in the mutant by

applying purified chaplins to the colony

surface 3, indicating that chaplins might

act as surfactants. Indeed, mixtures of

chaplins were shown to lower the water

surface tension from 72-28mJ m-2.

This surface activity is accompanied by

major conformational changes in the

proteins. At the water-air interface,

chaplins assemble into small amyloid-like

fibrils that are rich in ß-sheet 3. Chaplins

are the first reported example of

functional amyloid-like proteins in Gram-

positive bacteria and only the second in

the prokaryotic domains.

Future research will focus on metabolism

under oxygen limited conditions and on

the role of the light reflecting film formed

at the water-air interface. Identification of

the molecules that make up this film will

be the first step.

Streptomyces coelicolor in an oxygen-limited liquid environment:

adapt and escape

Dennis ClaessenHan AB Wösten

Lubbert DijkhuizenGeertje van KeulenUniversity of Groningen

Department of MicrobiologyKerklaan 30

9751 NN HarlenThe Netherlands

Tel: (31) 50 3632160Fax: (31) 50 3632154




DC and GVK are supported by a grant of

the Dutch Programme EET (Economy,

Ecology and Technology: EETK01031).

References1. van Keulen G, Jonkers HM, Claessen D,

Dijkhuizen L & Wösten HAB. Differentiation and

anaerobiosis in standing liquid cultures of

Streptomyces coelicolor. J. Bacteriol. 2003;

185:1455-1458.

2. Claessen D, Wösten HAB, van Keulen G, Faber

OG, Alves AMCR, Meijer WG & Dijkhuizen L. Two

novel homologous proteins of Streptomyces

coelicolor and Streptomyces lividans are

involved in the formation of the rodlet layer and

mediate attachment to a hydrophobic solid. Mol.

Microbiol. 2002; 44:1483-1492.

3. Claessen D, Rink R, de Jong W, Siebring J, de

Vreugd P, Boersma FGH, Dijkhuizen L & Wösten

HAB. A novel class of secreted hydrophobic

proteins is involved in aerial hyphae formation in

Streptomyces coelicolor by forming amyloid-like

fibrils. Genes Dev. 2003; 17:1714-1726.

4. Elliot MA, Karoonuthaisiri N, Huang J, Bibb MJ,

Cohen SN, Kao CM & Buttner MJ. The chaplins:

a family of hydrophobic cell-surface proteins

involved in aerial mycelium formation in

Streptomyces coelicolor. Genes Dev. 2003;

17:1727-1740.

Figure 2. The outer surface of aerial structures of S. coelicolor is characterised bya typical ultrastructure called the rodlet layer.

Figure 1. Standing liquid cultures of S. coelicolor demonstrate an extended life cycle, attachment to hydrophobic solids andnovel metabolic pathways.

Final Call for Abstracts

Close of abstract submission deadline: Friday 14 May 2004

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ASM 2004 National Conference

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ASM 2004

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Antimicrobial Resistance & Sunday 26/9/04 9.00am - 5.00pm Sydney University 25% sold

Mechanisms of Resistance

Clinical Mycology Workshop Sunday 26/9/04 9.00am - 5.00pm University of Technology 40% sold

Parasitology Workshop Sunday 26/9/04 9.00am - 1.00pm Sydney Super Dome 20% sold

Education Discussion Session: Sunday 26/9/04 10.00am - 4.00pm Sydney Super Dome

EDSIG Workshop

CDS Users Group Meeting: Tuesday 28/9/04 4.00pm-5.30pm Sydney SuperDome

Clinical CDS Users Update

Serology SIG Colloquium: Tuesday 28/9/04 4.00pm-5.30pm Sydney SuperDome

A Colloquia on the Serology of

Chronic Zoonosis

CDS Users Group Meeting: Wednesday 29/9/04 2.00pm-5.30pm Sydney SuperDome

The use of the CDS Antibiotic

Susceptibility Test in the

Veterinary Laboratory

Serology SIG Colloquium: Wednesday 29/9/04 11.15am-12.45pm Sydney SuperDome

Workshop on quality in serology

Serology SIG Colloquium: Wednesday 29/9/04 4.00pm-5.30pm Sydney SuperDome

Workshop on Uncertainty of

Measurement

Bacterial Identification - Thursday 30/9/04 9.00am - 5.00pm University of Technology 60% sold

Getting Back to Basics

Antibiotic Resistance & Thursday 30/9/04 1.30pm - 5.00pm Sydney SuperDome 20% sold

Pathogenesis in Bacterial

Populations Workshop

Mycobacteria Special Interest Thursday 30/9/04 2.00pm-5.30pm Sydney SuperDome

Group: Mycobacteria Update

Session

Cosmetic and Pharmaceutical schedule to be confirmed

Special Interest Group Workshops:

• Competency Based Training in

the Pharmaceutical Microbiology Lab

• Environmental Monitoring

Programmes

Cosmetic and Pharmaceutical schedule to be confirmed

Special Interest Group Colloquiums:

• Complementary Medicines Post

PAN Pharmaceuticals

• Sterile Process Development

Environmental schedule to be confirmed

How to Apply For Grants - questions, tips, handling rejection schedule to be confirmed

Water schedule to be confirmed

Industry WorkshopsThe trade will be organising and managing a number of workshops and demonstration during the conference. Registration is organised

through the trade and directly with the company holding the workshops. These will be scheduled repeatedly during the conference

and appointment is essential and can be organised through the company's booth in the exhibition area.

The emphasis will be on users and prospective users and will complement the scientific program. No charge is applicable but

appointment is essential.Accommodation & Flights

Discounted accommodation and flights to and from Sydney have been arranged for conference delegates so check out the website for

further info www.asm2004.org Conference Organisers

Australian Society for Microbiology, Suite 23, 20 Commercial Road, Melbourne VIC 3004

Tel: (03) 9867 8699 Fax: (03) 9867 8722 Email: [email protected] Conference website: www.asm2004.org



Despite not having evolved to grow

anaerobically, Streptomyces coelicolor

can nevertheless survive long periods of

oxygen deprivation, apparently by

metabolically ‘ticking over’. Remarkably,

this survival strategy has not been

adopted by all streptomycetes.

Streptomycetes are ubiquitous,

filamentous gram-positive soil bacteria

that have had a major impact in the

biotechnology and pharmaceutical

industries. The complex nature of soil

means that streptomycetes have had to

evolve a broad range of metabolic

pathways to enable them to survive in this

variable environment.

One of the aspects of streptomycete

metabolism that is of interest to us is their

capacity to survive long periods of

anaerobic stress. Despite their

remarkable metabolic diversity, most

Streptomyces species cannot grow under

laboratory conditions in the complete

absence of oxygen. It would be unusual if

the same holds true in the natural

environment, given the extremely

variable oxygen tensions experienced in

soil, with little to no oxygen in wet soil.

It is all the more surprising because the

genome sequence of Streptomyces

coelicolor reveals several enzymes that, in

facultative anaerobes such as Escherichia

coli, confer the ability to grow by

anaerobic respiration 1. Thus, S. coelicolor

has three respiratory nitrate reductase

gene clusters, all of which are expressed,

as judged by transcript analysis.

Two questions arise from these

observations: can S. coelicolor survive the

rapid onset of anaerobiosis and what

mechanisms does it have to survive short-

to long-term exposure to oxygen

deprivation?

S. coelicolor, but not S. avermitilis,survives long periods of anaerobic stress

We have determined that both resting

and pre-germinated spores of S.

coelicolor can survive for as long as 6

weeks under strictly anaerobic

conditions, and grow robustly once re-

exposed to air (Figure 1). In contrast,

Streptomyces avermitilis resting and pre-

germinated spores lose viability after a

few days’ exposure to anaerobic stress.

Although under these conditions there is

no direct evidence for growth, S.

coelicolor clearly is able to maintain

metabolic activity and consequently

viability 2.

Clues from the genomes?The 9Mb linear chromosome of S.

avermitilis and the 8.6Mb linear

chromosome of S. coelicolor share a

6.5Mb ‘core’ region with highly conserved

gene order and gene content 1. The ‘arm’

Streptomycetes and anaerobic stress survival

R Gary Sawers

Jesse Alderson

Janet WhiteDepartment of Molecular Microbiology

John Innes Centre, Norwich, UKE-mail: [email protected]

A. B.

C. D.

Figure 1. Streptomyces coelicolor, but not Streptomyces avermitilis, survives long-term anaerobic stress. S. coelicolor (A and B) and S. avermitilis (C andD) spores were germinated aerobically for 16h on rich medium.Colonies were then left to grow aerobically for 4 days (A and C) or theywere incubated anaerobically for 14 days and then exposed to air for afurther 4 days (B and D).



regions of the respective genomes,

however, exhibit considerable variation.

The enzymes and pathways of central and

RNA/DNA metabolism are conserved,

with the exception that S. avermitilis

encodes a second copy of the high-affinity

cytochrome d oxidase 3. Also, both

species have the NADH/NAD+-responsive

transcriptional regulator Rex, recently

identified in S. coelicolor as a sensor of

cellular redox status 4.

Intriguingly, however, S. avermitilis lacks

all three nitrate reductase operons, which

are present and expressed in the S.

coelicolor genome 3. Whether these

enzymes play a role in anaerobic stress

survival in S. coelicolor is currently under

investigation by gene knock-out

experiments. Apart from these anaerobic

enzymes, there are no obvious candidate

genes to provide a straightforward

explanation for this difference in stress

survival.

Maintenance of a membrane potential is

crucial to viability and nitrate reductase

clearly contributes to this in enteric

bacteria. If nitrate reductase is

dispensable for anaerobic stress survival,

then this suggests that another

mechanism governs this process in S.

coelicolor.

Future challengesS. avermitilis fails to survive long-term

anaerobic stress and therefore is a

‘mutant’ that can be used in

complementation assays to identify genes

in S. coelicolor required for maintenance

of viability. The use of whole genome

microarrays to identify genes with altered

expression in response to anaerobic

stress provides us with a further powerful

tool. The challenge will be to elucidate

the physiological and biochemical basis of

this survival strategy.

References1. Bentley SD et al. Complete genome sequence of

the model actinomycete Streptomyces coelicolorA3(2). Nature 2002; 417:141-147.

2. van Keulen G et al. Differentiation andanaerobiosis in standing liquid cultures ofStreptomyces coelicolor. J. Bacteriol. 2003;185:1455-1458.

3. Ikeda H et al. Complete genome sequence andcomparative analysis of the industrialmicroorganism Streptomyces avermitilis.Nature Biotechnol. 2003.

4. Brekasis D & Paget MSB. A novel sensor ofNADH/NAD+ redox poise in Streptomycescoelicolor A3(2). EMBO J 2003; 22:4856-4865.

Ongoing efforts in the development ofnew anti-infective drugs from nature arenecessary to overcome permanentresistance against clinically significantantibiotics, especially by pathogenicGram-positive bacteria. This problemcannot be solved by expanding existingchemical libraries because the chemicaldiversity in such libraries is narrower thanthat of natural products 1, and because thechemical diversity of natural productscannot be mimicked by organic chemists.

Nature is an almost inexhaustable sourceof novel microorganisms that arepotential producers of natural products,and of new diverse natural products.Actinomycetes still have the mostimportant role to play in screening fornovel bioactive metabolites. Neither themultitude of new actinomycete speciesnor the production of novel secondarymetabolites are limiting factors in searchand discovery programmes.

In the course of HPLC-diode arrayscreening for the detection of novelsecondary metabolites in actinomycete

cultures, we included a set of 29alkaliphilic and alkalitolerantstreptomycetes. The organisms wereisolated from sand-dunes at Warkworth(Northumberland, UK), pine forest soilsat Hamsterley Forest (County Durham,UK), and a steel waste tip soil fromConsett (County Durham, UK). All of thestrains were found to have a range ofchemical and morphological markersconsistent with their classification in thegenus Streptomyces 2.

Our approach has been to use reversedphase HPLC coupled with diode arraydetection (HPLC-DAD) to screenmicroorganisms for the production ofsecondary metabolites 3. Culture filtratesand extracts from culture filtrates andmycelia are analysed by HPLC-DAD, andthe UV-visible spectra of the resultingchromatographic peaks are comparedwith those of reference compoundsstored in our HPLC-UV-Vis-Database. Thedatabase contains about 750 referencecompounds, mostly antibiotics. Knownmetabolites are identified and newmetabolites are characterised accordingto their retention times and UV-visibleproperties. Hits coming out of thisscreening programme are investigatedintensively in scale-up fermentations, byoptimisation of the production, isolationand structure elucidation, and bydetermining biological activity inantibacterial, antifungal, antitumour,antiparasitic and phytotoxic assays.

The alkaliphilic and alkalitolerantstreptomycetes were cultivated in 100-mlshake flasks using two different complex

Alkaliphilic streptomycetes as a source ofnovel secondary metabolites

Prof Hans-Peter FiedlerMikrobiologisches Institut

Universität TübingenAuf der Morgenstelle 28

D-72076 Tübingen, GermanyE-mail: [email protected]

Prof Michael GoodfellowSchool of Biology

University of NewcastleNewcastle upon Tyne, NE1 7RU, UK




media at pH 7, pH 8.5 and pH 10. pH-static cultivation in 500-ml bioreactorsshowed that the growth maximum was atabout pH 9 (or higher); this did notcorrelate with the maximal production ofsecondary metabolites, which wasbetween pH 7 and pH 8. In some strainsthe production of known secondarymetabolites was observed, as exemplifiedby N-acetyltyramine, actinomycins,actiphenol, arylomycins, chromomycin,SEK 15, thiotetronic acid andtirandamycins. Three streptomycetestrains – AK 409, AK 623 and AK 671 –were the subject of closer scrutinybecause their metabolite peak patternscould not be identified by comparison oftheir UV-visible spectra with those ofreference compounds stored in thedatabase.

Streptomyces sp. AK 409This strain was isolated from a steel wastetip soil from Consett, County Durham as anew species. Comparison of an almostcomplete 16S rRNA gene sequence of the

strain with available corresponding

sequences of representative

actinomycetes showed that it forms a

distinct branch within the Streptomyces

griseus 16S rRNA gene subclade. The

strain became attractive because of the

detection of two prominent metabolites

in a culture extract which could not be

identified by means of the HPLC-UV-Vis-

Database.

Fermentation, isolation and structural

elucidation revealed that one of the

metabolites was pyrrole-2-carboxylic acid,

a known natural product. However, the

main compound was identical with

pyrocoll, which has been described as a

constituent of cigarette smoke. Pyrocoll

is known as a synthetic compound, but

until now it had not been isolated as a

natural product from microorganisms 4.

Its structure is shown in Figure 1.

Pyrocoll is the cyclic condensation

product of two molecules of pyrrole-2-

carboxylic acid.

The evaluation of the biological activitiesof pyrocoll brought astonishing results tolight. Pyrocoll showed biologicalactivities against various Arthrobacterstrains, filamentous fungi, severalpathogenic protozoa, and some humantumour cell lines 4. It also inhibited cellgrowth in the human tumour cell linesHM02, HepG2 and MCF 7 but did notexert cytotoxic effects. It exhibitedmoderate activities against Plasmodiumfalciparum, the pathogenic agent ofmalaria, against Leishmania donovani,the pathogen of visceral leishmaniasis(‘Kala Azar’) and against Trypanosomacruzi, the causative agent of Chagasdisease, and Tryponosoma bruceirhodesiense, the pathogen of Africansleeping sickness.

Streptomyces sanglieri AK 623Strain AK 623 was isolated from a sampleof the A2 mineral horizon of a pine forestsoil collected at Hamsterley Forest. Acombination of genotypic and phenotypicdata clearly showed that the organism

Figure 1. Structurally elucidated secondary metabolitesfrom alkaliphilic Streptomyces strains AK 409, AK623 and AK 671.

O

OH

HO OH

OH

N

N

O

O

O

H3CO

O

NO

O

OHCH3

O

OHO

O

O

H3C

OH

OH

O

O

HO O

CH3

O

O

CH3

COOH

OH

OH

pyrocoll

lactonamycin Z

4-hydroxyscytalone

(2-methyl-4-oxo-4H-chromen-5-yl)acetic acid

3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid

O

OHHO

HO

HOOC

OH3C

CH3

OOHOR

671-C1: R =

671-C2: R = H

Contact CrContact Cryosite for detailsyosite for details

phone phone ++61612 94202 9420 1 1400400emailemail atcc atcc@@crcryosite.comyosite.comwwwwww.cr.cryosite.comyosite.com

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belongs to the recently described species,Streptomyces sanglieri 5. Two prominentpeaks with congruent UV-visible spectrawere detected by HPLC-DAD analysis ofthe culture filtrate extract, and a furtherpeak was detected in the myceliumextract which could not be identified byour HPLC-UV-Vis-Database.

Both metabolites from the culture filtratewere structurally elucidated by the groupof Prof Günther Jung (Institut fürOrganische Chemie, Universität Tübingen)as lactonamycin Z, a new derivative oflactonamycin, and its aglycone,lactonamycinone. Lactonamycin Zshowed weak activity against Gram-positive bacteria but strongly inhibited theproliferation of gastric adenocarcinomacells in the G2/M cell cycle phase 6.

The metabolite in the mycelium extractwas structurally determined as 4-hydroxyscytalone by the group of ProfGerhard Bringmann (Institut fürOrganische Chemie, UniversitätWürzburg). 4-Hydroxyscytalone isdescribed in the literature as a phytotoxicsubstance produced by various fungi, butit has not previously been isolated frombacteria. The structurally elucidatedcompounds from strain AK 623 are shownin Figure 1.

Streptomyces sp. nov. AK 671Strain AK 671 was also isolated from asample of the F-horizon of pine forest soilcollected at Hamsterley Forest. An almost

Figure 2. Diversity of secondary metabolites produced by alkaliphilic Streptomycessp. nov. AK 671 detected in HPLC-DAD analyses.

complete 16S rRNA gene sequence ofstrain AK 671 was compared tocorresponding sequences ofrepresentatives of the genusStreptomyces. The resultant dataindicated that the organism forms adistinct phyletic line in the 16S rRNAStreptomyces gene tree and henceprobably belongs to a new species.

Strain AK 671 was highlighted during ourHPLC-DAD screening programmebecause of its variability in producingsecondary metabolites given changes inthe cultivation regime, particularly thecultivation medium. More than 20different secondary metabolites weredetected in the culture filtrate extractwhich could be related mainly topolyketide type 2-compounds by theirUV-visible spectra (Figure 2).

Metabolite 671-C2 was structurallyelucidated and postulated to be apreviously un-isolated intermediate in thebiosynthesis of chrysophanol, a typicalsecondary metabolite of various plantsand fungi. 671-C1 was determined as theglucuronide of 671-C2.

A further secondary metabolite, compound671-D, was structurally elucidated as (2-methyl-4-oxo-4H-chromen-5-yl) acetic acidwhich is a novel natural product.Compound 671-F was identified as 3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid, a rare member of the familyof α-methylanthraquinones.

Further metabolites produced by strainAK 671 are still the subject of structuralstudies being carried out by the group ofProf Gerhard Bringmann.

In summary, it can be said that alkaliphilicstreptomycetes are a highly interestingsource of secondary metabolites. Twentytwo out of 29 of the tested strains (76%)characterised by HPLC-DAD analysis wereshown to produce secondary metabolites,and, of those, only six (27%) were foundto produce known natural products. Inantibacterial and antifungal assays, 10 outof 29 strains (34%) showed inhibitoryactivity against Gram-positive bacteria,and 18 out of 29 strains (62%) exhibitedinhibitory activity against both Gram-positive bacteria and fungi. Pyrocollproduced by strain AK 409 showedpromising results in in vitro tumourcolony assays which must be nowconfirmed by in vivo tumour modelsraising hopes in the development of anew selective antitumour agent.

AcknowledgementsThe authors wish to thank the EuropeanCommission for financial support withinthe 5th framework (grant QLK3-CT-2001-01783, project Actapharm). The fruitfulcollaboration with Prof Dr GerhardBringmann, Universität Würzburg, andProf Dr Günther Jung, UniversitätTübingen, is gratefully acknowledged.

References1. Henkel TRM, Brunne RM, Müller H & Reichel F.

Statistische Untersuchung zur Strukturkomplexitätvon Naturstoffen und synthetischen Substanzen.Angew. Chem. 1999; 111:688-691.

2. Manfio GP, Zakrzewska-Czerwinska J, Atalan E &Goodfellow M. Towards minimal standards forthe description of Streptomyces species.Biotechnologia 1995; 7-8:242-253.

3. Fiedler HP. Biosynthetic capacities ofactinomycetes. 1. Screening for secondarymetabolites by HPLC and UV-visible absorbancespectral libraries. Nat. Prod. Lett. 1993; 2:119-128.

4. Dieter A, Hamm A, Fiedler HP, Goodfellow M,Müller WEG, Brun R, Beil W & Bringmann G.Pyrocoll, an antibiotic, antiparasitic andantitumor compound produced by a novelalkaliphilic Streptomyces strain. J. Antibiot. 2003;56:639-646.

5. Manfio GP, Atalan E, Zakrzewska-Czerwinska J,Mordarski M, Rodriguez C, Collins MD &Goodfellow M. Classification of novel soilstreptomycetes as Streptomyces aureus sp. nov.,Streptomyces laceyi sp.nov. and Streptomycessanglieri sp. nov. Antonie van Leeuwenhoek2003; 83:245-255.

6. Höltzel A, Dieter A, Schmid DG, Brown R,Goodfellow M, Beil W, Jung G & Fiedler HP.Lactonamycin Z, an antibiotic and antitumorcompound produced by Streptomyces sanglieristrain AK 623. J. Antibiot. 2003; 56:1058-1061.

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The Gause Institute of New Antibiotics,

Russian Academy of Medical Sciences, in

Moscow, Russia, was established in 1953

(Figure 1). For more than 25 years it was

headed by late Professor GF Gause and,

from 1986, it was directed by the late

Professor Yurii V Dudnik.

In the process of screening for new

biologically active substances, a number

of antibiotics were developed based on

original strains isolated in the Institute,

and these were produced on industrial

scale for the pharmaceutical industry.

These include antibacterial antibiotics

gramicidin S, monomycin (paromomycin),

colimycin (neomycin), albomycin, risto-

mycin, lincomycin, kanamycin, heliomycin

and representatives of nearly all-important

groups of antitumour antibiotics, e.g.

olivomycin, bruneomycin (streptonigrine),

rubomycin (daunorubicin), carminomycin

and bleomycetin (bleomycin A-5).

The main direction of the research

programme at the Institute involves

screening for antibacterial activity, in

particular screening for antibiotics active

against resistant bacteria, and screening

for antitumour and antiviral antibiotics. It

also includes studies on immuno-

modulators and chemical transformation

of antibiotics with the purpose of

generating new promising derivatives.

Furthermore, the programme also

contains methodologies to devise new

and improved methods for isolating rare

and novel organisms, revealing microbial

diversity; and investigations of cultivated

Basidiomycetes as a source of biologically

active compounds.

The screening of bioactive secondary

metabolites has traditionally focused

mainly on actinomycetes newly isolated

from natural sources and based on eco-

geographical and taxonomic approaches.

Filamentous fungi are also being screened

for bioactive metabolites.

For the isolation of actinomycetes from

soil, various methods are being applied

both conventional and selective. A set of

new isolation techniques has been

devised recently in the Institute for

selective isolation of rare actinomycetes

with the use of different kinds of

irradiation (UV-light, electrocurrent

pulses, SHF- and EHF-radiation) for the

pretreatment of soil samples (Figure 2).

A new procedure employing succession

analysis in combination with EHF

irradiation and other complex methods

combining different kinds of pretreatment

and subsequent plating of samples on

selective media supplemented with

antibiotics are also applied. The use of

various selective isolation procedures

allows the detection of microbial diversity

and the isolation of new and rare

actinomycetes for screening.

Besides physical factors, the effects of

biological factors on soil microbial

communities and interactions between

microorganisms of various taxons are

investigated. Studies carried out on the

endogenous differentiation regulator, the

A-factor, revealed that it also can act as an

exogenous regulator stimulating the

outgrowth of spores and differentiation of

some actinomycete species and also

stimulating the growth of nonfilamentous

bacteria 1.

The collection of microorganisms of the

Institute (INA) contains predominantly

actinomycetes, including type strains of

the species described by the Institute

taxonomists, producers of antibiotics, and

representatives of various rare genera

isolated in the Institute. The strains

maintained in the collection can be

exploited for basic scientific research and

in different biodiscovery programmes.

Biodiscovery programme conducted at theGause Institute, Moscow, Russia

Prof LP TerekhovaGause Institute of New Antibiotics

Russian Academy of Medical SciencesBolshaya Pirogovskaya, 11

119021 Moscow, RussiaTel: (7) 095 246 9980Fax: (7) 095 245 0295


Figure 2. Examples of Actinomycetes isolated for screening of bioactive compounds.

Figure 1. Gause Institute.



Early detection of producers of known

antibiotics among actinomycete isolates

and early identification of known

structures in crude culture extracts are

effectively carried out in the process of

screening. An efficient system of isolation

of antibiotics from culture broth with the

subsequent purification and structure

elucidation was developed. Among a

great variety of antibiotic compounds

obtained in the Institute from isolated

actinomycete strains, antibiotics

echinomycins, rifamycin, tobramycin,

apramicin, manumycin, ostreogrisins,

cervinomycins etc, should be mentioned.

Research is carried out on chemical

modifications of natural antibiotics obtained

earlier in the Institute such as antitumour

antibiotics daunorubicin, carminomycin,

bruneomycin (streptonigrine) and

antibacterial antibiotic eremomycin of the

vancomycin group.

A series (more than 400) of new semi-

synthetic derivatives of antitumour

antibiotics of anthracycline group was

obtained and investigated. It was found

that novel types of derivatives of

doxorubicin and carminomycin are active

against MDR tumour cells and they are

not substrates for P-glycoprotein (Pgp) 2.

The investigations on glycopeptide

antibiotics have been directed to

semisynthetic derivatives active against

MDR bacteria especially against VanA and

VanB enterococci and MRSA. The

introduction of hydrophobic moieties

into the peripheral regions of molecule

gives derivatives active against MDR

bacteria 3.

In the course of study of mechanism of

action of the glycopeptide derivatives

containing hydrophobic moieties, it was

shown that the activity against

vancomycin resistant enterococci was not

caused by antibiotic-D-Ala-D-Lactate

binding. It was found that the presence

of hydrophobic moieties in the molecule

of eremomycin and its derivatives much

more affects antibacterial activity than

their ability to dimerise (ESI mass-spectra

data). Recently antiretroviral activity of

glycopeptide antibiotics and their

semisynthetic derivatives against HIV-1

and HIV-2 has been revealed 4.

The most promising antibiotic for clinical

use discovered in the Institute is

eremomycin. Several other antibiotics

isolated at the Institute earlier, e.g.

esperamycins, chartreusin, anthracyclines,

illudin, streptonigrine and some others

are under investigation.

In the course of screening of antibiotics

active against MRSA, a number of

compounds suitable for studying in vivo

have been obtained. Some active

compounds with hypolipidemic and

antifungal properties have also been

isolated.

References1. Grusina VD, Efremenkova OV, Zenkova VA,

Reznikova MI & Dudnik YuV. A-factor as selective

agent for isolation of the soil Gram-negative

bacterium strain, producing antibacterial

antibiotic. Antibiotiki i Khimioterapiya 2003;

48:11-16 (in Russian).

2. Tevyashova A, Shtil A, Olsufyeva EN, Simonova

VS, Samusenko AV & Preobrazhenskaya MN.

Carminomycin, 14-hydroxycarminomycin and its

novel carbohydrate derivatives potently kill

human tumor cells and their multidrug resistant

variants. J. Antibiotics 2004 [In press].

3. Printsevskaya SS, Pavlov AY, Olsufyeva EN,

Mirchink EP, Isakova EB, Reznikova MI, Goldman

RC, Brandstrom AA, Baizman ER, Longley CB,

Sztaricskai F, Batta G & Preobrazhenskaya MN.

Hydrophobic derivatives of glycopeptide

antibiotic eremomycin and des-(N-methyl-D-

leucyl)eremomycin; chemistry and antibacterial

activity. J. Med. Chem. 2002; 45:1340-1347.

4. Balzarini J, Pannecouque C, DeClercq E, Pavlov

AY, Printzevskaya SS, Miroshnikova OV, Reznikova

MI & Preobrazhenskaya MN. Antiretroviral

activity of semisynthetic derivatives of

glycopeptide antibiotics. J. Med. Chem. 2003;

46:2755-2764.



Actinomycetes are soil microbes well

known for their ability to produce a wide

variety of bioactive compounds, including

antibacterial, antifungal, antitumour and

immunosuppressants agents. Close to

50% of the known microbial products are

produced by actinomycetes. In particular,

the discovery, development and clinical

use of antibiotics has been one of the

most significant medical advances in the

20th century, and antibiotics are probably

the most prescribed class of drugs.

However, the effectiveness of many

antibiotics has been severely diminished

by the insurgence and spreading of many

antibiotic-resistant pathogens, with the

consequent need for novel and better

antibiotics.

Discovery of novel antibiotics from natural

sources represents quite a challenge.

Streptomyces spp. have long been

recognised as the best antibiotic-producing

bacteria, and it can be estimated that

several million strains have been

extensively screened by the pharmaceutical

industry. Consequently, the chances of

isolating a novel Streptomyces strain have

substantially diminished. This implies that

the chances of discovering a novel

antibiotic from a Streptomyces strain by

traditional approaches will require a

substantially larger effort 1.

Therefore, in order to decrease the

probability of rediscovering known

compounds, novel strategies are required

in the search for new antimicrobial

products 2. These strategies must not

ignore the probabilistic nature of a

screening approach – a significant

number of microbes must be screened to

have a reasonable chance to discover a

new antibiotic with useful properties.

In the last decade, significant advances in

molecular genetics and genomics have

suggested alternative routes to antibiotic

discovery from natural sources. Current

estimates indicate that only 1% of

microbial strains are related to known

taxa, leading to the proposal that these

uncultured strains, or simply their DNA

expressed in a convenient host, could

represent a novel source of bioactive

compounds 3. In addition, genomic

studies have indicated that the potential

to produce secondary metabolites is not

uniformly distributed among bacteria,

with some taxa possessing few or no

genes for secondary metabolism.

Interestingly, it can be assumed that the

average Streptomyces strain 4, 5 and

possibly other actinomycete genera 6 may

have the genetic potential to produce a

dozen or so different secondary

metabolites. Since many of these clusters

are apparently unexpressed under normal

conditions, they could represent an

additional source for novel antibiotics 7.

An alternative approach, which is

currently pursued at Vicuron

Pharmaceuticals, would be to concentrate

efforts on unusual or difficult to isolate

microbes that are phylogenetically related

to good producers of secondary

metabolites. According to this strategy,

we have prepared a proprietary collection

of over 60,000 strains, mostly non-

Streptomyces actinomycetes and slow-

growing filamentous fungi. Since these

strains are hard to isolate, they are

unlikely to have been screened in large

numbers in the past.

Because they are phylogenetically related

to good producers of secondary

metabolites, they are likely to share the

same large genetic potential for

producing bioactive compounds. This

strategy rests on the assumption that the

ability to produce large numbers of

bioactive metabolites is a hallmark of

filamentous actinomycetes, and that

strains distantly related to cultured and

heavily exploited taxa offer a higher

probability of possessing clusters

containing novel combinations of

secondary metabolism genes, and hence

a higher probability of yielding novel

compounds.

In this respect, molecular tools can

greatly help in the identification of

promising sources of poorly described

actinomycete genera and in the quick

recognition of as-yet uncultured

representatives of these bacteria.

Isolation programmes can be oriented by

prescreening soil samples for the

presence of DNA derived from

uncommon genera of actinomycete 8,

while the extent of the genetic diversity of

newly isolated strains can be established

through rapid fingerprints 9. Our results

also indicate that the so-called ‘rare

actinomycetes’ are relatively abundant in

the soil, and they can be retrieved in large

numbers if a suitable isolation method is

available.

In the long run, however, if isolation

programmes are successful and

uncommon actinomycete strains are

isolated in large numbers, these taxa are

eventually going to become part of the

Exploiting and expanding actinomycetediversity for antibiotic discovery

Dr Margherita Sosio

Dr Stefano DonadioVicuron Pharmaceuticals

Gerenzano, ItalyE-mail: [email protected]



exploited groups of strains, leading to a

decreased result from equivalent effort.

Through analysis of soil DNA, we

observed 16S rRNA sequences ascribable

to as yet-uncultured groups of

actinomycetes 8. We reasoned that many

uncultured actinomycetes exist in the

environment, and they could be cultured

under appropriate conditions. To this

end, soil samples showing an interesting

diversity of actinomycete DNA can be

recognised and aliquots plated on a

variety of different conditions.

Morphologically unusual strain can be

rapidly classified through 16S rDNA

sequencing, leading to their phylogenetic

assignment within the Actinobacteria.

Next, their genetic potential to produce

secondary metabolites could be rapidly

established.

According to this scheme, illustrated in

Figure 1, strains belonging to new

actinomycete taxa were isolated and

identified (unpublished results).

Interestingly, many of them possess the

typical genes for secondary metabolism

that make Streptomyces strains successful

antibiotic producers, and thus these

strains constitute a potential source of

secondary metabolites worth of further

investigation. The success of this

approach depends on the use of isolation

methods that counterselect rapidly

growing actinomycete strains and on the

application of objective methods for

establishing strain identity.

In conclusion, opportunities exist to

exploit the genetic capability of microbes

for discovering valuable bioactive

metabolites. The success of new

approaches will ultimately depend on the

ability to rapidly assemble and effectively

screen a large diversity of gene clusters

for secondary metabolism. The

molecular structures observed today from

natural sources represent the results of

million of years of evolution. It is hard to

imagine that future drug discovery can be

effective without tapping into the rich

source of chemical diversity offered by

microbial products.

References1. Watve MG, Tickoo R, Jog MM & Bhole BD. How

many antibiotics are produced by the genusStreptomyces? Arch Microbiol 2001; 176:386-390.

2. Bull AT, Ward AC & Goodfellow M. Search anddiscovery strategies for biotechnology: theparadigm shift. Microbiol. Mo. Biol. Rev. 2000;64:573-606.

3. Rondon MR, Goodman RM & Handelsman J. TheEarth’s bounty: assessing and accessing soilmicrobial diversity. Trends Biotechnol 1999;17:403-9.


5. Omura S et al. Genome sequence of anindustrial microorganism Streptomycesavermitilis: deducing the ability of producingsecondary metabolites. Proc. Natl. Acad. Sci.USA 2001; 98:12215-20.

6. Sosio M, Bossi E, Bianchi A & Donadio S. Multiplepeptide synthetase gene clusters in Actinomycetes.Mol. Gen. Genet. 2000; 264:213-21.

7. Challis GL & Hopwood DA. Synergy andcontingency as driving forces for the evolution ofmultiple secondary metabolite production byStreptomyces species. Proc. Natl. Acad. Sci. USA2003; 2: 14555-61.

8. Monciardini P, Sosio M, Cavaletti L, Chiocchini C& Donadio S. New PCR primers for the selectiveamplification of 16S rDNA from different groupsof actinomycetes. FEMS Microbiology Ecology2002; 42:419-429.

9. Mazza P, Monciardini P, Cavaletti L, Sosio M &Donadio S. Diversity of Actinoplanes and relatedgenera isolated from an Italian soil. MicrobialEcology 2003; 45:362-372.

Figure 1. Scheme for identification of isolated ‘uncultured’ actinomycetes.

atypical isolation media

soil

soil fractions

morphologicalanalysis

known taxa unknowntaxa

16S rDNA sequencing

culturedtaxa

uncultured taxa

isolated “uncultured” strains

phylogeneticanalysis



Currently, the wealth of data available for

studying bacterial genotypes provided by

genome sequencing has resulted in an

increased interest in horizontal gene

transfer (HGT). It appears that there has

been considerably more gene flow

horizontally than was first thought 1. The

extent of HGT was reported in the case of

E. coli and Salmonella enterica, where

both lineages had each gained and lost

more than 3Mb of novel DNA since their

divergence some 100 million years ago 2.

Sequence analysis suggests that horizontal

gene transfer followed by gene

rearrangements has been involved in the

evolution of pathways with catabolic and

degradative functions. Biosynthetic

pathways such as those involved in

antibiotic production may equip organisms

with a selectable trait, which is adaptive in

certain environments such as soil.

The gene cluster involved in streptomycin

(Sm) production and resistance (SmR) has

undergone recent horizontal gene

transfer (HGT) and genes were recovered

in complete functional clusters and also in

partial clusters, which were not expressed3.

Previous work has demonstrated that the

resistance gene, strA, had undergone HGT

and in some cases was expressed but not

in others 4, 5. Some of the clusters

recovered appeared to consist of gene

mosaics, with some genes having high

similarity to homologues in Stretomyces

griseus, while other were more diverse

and did not group with any previously

characterised genes.

A new gene cluster was detected in

recently isolated strains of S. griseus (SmR)

and this requires further characterisation

to determine if it is responsible for

production of a novel aminoglycoside

with self resistance and resistance to

streptomycin. Analysis of a soil recovered

from a soft fruit orchard receiving

plantomycin (contains streptomycin as

major active component) to control fire

blight showed that S. griseus was the

dominant species isolated at all sites

(Table 1).

In addition, all strains not identified still

showed similarity to S. griseus, which was

Horizontal gene transfer within streptomycetes

Sahar Tolba

Prof Elizabeth MH WellingtonDepartment of Biological Science

University of WarwickCoventry, CV4 7AL, UKTel: (44) 24 76523184Fax: (44) 24 76523701


Soil sites % of identified Identification Nucleotide similarity StrA† StrB1†

isolates of isolates of 16S rDNA*

Apple 45 S. griseus 99-100 + +

22 S. platensis 99 + +

31 Streptomyces. spΔ 96 + –

Current 44 S. griseus 100 + +

28 S. platensis 99 + +

16 S. setonii 99 + +

11 S. roseoflavus 99 + –

Cotswold 45 S. griseus 98-99 + +


14 S. globisporus 100 + –

Coventry 67 S. griseus 100 + +


* % blast nucleotide similarity of 16S rDNA.† strA and strB1 detected by PCR and hybridisation. Δ Isolates with <97% nucleotide similarity to S. griseus DSM (40236) were identified as Streptomyces. Sp.

Table 1. Identification of selected isolates from the four soil sites according to partial sequencing of 16S rDNA including the γregion (% of isolates having the same identification and resistance level).



recorded as the closest species. Therewas a correlation between the lowerlevels of phenotypic resistance and theabsence of the biosynthesis gene strB1.

All isolates identified as S. griseuscontained the strB1 in addition to strA,and showed a high level of SmR. Isolatesidentified as S. platensis were found to beresistant to a high level of Sm and hadstrA genes homologous to S. griseus DSM40236 (GenBank accession numbers areAF510495 and AY114134), thus providingclear evidence of horizontal gene transferinvolving SmR.

This phenomenon was only observed inthe orchard soils. However, we have nowfound str genes homologous to those ofS. griseus in a number of other distinctspecies including S. limosus, S. coelicolorand S. cinnamoneus. The discovery ofmore highly diverse str genes in S. griseuswas unexpected but it has yet to beshown if the strA detected in these strainsis responsible for high level streptomycinresistance.

The streptomycin genes strA and strB1were widely distributed in isolatesrecovered from all sites; however, thepercentage containing strA was higher inthe streptomycin-treated soil isolatescompared with the control. Thissuggested that selection for SmR hadoccurred and the resistance generecovered from the S. griseusstreptomycin biosynthesis cluster waspredominant.

Previous studies provided evidence forhorizontal gene transfer (HGT) of both anintact functioning streptomycin genecluster and a partial gene cluster from S.griseus into a set of diversestreptomycetes. However, the recoveryof a new more diverse str cluster distinctfrom all know previously reportedclusters is intriguing especially as it wasrecovered in S. griseus. All the isolatesidentified as S. griseus by 16S rRNAsequence homology did show diversitywhen examined phenotypically and wererecovered in separate clusters as shown inFigure 1. The type strains groupedtogether in cluster B and C, whilst the soil

Figure 1. Phenogram of soil isolates and type strains based on UPGMA analysis of 41phenotypic characteristics 6. The type strains in cluster B and C werepreviously grouped as members of the Streptomyces albidoflavus cluster 1.

isolates formed a distinct cluster A. It is

possible that strains, once isolated and

cultured in the laboratory, can undergo

quite significant genetic and phenotypic

change.

References1. de la Cruz F & Davies, J. Horizontal gene transfer

and the origin of species: lessons from bacteria.Trend Microbiol. 2000; 8:128- 133.

2. Lawrence and Ochman. Amelioration ofbacterial genomes: rates of change andexchange. J. Mol. Evol. 1997; 44:383-397.

3. Egan S, Wiener P, Kallifidas D & Wellington EMH.Phylogeny of Streptomyces species and evidencefor horizontal transfer of entire and partialantibiotic gene clusters. Antonie vanLeeuwenhoek 2001; 79:127-33.

4. Wiener P, Egan S & Wellington EMH. Evidencefor transfer of antibiotic resistance genes in soilpopulations of streptomycetes. Mol. Ecol. 1998;7:1205-1216.

5. Egan S, Wiener P, Kallifidas D & Wellington EMH.Analysis of streptomycin biosynthetic geneclusters in streptomycetes isolated from soil.Appl. Environ. Microbiol. 1998; 64:5061-5063.

6. Williams ST et al. A probability matrix foridentification of some streptomycetes. J. Gen.Micro. 1983; 129:1815-1830.

1.00.90.80.70.6

36691

720 638 651

970 947

654 666

973

705

708

709

704

751

736

724

726

944

985

745 759

S. gr . farinosus 40932

744

S. bacillaris

S. griseus 40693 S. griseus 40236

S. roseochromogenes 40463

S. griseus 40855 S. vinaceus

S. griseus 40707

S. griseus 40654 S. californicusS. griseus 40657 S. griseus 40659

S. griseus 40660 S. olivoveridis

S. griseus 40939

S. citreofluorescensS. reticuli

S. mediocidicus

S. albidusS. griseus 40817

S. albovinaceus

S. acrimycini

S. fradiaeS. cretaceus

S. willmoreiS. lipmanii

S. griseus 40670

S. alboviridisS. fluvissimus

S. roseochromogenes 40856S. floridaeS. halstedii

S. griseobrunneus

1.00.90.80.70.6

676

718

104

983

632

712

965

967

97

A

C

B



Search and discoveryBiotechnology is based on the search for,and discovery of, exploitable biologicalresources. The course of biotechnologysearch and discovery starts with theassembly of appropriate biologicalmaterials. It then moves throughscreening for a desired attribute andselecting the best option from among ashort list of positive screening hits, andculminates in the development of acommercial product or process 1.

In this search, the screening of microbialnatural products continues to representan important route to the discovery ofnovel chemicals for development of newtherapeutic agents, and the evaluation ofthe potential of lesser-known and/or newbacterial taxa is of increasing interest.However, selection of novel bioactiveproducing microorganisms from naturerequires a sound microbial taxonomicalknowledge and a full understanding ofmicrobial ecology and physiology asmeans for revealing novelty 2.

Approaches to the isolation of potentiallyvaluable bacteria have been, and still are,largely empirical and restricted to thesampling of a tiny fraction of themicrobial community found in manyenvironments. Consequently, it stillremains unclear which components ofthe isolated bacteria and fungi representan indigenous microflora, in particularrare taxa, in most soils. In order toimprove the detection and isolation ofthe rare and diverse microorganisms fromnatural environments, new and moreobjective selective isolation procedureshave to be designed using theinformation generated through microbialphysiology and ecology 1-3.

Discovery of new molecules fromactinomycetes, beginning with

Streptomycin in 1944, has marked anepoch in antibiotic research andsubsequent developments in antibioticchemotherapy. Since then, actinomyceteshave provided about two thirds of thenaturally occurring antibiotics whichcurrently number more than 10,000 4.

In the search for novel antibiotics,attempts have been made to designisolation programmes to recover the so-called ‘rare actinomycetes’ from naturalenvironments. The development andapplication of highly selective isolationtechniques has given a significant impetusto the discovery of new actinomycetederived compounds of medicalimportance 4.

Continuing industrial need for thediscovery of new products will alwaysdrive the design of highly specificselective isolation techniques 5. However,in an era, which tends to neglect theimportance of designing objectiveselective isolation protocols and classifiesmost organisms as ‘unculturable’, it isvital that the following points are takeninto consideration in the search forindustrially important actinomycetes:

• Increased knowledge of microbialecology and physiology.

• Study of neglected habitats and moreintensive investigations of the betterstudied ones.

• Improved sampling procedures,particularly for marine habitats.

• Development of more objective, lessconservative isolation procedures.

• Provision of more efficient identificationsystems to determine novelty.

• Understanding of the natural roles ofsecondary metabolites.

Additionally, in order to obtain newstrains likely to produce novelmetabolites, examination of samples fromdiverse habitats as well as thoseinhabiting extreme environments such asacidophilic, alkalophilic, neutrophilic,mesophilic and osmophilic strains isnecessary 2. These studies should becoupled together with investigationsconducted towards the functionaldiversity of the rare actinomycete taxa inthose environments.

Unique Australianbiodiversity

In the above context, Australia could be

one of the ‘hot spots’ for search and

discovery. It is one of the world’s most

biodiverse continents. It has been

geologically separated from other

continents for over 20 million years,

which has allowed a period of extensive

evolutionary divergence. As a result,

Australia has a very high rate of endemism

in both its flora and fauna.

Queensland is one of the most diverse

States of Australia. However, little is

known about the functional diversity of

its actinomycete communities. Although

some isolations have been done for

natural screening programmes by various

research institutes and companies, the

true taxonomic relationships between

microorganisms and their functional roles

within Queensland’s unique biota are still

lacking.

The State Government of Queensland isnow fully supportive of the generation ofa Microbial Genetic Resources Centre.

Uniqueness of the ‘Smart State’s’ microbial diversity

From an Actinomycete collection to biodiscovery at the University of the Sunshine Coast

Dr Ipek KurtbökeUniversity of the Sunshine Coast

Faculty of Science,Queensland, 4558




Figure 1. Phylogenetic analysis of an University of the Sunshine Coast isolate.

Actinomadura coerulea U49002

Actinomadura citrea U49001

Actinomadura luteofluorescens U49008

Actinomadura glucoflavus AF153881

Actinomadura mexicana AF277195

Actinomadura verrucosospora D50667

Actinomadura fibrosa AF163114

Actinomadura formosensis strain DSM 4399

Actinomadura pelletieri AF163119

Actinomadura cremea subsp. cremea AF1340

Actinomadura cremea rifamycini U49003

Actinomadura madurae D50668

Actinomadura latina AY035998

Actinomadura macra U49009

Actinomadura nitritigenes AY035999

Actinomadura fulvescens U49005

Actinomadura atramentaria U49000

Actinomadura rugatobispora U49010

Actinomadura oligospora AF163118

USC 427

Actinomadura rubrobrunea AF134069

Actinomadura hibisca AF163115

Actinomadura kijaniata U49006

Actinomadura namibiensis strain DSM 4419

Actinomadura vinacea AF134070

Actinomadura viridis strain DSM 43175T.

Actinomadura yumaensis AF163122

Actinomadura catellatospora AF154127

Actinomadura livida AF163116

Actinomadura umbrina AF163121

Actinomadura echinospora U49004

Actinomadura glomerata AF134068

Actinomadura longicatena AF163117

Actinomadura aurantiaca AF134066

Actinomadura libanotica U49007

Actinomadura spadix AF163120

% Difference



Accessing the diverse, endemic and

largely ‘unscreened’ microbial

biodiversity of Queensland, which is

strategically positioned with respect to

other regions of high biodiversity in

South East Asia and Oceania, will provide

a strategic edge for the ‘Smart State’.

Actinomycete library at theUniversity of theSunshine Coast

The University of the Sunshine Coast

(USC) in Queensland has established a

actinomycete library utilising the

microbial genetic resources of the most

biodiverse State of Australia in the search

for novel bioactive compounds. This

library now will be part of the large

microbial library, which is now being

established in the State with participation

of the State’s other expert Institutes.

Through the use of effective conventional

and molecular detection procedures and

focussing principally on the State’s unique

habitats and microenvironments, the USC

has isolated rare and novel actinomycetes

as potential sources of novel therapeutic

agents. The USC now holds a collection of

over 3000 actinomycetes containing over

75% novel strains (Figures 1 & 2) and

showing strong antimicrobial activity

against vancomycin and methycillin

resistant bacteria.

The library has already attracted

international partners and compounds

are being studied towards different

targets such as antimicrobials, immuno-

suppressants and anti-cancer compounds

as well as enzymes and agrobiologicals.

Microbial Genetic ResourcesCentre at the Smart State

The USC’s microbial library is expected

to:

• Contribute to the ongoing efforts of

the Queensland government to map

Queensland’s unique microbial

diversity to attract biotechnological

investment.

• Complement the studies conducted

by other research institutes in the

State towards the establishment of the

State’s new ‘Microbial Genetic

Resources Facility’.

• Provide a reservoir of strains for

future work on the production of

useful antibiotics, enzymes, and other

biologically active compounds, which

can be potentially commercialised in

the State.

References1. Bull AT, Ward AC & Goodfellow M. Search and

discovery strategies for biotechnology: the

paradigm shift. Micro & Mol. Biology Rev 2000;64:573-606.

2. Goodfellow M & Williams ST. New strategies forthe selective isolation of industrially importantbacteria. Biotech. & Gen. Eng. Rev. 1986; 4:213-262.

3. Kurtböke DI. Exploitation of host-phageinteractions for the selective isolation of theindustrially important bacteria. Med. Chem. Res.1996; 6:248-255.

4. Lazzarini A, Cavaletti L, Toppo G & Marinelli F.Rare genera of actinomycetes as potentialproducers of new antibiotics. Antonie vanLeeuwenhoek 2000; 78:388-405.

5. Kurtböke DI. Selective Isolation of RareActinomycetes. Queensland Complete PrintingServices, Australia, 2003.

Saccharopolyspora hirsuta X53196

Saccharopolyspora hirsuta M20388

USC 426

Saccharopolyspora sp. IM-8155 AF131491





Saccharopolyspora hordei X53197

Saccharopolyspora sp. (A215) X76967

Saccharopolyspora flava AF154128

Saccharopolyspora spinosa AF002818

Saccharopolyspora rectivirgula X53194

Saccharopolyspora thermophilus AF127526

Saccharopolyspora gregorii X76962

Saccharopolyspora taberi AF002819

Saccharopolyspora erythraea X53198

Saccharopolyspora spinosporotrichia Y095

% Difference

Figure 2. Phylogenetic analysis of an University of the Sunshine Coast isolate.


Selective Isolation of Rare ActinomycetesEdited by: Dr Ipek Kurtböke

Actinomycetes, in particular streptomycetes, have been described as the greatest source of antibiotics since the discovery

of Streptomycin from Streptomyces griseus in 1944. Since then, actinomycetes have provided about two thirds of the

naturally occurring antibiotics which currently count more than 9000. These include aminoglycosides, anthracyclines,

chloramphenicol, β-lactams, macrolides, tetracyclines and other industrially important secondary metabolites.

Over the years, most antibiotics and other useful secondary metabolites from these common genera have been studied,

identified and applied in industry. Consequently, the search for novel compounds has concentrated on rarely isolated

genera. However, the detection and isolation of these genera is impeded by bacteria and faster growing large

streptomycete colonies on isolation plates. Specific isolation procedures are required to detect these organisms.

This book deals with the isolation procedures against this background. Authors describe methods, which have led to the

successful isolation of bioactive compound producing rare actinomycetes from different environments, with the belief

that these methods will complement the ongoing efforts in the industry and research institutions, and facilitate the rapid

detection and isolation of industrially important rare actinomycetes.

Contributing Authors and Chapters:

Dr Ipek Kurtböke, University of the Sunshine Coast, Australia

Use of Bacteriophages for the selective isolation of rare actinomycetes

Prof. Masayuki Hayakawa, Yamanashi University, Japan

Selective isolation of rare actinomycete genera using pretreatment techniques

Prof. Larissa Terekhova, Gause Institute of New Antibiotics, Russia

Isolation of Actinomycetes with use of microwaves and electric pulses

Dr. Takao Okazaki, Sankyo Co. Ltd., Japan

Studies on Actinomycetes isolated from plant leaves

Published by:Queensland Complete Printing Services, Nambour, Australia

ISBN: 0 646 429 10-8

Enquiries to [email protected]

Actinomycete WebsitesFor the new John Innes Streptomyces Manual (with users’ testimonials) see:

http://www.jic.bbsrc.ac.uk/SCIENCE/molmicro/Strepmanual/Manual.htm

For the Streptomyces coelicolor genome database (ScoDB II) and genome-related tools see:

http://jic-bioinfo.bbsrc.ac.uk/S.coelicolor/

Website of the S. coelicolor genome project:

http://www.sanger.ac.uk/Projects/S_coelicolor/

The Digital Atlas of Actinomycetes by Shinji Miyadoh, Japan

http://www.nih.go.jp/saj/DigitalAtlas/


Emerging Microbiologists

Catherine GangellPh.D student

Respiratory MedicinePrincess Margaret HospitalRoberts Road, Subiaco, WA


Neil WilsonEMMA Laboratory E8A 260

Department of Biological SciencesMacquarie University, NSW 2109

Tel: (02) 9850 6977E-mail: [email protected]

Philip ButtonPhD student (The University of Melbourne)

Food Science AustraliaPrivate Bag 16, Werribee, VIC 3030

E-mail: [email protected]@foodscience.afisc.csiro.au

In this issue of Microbiology

Australia we showcase the work of

two young microbiologists who are

just starting their careers.

Future editions of Emerging

Microbiologists will present the

‘work in progress’ of honours

students, graduate students and

post doctoral researchers, as well as

focussing on career opportunities

in microbiology.

By doing this we hope todemonstrate the incredible diversityof microbiological research beingdone in Australia as well as givingstudents a forum in which todescribe their work.

These contributions are notreviewed and do not have to be acompleted project in order to beincluded in this section – it is morean opportunity for graduatestudents to present a ‘seminar’ on

their work in order to inform

students in other parts of Australia

about the range of research being

carried out in laboratories in

CSIRO, universities, hospitals and

other institutions.

If you would like to be included in

this section, please contact your

nearest State representative (above)

or the editor of Microbiology

Australia, p.bishop@ usyd.edu.au

Abbott Diagnostics Division

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bioMerieux Australia Pty Ltd

Bio-Rad Laboratories

Blackwell Publishing Asia

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Dade Behring Diagnostics

Diagnostic Technology

The Kelly Company Pty Ltd

In Vitro Technologies Pty Ltd

Millipore Australia Pty Ltd

Ortho – Clinical Diagnostics

Oxoid Australia Pty Ltd

Sabac Pty Ltd

TECRA International Pty Ltd



Lynne Dailey

Department of Public Health

Curtin University of Technology, WA

Tel: (08) 9246 4114


About Lynne

I have just completed a Master of Public

Health degree, majoring in epidemiology

and biostatistics from Curtin University of

Technology, following on from a science

degree from the University of Western

Australia majoring in microbiology.

Currently I am enrolled in a PhD at Curtin

researching syndromic surveillance for

the early detection of infectious disease

outbreaks. My main motivation for this

line of research stems from my interest in

emerging infectious diseases and the

opportunity to work with, and learn from,

my supervisors. I also feel this project will

build on knowledge developed during my

undergraduate and postgraduate studies.

Research wise, I would like to remain in

the area of emerging infectious disease,

with a focus on the epidemiology and

public health aspects of this subject.

Following on from a PhD, I would like to

continue with a post doc in this line of

research and travel overseas.

IntroductionGram-negative septicaemia (GNS) is an

important cause of morbidity and

mortality in hospitalised patients. An

increase in the incidence of GNS over the

past few decades has been connected

with the widespread use of broad-

spectrum antibiotics and advances in

medical treatments for severe conditions.

In this study, the frequency of Gram-

negative organisms associated with

septicaemia at a large teaching hospital

was determined. Particular emphasis was

placed on the type of organism isolated

from community- and hospital-acquired

septicaemia, antimicrobial susceptibility

patterns, possible risk factors and

outcomes.

Methods

Reports of all blood cultures taken at Sir

Charles Gairdner Hospital (SCGH), in the

period from January 1998 to December

2002 were analysed retrospectively to

identify reports of Gram-negative

organisms. Cases of microbiologically

documented septicaemia caused by

Gram-negative organisms were linked

and merged with hospitalisation data for

these episodes. This data set was

analysed to determine the incidence of

GNS and risk factors associated with

demographic characteristics. In-hospital

mortality was the principal outcome

variable evaluated. Secondary outcomes,

including length of stay (LOS) and

readmission for GNS within 30 days of

hospital separation, were also assessed.

Results and discussion

The incidence of GNS remained stable

over the 5 year period, with an average of

3.4 episodes per 1000 separations. A total

of 1270 episodes of septicaemia were

documented from 1998-2002, of which

29% were considered community-

acquired and 71% hospital-acquired.

Escherichia coli (34.9%), Klebsiella

pneumoniae (17.6%), Pseudomonas

aeruginosa (9.4%), Enterobacter species

(9.4%) and Stenotrophomonas

maltophilia (6.3%) were the predominant

Gram-negative bacteria isolated.

Polymicrobial infections accounted for

18% of episodes of GNS. Risk factors

associated with the development of GNS

included increasing age, male gender,

underlying comorbidities, a surgical

procedure, or admission to an intensive

care unit.

The LOS for patients with GNS was on

average 9.1 days longer than all other

patients at SCGH. The overall mortality

of patients with GNS was 14.7%. Higher

mortality rates were associated with

increased age, male gender, transplantation,

intensive care stay and P. aeruginosa.

The non-fermentative species P.

aeruginosa and S. maltophilia displayed

the highest levels of resistance to tested

antimicrobials in the study.

The current study identified risk factors

and outcomes similar to those reported

elsewhere. The incidence of S.

maltophilia infections was higher than

reported in other studies. Overall, GNS

was associated with significantly longer

hospital stays compared with all patients

at the hospital and high GNS readmission

rates within 30 days of discharge. From a

public health perspective, this condition

is costly to both the hospital and the

patient.

Identification of patients at greatest risk,

host factors of greatest importance, and

awareness of ecologic and epidemiologic

aspects of GNS, are essential for the

development of adequate preventative

measures and early clinical recognition.

Hence, these data may allow clinicians to

identify patients at risk and better target

empirical therapy for hospital-acquired

cases of septicaemia.

Acknowledgements

This project was under the supervision of

Professor Thomas Riley, Professor Aileen

Plant and Dr Tim Inglis. I would like to

thank them for their support, suggestions

and invaluable knowledge. I would like to

thank Ms Alison Sewell of the

Management Information Services at

SCGH and Mr Brett Richards in the

Information Systems Department at

SCGH/Path Centre for their technical

assistance.

Trends in Gram-negativeSepticaemia at a Perth

teaching hospital, 1998-2002



through this association with Food

Science Australia, I was offered the

opportunity to perform research for my

PhD in the related area of HPP.

IntroductionBivalve molluscan shellfish, such as

oysters, clams and mussels, have been

frequently implicated in incidents of

foodborne viral disease in Australia and

throughout the world, most commonly

by the Norovirus (NV, previously known

as the Norwalk-like virus) and the

Hepatitis A virus (HAV).

These viruses are introduced into the

aquatic environment via contaminated

sewage, by either surviving the treatment

process or being flushed into waterways

with untreated wastes following heavy

rainfalls.

The filter-feeding nature of bivalve

molluscs allows contaminants in the

water to be ingested and concentrate

within the shellfish tissues. Whilst

depuration techniques are efficient at

removing bacterial contamination from

shellfish, viruses are retained within

tissues for a longer period, posing a

public health risk to consumers when the

raw product is consumed.

HPP is a non-thermal process that can

inactivate spoilage and pathogenic

microorganisms, whilst having little effect

on the organoleptic and nutritional

qualities of foods. HPP has been used to

extend the shelf life of a variety of foods

throughout the world, including oysters,

which are also shucked during the

process. HPP-treated South Australian

Pacific oysters, with an extended shelf life,

are already on the market in Australia.

However, before HPP is applied to

shellfish for the purpose of virus

inactivation, systematic inactivation

kinetic data is required to measure the

susceptibility of the viruses to a range of

pressure treatments. This information for

human enteric viruses, such as HAV and

NV, is currently not available.

AimThe aim of this research is to obtain

inactivation kinetic data of HAV and feline

calicivirus (FCV), a surrogate to the non-

culturable NV, following treatments with

high pressure. Pure cultures of FCV and

HAV in buffered suspensions will be

treated with HPP under a range of

environmental conditions.

The inactivation data obtained can

subsequently be used to create a HPP

kinetic inactivation model that can be

validated in not only Australian bivalve

molluscs, but in many ready-to-eat foods

that may be at risk of contamination with

these viruses.

To investigate virus retention in oysters,

viruses will be accumulated by the natural

filter feeding process of oysters in

custom-built tanks containing artificially

contaminated seawater, and compared to

the direct injection of viruses into

homogenised oyster tissue.

Various methods to extract virus from

shellfish tissue have been described in the

literature, each with a varied degree of

effectiveness. An optimal method for the

extraction of virus from oyster tissue will

be determined, based on its reliability,

efficiency, labour, cost, etc. Similarly,

methods for quantitation of the purified

virus will be compared, and a method of

optimising real time RT-PCR to detect

only whole, viable virus particles will be

sought to improve the sensitivity of this

quantitative technique.

The futureThe design of a HPP inactivation model

for these viruses is just the beginning of

this research. Once created, the model

may be validated in many foods, and

expanded to increase its capabilities.

I would like to continue my involvement

with non-thermal food processing

technologies, as well as in the

development of inactivation models for a

variety of pathogenic microorganisms.

About Stephen

My undergraduate studies were based at

RMIT University, where I completed the

Bachelor of Applied Science (Applied

Biology/Biotechnology) in 2001, and

honours in 2002. My honours research

project investigated the application of

high power ultrasonics to inactivate

Campylobacter species for use in poultry

processing.

The majority of experimental work during

my honours year was undertaken at Food

Science Australia, a joint venture between

CSIRO and the Victorian Department of

Primary Industries, where research into

the use of novel food processing

techniques such as ultrasonics and high

pressure processing (HPP) is carried out.

This is where my interest in alternative

food processing techniques began and,

Stephen GroveSchool of Agricultural Science/

Tasmanian Institute ofAgricultural Research, University of

Tasmaniain conjunction with

Food Science Australia671 Sneydes Road (Private Bag 16),

Werribee VIC 3030

Tel: (03) 9731 3361

Fax: (03) 9731 3250


Development of a highpressure processing

inactivation model for humanenteric viruses and itsapplication to bivalve

molluscan shellfish processing


ASM Affairs

Award nominations invited

For further informationconcerning these awards, pleasecontact the National OfficeTel: (03) 9867 8699E-mail: [email protected]

New MembersNew South Wales

Bronwyn BeavanHelen BoydShear Ch’ng

Ruth CornforthManuela Dieckelmann

Jacqueline FaltasKatherine Ferguson

Andrew GinnJennifer Hitchcock

Raquel IbanezJulie Irish

Jenna IwasenkoCandy Jacques

Tass KaralisDebbie KoShiuan KohJanice LamClaus LangAmie Lau

Jennifer MakAnne-Laure Markovina

Naomi MaslenMeryta May

Penelope McCartney

David NewsomeSophie OctaviaSandra Oliver

Ferdousi RelwanNicola RogersGlenn RoseAnna SalimNathan SaulVira SophaJoanne Tan

Chin Yen TayElise Tu

QueenslandIbrahim Diallo

Christopher FrancoJosie Hayward

Esther HodgsonBevan Kennedy

Deborah KrishnaWendy Lardner

Tamar LawLyle McMillen

Simon MorrowDaniel Powell

Pracscilla TagoreKim WestonDavid Whiley

South AustraliaPaul Costello

Carissa CourtneySusan Semple

TamaniaTara Carswell

Katrina McFadyen

VictoriaDeborah BaldiLauren BingeKathryn DavisDavid Franken

Samantha GeorgeStephen Grove

Owen HarrisAnne Hendtlass

Anthony KeyburnElizabeth Lukaczynski

Ellisa McFarlaneCatherine Osborne

Western AustraliaBlackaby Diagnostics Pty Ltd

Fiona EdwardsTara Fernandez

Jasmin HergChelsea Longbottom

Maha ShihataAmy StrachanTrina-Jean TanTessa VanzettiJulius Varano

Elizabeth Watkin

OverseasDavinder Dhillon – Malaysia

Thomas Henderson – New Zealand

Alexandre Esteban – SpainJeffrey Driscoll – USA

Gifty Immanuel – India

ASM Distinguished Service Award

The ASM Distinguished Service Awardprovides a mechanism whereby theAustralian Society for Microbiologyrecognises outstanding service of, orcontributions by, individuals ororganisations to the Society.

It is intended that both individuals andcorporations be eligible for the award. Theselection criteria for an individual and foran organisation are different. Membershipof the Society is not a requirement.

Individuals or organisations deemedworthy of recognition for DistinguishedService to the Society may be nominatedby members or branches to the Presidentof the Society. The informationaccompanying the nomination shouldinclude a summary of the individual’s ororganisation’s contribution to the Society.

The selection committee will consist of theExecutive Committee of the NationalCouncil. Recommendations concerningthe Distinguished Service Award will bemade to the National Council, at whichtime a decision concerning the award willbe made.

Honorary Life MembershipHonorary status is the highestmembership recognition given by theSociety and carries with it all the rightsand privileges of a Member or Fellow,together with exemption from paymentof the annual subscription.

Nominations for election to Honorary LifeMembership may be forwarded toCouncil by 30 June in any calendar year.At this time, nominations will be groupedand presented to the HonoursSubcommittee for assessment and review.Based upon the recommendation of theHonours Subcommittee, nominees maybe elected to Honorary Life Membershipby National Council, if feasible. No morethan three Honorary Life Members shallbe elected in any 1 year.

The Council may elect a person as anHonorary Life Member who, in itsopinion, has rendered distinguishedservice to the science of microbiology, tothe Society, or to both. A person shall notbe considered for election as an HonoraryLife Member unless the Council hasreceived from the proposer:

• A nomination for Honorary LifeMembership signed by the proposer,a seconder, and 10 other signatorieseach of whom shall be a Member orFellow of the Society.

• A complete curriculum vitae, togetherwith a condensed summary andphotograph suitable for publication inMicrobiology Australia.

• A statement summarising thenominee’s major contribution to thediscipline and/or practice ofmicrobiology, to the Society, or toboth, together with informationattesting to the high personal andprofessional standards of conduct ofthe candidate; and

• A bibliography of scientificpublications and/or contributions tothe relevant area of microbiology.

This column will make a

regular appearance in

Microbiology Australia to

keep members abreast of

what this Standing

Committee of ASM

is doing.

For further details contactJanice Stavropoulos,National convenor


Join Now!Join Now!

Parasitology andTropical Medicine

SIG


ASM Affairs

The current members of the committee

are:

• Stephen Graves (chair)

• David Ellis

(ex-officio, as President of ASM)

• Jock Harkness (NSW)

• Jan Lanser (NSW)

• David Looke (QLD)

• Jenny Robson (QLD)

• Alister McGregor (TAS)

• Peter Ward (VIC)

• Michael Leung (WA)

• Arthur Morris (NZ)

This new committee has been in

operation for approximately 1 year and

has as its role:

• To advise ASM on issues relevant to

the conduct of clinical microbiology

in Australia.

• To encourage the development of

standards in the scientific practice of

microbiology in clinical diagnostic

laboratories.

• To contribute to the scientific

activities of the ASM in the area of

clinical microbiology.

• To liaise with other groups and

societies in Australia in relevant areas

of clinical microbiology.

The members of the committee all have

expertise in clinical microbiology and

have been invited to join the committee

Standing Committee on Clinical Microbiology

Dr Stephen GravesDirector of Microbiology

Hunter Area Pathology Service (HAPS)John Hunter Hospital,

Newcastle, NSWTel: (02) 4921 4420

Mobile: 0407 506 380Fax: (02) 4921 4440


for a 2 year term. A attempt has been

made to have persons:

• From different parts of Australasia

(only SA & NT are currently

unrepresented).

• From public and private laboratories.

• Who are either medically qualified or

scientifically-qualified in microbiology.

We communicate by e-mail as issues arise

and meet once a year in person at the

Annual Scientific Meeting. The following

issues were dealt with during 2003:

• The Therapeutic Goods Administration

(TGA) discussion paper A proposal for

a new regulatory framework for in

vitro diagnostic devices (2003) was

discussed and a formal submission made to

the review committee of TGA.

• Request from the National Association

of Testing Authorities (NATA) about

the need for separate incubators for

uninoculated tissue cultures and

inoculated flasks in virology

diagnostic laboratories.

• Request form the Pharmaceutical

Benefits Advisory Committee for advice

on proposed restrictions on the use of

antiviral drugs in the treatment of

genital herpes by the Pharmaceutical

Benefits Scheme (PBS).

• Request from NATA for advice about

quality control (QC) of tissue culture

cell lines, viral culture medium, sub-

culture of QC microbes and passaging

of cell lines (numbers of passages).

The first issue for 2004, currently under

consideration, pertains to the new

pathology agreement (1 July 2004 to 30

June 2008) between the Commonwealth

government and the pathology

profession, represented by:

• The Royal College of Pathologists of

Australia (RCPA).

• The Australian Association of

Pathology Practice (AAPP).

• The National Coalition of Public

Pathology (NCOPP).

There are some issues in the proposed

Pathology manpower, quality and outlay

agreement that are relevant to ASM

Members.

If any ASM member would like to see the

response of the Standing Committee on

Clinical Microbiology, to any of the above

issues, they should contact me.


ASM Affairs

Dr Eric French has made a mostsignificant contribution to science ingeneral and veterinary virology inparticular. His pioneering efforts inveterinary virology in Australia haveresulted in immense benefits to thiscountry and brought him great honourand respect.

He joined the CSIRO Division of AnimalHealth in 1958 to establish a newprogramme of veterinary virology at itsAnimal Health Research Laboratory atParkville, Melbourne, and retired asOfficer-in-Charge of that laboratory andAssistant Chief of the Division in 1977.

Eric was born at Jamestown, SouthAustralia and, after he matriculated at nightschool while working at various part timejobs, he obtained a position as a laboratorytechnician with FH Faulding. In 1938 hejoined the Bacteriology Department at theInstitute of Medical and Veterinary Sciencein Adelaide as a senior laboratorytechnician and commenced studies parttime for a science degree, which hecompleted in 1942.

In the same year he was commissioned alieutenant in the Australian Army MedicalCorps of the AIF and, after serving in theSouth West Pacific area and in armyhospitals in Australia during World War II,he was demobilised in 1946, and took upa teaching position in the BacteriologyDepartment at the University of Adelaide.He immediately commenced studying foran MSc, under the guidance of Dr NancyAtkinson, which he obtained in 1947.

In late 1946 he was offered a position atthe Walter and Eliza Hall Institute forMedical Research by Sir MacfarlaneBurnet, which he took up in 1947.During his 11 years at the Institute, hismain investigations centred on aspects ofinfluenza, Murray Valley encephalitis(MVE) (the topic of his PhD thesis) and

Obituary

Dr Eric Lancelot French AO1 June 1914 – 12 February 2004

cot deaths. His successful isolation and

characterisation of the MVE virus ruled

out any connection of MVE with

myxomatosis which was decimating

rabbit populations in the Murray Valley at

the time.

After being awarded a CJ Martin

Fellowship in 1953 and a Rockefellow

travel grant in 1954, he spent 18 months

on post graduate study in the UK and

USA. Soon after returning to Melbourne

he was invited by Dr TS Gregory to

establish the Veterinary Virology

Programme at CSIRO. It was a formidable

task in the late 1950s to establish a

veterinary virology laboratory as there

were few people in Australia at that time

who were trained to do such work. This

meant that staff, untrained in virological

techniques, had to be employed and

trained in the basic skills. This became

Eric’s responsibility and it was a role in

which he excelled.

At the time the programme was

established, Australia was considered

virtually free of virus diseases of livestock;

however, in a very short time, a number

of viruses were isolated and identified,

many of which were linked to known or

newly recognised clinical conditions.

Hence, from the outset and until he was

appointed Assistant Chief of the Division

in 1968, he carried out research into a

number of indigenous and exotic

livestock diseases.

Eric developed an intense interest in

exotic livestock diseases that could have

catastrophic effects if introduced into

Australia. He acted as an expert advisor in

those forums where the diagnosis and

control of exotic diseases were discussed,

chaired an organising committee that

conducted schools on their diagnosis and

co-authored a diagnostic manual. He also

provided pivotal advice to the Chief of the

Division on the need for the Australian

Animal Health Laboratory.

Eric had broad scientific interests thatencompassed not only virology but alsopathology and epidemiology of bothhuman and veterinary diseases. Atvarious times he was President of theVictorian Society of Pathology andExperimental Medicine, Victorian Societyfor Microbiology, Cell Culture Society ofVictoria and Section 14 of ANZAAS. Hewas a founding member of ASM,President in 1976 and 1977 and waselected an Honorary Life Member in 1988.

The impact that Eric had in the area ofanimal health in Australia, andinternationally, can best be gauged by thenumerous honours that he has receivedover the years. He was elected anassociate member of the of the AustralianVeterinary Association in 1970 and anhonorary member in 1977. He waselected an honorary Fellow of theAustralian College of Veterinary Scientistsin 1975, and received honorary doctoratesin veterinary science from the universitiesof Hanover, Germany and Melbourne,Australia. He received an AO in theAustralia Day Honours in 1997 forscientific research, particularly in the fieldsof veterinary microbiology and virology.

His wife, Monsie, predeceased him andhe is survived by his daughter, Jennifer,son Gregory, and their families.

Bill Snowdon

Photo reproduced with permission of the AustralianVeterinary Journal 2004; 82:240.


ASM Affairs

In 2004 the Annual Scientific Meeting of ASM is in Sydney and EDSIG, through

Julian Cox, Kathy Takayama and others, is planning a pre-conference Educators’

Workshop. This is an experiment to see if we can attract more people to

participate in our group and talk teaching, by taking away the need to choose

between our sessions and sessions they feel they need to attend to update their

teaching.

The workshop will be on the Sunday before the conference begins and there will

only be a minimal charge to cover catering costs. Something to keep in mind.

If you have any ideas at all about what you’d like to hear, see or even do at the

workshop, please contact Julian Cox ([email protected]) or register for

AMEN and give your opinion there. Details will appear in AMEN and on the

EDSIG website (http://www.foodscience.unsw.edu.au/edsig/index.html) as they

come to hand.

VictoriaThe New Year has started with a wave of

activity for the Victorian Branch

committee. We are busy planning the

year’s calendar of events, which includes

some new activities along with others that

have become regulars.

The year will kick off with a ‘double-

header’ based around David Ellis’

onychomycosis seminar. The Melbourne

event will be followed by a similar session

hosted by Ballarat Hospital. This will give

Victorian country members the

opportunity to meet our National

President and members of the State

branch committee. The branch intends

to organise rural meetings on a regular

basis, an idea that has met with

overwhelming approval.

The rest of the calendar is full of a variety

of meetings that should keep members

(and the committee) busy, entertained

and informed.

The ever-popular Southern and Northern

News from the Hospitals are planned for

May and November, respectively. We have

been fortunate to attract sponsorship

from BD and Oxoid for these events, and

are very grateful for their generous

support.

Other events include career seminars

aimed at undergraduate and

postgraduate students and experienced

hospital scientists who might be looking

for career alternatives. Our student

careers night last year was a very

successful event, which has encouraged

us to organise two separate events: one

for post-graduates entitled “Where will by

PhD in microbiology take me?” and

another undergraduate careers night.

The Students Awards Night is also in the

planning stages. The committee recently

accepted the resignations of two

members: Stuart Smith and Wendy

MacDonald. Stuart has served on the

committee for 10 years and made

important contributions, most recently as

past-chair and treasurer. Wendy, who has

been involved over the last few years as

the branch’s student representative, has

crossed the Tasman for a post-doc

position in NZ. Sue Cornish, who has

served as branch secretary in recent years,

has nominated to take over as chair at the

next AGM, a significant change to the

committee for next year.

Enzo Palombo

Chair, Victorian Branch

TasmaniaThe year’s activities have started slowly, with

a deferred Christmas Party for southern

(Hobart) members in early March and

another planned for northern members

(Launceston) about mid-year. The reason

for the deferral was that there was just too

Branch reports

much activity in that month of December; a

quieter time seemed appropriate.

Assoc. Prof David Ellis will speak on his

mycotic speciality in both Launceston (mid-

day) and Hobart (evening) on March 29,

and our BD travel-to-conference

competition will take place in April by

video-conference, Hobart/ Launceston.

The State branch will provide additional

funds to enable a further postgraduate

student plus a clinical microbiologist to

attend the annual meeting in Sydney.

Following the success of last year’s

speciality microbiology talks in a local

Hobart hotel, we will begin a new round

of sessions in April/May. We will also trial

a competitively-based branch-funding

(matched by industry funding) of a newly-

graduated ASM microbiology student

over the long vacation, on the

understanding that she/he will progress

to a higher degree in microbiology.

Martin Line, Convenor


ASM Affairs

Level Requirements Advantages

Student • Pursuing course of study • Network with scientists and international experts

at local branch meetings • Access to awards and prizes

• Not full-time employed • Free journal 5 times a year

• Interested in microbiology • Access to members’ lounge website

or ASM activities • Discounted annual subscription

Associate • Interested in microbiology • Network with scientists and international experts

or ASM activities at local branch meetings

• Access to awards and prizes

• Free journal 5 times a year

• Access to members’ lounge website

Senior Associate • Diploma or equivalent in microbiology • Privileges of an Associate

(SASM) • 5 years appropriate full-time or • Peer recognition of achievement

equivalent employment post-qualification • Society recognition of corporate grade

• ASM voting rights

MASM • Degree or equivalent in microbiology • Privileges of an Associate

• 2 years appropriate full-time or • Peer recognition of achievement

equivalent employment post-qualification • Society recognition of corporate grade

• ASM voting rights

• Recognition by industrial awards

FASM • Membership of ASM • Privileges of a Member

• 10 years appropriate full-time or • Peer recognition of achievements

equivalent employment post-qualification • Recognition by industrial awards at a senior level

• Examinations and dissertation or

equivalent as assessed by the

ASM National Examinations Board

Honorary Life • Distinguished service to the science • Privileges of a Member

of microbiology or to ASM • Highest recognition by Society

• Exempt from annual subscription

Fees Associate, SASM, MASM, AND FASM – $155 per annum

Students – $45 per annum

To apply for change of membership status or to obtain further information, contact ASM National Office or download the

forms from the ASM website at www.theasm.com.au

MEMBERSHIP MEMBERSHIP MEMBERSHIP MEMBERSHIP

Senior Associate Level... now availableAre you eligible for a different level? Be active in your career

ASM now has 6 levels of membership for individuals:Student, Associate, Senior Associate (SASM),

Member (MASM), Fellow (FASM) and Honorary Life Membership. Advance your career by progressing through these levels


What’s On

Contributions listing relevant meetingsare welcome. Please send to:<[email protected]>

200424-26 MayCrystal Gateway Marriott HotelArlington, Virginia

The National Foundation forInfectious Diseases (NFID)Seventh Annual Conference OnVaccine Research

Web: www.nfid.org/conferences/vaccine04/

6-10 June 2004Phoenix, Arizona, USA

The Association for Professionalsin Infection Control andEpidemiology (APIC) – APIC ’04 –31st Annual Education Conferenceand International Meeting

Contact: APIC, 1275 K StreetNW, Suite 1000Washington DC, 20005-4006, USATel: (1) 202 789 1890Fax: (1) 202 789 1899E-mail: [email protected]: annual.apic.org/phoenix2004/

9-11 JuneHobart, TAS

Australian Infection Control Assoc3rd Biennial ConferenceInfection control:The clean green approach

Major themes include:• New pathogens – new problems• Alternative approaches to the

management of infection• Out of hospital but still in control• Surveillance• Perioperative issues• Health care worker vaccination• Infection control – nuts & bolts• Approaches to the control of MROs• Environmental cleaningContact the conference manager to register your interestAustralian Infection Control Assoc.Third Biennial Conference 2004Intermedia Convention & Event MgmtPO Box 1280, Milton QLD 4064Tel: (07) 3858 5532Fax: (07) 3858 5510E-mail: [email protected]

28-30 June

Hyatt Regency Bethesda

Bethesda, Maryland

The National Foundation forInfectious Diseases (NFID)2004 Annual Conference OnAntimicrobial Resistance

E-mail [email protected]:www.nfid.org/conferences/

resistance04/

1-3 July 2004

Carrington Hotel,

Katoomba, Blue Mountains NSW

Come catch Viruses in July!

“Viruses in July” is a unique trainingevent bringing together medicalvirologists, clinicians and scientistsfrom around Australia, in order todiscuss diagnostic and managementissues related to virology.Topics covered will include• Fundamentals of diagnosis –

molecular & serological testing• Viral vaccines• Paediatric virology & diagnosis• Congenital virology & diagnosis• Emerging infectious diseases• Viral chemotherapy and resistanceMOPS accreditation points are availablefor registrants on application to theRACP.For further information please contactDr Cristina BaleriolaE-mail: [email protected] FordE-mail: [email protected]

24-27 July

Banff, Alberta, Canada

American Society for MicrobiologyASM Conference on Cell-CellCommunication in Bacteria

Web: www.asm.org/Meetings/Because of the recent explosion inresearch in the area of cell-cellcommunication in bacteria and itsnewly discovered role in elicitinghuman disease, the ASM hosted aconference devoted to cell-cellsignalling during the summer of 2001.This meeting was so well received by itsparticipants that the ASM becamecommitted to provide an ongoingvenue for this topic, and will host asecond conference in July of 2004.

31 August – 3 September 2004

National Convention Centre,

Canberra

4th Australasian Hepatitis CConference

2-4 September 2004National Convention Centre,Canberra

The 16th Annual Conference of theAustralasian Society for HIVMedicine

Details for both of the above can beobtained atWeb: www.ashm.org.au/conference2004

19-23 SeptemberChesapeake Bay, Maryland

An ASM ConferenceExtremophiles 2004:5th International Conference onExtremophiles

26 September – 1 October Sydney SuperDome

ASM 2004 National Conference

Conference Manager: Janette SofronidisAustralian Society for MicrobiologyE-mail: [email protected], Local Organising Committee: Tom OlmaE-mail: [email protected]: www.ASM2004.org

6-9 OctoberPortland, Oregon

ASM Conference onFunctional Genomics andBioinformatics Approaches toInfectious Disease Research

This ASM conference will• Highlight new developments

in genomics & bioinformatics technologies.

• Address the challenges of datastorage, interpretation and sharing.

• Describe recent application of such technologies to infectious disease research.

The conference will bring leaders in thefunctional genomics and bioinformaticsfields together with microbiologists,virologists and immunologists who useor intend to use such approaches.

200615-18 October 2006Amsterdam, Netherlands

6th International Conference ofthe Hospital Infection Society

Contact: Congress SecretariatHIS 2006, Concorde Services Ltd4B/50 Spiers Wharf, Glasgow G4 9TBTel: (44) 141 331 0123Fax: (44) 141 331 0234E-mail: [email protected]: www.his2006.com

Meetings

Documents

OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR ... · May 2004 Volume 25 Number 2 OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC. A. B. C. D. Page 26 Page 23 1 2