Introduction and Review of Literatureshodhganga.inflibnet.ac.in/bitstream/10603/40095/10/10_chapter 1.pdfIntroduction and Review of Literature 4 Table 1.1. Approximate number of bioactive

Chapter 1

Introduction and

Review of Literature

Introduction and Review of Literature

2

1.1. BIOGEOGRAPHY OF MICROBIAL COMMUNITIES

The fundamental concept of biogeography, “Everything is everywhere, but the

environment selects” (Baas Becking 1934) has been widely propagated in ecological

studies. Traditionally, the field encompasses studies of spatial distribution of plants

and animals with relation to time. This concept is undergoing rapid changes with the

inclusion of microorganisms in this discipline.

With reference to microbial community, the concept has been interpreted as „most

bacteria are widely distributed (everything is everywhere) and that different

ecosystems select for the bacteria that are best adapted, which leads to relatively

greater abundance of these bacteria (the environments selects)‟ (Wawrik et al.

2007). Several findings have supported the biogeographical patterns in microbial

distribution (Cho and Tiedje 2000; Hanson et al. 2012; Jensen and Mafnas 2006;

Nemergut et al. 2011; O‟Malley 2008; Pasternak et al. 2013; Wawrik et al. 2007).

One such instance of bacterial geographical distribution is the distribution of

Roseobacter cluster in polar and temperate regions but not in tropical and subtropical

regions (Selje et al. 2004). Another marine bacterium, Prochlorococcus marinus, was

isolated from subtropical and tropical surface waters of the world‟s oceans but not

from temperate and polar regions (Campbell et al. 1994). Different ecotypes of

Prochlorococcus (populations adapted to high- and low-light conditions) have been

found in various light regimens in the ocean (Moore et al. 1995; Morel et al. 1993;

Partensky et al. 1993).

Among the domain Bacteria, Actinobacteria form the largest phylum (Gao and

Gupta 2012; Ventura et al. 2007). They have been reported from various ecosystems:


3

soil (Hopwood 2007), lake (Terkina et al. 2002); river (Ningthoujam et al. 2011);

marine surface and sediment (Egan et al. 2008; Fenical and Jensen 2006), marine

sponge (Zhang et al. 2008), estuary (Li and Qin 2005); thermal spring (Barabote et al.

2009), plant root (Normand et al. 2007), human gut (Hoyles et al. 2013), granite rock

(Li et al. 2013), and Antarctic ice (Adams et al. 2006; Jadoon et al. 2012). The

ubiquitous nature of the phylum Actinobacteria makes it an appropriate candidate for

their biogeographical studies.

1.2. PROPOSED Ph. D. RESEARCH PROJECT AND ITS IMPORTANCE

The proposed Ph.D. research project aims to study the actinomycete diversity of a

limestone habitat (an unexplored region for microbial studies) located at Hundung,

Ukhrul district, Manipur and explore the biotechnological potential of promising

isolate(s) especially with reference to their antimicrobial activities. The importance of

this research project arises from three crucial elements: i) Natural Products ii)

Actinobacteria, and iii) Limestone habitat.

The natural product diversity has been estimated to be more than 105 (Watve et al.

2001). Table 1.1 indicates the approximate number of bioactive compounds of

microbial origin from 1940 to 2010. Majority of the reported bioactive compounds are

either produced by actinomycetes or fungi (Bérdy 2012). Actinomycetes have been

reported to produce 13,700 natural products (Bérdy 2012). However, only a small

portion of the huge microbial bioresources has been explored till date.


4

Table 1.1. Approximate number of bioactive microbial metabolites in periods from 1940 to

2010 according to their producers (Reproduced from Bérdy 2012)

Periods 1940-1974

Early years %

1975-2000

Mid-era %

2001-2010

New age % Total

Species

Actinobacteria

Streptomyces sp.

Other actinobacteria

All microscopic bacteria

Myxobacteriales

Cyanobacteria

All Fungi

Microscopic fungi

Basidiomycetes

Other fungi

3400

2900

500

800

25

10

1300

950

300

20

62

15

23

7200

5100

2100

2300

400

30

7700

5400

1800

200

42

13

45

3100

2400

700

1100

210

1250

6600

4900

1500

160

28.5

10

61

13700

10400

3300

4200

635

1290

15600

11250

3600

380

Total per year 5500/180 1700/690 10800/1100 33500

The phylum Actinobacteria is notable for containing organisms producing diverse

natural products. The genus Streptomyces within the order Actinomycetales produces

~75% of the characterized actinomycete natural products (Bérdy 2012). Whole

genome sequencing of several bioactive strains of actinomycetes have also indicated

the rich potential of actinomycetes for production of bioactive metabolites (e.g.

Bentley et al. 2002; Ikeda et al. 2003; Zhang et al. 2013). Abyssomicins (anti-MRSA

antibiotics) from Verrucosispora strain AB-18-032 (Keller et al. 2007; Riedlinger et

al. 2004), Anthracimycin (potent anthrax antibiotic) from Streptomyces strain

CNH365 (Jang et al. 2013), Daptomycin (anti-MRSA) from Streptomyces

roseosporus NRRL 11379 (Miao et al. 2005; Steenbergen et al. 2005; Tally and

DeBruin 2000; Thorne and Alder 2002), Marinomycins (antitumour) from

Marinispora strain CNQ-140 (Kwon et al. 2006) and Salinisporamide A (antitumor)


5

from Salinispora strain CNB-392 (Feling et al. 2003) are just a few examples of rich

bioactive compounds hidden in actinomycetes indicating that actinomycetes still

harbor unique compounds (Czárán et al. 2002; Farnet and Zazopoulos 2005; Jenke-

Kodama et al. 2006).

Reports on actinomycete diversity in limestone habitats are still scanty, though some

studies have been carried out on microbial community of the caves, especially the

limestone caves (Barton et al. 2004; Groth et al. 2001; Konkol et al. 2008; Laiz et al.

1999, 2003; Macalady et al. 2006; McNamara et al. 2006; Muyzer et al. 1993; Ortiz et

al. 2013; Schabereiter-Gurtner et al. 2002; Wong et al. 2010). Actinobacteria

constitute a major portion of the microbial communities in these habitats. The

metagenomic studies of these habitats also indicate actinobacteria as a major

community among the biofilms of caves. There are, however, very few reports on

studies of bioactive metabolites from this habitat (Herold et al. 2004, 2005).

1.3. THE PHYLUM ACTINOBACTERIA

The phylum Actinobacteria represents one of the largest taxonomic units among the

18 major lineages currently recognized within the domain Bacteria (Gao and Gupta

2012; Stackebrandt et al. 1997; Ventura et al. 2007). The phylum is well known for its

high G+C content and unparalleled capacity for production of secondary metabolites

(Ventura et al. 2007). Members of this phylum exhibit a wide variety of

morphological, physiological and metabolic properties, i.e., from coccoid or rod-

coccoid to fragmenting hyphal forms or permanent and highly differentiated branched

mycelia (Atlas 1997); production of various extracellular enzymes (Schrempf 2001)

and diversity of secondary metabolites (Lechevalier and Lechevalier 1967); and from


6

being pathogens to soil saprophytes or symbionts. The G+C content ranges from 42%

(Gardnerella vaginalis; Greenwood and Pickett 1980) to 74.4% (Kineococcus

radiotolerans; Phillips et al. 2002). Low G+C Actinobacteria have also been reported

from metagenomic studies of freshwater ecosystems (Ghai et al. 2012). The genomes

of Actinobacteria are either circular (Mycobacterium, Corynebacterium) or linear

(Streptomyces) with sizes ranging from 0.93 Mb (Tropheryma whipplei; Raoult et al.

2003) to 11.9 Mb (Streptomyces bingchenggensis; Wang et al. 2010).

With the development of molecular techniques, the phylum has undergone several

taxonomic amendments. On the basis of 16S rRNA gene sequences alone, there have

been at least 4 amendments (Garrity et al. 2005; Normand 2006; Zhi et al. 2009;

Ludwig et al. 2012). As per the latest update (Ludwig et al. 2012), the phylum

encompasses 6 classes (Acidimicrobiia, Actinobacteria, Coriobacteriia,

Nitriliruptoria, Rubrobacteria and Thermophilia), 50 families and 221 genera.

Subsequently, more than 30 new genera have been added (Euzéby 2014). However,

due to ambiguity of the origin of 16S rRNA genes and the related problem of defining

the phylogenetic positions of some actinomycetes, alternative markers especially the

single copy number housekeeping genes such as recA (synthesizing DNA repair and

recombination proteins), rpoB (DNA-directed RNA polymerase subunit B), gyrB

(DNA gyrase subunit B) and dnaK (molecular chaperone) have been proposed for

actinomycete systematics. This has helped in redefining the phylogenetic position for

some strains of the genus Thermobispora which possess two distinct sets of 16S

rRNA genes. However, the use of alternative markers is still restricted to certain

groups and often generates unstable phylogenetic data for complex groups (Alam et


7

al. 2010; Rokas et al. 2003). The molecular systematics of actinomycetes, therefore,

can be considered to be in its infant stage. Hence, more studies of the genomics and

proteomics of the organisms are necessary for proper assignment of their taxonomic

positions (Gao and Gupta 2012; Verma et al. 2013).

Biotechnologically, the phylum Actinobacteria occupies a unique position among the

microbial world. Actinomycetes are the major producers of bioactive metabolites

including antibiotics. Table 1.2 gives a summary of some major antibiotics produced

by Actinobacteria (adapted from Keiser et al. 2000). They also play a crucial role in

bioremediation and in humus formation (Goodfellow and Williams 1983; Stach and

Bull 2005). Furthermore, many Bifidobacteria are used as active ingredients in a

variety of foods due to their perceived health-promoting or probiotic properties

(Lievin et al. 2000; Ouwehand et al. 2002; Staton et al. 2005).

1.4. ECOLOGY OF ACTINOMYCETES

Actinomycetes are the most widely distributed group of microorganisms in nature

(Takizawa et al. 1993). The strong odour in the air when rain falls after a dry spell of

weather is due to production of geosmin by the soil actinomycetes (Gust et al. 2003).

In natural habitats, Streptomyces species are common inhabitants and are usually a

major component of the total actinomycete population. Till date, there are over 600

Streptomyces type species cited in literature (Euzéby 2014). Other actinomycete

genera such as Actinoplanes, Amycolatopsis, Catenuloplanes, Dactylosporangium,

Kineospora, Microbispora, Micromonospora, and Nonomuraea which are often very

difficult to isolate and cultivate due to their slow growth, are called „rare

actinomycetes‟ (Hayakawa 2008).


8

Table 1.2. Useful actinomycete antibiotics (Adapted from Keiser et al. 2000)

Antibiotic Producer Application

Avermectin Streptomyces avermitilis Antiparasitic

Bialaphos Streptomyces hygroscopicus Herbicidal

Erythromycin Saccahropolyspora erythraea Antibacterial

Gentamicin Micromonospora sp. Antibacterial

Mitomycin C Streptomyces caespitosus Antitumour

Monensin Streptomyces cinnamonensis Anticoccidial; growth promotant

Nikkomycin Streptomyces tendae Antifungal; insecticidal

Nocardicin Nocardia uniformis Antibacterial

Nystatin Streptomyces noursei Antifungal

Paromomycin Streptomyces rimosus Antiamoebal

Rapamycin Streptomyces hygroscopicus Immunosuppressant

Rifamycin Amycolatopsis mediterranei Antibacterial (tuberculosis and leprosy)

Ristocetin Amycolatopsis lurida Antibacterial

Spinosyns Saccharopolyspora spinosa Insecticidal

Streptomycin Streptomyces griseus Antibacterial

Validamycin Streptomyces hygroscopicus Plant protectant

Vancomycin Amycolatopsis orientalis Antibacterial

Virginiamycin Streptomyces virginiae Growth promotant

Majority of the actinomycetes are free living and are widely distributed in natural

habitats such as soil, freshwater ecosystems, marine habitats and plant tissues.

1.4.1. Actinomycetes in terrestrial environments

The initial work on soil actinomycetes began in the last decades of the 19th

century.

But intensive studies on actinomycetes started only in 1943 with the discovery of

streptomycin, the first effective cure against tuberculosis, from Streptomyces griseus

(Hopwood 2007). Many actinomycetes have since been isolated mostly with the

objective of discovering new antibiotics.


9

Majority of the actinomycetes have been isolated from soils (Gomes et al. 2000; Ilic

et al. 2007; Jayasinghe and Parkinson 2008; Lee and Hwang 2002; Shirokikh et al.

2006). Rhizospheric soil forms a good host for many actinomycetes (Nimnoi et al.

2011). However, the distribution of actinomycetes, especially the population density

and diversity, seems to differ between rhizophere and other plant-free soil litter (Keast

et al. 1984). Rhizophere soil seems to contain more diverse actinomycetes compared

to non-rhizosphere soil (Lynch 1990). This high diversity may be due to the type of

symbiotic interactions with the plant (which may be beneficial, harmful or neutral)

and the edaphic conditions (Smith and Read 1997). The plant type may also influence

the diversity of actinomycetes in the soil (Ladygina and Hedlund 2010).

Nimnoi et al. (2011) and Wawrik et al. (2007) studied soil actinomycete community

using molecular biology tools. They found that majority of them are unculturable

actinobacteria, and among the culturable actinobacteria the genus Streptomyces is the

predominant genus (Abdulla 2009; Boudemagh et al. 2005; Keast et al. 1984;

Priyadharsini and Dhanasekaran 2013; Semêdo et al. 2001). Similar findings have

been reported by Xu et al. (1996) who have studied actinomycete diversity in 4,200

soil samples collected from different parts of Yunnan province (PR China).

1.4.2. Actinomycetes in aquatic environments

Aquatic environments form the largest ecosystem on the earth. Marine habitats cover

nearly two-thirds of the earth‟s surface. These large but unexplored habitats have been

reported to contain diverse actinomycete communities (Anzai et al. 2008; Bel‟kova et

al. 2003; Fenical and Jensen 2006; Pathom-aree et al. 2006; Singh et al. 2006; Valli et

al. 2012). Streptomyces has been reported as the dominant genus in water bodies


10

while Micromonospora is predominant in sediments (Anzai et al. 2008; Eccleston et

al. 2008; Jensen et al. 2000, 2005; Rifaat 2003; Sharma and David 2012; Solano et al.

2009). Table 1.3 lists the major actinomycetes obtained from marine habitats

(reproduced from Goodfellow and Fiedler 2010). The genera Marinispora,

Salinibacterium, Salinispora, Serinicoccus and Solwaraspora are unique to marine

habitats (Goodfellow and Fiedler 2010; Hodges et al. 2012; Ward and Bora 2006).

Freshwater ecosystems have been reported as promising source of bioactive

actinomycetes (Ningthoujam et al. 2011; Sanasam et al. 2011).

Other important marine ecosystems for exploration of actinomycetes are the marine

organisms and corals. Different processing methods have been proposed for isolation

of actinomycetes from these unique ecosystems including mechanical, physical and

chemical pretreatment methods (Bredholdt et al. 2007; Eccleston et al. 2008; Gontang

et al. 2007; Jensen et al. 1991, 2005; Kim et al. 2005; Maldonado et al. 2005; Mincer

et al. 2002; Selvin et al. 2009). Sun et al. (2010) and Vicente et al. (2013) have

reported various symbiotic actinomycete genera such as Cellulosimicrobium,

Gordonia, Microbacterium, Micromonospora, Nocardia, Rhodoccus, Salinispora,

Solwaraspora, Streptomyces and Verrucosispora from marine sponges.

Brevibacterium, Cellulomonas, Corynebacterium, Dermacoccus, Dermatiphilus,

Dietzia, Gordonia, Janibacter, Kocuria, Leucobacter, Microbacterium, Micrococcus,

Micromonospora, Microthrix, Mycobacterium, Nocardioides, Rhodococcus,

Serinicoccus and Streptomyces are major symbiotic actinomycete genera reported

from corals (Yang et al. 2013).


11

Table 1.3. Culturable actinomycetes isolated from marine habitats (Reproduced from

Goodfellow and Friedler 2010)

Isolates assigned to known genera

Actinocorallia

Amycolatopsis

Corynebacterium

Gordonia

Microbacterium

Mycobacterium

Nonomuraea

Saccharopolyspora

Streptomyces

Actinomadura

Arthrobacter

Dermacoccus

Isoptericola

Microbispora

Nocardia

Prauserella

Sanguibacter

Tsukamurella

Actinoplanes

Arsenicococus

Dietzia

Knoella

Micrococcus

Nocardioides

Pseudonocardia

Streptosporangium

Verrucosispora

Aeromicrobium

Brevibacterium

Glycomyces

Kocuria

Micromonospora

Nocardiopsis

Rhodococcus

Tessaracoccus

Williamsia

Isolates assigned to novel genera

Actinoaurantispora

Marinactinispora

Salinibacterium

Demequina

Marisedimenicola

Salinispora

Euzébya

Miniinunas

Sciscionella

Iamia

Phycicola

Serinicoccus

1.4.3. Actinomycetes in special habitats

An important but previously unexplored habitat is the living internal tissues of plants.

Many actinomycetes have been recently found to colonize the living plant tissues

(referred to as endophytic actinomycetes) with beneficial effects as biocontrol agents

and plant growth promoters (Hardy and Sivasithamparam 1995; Goudjal et al. 2013;

Poovarasan et al. 2013). Genetic diversity and community profiling of endophytic

actinomycetes indicate that they exhibit a biogeographical pattern of distribution in

different parts of the plant (Nimnoi et al. 2010). The highest diversity was reported to

be found in roots (El-Shatoury et al. 2013) followed by stems and leaves (Tian et al.

2007; Verma et al. 2009). The dominant endophytic actinomycetes found in roots are


12

Streptomyces, Micromonospora, Kitasatospora and Nocardiodes. Micromonospora

and Frankia have been reported to be dominant colonizers in nodules and play

important roles in nitrogen fixation in soil (Benson and Silvester 1993; Carro et al.

2012; Clawson et al. 2004; Faure-Raynaud et al. 1991; Nazaret et al. 1989).

Actinomycete diversity has also been studied in extreme habitats. These environments

have unusual growth conditions such as high and low temperatures, high salt

concentrations, alkaline and acidic pH, high radioactivity and high pressures. Jiang et

al. (2006) have reported novel actinomycete genera Streptomonospora, Jiangella,

Myceligenerans and Naxibacter from alkaline salt mines of Xinjiang and Qinghai, PR

China. Meklat et al. (2011) studied the halophilic actinomycetes among the Saharan

soils of Algeria. Okoro et al. (2009) found that actinomycetes were the only culturable

bacteria in some of the hyper-arid soils of Atacama Desert in Chile. Pasternak et al.

(2013) studied the microbial biogeographical patterns in arid and semiarid regions of

Isreal with special reference to Actinobacteria. Gayathri et al. (2011) have

investigated the diversity of actinomycetes in salt pans of Kodiakarai in Tamil Nadu,

India.

Another extreme habitat is the polar region. Adams et al. (2006) and Encheva et al.

(2013) studied actinomycete diversity in Antartica. Biogeographical notes on Antartic

microflora were reported by de Pascale et al. (2012) and Jadoon et al. (2012).

Cave environments, characterized by low nutrients, low temperatures and poor

illumination along with high humidity, are another promising underexplored

environment for microbial studies (Schabereiter-Gurtner et al. 2002). Several new

species of actinomycetes have been isolated from caves, e.g. Actinocorallia cavernae,


13

Amycolatopsis halotolerans, Amycolatoptis jejuensis, Catellatospora koreansis,

Nocardia jejuensis, Pseudonocardia kongjuensis, Saccharothrix violacea and

Saccharothrix albidocapillata from a gold mine in Korea (Lee et al. 2000a, b, 2001;

Lee 2006a, b, c), Beutenbergia cavernae from Reed Flute Cave in China (Groth et al.

1999a), Agromyces salentinus from Grotta Dei Cervi Cave in Italy (Jurado et al.

2005a) and Agromyces subbeticus from a bat-inhabited cave in Spain (Jurado et al.

2005b).

The insect world is another unexplored environment for actinomycetes. Attine ants

are often found to be associated with antibiotic-producing actinomycetes (Currie et al.

1999; Mueller et al. 2008). Two compounds produced by the ant associated

actinomycetes have been identified as „Dentigerumycin‟ (a previously unknown

antifungal) and „Candicidin‟ (Haeder et al. 2009; Oh et al. 2009).

Limestone habitats with high deposition of CaCO3 salts may be considered as another

type of unique habitat. Meagre studies have been done on actinobacterial diversity in

limestone habitats (Groth et al. 1999b, 2001; Jurado et al. 2009; Kim et al. 1998;

Nakaew et al. 2009; Niyomvong et al. 2012). To date, four new genera Beutenbergia,

Fodinibacter, Hoyosella and Knoellia have been reported from limestone habitats and

related limestone ecosystems such as cave biofilms (Groth et al. 1999a, 2002; Jurado

et al. 2009; Wang et al. 2009).


14

1.5. MOLECULAR TOOLS FOR STUDYING ACTINOMYCETE

DIVERSITY AND PHYLOGENY

To assess the bacterial diversity and phylogeny, DNA sequence analyses of

evolutionarily stable marker genes are considered a potentially useful strategy (Tringe

and Hugenholtz 2008). In bacteria, the three ribosomal genes (16S, 23S, 5S) are

conserved within the species (Maidak et al. 1997), among which 16S rRNA is the

most conserved. For the past two decades, microbiologists have relied on 16S rRNA

gene sequence analysis for identification and classification of bacteria. Hence, the 16S

rRNA gene has been proposed as an “evolutionary clock” for the construction of the

tree of life (Woese 1987).

1.5.1. Actinomycete diversity

The basic approach for diversity studies involves the analysis of physico-chemical

differences in the genome and/or rRNA operon. DNA fingerprinting techniques based

on 16S rRNA genes are summarized in Table 1.4 (reproduced from Rajendhran and

Gunasekaran 2011). Each of these techniques has its own strengths and weaknesses.

Among these techniques, Denaturing Gradient Gel Electrophoresis (DGGE) has been

predominantly used to study actinomycete diversity associated with different samples

(soil, Nimnoi et al. 2011; human gut, Hoyles et al. 2013; plant tissues, Nimnoi et al.

2010; continuous cotton cropping field, Zhang et al. 2013 etc.). Findings from these

diversity studies have indicated that only about 1% of the total diversity is amenable

to cultural approach. This, in turn, led to the development of improved culturing

methods (Janssen et al. 2002).


15

1.5.2. Phylogeny of actinomycetes

Actinomycetes being the largest phylum among the domain Bacteria, the placement

of new or existing species has become problematic due to the low congruence level of

the various metabolic and genomic properties. With the advent of rRNA gene

sequencing technology, molecular phylogeny could finally find a place in taxonomy.

Identification of new actinomycete species is based on 16S rRNA gene sequence

homologies with existing sequences in databases. For homologs of actinomycete

strains with rRNA gene identities of 97% and below, the corresponding DNA-DNA

hybridization (DDH) values was never higher than 70% (Figure 1.1) which is

considered the threshold limit for species delineation (Stackebrandt and Goebel 1994;

Wayne et al. 1987). This finding led to the recommendation to abolish the need to

perform DNA-DNA reassociation in those cases in which putative novel strains

showed only moderate rRNA similarities (<97%) with their nearest neighbours

(Stackebrandt and Goebel 1994). This threshold value has subsequently been revised

to 98.5% (Stackebrandt and Eber 2006; Stackebrandt 2011). The taxonomic

subcommittes working under the auspices of the International Committee on

Systematics of Prokaryotes have laid down minimal standards for the description of

new taxa (Trujillo 2011).


16

Table 1.4. Microbial typing methods based on rRNA sequences (Reproduced from Rajendhran

and Gunasekaran 2011)

Method Principle Reference

Ribotyping Polymorphism in the hybridization with 16S rRNA gene-

based probes in total genomic DNA digested with restriction

enzymes. Ribotying represents position of RNA operon(s) in

the whole genome. However, it is based on the variations

associated with restriction sites only

Grimont and Grimont

(1986)

Amplified ribosomal DNA

restriction analysis

(ARDRA)

Restriction fragment length polymorphism (RFLP) of the

amplified or cloned 16S rRNA genes. It is faster and it

represents variations associated with restriction sites only

Gurtler et al. (1991)

Terminal restriction

fragment length

polymorphism (T-RFLP)

Polymorphism in the length of fluorescently labeled terminal

restriction fragment of 16S rRNA gene. It is a high-throughput

method useful for metagenomic studies and the variation is

based on the size of the terminal restriction fragment only

Liu et al. (1997)

The 16S-23S internally

transcribed spacer (ITS)

typing

Polymorphism in the length, RFLP pattern or sequences of

the ITS region. It exhibits greater variation than 16S rRNA

gene sequence and thus, useful for the typing of closely

related organisms

Jensen et al. (1993)

Automated ribosomal

intergenic spacer

analysis (ARISA)

Polymorphism in the length of fluorescently labeled ITS

regions. It is a high throughput method useful for

metagenomic studies and the variation is based on the size

of the amplified fragments

Fisher and Triplett

(1999)

Long PCR-RFLP RFLP of the entire rRNA operon. It has more discrimination

power than the analysis of 16S rRNA gene sequence alone.

However, it is based on the variations associated with

restriction site only

Smith-Vaughan et al.

(1995)

DGGE Polymorphism based on the separation of partially melted

16S rDNA in a linear denaturing gradient gel. It represents

the sequence variations other than the restriction sites also.

However, it covers only less than 400 bp of 16S rRNA gene

Muyzer et al. (1993)

TGGE Polymorphism based on the separation of partially melted

16S rDNA in a linear temperature gradient. It represents the

sequence variations other than the restriction sites also.

However, it covers only less than 400 bp of 16S rRNA gene

Nűbel et al. (1996)

SSCP Polymorphism based on the single-stranded 16S rDNA in

polyacrylamide gel. It represents the sequence variations

other than the restriction sites also.

Lee et al. (1996)

16S pyrotags Pyrosequencing of 16S rDNA. It is a high-throughput method

useful for metagenomic studies. However, it gives only a

small tag of the 16S rRNA gene, approximately 200 bp.

Ronaghi and Elahi

(2002)

16S rRNA gene

sequencing

Sequencing of PCR amplified or cloned 16S rRNA gene.

Complete 16S gene sequence (~1500 bp) has more

discriminatory power

Woese (1987)


17

Fig. 1.1. Comparison of DNA-DNA relatedness and 16S rRNA gene similarities. The orange-coloured box indicates the potential “genetic space” around the type strain of one particular species as

defined by the current bacterial species concept (Reproduced from Rossello-Mora and Amann 2001)

The phylogenetic relationships of the various families within the class Actinobacteria

based on the 16S rRNA sequences is illustrated in Figure 1.2 (Adapted from Zhi et al.

2009). Ladeda et al. (2012) presented an overview of the phylogeney of different

species included in the family Streptomycetaceae and classified the genus

Streptomyces into 130 clades based on the 16S rRNA gene sequence analyses. Figure

1.3 (Adapted from Gao and Gupta 2012) depicts the latest update on the classification

of the phylum Actinobacteria which divides the phylum into 6 classes. This

classification system has been used in the Bergey’s Manual of Systematic

Bacteriology 2nd

edition: Volume 5 (Ludwig et al. 2012).


18

Fig. 1.2. Intraclass relationship of the class Actinobacteria showing the presence of five orders

based on 16S rRNA gene sequence comparisons (Reproduced from Zhi et al. 2009)


19

Fig. 1.3. The current classification of phylum Actinobacteria (Reproduced from Gao and Gupta 2012)

Another problem in understanding the phylogeny of actinomycetes is the presence of

multiple copies of the rRNA operon and intra-genomic heterogeneity of the 16S

rRNA gene. This problem has been solved partially with the use of single copy

housekeeping genes as alternatives to the 16S rRNA gene. Other approaches include

multilocus sequence analysis (MLSA; Kampfer and Glaeser 2011) and whole genome

comparison. Verma et al. (2013) presented a new approach for phylogenetic analyses

of actinobacteria through generation of a consensus tree containing the information

from single gene and genome based phylogenies. It was found that whole genome

based approaches (i.e. alignment-free method and use of overlapping gene content

and gene order) may help overcome the shortcomings in single gene based

phylogenies.


20

1.6. BIOPROSPECTING OF ACTINOMYCETE NATURAL PRODUCTS

Soil actinomycetes represent a rich source of biologically active compounds (Bérdy

2005, 2012; Demain and Sanchez 2009; Doroghazi and Metcalf 2013; Solecka et al.

2012; Watve et al. 2001). Many of these compounds find applications in medicine,

veterinary practice, agriculture and industry. The genus Streptomyces alone accounts

for nearly 75% of the metabolites produced by actinomycetes (Bérdy 2012). Table

1.5 indicates the number of actinomycete species producing bioactive metabolites

(Reproduced from Bérdy 2005).

During the last decade, the focus of biodiscovery of microbial metabolites has been

shifted to the marine and other unexplored ecosystems (Bull and Stach 2007;

Williams 2008). Marine natural products have been reviewed each year since 2003 by

Blunt and co-workers for the marine-derived new compounds from 2001 onwards

(Blunt et al. 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013). As

per their latest review (Blunt et al. 2013), 1152 compounds have been reported during

the year 2011, for which the majority of the reported microbial sources are

actinomycetes. Some important marine actinomycete metabolites are Abyssomicin C,

Marinomycin, Proximicin, Salinosporamide A and Thiocoline (Bister et al. 2004;

Feling et al. 2003; Kwon et al. 2006; Romero et al. 1997; Schneider et al. 2008) which

have found applications as antitumour compounds and as antibacterial agents against

methicilin-resistant Staphylococcus aureus (MRSA) (Williams 2008).


21

Table 1.5. Number of some actinomycetales species producing bioactive microbial metabolites

(Reproduced from Bérdy 2005)

Streptomycetaceae: Streptomyces Streptoverticillium Kitasatosporia Chainia Microellobosporia Nocardioides

Micromonosporaceae:

(Actinoplanetes) Micromonospora Actinoplanes Dactylosporangium Ampullariella Glycomyces Catenuloplanes Catellatospora

Pseudonocardiaceae: Saccharopolyspora Amycolatopsis/Nocardia Kibdellosporangium Pseudonocardia Amycolata Saccharomonospora Actinopolyspora

Streptosporangiaceae: (Maduromycetes) Streptosporangium Streptoalloteichus Spirillospora Planobispora Kutzneria Planomonospora

~8,000

258 37 30 11

9

740 248 58

9 2 3 1

131 120/357

34 27 12

2 1

79 48 11 10

4 2

Thermomonosporaceae: Actinomadura Saccharothrix Microbispora Actinosynnema Nocardiopsis Microtetraspora/Nonomuria Thermomonospora Micropolyspora/Faenia Thermoactinomyces Thermopolyspora Thermoactinopolyspora

Mycobacteriaceae: (Actinobacteria) Nocardia Mycobacterium Arthrobacter Brevibacterium Proactinomyces Rhodococcus

Other species: Actinosporangium Microellobosporia Frankia Westerdykella Kitasatoe Synnenomyces Sebekia Elaktomyces Excelsospora Waksmania Alkalomyces Catellatospora Erythrosporangium Streptoplanospora Microechinospora Salinispora

345 68 54 51 41

26/21 19

13/3 14

1 1

(357) 57 25 17 14 13

30 11

7 6 5 4 3 3 3 3 1 1 1 1 1 1

Besides marine actinobacteria, endophytic actinomycetes also find potential

applications in agriculture. The genera Frankia and Micromonospora are found to

colonize nodules of plants (Carro et al. 2012; Normand et al. 2007). Many of these

endophytic actinomycetes protect their host plants from attack by phtyopathogens by


22

secreting antifungal compounds, and also by enhancing plant growth (Goudjal et al.

2013; Hardy and Sivasithamparam 1995; Poovarasam et al. 2013). Apart from agro-

active metabolites, several other clinically significant metabolites have also been

obtained from endophytic actinomycetes. Antitumour anthraquinone compounds,

Lupinacidins A and B, have been obtained from Micromonospora lupini sp. nov., an

endophytic actinomycete isolated from the root nodules of blue lupin (Lupinus

angustifolius; Igarashi et al. 2007).

1.6.1. Bioactive secondary metabolites from actinomycetes

Microbial antibiotics are usually synthesized from intermediates of primary

metabolites such as amino acids, aliphatic acids, sugars, nucleotides and lipids. The

major groups of secondary metabolites include the peptide and polyketide antibiotics.

1.6.1.1. Peptide antibiotics

Peptide antibiotics form a diverse and clinically important class of antibiotics. Most

peptide antibiotics are synthesized from amino acids by a group of enzymes called the

non-ribosomal peptide synthetases (NRPS) (Konz and Marahiel 1999; Marahiel

1997). Figure 1.4 represents some of the peptide antibiotics produced by

actinomycetes. The NRPS enzyme has the potential to develop new libraries of

antimicrobial therapeutics by joining together the amino acid precursors into diverse

chemical structures. These enzymes are very large and modular proteins, the order of

which corresponds with that of the amino acids incorporated in the final product

(Metsä-Ketelä et al. 2003).


23

Fig. 1.4. Representative peptide antibiotics from actinomycetes

1.6.1.2. Polyketides

Polyketide natural products include antibiotics, antifungals, anthelminthics,

immunosuppressants, anticholesterolemics, antiparasitics and natural insecticides.

Figure 1.5 represents some polyketides obtained from actinomycetes. They are

synthesized by multifunctional enzyme complexes known as polyketide synthases

(PKSs). Assembly of the initial carbon skeleton of a polyketide is catalysed by a large

enzyme known as polyketide synthase (PKS). Based on their gene sequences and the

conformation of their constituent proteins, PKSs can be divided into two groups:

modular PKSs (type-I PKSs) and iterative PKSs (type II PKSs).

Type I polyketides

Type I polyketides (or macrocyclic polyketides) are synthesized by the condensation

of acetates, propionates and butyrates using the multienzyme complexes known as

modular PKSs (PKS-I). Macrocyclic polypeptides are highly significant because of

their clinical properties. For example, rifamycin and erythromycin are useful

antimycobacterial antibiotics. Tacrolimus (FK506) is an immunosuppressant and

avermectin is an antihelminthic compound.

Actinomycin

Bialaphos


24

Fig. 1.5. Representative polyketides from actinomycetes (Reproduced from Zhang and Tang 2009)

Type II polyketides

Many Streptomyces spp. produce type II polyketide antibiotics, e.g. actinorhodin (S.

coelicolor A3(2), Fernandez-Moreno et al. 1992) and griseorhodin (Streptomyces sp.

JP95, Li and Piel 2002). Type-II PKS (PKS-II) is an aggregate of monofunctional

proteins. They build the polyketides mainly from acetate units through a series of

reactions involving formation and aromatization of the polyketide chain, and final

derivatization including methylation and oxygenation.

The advent of molecular techniques has accelerated the discovery of new polyketide

and peptide antibiotics by using primers synthesized from PKS and NRPS genes (Bull

et al. 2000; van Wezel et al. 2009). Metsä-Ketelä et al. (2003) used specific primers to


25

screen for type-II PKS genes in Streptomyces and found new polyketide compounds.

Specific primers were successfully used for screening PKS-I and NRPS in a group of

actinomycetes (Ayuso-Sacido and Genilloud 2005; González et al. 2005; Metsä-

Ketalä et al. 1999; Savic and Vasiljevic 2006).

1.6.2. Drug discovery in actinomycetes

Despite the manifold attractive features of natural products, majority of large

pharmaceutical companies have shifted the focus away from screening of natural

products for drug discovery in favour of synthetic compound libraries. The major

reason for this is the incompatibility of natural product libraries with high-throughput

screening (HTS, a technical advancement over the traditional assay method). Core

technologies for discovering natural products have not evolved substantially yet and

new technologies that could improve the natural product drug discovery efforts have

not advanced yet to the degree at which the discovery rate of natural product-derived

drugs meets the demands of the industry (Lam 2007). Other reasons are the

rediscovery of known compounds in the natural product libraries and incompatibility

of the time-consuming process of dereplication and purification with HTS. During the

last few decades, only two antibiotic groups, Lipopeptides and Diarylquinolines, were

discovered as compared to the period from the 1940s to 1960s during which nearly 20

groups of major antibiotics were discovered (Butler and Buss 2006; Coates et al.

2011, Hair and Kean 2007; Lewis et al. 2013; Zappia et al. 2007). New innovations

are required to enable chemical library construction and purification to go hand in

hand with HTS technologies (Lam 2007).


26

1.6.2.1. Genomics in drug discovery

Biosynthetic gene clusters

A major contribution towards the elucidation of secondary metabolite biosynthesis

came from the application of DNA sequencing in studies of secondary metabolism.

This was possible because microorganisms carry all the secondary metabolite related

genes in contiguous DNA segments known as the biosynthetic gene clusters. The

genomes of actinomycetes posses a large number of these gene clusters. The genome

of the strain Streptomyces coelicolor A3(2) (a genetic workhorse for more than 40

years before having its genome sequenced) indicates the presence of 20 biosynthetic

gene clusters (Bentley et al. 2002). Avermectin producing strain Streptomyces

avermitilis has at least 30 gene clusters (corresponding to 6.6% of the genome)

involved in the synthesis of melanin, carotenoid, siderophore, polyketide and peptide

compounds (Ikeda et al. 2003; Omura et al. 2001). The draft genome sequence of

Streptomyces bottropensis ATCC 25435 (a bottromycin-producing actinomycete)

indicates the presence of 21 secondary metabolites (4 siderophores, 5 terpenes, 1

lantibiotic, 1 bacteriocin, 2 PKS-I, 2 PKS-II, 3 NRPS and 3 hybrid NRPS-PKS)

biosynthetic gene clusters (Zhang et al. 2013). Till date, more than 90 actinomycete

genomes have been sequenced (Verma et al. 2013).

Regulation of biosynthesis

Despite the available information on biosynthetic gene clusters, it has been difficult so

far to correlate the information with the synthesized natural products (Doroghazi and

Metcalf 2013). For example, the strain Streptomyces coelicolor A3(2) is known to


27

make only four secondary metabolites but its genome revealed an additional 18

cryptic biosynthetic gene clusters (Bentley et al. 2002).

The biosynthetic gene clusters often include the information for regulation and

transport of biosynthesized metabolites. This facilitates the manipulation of such

pathways by molecular genetic techniques. For instance, Streptomyces spp. have been

shown to contain multiple regulatory genes for the biosynthetic gene clusters (Bate et

al. 1999; Knirschová et al. 2007; Martin 1992). One way to enhance the secondary

metabolite production is by manipulating pathway regulation (Chen et al. 2010).

Figure 1.6 indicates the strategies to increase secondary metabolite titres by

manipulating pathway regulation (Adapted from Chen et al. 2010). Other means

include manipulating regulators in heterologous hosts (Chen et al. 2010; Komatsu et

al. 2010), and increasing the limiting precursor or inducing biosynthetic enzymes by

intracellular and extracellular signaling molecules (Bibb 2005; Demain 1998). It has

also been shown that biosynthetic gene clusters can be heterologously expressed in

genome-minimized host (i.e. mutant host whose nonessential genes have been

systematically deleted) (Komatsu et al. 2010). Therefore, expression in heterologous

hosts can be assumed to be a good method for expression of the cryptic biosynthetic

genes (Lal et al. 2000).


28

Fig. 1.6. Strategies to increase secondary metabolite production by manipulating pathway

regulations (Reproduced from Chen et al. 2010)

Another method for expression of cryptic or sleeping biosynthetic gene clusters is the

combined culturing of an antibiotic producing actinomycete with a competitor. Onaka

et al. (2011) first reported that mycolic acid-containing bacteria induce natural

product biosynthesis in Streptomyces species. More recently, Traxler et al. (2013)

have applied the techniques of nanospray desorption electrospray ionization

(NanoDESI) and microbial matrix-assisted laser desorption ionization-time of flight

(MALDI-TOF) imaging mass spectroscopy (IMS) to detect and characterize minute

quantities of metabolites produced by Streptomyces coelicolor A3(2) on co-culturing

with another actinomycete strain. In this particular experiment, they have used one of

5 different Streptomyces strains or one Amycolatopsis strain for co-culturing with

Streptomyces coelicolor A3(2). Figure 1.7 depicts the methodological workflow used

by Traxler et al. (2013) for determining the metabolite production by Streptomyces

coelicolor A3(2). It was found that the strain produced numerous chemical

compounds that were not made when they were grown alone. These include, at least,

12 different versions of a molecule called desferrioxamine (a siderophore).


29

Fig. 1.7. Methodological workflow for detecting metabolite formation during co-culturing

(Reproduced from Traxler et al. 2013)

Synthetic biology also offers a new perspective for expression of cryptic biosynthetic

gene clusters by either engineering precursor supply to optimize antibiotic production

(Wohlleben et al. 2012) or using metabolomic approaches (Nguyen et al. 2012).

Metagenomics-based drug discovery

The area of metagenomics has opened up a new era in the study of microbial chemical

diversity by enabling direct access to the genomes of numerous unculturable

microorganisms (Li and Qin 2005). Takagi and Shin-ya (2012) have exploited

metagenomic data to generate a natural product library containing secondary

metabolites from actinomycetes. This has enabled efficient discovery of many new


30

metabolites which were not previously isolated. However, the massive data on natural

products, even with the help of functional metagenomics, have so far failed to

produce significant numbers of new products (Ekkers et al. 2012). Figure 1.8 depicts

the flowchart for metagenome sequence based biosynthetic gene discovery.

1.6.2.2. Systematics in drug discovery – need for novel strains

A possible reason for bacterial speciation is the constant chemical warfare among

microorganisms for niche occupation resulting in evolution of secondary metabolites

for defense, niche competition and evolutionary success (Czárán et al. 2002). There

are potentially thousands of new species awaiting isolation (Stach and Bull 2005;

Stach et al. 2003). Each of these novel species may contain unique natural products.

Several techniques have been proposed for isolation of previously uncultured

actinomycetes. These techniques mainly involve physical and chemical pre-treatment

of the samples, addition of selective antibiotics and antifungal compounds and use of

various selective isolation media (Hayakawa 2008). One such selective medium is

Humic acid-Vitamin (HV) agar medium (Hayakawa and Nonomura 1987). The genus

Actinomadura was isolated in HV agar medium after dry heating the soil samples at

110 °C for 1 h followed by pretreatment with 1% phenol (Hayakawa et al. 1995).

Members of the genus Herbidospora, Microbispora, Microtetraspora, Nonomuraea

and Streptosporangium were isolated in HV agar supplemented with nalidixic acid in

a method incorporating treatment of soil with Chloramine-T (Hayakawa et al. 1997).


31

Fig. 1.8. Workflow of metagenome sequence-based gene discovery:

Metagenomics (A), Metatranscriptomics (B), Metaproteomics (C) or A Combination of Approaches can

be used to identify target genes (Reproduced from Chistoserdova 2010)


32

Other techniques involve exploration of unexplored and underexplored habitats.

Manipur, a state in the Indo-Burma hotspot region, is rich in diversity. Unlike the

floral and faunal diversity, microbial diversity is relatively unexplored. The region has

several diverse ecosystems including pristine forests, fresh water lakes, rivers,

limestone deposit sites, and salterns, etc. This region, especially the unique habitats

such as the limestone deposit sites, could be a potential source for novel

actinomycetes and possibly novel metabolites.

1.7. OVERVIEW OF THE LIMESTONE DEPOSIT SITES IN MANIPUR

Manipur has a huge reserve of good quality limestone suitable for use in the

manufacture of cement. These limestones are of light grey to brown colour (Bhatt and

Bhargava 2005). The major limestone reserves have has been located by Geological

Survey of India near Ukhrul. Other limestone deposit sites include areas in Hundung,

Phungyar, Meihring, Mova, Khonggoi, Lambui and Paoyi.

In the Ukhrul area, a potential reserve of 579 million tonnes has been estimated by

drilling to a depth of 105 m. Other limestone deposits are those located at Khonggoi

(0.26 million tonnes) and Hundung (1.88 million tonnes). A reserve of 6.35 million

tonnes of cement grade limestone has been estimated from Phungyar. Meihring may

provide another reserve with an estimate of 5.76 million tonnes of limestone (Lisam

2011; Sadangi 2008).

The State Government of Manipur had previously commissioned a cement factory on

Oct 15, 1989 at Hundung (Figure 1.9), a village in Ukhrul district (25.05°N,

94.33°E). This factory has, however, been closed down since 2003.


33

.

Fig. 1.9. Limestone deposit sites at abandoned Hundung cement factory site (a, c, d) and quarry (b)

1.8. OBJECTIVES OF THE RESEARCH PROJECT

The major objectives of this Ph.D. research project are:

1. Studies of actinomycete diversity in Hundung Limestone habitats

2. Characterization of selected novel actinomycete strains

3. Studies of antimicrobial activities of Hundung actinomycetes

4. Partial characterization of antimicrobial metabolites from the most promising

actinomycete strain

5. Biocontrol and plant growth promoting potential of the selected strain


34

1.9. CHAPTER LAYOUT OF THE DISSERTATION

The Ph.D. dissertation has been arranged in the following layout: Chapter 1 highlights

the importance of the studies of actinomycetes and their biotechnological potential. It

reviews ecology, systematics and secondary metabolism of actinomycetes. The

chapter ends with the objectives of the research project.

Chapter 2 analyzes the diversity of actinomycetes from Hundung Limestone quarry

sites using Amplified Ribosomal DNA Restriction Analysis (ARDRA), followed by

sequencing of 16S rRNA genes of representative strains. Chapter 3 incorporates the

characterization of novel strains among the Hundung actinomycetes. The

antimicrobial profile of Hundung isolates against indicator bacterial and yeast

pathogens, and their biocontrol potential against selected rice phytopathogens are

summarized in Chapter 4. This chapter also discusses the possible involvement of

biosynthetic genes in antimicrobial activity.

Partial characterization of the secondary metabolite for the most promising strain was

also done during the current investigation. These findings have been incorporated in

Chapter 5. The biocontrol potential and rice growth promoting activities of the

selected bioactive strain (against rice fungal pathogens) are described in Chapter 6.

The dissertation concludes with overall summary and conclusions (Chapter 7).

Relevant bibliographies have been added at the end of each chapter.


35

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Documents

Introduction and Review of Literatureshodhganga.inflibnet.ac.in/bitstream/10603/40095/10/10_chapter 1.pdfIntroduction and Review of Literature 4 Table 1.1. Approximate number of bioactive