Development and validation of a diagnostic microbial microarray … · 2017-08-12 · of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase

Environmental Microbiology (2003)

5

(7) 566ndash582

copy 2003 Society for Applied Microbiology and Blackwell Publishing Ltd

Received 21 January 2003 accepted 27 February 2003 Forcorrespondence E-mail leventebodrossyarcsac Tel(

+

43) (0) 50550 3548 Fax (

+

43) (0) 50550 3444

Development and validation of a diagnostic microbial microarray for methanotrophs

Levente Bodrossy

1

Nancy Stralis-Pavese

1

J Colin Murrell

2

Stefan Radajewski

2

Alexandra Weilharter

1

and Angela Sessitsch

1

1

Department of Biotechnology Division of Life and Environmental Sciences ARC Seibersdorf research GmbH A-2444 Seibersdorf Austria

2

Department of Biological Sciences University of Warwick Coventry CV4 7AL UK

Summary

The potential of DNA microarray technology in high-throughput detection of bacteria and quantitativeassessment of their community structures is widelyacknowledged but has not been fully realised yet Agenerally applicable set of techniques based onreadily available technologies and materials wasdeveloped for the design production and applicationof diagnostic microbial microarrays A microarraytargeting the particulate methane monooxygenase(

pmoA

) gene was developed for the detection andquantification of methanotrophs and functionallyrelated bacteria A microarray consisting of a set of59 probes that covers the whole known diversity ofthese bacteria was validated with a representative setof extant strains and environmental clones Thepotential of the

pmoA

microarray was tested withenvironmental samples The results were in goodagreement with those of clone library sequence anal-yses The approach can currently detect less domi-nant bacteria down to 5 of the total communitytargeted Initial tests assessing the quantificationpotential of this system with artificial PCR mixturesshowed very good correlation with the expectedresults with standard deviations in the range of 04ndash172 Quantification of environmental samples withthis method requires the design of a reference mix-ture consisting of very close relatives of the strainswithin the sample and is currently limited by biasesinherent in environmental DNA extraction and univer-sal PCR amplification

Introduction

Methanotrophs are bacteria capable of utilizing methaneas their sole source of carbon and energy They are ubiq-uitous in nature and represent the largest biogenic sinkfor the greenhouse gas methane They oxidize methanevia methanol and formaldehyde to carbon dioxide or incor-porate carbon from methane into cell biomass at the oxi-dation level of formaldehyde The first step in the pathwayis catalysed by one of the two types of the enzyme meth-ane monooxygenase (MMO) The soluble MMO is foundin only some of these bacteria whereas the particulateMMO (pMMO) is present in all known methanotrophs(Hanson and Hanson 1996) except for one

Methylocellapalustris

(Dedysh

et al

2000) The sequence of the

pmoA

gene encoding the 27 kDa subunit of pMMO has beenshown to reflect evolutionary relationships amongst thecarrying organisms The ammonia monooxygenase(AMO) of autotrophic ammonia oxidizing bacteria (AOB)is evolutionarily related to pMMO and

pmoA

the geneencoding for the corresponding subunit of the AMO has ahigh degree of identity with

amoA

genes (McDonald andMurrell 1997) Both

pmo

A and

amo

A genes can bepresent in one to three generally highly similar copies inthe genomes of methane and ammonia oxidisers (McTav-ish

et al

1993 Semrau

et al

1995 Purkhold

et al

2000Bourne

et al

2001) There are

pmoA

amoA

related genescloned from environmental samples where the nature ofthe encoded enzyme is not clear (Holmes

et al

1999Henckel

et al

2000a Bourne

et al

2001) There is arapidly growing database (with over 700 entries) of

pmoA

amoA

and related gene sequences from cultivated strainsand lsquoenvironmentalrsquo clones retrieved directly from the envi-ronment by PCR Methanotrophs play an essential role inmitigating the greenhouse effect by metabolizing most ofthe biogenically produced methane Understanding thefactors influencing their diversity is thus of crucial impor-tance Denaturating gradient gel electrophoresis (DGGE)and clone library analysis have been used to monitorseasonal changes in methanotroph diversity or changesinduced by environmental impacts such as drainage oraeration (Henckel

et al

2000b Henckel

et al

2001Reay

et al

2001) The most comprehensive oligonucle-otide probe set for methanotrophs so far was designed byGulledge

et al

(2001) and targeted the 16S rRNA geneDNA microarrays are a powerful tool for the parallel

high-throughput detection and quantification of many

Diagnostic microarray for methanotrophs

567


Environmental Microbiology

5

566ndash582

genes Originally developed for whole genome geneexpression analyses (Schena

et al

1996 Tao

et al

1999) DNA microarrays have very strong applicationpotential in many areas of microbiology Upon availabilityof corresponding probe sets they enable the detection ofup to several thousand microbial strains species generaor higher clades (depending on the design of the probe)in a single assay In clinical veterinary and plant microbi-ology food and water quality control this means that asingle test can be developed to detect all pathogenicbeneficialcontaminating bacteria which might be presentin the sample being investigated The potential for envi-ronmental microbiology is even stronger By applyingnested sets of oligonucleotide probes (Behr

et al

2000)which target genes reflecting the phylogeny of the targetorganism it becomes possible to assess the wholeprokaryotic diversity of an environment The most obvioustarget for such studies are the 16S and 23S rRNA genesOther (often referred to as lsquofunctionalrsquo ie encoding forrelated enzymes carrying out a defined function) possibletarget genes can also be used at least for a physiologi-cally restricted group of microorganisms The applicationof functional genes narrows down the analysis to a func-tionally (sometimes also phylogenetically) defined groupof microbes The main advantage of this approach is thatit also enables the detection and analysis of uncultivatedmembers of the microbial groups being investigated(Raskin

et al

1994 McDonald and Murrell 1997Purkhold

et al

2000 Radajewski

et al

2000 Lovell

etal

2001 Lueders

et al

2001) In contrast to 16S rRNAenvironmental sequences belonging to a novel but func-tionally related phylum can easily be recognised andincluded in the analysis

Oligonucleotide probe sets spotted onto nylon or nitro-cellulose membranes (lsquomacroarraysrsquo) have been used forthe diagnosis of bacteraemia (Anthony

et al

2000) food-contamination (Rudi

et al

2002) detection of enterococci(Behr

et al

2000) or cyanobacteria (Rudi

et al

2000) Aspecial microarray format consisting of individual poly-acrylamide gel micropads with immobilized oligonucle-otides (Liu

et al

2001) was used for the characterizationof aromatic hydrocarbon degrading consortia (Koizumi

et al

2002) and identification of rifampicin resistantstrains of

Mycobacterium tuberculosis

(Mikhailovich

et al

2001) A lsquotraditionalrsquo glass microarray consisting ofseven probes was developed for

Staphylococcus

diagno-sis (Hamels

et al

2001) Different genotypes of rotavi-ruses were specifically detected by an oligonucleotidemicroarray (Chizhikov

et al

2002) Cho and Tiedje (2001)applied whole genome DNAndashDNA hybridization for thedetection and community analysis of microorganisms Ahigh-density Affymetrix GeneChip containing over 30 00016S rRNA targeting oligo probes was used to identifyculture collection species and subsequently to character-

ize populations of airborne bacteria at the level of higherphylogenetic taxa (Wilson

et al

2002) A 16S rRNA genebased oligonucleotide microarray targeting and coveringthe entire known diversity of sulphate reducers was devel-oped and successfully validated with environmental sam-ples (Loy

et al

2002)In most cases the target consists of labelled large gene

fragments (several hundred nucleotides long) increasingthe potential for the accumulation of background signalarising from a low rate of non-specific hybridization Alter-native labelling approaches such as terminal transferaselabelling of specific oligonucleotides (Rudi

et al

2002)and the application of end labelled specific stackingprobes in conjunction with immobilized capture probes(Small

et al

2001) may improve the detection limit ofdiagnostic microbial microarrays by decreasing the abovementioned non-specific background hybridization

Microbial community structures can be assessed by thequantitative analysis of micro or macroarray results Asimple and elegant method to quantify specific microbialgenes from a sample has recently been published (Choand Tiedje 2002) In this method gene fragments (500ndash900 bp in length) were applied as probes Each spot con-sisted of a mixture of an individual probe and a commonreference gene fragment Hybridization was done with aCy3-labelled environmental mixture and a Cy5-labelledreference DNA Quantification was based on the Cy3Cy5ratios Unfortunately the same principle cannot be appliedto the quantification of oligonucleotide chip results becauseof the inherent differences in hybridization efficienciesbetween oligonucleotide probes In practice a differentreference oligo for each probe should be designed

Even though high expectations exist for diagnosticmicrobial microarrays applications utilizing their fullpotential (high throughput detection of large numbers ofmicrobes and quantitative assessment of their communitystructures) are still very limited (Loy

et al

2002) Part ofthe reason for this is that a technical platform of reason-able cost based on readily available consumables andequipment with demonstrated potential in high-through-put quantitative microbial diagnostics is missing

Here we describe the development and validation of adiagnostic microbial microarray for the high-throughputdetection and community structure analysis of methan-otrophic and functionally related bacteria A set of tech-niques based on well established and widely appliedtechnologies and commercially available consumablesand equipment is reported

Results and discussion

Protocol optimization

One of the main problems when designing oligonucleotidemicroarrays is to achieve nearly identical melting temper-

568

L Bodrossy

et al



5

566ndash582

atures for all the probes on the array One potentialapproach to achieving this is the use of hybridization buff-ers containing tertiary amine salts Tetramethyl ammo-nium chloride (TMACl) or tetraethyl ammonium chloride(TEACl) have been successfully applied to enable GC-content independent hybridization on nitrocellulose ornylon membranes (Wood

et al

1985 Spiro

et al

2000)The present weakness of this approach is the lack ofdetailed information on the thermodynamics of hybridiza-tions in such solutions making it impossible to predict theeffects of further factors (as detailed later in this paper)on the behaviour of a given oligo probe Further the effectof tertiary amine salts on the different present and futuresurface chemistries is largely unknown Thus we choseanother approach by designing oligos which should havenearly identical melting temperatures in lsquotraditionalrsquo hybrid-ization buffers

Many independent studies have shown that stericeffects (interference of the solid support on the hybridiza-tion properties of the immobilized oligos and steric hin-drance resulting from the crowding of immobilized oligos)can seriously hinder the accessibility of immobilized oli-gonucleotide probes (Gou

et al

1994 Shchepinov

et al

1997 Brown and Anthony 2000) These effects are suc-cessfully mitigated by the application of spacer moleculesIn our hands a C12 linker and an extra five thymidineresidues at the 5

cent

end provided optimal spacing additionof further thymidine residues had no significant effect onthe hybridization capacity (accessibility) of the oligostested (data not shown)

Dimethylsulphoxide (DMSO) (50 vv in dH

2

O) wasselected as the printing buffer because it did not dry outduring long spotting rounds and provided uniform spotson the slides Standard deviations in signal intensitiesbetween replicate spots were 10ndash15 as opposed to 20ndash30 for arrays printed from 3

yen

SSC (data not shown)Spotting was done with a single pin to avoid variationsinherent in spotting with multiple pins

Several alternative approaches (methods are availablefrom the authors upon request) were tested for targetpreparation These included direct incorporation of Cy-labelled dNTPs into dsDNA during PCR application of alabelled PCR primer for the generation of dsDNA targetsand application of a labelled and a biotinilated PCR primerfor the generation of ssDNA targets via subsequent sep-aration of the two strands using streptavidin coated mag-netic beads As the secondary structure of the target playsa very significant role in determining the maximal hybrid-ization signal obtainable with a given probe (lsquohybridizationcapacityrsquo) it was an absolute necessity to fragment thetarget before hybridization RNA targets were generatedbecause RNA can be fragmented in a random manner viachemical fragmentation (Hughes

et al

2001) A furtheradvantage of using RNA targets is that the direct incorpo-

ration of the Cy-labelled nucleotides by the T7 RNA poly-merase is very efficient (Only a fourfold decrease wasobserved in RNA yield when 50 of UTP was replacedwith Cy3-UTP during

in vitro

transcription) Yields of targetpreparation were in the range of 50 ng

m

l

-

1

concentration(2500 ng) with every 10th to 12th nucleotide beinglabelled Fragmentation of the target RNA to an averagefragment size of 50 nucleotide (Hughes

et al

2001)resulted in a significant enhancement of hybridization Inspite of the sensitivity of the Cy dyes to nucleophilic attackapplied for RNA fragmentation an increase by over anorder of magnitude in the Cy3 as well as in the Cy5signals was observed (compared to signals obtained withunfragmented target RNA) Furthermore in many casesfragmentation decreased the differences between thehybridization capacities of probes (data not shown)Hybridization conditions were chosen which were relaxedenough to enable the hybridization of both perfect match(PM) and single mismatch (1 MM) targets

As the hybridization between oligonucleotides and anyother type of nucleic acid (oligonucleotide gene fragmentRNA etc) is a reversible process all hybridizations werecarried out overnight (16 h) to ensure complete hybridiza-tion In static microarray hybridizations diffusion is the onlyprocess providing mixing Statistical errors in the finalsignal are inherent in such a diffusion limited systemFurthermore such circumstances usually result in theedges of probe sets hybridizing more efficiently thus yield-ing a relatively strong signal for the edge of the spot anda much weaker one for the centre To overcome theselimitations a custom modified BellyDancer laboratoryshaker and sticky hybridization chambers with significantlyhigher volumes than those which exist between a microar-ray and a traditional coverslip were used The tiny bubblesunavoidably formed within this chamber were slowlymoved to the edge due to the motion of the BellyDancerand this provided enough extra mixing to ensure a rea-sonably uniform hybridization across the whole microarray

Standard deviation of results for (triplicate) spots ofindividual probes was 3 to 30 between parallel slideshybridized with targets prepared in parallel (slide-to-slidevariation)

The extent to which the application of different

pmoA

amoA

specific PCR primers influenced the results wasinvestigated Two alternative reverse primers were usedfor target amplification The primer

pmoA

682 can amplify

pmoA

amoA

and similar sequences and yields a productof 531 bp with the forward primer

pmoA

189 The primermb661 is specific for

pmoA

-type sequences and yields aslightly shorter (508 bp) product (Bourne

et al

2001) Asmany of the environmental

pmoA

clones were obtainedwith the latter primer both primer pairs were appliedStandard deviations between results generated with thedifferent primer pairs were 10ndash30 not exceeding that


569



5

566ndash582

between parallel experiments using the same primerpairs

Probe set design and validation

A database of over 700

pmoA

amoA

sequences wasestablished from public database entries and in part fromunpublished sequences (courtesy of A Auman S

Dedysh P Dunfield W Liesack I McDonald S Morrisand S Nold) Full-length and nearly full-length [ie cover-ing positions 206ndash661 of the

Mc capsulatus

(Bath)

pmoA

gene] as well as some of the (unique) shorter sequences(514 entries) were used to create a phylogenetic tree

One hundred and eighteen

pmoA

amoA

based oligonu-cleotide probes were designed A nested set of 68 probes(Table 1) was selected by omitting redundant probes The

Table 1

Set of oligonucleotide probes synthesized and spotted for evaluation Positions of mismatches with target sequences are indicated byboldfaced and underlined characters Melting temperatures were calculated by the nearest neighbour method

Name

a

Intended specificity Sequence 5

cent

AElig

3

cent

b

Length T

m

Selected

c

MM

d

Mb460

Methylobacter

GACAGTTACAGCGGTAATCGGTGG

24 609

+

Mb478

Methylobacter

TGGTATGGC

A

TGGGGTCTGT

20 597

+

TMb271

Methylobacter

TTGTGGTGGCGTTACCGT

18 580

+

511ndash436

Methylobacter

GTTTTGATGCTGTCTGGCAG

20 555

+

BB51-299

Methylobacter GCGCGGTTGTTTGTGTCT 18 574 ndashMb292 Methylobacter CCGTTACCGTCTGCCTTTCG 20 591 + T ATMm275 Methylomonas GTGGTGGAGATACCGTTTGCC 21 592 +Mm391 Methylomonas ATTTGCTTCCCATCTAACCTG 21 540 ndashPS80-291 clone PS-80 ACCAATAGGCGCAACACTTAGT 22 583 +peat1ndash3-287 Metylomonas-related peat clones AACTGCCTTTAGGCGCTACC 20 586 +Mb_SL1ndash421 soda lake Methylobacter isolates and clones GCGATCGTATTAGACGTTATCCTG 24 564 ndashMb_SL-299 soda lake Methylobacter isolates and clones GGGGTGCAACTCTGTGTATCTTAGG 25 605 + TJpn284 clone Jpn 07061 ACCGTATCGCATGGGGTG 18 580 +Mm_pel467 Methylomicrobium pelagicum ACTGCGGTAATCGATGGTTTGGC 23 616 +Est514 Methylomicrobium-related clones AATTGGCCTATGGTTGCGCC 20 599 +LP20-644 Methylomicrobium-related clones GGTACACTGCGTACTTTCGGTAA 23 582 +Mmb303 Methylomicrobium album CAATGCTGGCTGTTCTGGGC 20 603 +Ia193 Type I a (M bacter-M monas-M microbium) GACTGGAAAGATAGACGTCT 20 519 ndashIa577 Type I a (M bacter-M monas-M microbium TGGCTGACTTGCAAGGTTACC 21 589 + ANc_oce426 Nitrosococcus oceani CTTGGATGCCATGCTTGCGA 20 598 +Mth413 Methylothermus CACATGGCGATCTTTTTAGACGTTG 25 583 +Mc396 Methylococcus CCCTGCCTCGCTGGTGC 17 619 + C A501ndash286 Methylococcus-related clones GTCAGCCGTGGGGCG 15 590 ndashfw1ndash639 Methylococcus-Methylocaldum related clones GAAGGGCACGCTGCGTACG 19 620 + T CM90-201 Methylocaldum-related clones CGGCTGCTGTACAGGCGTTC 20 618 +Mcl408 Methylocaldum GGTTCCGGGTGCGATTTTG 19 578 +Ib453 Methylococcus-Methyocaldum and related GGCAGCTACCTGTTCACCGC 20 617 + GIb559 Methylothermus-Methylococcus-Methyocaldum

and relatedGGCATGCTGATGTCGATTGCCG 22 605 + C C C

Mcy262 Methylocystis CAGGCGTTCTGGTGGGTGAA 20 610 + T TMcy409 Methylocystis and peat clones ATCGTTCCGGCGATCTGGC 19 610 + U C

+hairpinPeat264 peat clones GGCGTTTTTCTGGGTCAACTTCC 23 603 +Msi520 Methylosinus GCGATCGCGGCTCTGCA 17 616 +Msi_tri309 Methylosinus CGCGGTTCTGGGTCTGCTC 19 614 + C C A GMsi270 Methylosinus GTTCTTCTGGGAGAACTTCAAGC 23 571 ndashMsi232 Methylosinus CCTGGGCGTGACCTTCGC 18 610 + T C G TGII510 Type II methanotrophs CGAACAACTGGCCGGCG 17 600 +II630 Type II methanotrophs CATGGTCGAGCGCGGC 16 597 +RA14-598 RA14 related clones AACGTTCGTACCTCGATGCC 20 583 + TT C CB2rel260 Methylocapsa-related clones GCCCAGTATTATTTCTGGACCCCAT 25 604 + Most of GC

at theends

B2-400 Methylocapsa ACCTCTTTGGTCCCGGCTG 19 605 +B2all343 Methylocapsa and related clones AACCGCTACACCAATTTCTGGCG 23 618 + CpmoAMO3-400 clone pmoA-MO3 CCCAGATGATCCCGTCGGC 19 608 +xb6ndash539 Methanotroph-related clones AGGCCGCCGAGGTCGAC 17 630 +LP21-190 Methanotroph-related clones ATCGACTTCAAGGATCGCCG 20 582 ndashLP21-232 Methanotroph-related clones ATCGTCGCCATGTGCTTCGC 20 619 +mtrof173 Universal GGbGACTGGGACTTCTGG 18 582 +mtrof362-I Methanotrophs TGGGGCTGGACCTACTTCC 19 595 ndashmtrof656 Methanotrophs ACCTTCGGTAAGGACGT 17 532 +mtrof661 Methanotrophs GGTAARGACGTTGCKCCGG 19 619 +mtrof662-I Methanotrophs GGTAAGGACGTTGCGCCGG 19 619 ndashNmNc533 Nitrosomonas-Nitrosococcus CAACCCATTTGCCAATCGTTGTAG 24 586 + G C

570 L Bodrossy et al

copy 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Environmental Microbiology 5 566ndash582

probes targeted different species groups of species gen-era as well as higher taxonomic groups of methanotrophsand related bacteria Several broad specificity probes tar-geting AOBs were also designed and included in order toimprove the potential of the array for analysing variousenvironments including those potentially dominated byAOBs Two probes (mtrof173 and mtrof661) weredesigned to target the PCR primers pmoA189 and mb661respectively A third probe lsquouniversalrsquo to methanotrophs(mtrof362-I) was designed for a region in the middle of thepmoA sequence which is reasonably conserved amongstmethanotrophs

The most critical step of the probe design process is tofine tune the probe set in a way that all probes in the setdisplay hybridization behaviour as identical as possible Inthe first stage an attempt was made to design oligos withpredicted melting temperatures [according to the nearestneighbour model (Breslauer et al 1986)] of 60 plusmn 2infinC Insome cases this was not possible because of the limitedlength of differentiative sequence regions When no alter-native probe sites were found the probes with suboptimalmelting temperatures were accepted and synthesized Aspresent models can only predict melting temperatures offree oligos but not of those bound to solid surfaces (seedetailed discussion below) probes with suboptimal pre-dicted melting temperature do not necessarily performsuboptimally

The hybridization behaviour of oligonucleotide probesimmobilized onto a solid surface depends on several fac-tors Length GC content and exact sequence of theprobe these together are considered when predicting Tm

for the oligos by using the nearest neighbour method

(Breslauer et al 1986) Position of GC and AT pairs themiddle of the probe is more important in stabilizing hybrid-ization thus a probe with most of its GC content in themiddle binds to its target more strongly than another onewith homogenous GC distribution (but with identical lengthand GC content) (Guo et al 1994 Shchepinov et al1997 Hughes et al 2001) Secondary structures of theprobe and of the corresponding target when any of thesetwo are of significant strength compared to the strengthof hybridization between the probe and the target a sig-nificant drop in hybridization efficiency is expected Theexact nature of the overhanging nucleotides on the target(nucleotides immediately next to the area targeted by theprobe) this comes from the nearest method model butisnrsquot normally accounted for because the overhangs of thetarget sequence are not considered Number and type ofmismatches some mismatches have little while othershave very strong destabilizing effect (Sugimoto et al2000) Position of mismatches mismatches in the middleare more destabilizing than mismatches at end positions(Fotin et al 1998) Factors arising from the immobilizednature of the probes steric effects can hinder the forma-tion of hybrids between the target and the bound probeThis effect is much stronger for the immobilized end of theprobe Thus the bound end of the probe plays a lesserrole in the hybridization than the free end (Guo et al1994 Shchepinov et al 1997 Hughes et al 2001) Thisapplies for the position of GC and AT pairs as well as forthe position of mismatches Hybridization between DNAoligos and RNA fragments as in our case has slightlydifferent thermodynamics to that of DNAndashDNA hybridiza-tion (Hung et al 1994 Sugimoto et al 2000)

Nsm_eut381 Nitrosomonas eutropha CCACTCAATTTTGTAACCCCAGGTAT 26 590 +Pl6ndash306 Nitrosomonas-Nitrosococcus related clones GGCACTCTGTATCGTATGCCTGTTAG 26 605 +PS5-226 Nitrosomonas-Nitrosococcus related clones ACCCCGATTGTTGGGATGATGTA 23 599 +NsNv207 Nitrosospira-Nitrosovibrio TCAATGGTGGCCGGTGG 17 585 + GNsNv363 Nitrosospira-Nitrosovibrio TACTGGTGGTCGCACTACCC 20 596 + A T TNit_rel223 AOB related clones GTCACACCGATCGTAGAGGT 20 569 +Nit_rel351 AOB related clones GTTTGCCTGGTACTGGTGGG 20 592 +Nit_rel470 AOB related clones CGATATTCGGGGTATGGGCG 20 584 + ANit_rel304 AOB related clones CGCTCTGCATTCTGGCGCT 19 618 +M84P105-451 environmental clones of uncertain identity AACAGCCTGACTGTCACCAG 20 581 +WC306ndash54ndash385 environmental clones of uncertain identity AACGAAGTACTGCCGGCAAC 20 592 +M84P22-514 environmental clones of uncertain identity AACTGGGCCTGGCTGGG 17 610 +gp23ndash454 environmental clones of uncertain identity AACGCGCTGCTCACTGCG 18 623 +MR1-348 environmental clones of uncertain identity AATCTTCGGTTGGCACGGCT 20 611 +gp391 environmental clones of uncertain identity ATCTGGCCGGCGACCATG 18 611 +gp2ndash581 environmental clones of uncertain identity ACATGATCGGCTACGTGTATCCG 23 600 +RA21-466 clone RA21 ndash environmental clone of

uncertain identityCGGCGTTCTTGGCGGCAT 18 624 +

Namea Intended specificity Sequence 5cent AElig 3centb Length Tm Selectedc MMd

a Numbers at the end of the probe names refer to their relative positions on the Mc capsulatus (Bath) pmoA geneb Sequences are of the sense strandc Oligonucleotide probes of the final probe set are indicated by +0 Probes not selected are indicated by ndashd Nucleotide residue(s) at mismatch position(s) Other factors included in the calculation of weighed mismatches are also indicated

Table 1 cont

Diagnostic microarray for methanotrophs 571


Based on the above criteria on initial results from test-ing the hybridization behavior of our probes and on pub-lished data (Shchepinov et al 1997 Fotin et al 1998Meroueh and Chow 1999 Sugimoto et al 2000) a set ofsimple findings was compiled which significantly improvedthe prediction of the hybridization behaviour of the probesThese findings are

(i) UAEligC changes in the target sequence leads to an rG-dT bond which is almost as strong as the original rA-dT bond (for perfect match cases) These are notconsidered as mismatches

(ii) GAEligA changes in the target sequence leads to an rU-dG bond which is almost as strong as the original rC-dG bond (for perfect match cases) These mis-matches are considered only if they are presenttogether with other types of mismatches

(iii) Mismatches in the end positions are not consideredMismatches adjacent to the end positions are consid-ered only if they are present together with other typesof mismatches

(iv) If most of the GC content of a probe is close to the5cent (immobilized) end the probe will display a signifi-cantly lower melting temperature than originally pre-dicted The same is true for a G or C residue in the3cent end position

(v) Probes with strong hairpin structures (DG ge 20) wereconsidered as having an extra mismatch to the target

(vi) High GC content probes shorter than 17 nucleotidesin length display unreliable hybridization behaviorunder the experimental conditions applied

Considering the above points another nine probeswere excluded and mismatch values were updated toweighed mismatch values The resulting set of 59 oligo-nucleotide probes was validated with a reference set of59 pure strains and environmental clones covering almostthe entire known diversity of methanotrophsand bacteriacarrying pmoAamoA homologues (listed in Fig 2) Sev-eral AOB clones were also included in the validationHybridizations were carried out at 55infinC with the aim ofallowing for perfect match and single (weighed) mismatchprobe-target pairs to hybridize Figure 1 shows some typ-ical hybridization results Hybridization between a probeand a target was considered positive if the signal was atleast 5 of the signal obtained for mtrof173 on the samearray There were unfortunately groups of clones forwhich no representative was available Figure 2 shows thepredicted hybridization behaviour of the probe set and theresults obtained

Out of 59 probes in the probe set we were unable toobtain reference targets for seven which were thus notpossible to validate Most (42) probes displayed hybridiza-tion behavior as predicted Two probes II510 and II630were expected to have unreliable hybridization behaviour

because of their shortness (in combination with a strongsecondary structure in case of II510) These two probeswere left in the probe set as we were unable to identify abetter region for a probe specific to the Type II methan-otrophs Eight probes displayed some unpredicted resultsThe unexpected positive result of Mcy409 came from acombination of a strong secondary structure and a mis-match The unexpected negative results of Msi232 wasobtained with targets displaying two adjacent mismatchesright at the 3cent end plus a third one at different internallocations of the probe Probe Ia577 displayed unexpectednegative results to three targets all having the a singlecentral mismatch U(r)A change resulting in an rU-dT pairreplacing the perfect match rA-dT pair

Fifty out of the 59 probes were succesfully validated(seven probes with no reference targets available and twosuboptimal probes II510 and II630 were not) Of the 2950individual hybridization reactions (50 validated probes yen59 reference strainssequences) 2931 (993) yieldedthe expected result by either showing detectable signalwhere expected or by no hybridization where a negativeresult was predicted Only 19 of the hybridization reac-tions (07) resulted in false negative or positive hybrid-ization Forty-two out of the 50 probes consideredbehaved 100 as predicted (in all of the hybridizationreactions) This success rate is acceptable when redun-dant probe sets (three or more probes for each speciesor higher taxonomic group targeted) are and can bedesigned There is however a need for an improvedmethod to predict hybridization behaviour of oligonucle-otide probes About half of the unpredicted results wereassociated with complicated cases where additionalparameters influencing hybridization behaviour had to beconsidered together with mismatches Mismatches espe-cially when their relative positions are also consideredcan reliably be accounted for only by a nearest-neighbourmethod based algorithm Software applying such an algo-rithm which considers further effects arising from theimmobilized nature of the probes as well as the second-ary structure of the probe and the target via user definedparameters is badly needed A computer program underdevelopment called CALCOLIGO is aiming exactly at fillingthis gap (J Csontos Bay Zoltaacuten Institute for Biotechnol-ogy Szeged Hungary pers comm)

Despite its apparent shortcomings the probe set candiagnose almost the entire known diversity of methanotro-phs and bacteria carrying pmoAamoA homologues Thegaps in the validation of probe set represent probesagainst unique clones or small groups of clones whichseem to be very poorly represented in the environmentsinvestigated so far Results from environmental samplesneed to be referred to the validation results with the ref-erence set rather than to the predicted ones thus mini-mizing the chances of false interpretation of results



Evaluation of the microarray with environmental samples

To assess the applicability of the developed methanotrophmicroarray in environmental studies two different environ-mental samples were analysed

The first experiment was carried out with a soil samplefrom a landfill site collected at the end of the summerMicroarray results indicated that strains related to thegenera of Methylocaldum and Methylocystis were mostabundant in this sample In addition to Methylocaldum andMethylocystis pmoA specific probes general pmoAprobes for the B subgroup of Type I methanotrophs andfor the Type II methanotrophs also showed positive hybrid-ization Two further probes Mb478 and Msi232 were pos-itive Mb478 was known from the validation set to

hybridize strongly to pmoA of Methylocaldum tepidumthus this signal was accounted for as arising from Methy-localdum-related bacteria Msi232 is a probe with a singlemismatch towards pmoA from most Methylocystis strainsDuring validation experiments it was shown to displaystrong hybridization signal with pmoA from one clade ofMethylocystis Thus (weak) positive results with Msi232were accounted for as arising from Methylocystis relatedbacteria To confirm these results a pmoA clone librarywas constructed Out of 100 clones sequenced 91 con-tained inserts with high homology to pmoA sequencesSixty-five per cent of these clones were related to pmoAfrom Methylocaldum and 31 to pmoA from Methylocys-tis confirming the results of the microarray analysis Afurther 3 of the clones showed highest similarity to

Fig 1 A Schematic diagram of the microarray design Arrays were spotted in triplicate Frames indicate universal (lsquomtrofrsquo) probes spotted in multiple copies and spots with an external positive control probe (lsquohyaBprsquo results of this were not considered or used in the present study)B Detailed design of a single array with exact positions for each probeC Representative hybridizations with reference strains or environmental clones Probe hyaBp targets an independent gene (hyaB of E coli) It can be applied as an alternative control spot for normalization after spiking of the in vitro transcription reaction with hyaB PCR product Note that results of probe hyaBp were not considered throughout the work presented here Microarray images were adjusted for best viewing (quantitative conclusions drawn from the image may be misleading)



Fig

2

Ran

ge o

f st

rain

cov

erag

e fo

r ol

igon

ucle

otid

e pr

obes

tar

getin

g pm

oAa

moA

gen

es o

f m

etha

notr

ophs

A

OB

s an

d ba

cter

ia c

arry

ing

pmoA

am

oA h

omol

ogue

s A

sim

ilar

tabl

e w

ith a

ll 51

4 se

quen

ces

cons

ider

ed (

with

out

hybr

idiz

atio

n re

sults

) is

ava

ilabl

e fr

om t

he a

utho

rs u

pon

requ

est

Und

er lsquoP

robe

srsquo b

lack

fill

indi

cate

s ex

pect

ed p

ositi

ve r

esul

ts

grey

fill

indi

cate

s po

sitiv

e re

sults

not

pr

edic

ted

and

thic

k bl

ack

fram

ing

indi

cate

s ne

gativ

e re

sults

whe

re h

ybrid

izat

ion

was

pre

dict

ed W

hite

num

bers

insi

de b

lack

and

gre

y bo

xes

indi

cate

the

num

ber

of lsquom

ism

atch

equ

ival

ents

rsquo as

desc

ribed

in t

he r

elev

ant

sect

ion

of R

esul

ts L

ette

rs in

gre

y bo

xes

indi

cate

pro

bes

of u

nrel

iabl

e hy

brid

izat

ion

beha

viou

r lsquoA

cent a

shor

t pr

obe

with

sig

nific

ant

seco

ndar

y st

ruct

ure

lsquoBcent a

sho

rt p

robe

N

ames

of

envi

ronm

enta

l clo

nes

are

prec

eded

by

an in

dica

tion

of t

heir

pred

icte

d im

med

iate

phy

loge

netic

rel

atio

nshi

p H

ighe

st a

nd lo

wes

t si

gnal

val

ues

( o

f th

at o

f m

trof

173

) ob

tain

ed w

ith f

ull

mat

ch ta

rget

s ar

e in

dica

ted

(lsquomax

val

rsquo an

d lsquom

inv

alrsquo)

Gre

y bo

xes

indi

cate

dat

a w

hich

are

not

rep

rese

ntat

ive

(Jpn

284

Est

514

Nc_

oce4

26 g

p23ndash

454

and

MR

1-34

8 n

o fu

ll m

atch

ref

eren

ce ta

rget

av

aila

ble

II5

10 a

nd I

I630

sho

rt p

robe

s of

unr

elia

ble

hybr

idiz

atio

n be

havi

our

B2r

el26

0 an

d N

sNV

207

dat

a fr

om s

ingl

e m

ism

atch

tar

gets

mtr

of 1

73 r

efer

ence

pro

be m

trof

661

tar

getin

g th

e re

vers

e pr

imer

app

lied

in m

ost

ampl

ifica

tions

) A

pm

oAa

moA

DN

A n

eigh

bour

tre

e is

add

ed t

o th

e le

ft pr

ovid

ing

furt

her

insi

ght

into

the

phy

loge

netic

rel

atio

nshi

ps o

f th

e re

fere

nce

set

The

sca

le

bar

indi

cate

s th

e es

timat

ed n

umbe

r of

bas

e ch

ange

s pe

r nu

cleo

tide

sequ

ence

pos

ition

Str

ain

C

lon

eP

r o

b e

s

Mm275

PS80-291

Mb460

Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

Mcy409

Peat264

Msi520

Msi_tri309

Msi232

II510

II630

RA14-598

B2rel260

B2-400

B2all343

pmoAMO3-400

xb6-539

LP21-232

NmNc533

Nsm_eut381

Pl6-306

PS5-226

NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

6261

6160

6058

6061

6261

6362

5959

6160

5960

5759

5862

5859

6162

6161

6062

5860

62

Min

val

130

26

389

353

14

16

50

NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

NA

192

87

189

91

373

112

12

335

49

103

29

88

111

17

116

NA

NA

22

34

58

100

84

NA

Max

val

180

26

389

353

72

16

80

NA

108

NA

31

NA

166

41

50

NA

41

175

312

10

47

158

371

194

38

75

34

46

167

39

29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

29

88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

e M

90-P

12+

33

1M

eth

ylo

mo

nas

clo

ne

mv1

9pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv2

1pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv9

pa

+2

++

1M

eth

ylo

mo

nas

met

han

ica

clo

ne

D1

+2

++

2++

clo

ne

PS

-80

2+

33

Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

b2

+3

1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

3+

3M

eth

ylo

bac

ter

clo

ne

5hm

-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

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eth

ylo

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ter

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ne

RB

-100

1+

33

2+

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Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

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eth

ylo

cyst

is c

lon

e F

12

+1

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B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

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B1

3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

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B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

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B3

1M

eth

ylo

sin

us

tric

ho

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riu

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b3b

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B3

+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+



pmoA from Methylomicrobium album strains The pres-ence of the Methylomicrobium album related strains wasnot detected by the microarray (relevant specific probeMmb303) This is due to the current detection limit whichis about 5 of cells in relation to the total bacterial com-munity analysed (ie containing pmoAamoA genes)There is also a statistical uncertainty in the percentagevalues derived from the analysis of only 91 clonesMicroarray and clone library results are shown on Fig 3

The second experiment was done using a sample froma soil microcosm incubated with CH4 as described byRadajewski et al (2002) Microarray experiments indi-cated the presence of methanotrophs belonging to twoclades The first clade the lsquopeat clonesrsquo group belongs toType II methanotrophs and is only known from acidic(peat) environments so far There are no cultured repre-sentatives of this group yet The second clade the LP21group is related to the Methylocapsa and to the RA14groups This clade consists of environmental clones ofvarious origins and of the lsquounusualrsquo second pmoA copiesfound in some Type II methanotrophs (Dunfield et al2002) This environmental sample has already beenanalysed by clone library construction and sequencing(Radajewski et al 2002) Fifty pmoAamoA clones wereanalysed (Fig 4) Eighteen per cent of the clonesbelonged to the peat clones group and 70 to the LP21group confirming the microarray results detecting thesetwo groups as the dominant ones Four per cent of theclones analysed belonged to the NitrosomonasNitroso-coccus group and 8 to the gp2 group The latter twogroups were not detected by the microarray analysis (rel-evant probes were NmNc534 for the NitrosomonasNitrosococcus group and gp391 and gp2ndash581 for the gp2group) The abundance of these groups was very near tothe detection level Furthermore the clone library and themicroarray analyses were done from different PCR reac-tions carried out from the same environmental DNAstock but in different laboratories Thus different PCRbiases may account for the discrepancy between themicroarray and clone library analysis results for these lowabundance sequences

Quantification of methanotrophs and related bacteriabased on pmoAamoA sequences is potentially biased bythe different number of pmoAamoA gene copies per cellThis has to be taken into account when interpreting suchresults

Quantification potential

Quantification potential of the developed microarray wastested with artificial mixtures of pmoA sequences Thisway it was possible to avoid the introduction of biasesinherent in DNA purification from environmental samplesand PCR with degenerate primers (Reysenbach et al

1992 Witzingerode et al 1997 Polz and Cavanaugh1998 Ishii and Fukui 2001) and the results show thepotential of the array to reflect the composition of the PCRmixture (rather than that of the original environmentalDNA or the original microbial community) By employinga reference mixture of known composition it was possibleto normalize variations in spot morphology and local dif-ferences in hybridization efficiency as well as for the sig-nificant variation in hybridization capacities betweendifferent probes

Our results shown in Fig 5 showed very good corre-lation between the true composition of the artificial mix-tures and the results of quantitative analysis of thehybridization results Standard deviation from expectedratios were in the range of 04ndash172 These results showthe potential of the microarray approach to reflect theratios within the PCR product (used as template for targetpreparation) As the first steps of the procedure includeenvironmental DNA purification and PCR with universalprimers the microarray approach is also prone to the biasinherent in these techniques

Quantification potential was further tested with the land-fill site and microcosm environmental samples (Fig 6)Results from the first analyses (hybridization with Cy3-labelled target prepared from environmental DNA) wereused to gain a rough estimate of the relative abundanceof methanotrophs in these samples Based on theseresults a mixture of reference sequences covering theobserved diversity was designed and labelled with Cy5Competitive (lsquotwo-colourrsquo) hybridization with the Cy3-labelled environmental and the Cy5-labelled reference tar-get was used to refine quantitative assessment of meth-anotroph community composition

In this quantification scheme a one-colour hybridizationis carried out first giving a rough estimation of the com-munity structure by referring back the relative intensitieson the array to the results from the reference straincloneset This information can also be used to select a subsetof reference strainsclones to be used in the next stagewhere the same target is then hybridized against theselected reference set and quantitative data are drawnfrom the ratios of the two signals The basic requirementof such a two-colour quantification approach is the abilityto identify and create an appropriate reference set Thisshould consist of sequences as similar to those in thesample as possible Failure to do so will result in skewedpredicted ratios Even though this limits the applicationpotential of this approach it can be very useful in studieswhere the same community is analysed over time or underdifferent conditions

By comparing the results of the two-colour microarrayhybridizations to the composition of the correspondingclone libraries (landfill site 14ndash30 predicted for Methy-localdum and 21ndash28 for Methylocystis versus 65 and



Fig 3 A pmoAamoA DNA neighbour tree showing the phylogenetic positions of the clones obtained from the landfill site sample The number of clones (out of 91 analysed) identical to the displayed representatives is indicatedB Application of the diagnostic microarray to analyse the diversity of methanotrophs in the landfill site sample One slide contained three replicates of the array thus each probe was printed in triplicate For each microarray position the name and the sequence of the probe is indicated in Fig 1A and in Table 1 respectively Probe spots having a normalized signal value (reference mtrof173) greater than 5 of the maximum value obtained with reference sequences were considered as positive Microarray image was adjusted for best viewing (quantitative conclusions drawn from the image may be misleading)

Methylomonas sp LW 15Methylomonas methanica

Methylobacter sp LW 12Methylobacter sp BB51

82

67

Soda lake strain 5b

15

Methylomicrobium pelagicum

Nitrosococcus oceanus

31

66

Methylomicrobium album

Methylobacter sp LW 1M2 78 (2 clones)

M2 105 (1 clone)

47

88

Methylobacter sp LW14

88

55

Methylothermus sp HB

Methylocaldum tepidum

M2 11 (30 clones)M2 9 (6 clones)

M2 80 (1 clone)M2 19 (2 clones)

80

66

80

M2 32 (2 clones)Methylocaldum gracile

M2 37 (1 clone)

88

66

M2 4 (9 clones)Methylocaldum szegediense

M2 69 (1 clone)

80

M2 66 (1 clone)

99

56

M2 10 (2 clones)M2 83 (1 clone)

88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54

Methylococcus capsulatus

CloneFW-18

78

43

36

46

Type II methanotroph AML-A6


88


80

Methylocystis sp LW 5

39

Methylocystis sp M

M2 35 (3 clones)M2 53 (1 clone)M2 29 (1clone)

80

Methylocystis echinoides strain 491

88

99

Methylocystis parvus OBBP

23

37

25

Clone peat1-2

69

Methylosinus trichosporium Ob3b

Clone SL 5101

Methylosinus sporium strain SE 2

41

92

94

Methylocapsa acidophila strain B2

Clone RA 14

96

89

86

Nitrosococcus mobilis

Nitrosomonas eutropha

Nitrosolobus multiformis

Nitrosospira briensis

42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B



Fig 4 A pmoAamoA DNA neighbour tree showing the phylogenetic positions of the clones obtained from the microcosm sample The number of clones (out of 50 analysed) identical to the displayed representatives is indicatedB Application of the diagnostic microarray to analyse the diversity of methanotrophs in the microcosm sample One slide contained three replicates of the array thus each probe was printed in triplicate For each microarray position the name and the sequence of the probe is indicated in Fig 1A and in Table 1 respectively Probe spots having a normalized signal value (reference mtrof173) greater than 5 of the maximum value obtained with reference sequences were considered as positive Microarray image was adjusted for best viewing (quantitative conclusions drawn from the image may be misleading)

Methylomonas sp LW 15

Methylomonas methanica

Methylobacter sp LW 12

Methylobacter sp BB51

82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18

Methylocaldum szegediense

Clone M90-P4

Clone FW-1

54

43

78

36

46




39

Methylocystis echinoides 491

Methylocystis sp M


23

37

25

Clone peat1-2

Clone P129 (8 clones)

69

29


Clone SL 5101

Methylosinus sporium SE 2

41

92

94

Methylocapsa acidophila B2

Clone RA 14

96

Clone P128 (35 clones)Clone P1212 (1 clone)

Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88

Nitrosomonas europaea



11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232

NmNc533NsNv207NsNv363

Gp2-581 Gp391

A

B



Fig 5 Quantification of artificial mixtures of pmoA sequences using the diagnostic microarray Relative abundance values were calculated from each positive probe the two-colour calculation method Cy3Cy5 ratios were used where the Cy3 signal arose from artificial mixtures of varying composition (column lsquoexpected ratiosrsquo) and the Cy5 signal from the artificial reference mixture containing all 5 sequences in equal amount (20) Standard deviations are indicated next to measured ratios Microarray images were adjusted for best viewing (quantitative conclusions drawn from the image may be misleading)

Fig 6 Quantitative analysis of environmental samples using the diagnostic microarrayA Landfill site sampleB Microcosm sampleImages of two-colour hybridization results are shown Relative abundance values were calculated from each positive probe both using the single-colour and the two-colour calculation methods In single colour calculations normalized ratios were divided by the highest values obtained with reference sequences and the resulting values were taken as indications for the relative abundance of the carrying bacteria In two-colour hybridizations the Cy3Cy5 ratios were used where the Cy3 signal arose from the environmental sample and the Cy5 signal from artificial reference mixtures Microarray images were adjusted for best viewing (quantitative conclusions drawn from the image may be misleading)



31 in the clone library respectively microcosm 25ndash35predicted for the lsquopeat clonesrsquo group and only 3ndash5 forthe LP21 group versus 18 and 70 in the clone libraryrespectively) it becomes obvious that further work isneeded before diagnostic microbial microarrays can beused for quantitative environmental work Obvious culpritsfor these discrepancies are biases in the PCR and in thecloning of the PCR mixes

Conclusions

The set of techniques presented here enables the designproduction and application of diagnostic microbialmicroarrays by an average microbiology laboratory withaccess to standard molecular biology equipment a com-mercially available spotter and scanner Even though thepilot array developed is targeting a functional gene(pmoA) the techniques and experience described hereare also directly applicable to 16S rRNA based arrays

The current detection limit of the technology is about5 of the total population analysed If cells are present ata lower ratio they may be missed ndash this depends on thenarrow specificity probes targeting them This bottleneckcan be improved by employing more stringent hybridiza-tion conditions however that way one gets limited to PMprobes thereby losing the potential for designing widerspecificity probes New higher binding capacity surfaceswill improve the performance of the approach Finallyalternative labelling techniques (Small et al 2001 Rudiet al 2002) ndash where only a short oligonucleotide islabelled instead of the entire length of the PCR product ndashmay decrease background arising from non-specifichybridization thus improving the system

There are many advances taking place in the field ofmicroarrays which will result in novel technologies thatmay significantly improve the power of this technologyNovel platforms such as new three dimensional slidesurfaces electrically addressed microarrays bead-arraysand lab-on-the-chip techniques are being developed Thecornerstone of diagnostic microbial microarrays thedesign and behaviour of the oligonucleotide probes willhowever not differ too much between the different plat-forms Thus most of the techniques and guidelines pre-sented here will be easily transferable to emerging noveltechnologies

A semi-quantitative analysis of environmental samplesis possible in two stages a first single-colour hybridiza-tion is used to develop a rough estimation of thecommunity structure followed by a second two-colourhybridization with a custom-made reference set based onthe initial results Biases inherent in the preceding molec-ular biology procedures impose limitations upon thisapproach Even after a careful optimization of these stepssuch results must be interpreted with caution Perhaps the

most promising field for diagnostic microbial microarraybased quantification is the analysis of temporal and spa-tial changes within one environment Before applying thedeveloped pmoA microarray in high-throughput analysisof environmental samples for methanotroph diversity it isnecessary to optimize DNA extraction purification andlsquouniversalrsquo pmoAamoA PCR protocols to minimize thebias introduced This aim will also be made easier by thismicroarray

Experimental procedures

Environmental samples

A landfill site sample (Seibersdorf Austria) was collected atthe end of August 2002 The sample collected from the top20 cm of the landfill site cover soil had a pH of 705 and atemperature of 10infinC above ambient air temperature Thesample was lyophilized and stored at - 80infinC until use A soilmicrocosm incubated with 12C-CH4 was also used to evaluatethe microarray Details of this microcosm are already pub-lished (Radajewski et al 2002)

Oligonucleotide probe design

Database and phylogenetic trees were constructed and oli-gonucleotide probes were designed using the phylogeneticsoftware package ARB (Strunk et al 2000) A comprehensivedatabase containing all published pmoAamoA and relatedsequences as well as many unpublished ones was estab-lished Alignments were made using Old Aligner function inARB_EDIT Parsimony DNA and protein trees were constructedand used to guide the probe design process Probes weredesigned using the Probe Design and Probe Match functionsaccessing a PT-server database created from the above ARB

database Outputs of the Probe Match function were importedinto Excel and a pivot table was constructed indicating thenumber of mismatches between each probe-target pair Thistable was refined by applying a set of empirical rules asdescribed in the relevant section of Results and discussionMelting temperatures of the probes were predicted usingthe nearest neighbour method using the public web sitehttpbiotoolsidtdnacomanalyzer Weighed mismatch val-ues were calculated from the number of mismatches asdescribed in the Results section

Microarray preparation

Oligonucleotides for immobilization were custom synthesized(VBC Genomics Vienna Austria) with a 5cent NH2 group fol-lowed by a C12 spacer and five thymidines residues precedingthe probe sequence A 384-well flat bottom plate was pre-pared with 30 ml of 50 mM oligonucleotide solutions in 50DMSO Samples were spotted with an OmniGrid spotter (1TeleChem SMP3 pin) at 50 relative humidity (using thehumidity controller of the spotter) and 22infinC onto silylatedslides (with aldehyde chemistry Cel Associates Houston)Arrays were always spotted in triplicate to enable a statisticalcorrection for errors Spotted slides were incubated overnight



at room temperature at lt30 relative humidity rinsed twicein 02 (wv) SDS for 2 min at room temperature with vigor-ous agitation to remove the unbound DNA Slides were thenrinsed twice in distilled water (dH2O) for 2 min at room tem-perature with vigorous agitation transferred into dH2O pre-heated to 95ndash100infinC for 2 min and allowed to cool at roomtemperature (~ 5 min) Slides were treated in a freshly (imme-diately before use) prepared sodium borohydride solution for5 min at room temperature to reduce free aldehydes Prepa-ration of the sodium borohydride solution 05 g NaBH4 wasdissolved in 150 ml phosphate-buffered saline (PBS 8 gNaCl 02 g KCl 144 g Na2HPO4 024 g KH2PO4 in 1000 mlH2O pH 74 autoclaved) then 44 ml of 100 ethanol wasadded to reduce bubbling Slides were rinsed three times in02 (wv) SDS and once in dH2O for 1 min each at roomtemperature Finally slides were dried individually using anairgun fitted with a cotton wool filter inside (to keep oil micro-droplets away from the slide surface) Dried slides werestored at room temperature in the dark before use

Environmental DNA preparation

The DNA isolation was performed as previously described(Sessitsch et al 2001) Briefly freeze-dried soil was resus-pended in 012 M sodium phosphate buffer (pH 80) andtreated with lysozyme Cells were disrupted by bead beatingand cell lysis was completed by SDS treatment Impuritieswere removed by phenol-chloroform extraction and precipita-tion of humic acids by potassium acetate DNA was precipi-tated by isopropanol washed in 70 ethanol dried andresuspended For final purification spin columns that con-tained Sepharose CL-6B (Pharmacia) and polyvinylpyrroli-done (Sigma 20 mg ml-1) were used

Target preparation

pmoAamoA genes were amplified using the forward primerpmoA189 (5cent-GGBGACTGGGACTTCTGG-3cent) and either oneof the reverse primers T7-mb661 (5cent-TAATACGACTCACTATAGCCGGMGCAACGTCYTTACC-3cent) or T7-A682 (5cent-TAATACGACTCACTATAGGAASGCNGAGAAGAASGC-3cent) where B =(CGT) M = (AC) Y = (CT) S = (CG) and N = (ACGT) Prim-ers T7-mb661 and T7-A682 were specific for methanotrophsand for methanotrophsAOBshomologous genes from envi-ronmental libraries respectively (Bourne et al 2001) Thereverse primers contained the T7 promoter site (5cent-TAATACGACTCACTATAG-3cent) at their 5cent end which enabled T7 RNApolymerase mediated in vitro transcription using the PCRproducts as templates For each target three PCR reactionsof 50 ml volume each consisting of 1yen PCR buffer 15 mMMgCl2 50 nM for each four dNTPs 15 pmoles of both prim-ers 1 ng genomicenvironmental DNA or 01 ng cloned PCRproduct as template and 1 U of Taq polymerase (Invitrogen)were performed in a Hybaid Combi Thermal Reactor TR2using Taq DNA polymerase in accordance with the manufac-turerrsquos instructions Amplification conditions were 95infinC for5 min before template was added then 32 cycles of 1 minat 95infinC 1 min at the annealing temperature 1 min at 72infinCfollowed by a final elongation step of 10 min at 72infinC Poly-merase chain reaction products were pooled and purified

using the HighPure PCR purification kit (Macherey-Nagel)according to manufacturerrsquos instructions Purified DNA wasdissolved in ultrapure water to a DNA concentration of 50 ngml and stored at - 20infinC

Working under RNAse-free conditions in vitro transcriptionwas carried out as follows 8 ml purified PCR product (50 ngml-1) 4 ml 5yen T7 RNA polymerase buffer 2 ml DTT (100 mM)05 ml RNAsin (40 U ml-1) (Promega) 1 ml each of ATP CTPGTP (10 mM) 05 ml UTP (10 mM) 1 ml T7 RNA polymerase(40 U ml-1) (Gibco BRL) and 1 ml Cy3 or Cy5-UTP (5 mM)were added into a 15 ml microcentrifuge tube and incubatedat 37infinC for 4 h RNA was purified immediately using theQuiagen RNeasy kit according to manufacturerrsquos instructionsPurified RNA was eluted into 50 ml dH2O RNA yields and dyeincorporation rates were measured by spectrophotometry

Purified RNA was fragmented by incubating with 10 mMZnCl2 and 20 mM TrisCl (pH 74) at 60infinC for 30 min Frag-mentation was stopped by the addition of 10 mM EDTApH 80 to the reaction and putting it on ice RNAsin (1 ml40 U ml-1) was added to the fragmented target Fragmentedlabelled RNA targets were stored at -20infinC Length of thefragmented RNA target was measured by running the sampleon an ABI capillary sequencer as well as running on a thin2 agarose gel applied onto a standard microscope slideand subsequent scanning in a GenePix 4000 A scanner

Reference targets and artificial target mixtures for testingthe quantification potential were synthesized by mixing knownamounts of purified PCR products and carrying out in vitrotranscription and target fragmentation as described above

Hybridization

No prehybridization was done Hybridization was carried outin a custom tailored aluminum block used as an insert for atemperature controlled Belly Dancer (Stovall Life SciencesGreensboro NC) set at maximum bending (about 10infin) Thehybridization block was preheated to 55infinC for at least 30 minto allow the temperature to stabilize An Eppendorf incubatorwas also preheated to 65infinC HybriWell (Grace BioLabs) stick-on hybridization chambers (200 ml in volume) were appliedonto the slides containing the arrays Assembled slides werepreheated on top of the hybridization block For each hybrid-ization 124 ml DEPC-treated water 2 ml 10 SDS 4 ml 50yenDenhardtrsquos reagent (Sigma) 60 ml 20yen SSC and 10 ml targetRNA were added into a 15 ml Eppendorf tube and incubatedat 65infinC for 1ndash15 min Preheated hybridization mixtures wereapplied onto assembled slides via the port in the lower posi-tions (to minimize risk of air bubbles being trapped within thechamber) Chambers were sealed with seal spots (GraceBioLabs) and incubated overnight at 55infinC at 30ndash40 rpmcirculation and maximum bending

Following hybridization HybriWell chambers wereremoved individually and slides were immersed immediatelyinto 2yen SSC 01 (wv) SDS at room temperature Slideswere washed by shaking at room temperature for 5 min in2yen SSC 01 (wv) SDS twice for 5 min in 02yen SSC andfinally for 5 min in 01yen SSC Slides were dried individuallyusing an airgun with a cotton wool filter inside Slides werestored at room temperature in the dark and scanned thesame day



Scanning and data analysis

Hybridized slides were scanned at three lines to average10 mm resolution with a GenePix 4000 A laser scanner(Axon Foster City CA) at wavelengths of 532 nm and635 nm for Cy3 and Cy5 respectively Fluorescent imageswere captured as multilayer tiff images and analysed with theGENEPIX PRO 30 software (Axon) Microsoft Excel was usedfor statistical analysis and presentation of results

Results were normalized to a positive control Hybridizationsignal for each probe was expressed as percentage of thesignal (median of signal minus background) of the positivecontrol probe mtrof173 on the same array As each slidecontained triplicate arrays normalized signal intensities ofthe triplicate spots on a slide were used to determine averageresults and standard deviations Non-specific signalsreached in some cases a value of up to 7 ( of that ofmtrof173) Such high non-specific signals were found forprobes which had a high binding capacity as reflected by thesignals obtained with perfect match targets Thus a cutoffvalue of 5 in relation to the highest signal (obtained withperfect match targets) was chosen to define positive andnegative calls

For assays where a (Cy3)-labelled target and a referencetarget (labelled with Cy5) were applied (lsquotwo-colour hybridiza-tionsrsquo) the median of ratios were used Results were normal-ized to mtrof173 and corrected for the relative amounts ofdifferent sequences added into the reference target mixture

Even though no dedicated negative controls were appliedfor each individual hybridization over 70 of all probespresent on the array were negative controls

All results reported represent the average of at least threereplicates

Quantification potential assessment

Mixtures with known compositions were made from referencesequences (PCR products ready for IVT labelling) and werelabelled with Cy3 and Cy5 Cy5-labelled mixtures were usedas reference Median of ratios (Cy3Cy5) values from Gene-Pix were taken and normalized to that of the probe mtrof 173For each reference sequence the average of these valuesfrom all the positive spots were taken as an estimate of therelative abundance of the given sequence in the artificialmixture

Cloning and sequencing of PCR products

pmoA PCR amplicons obtained from environmental sampleswere ligated into the pGEM-T plasmid vector (Promega) andtransformed into E coli DH5a competent cells Screeningfor positive clones was done by the a-complementation asdescribed by Sambrook et al (1989) Plasmid DNA was pre-pared from positive clones using the NucleoTrap kit (Mach-erey-Nagel) according to the manufacturerrsquos instructions andused as a template in sequencing reactions DNA sequenc-ing was performed with an ABI 373 A automated DNAsequencer (PE Applied Biosystems Foster City CA) and theABI PRISM Big Dye terminator cycle sequencing kit (Perkin-Elmer) All DNA sequences reported were sequenced onboth strands

Nucleotide sequence accession numbers

The partial pmoA sequences used in this study to validatethe probe set are available under accession nos AB484595AB484597 AB484601 AB480948 AF148521 AF148522AF150764 AF177325 AF211872 AF211879 AF211883AF211889 AF264115 AF239884 AF264136 AF358040AF358041 AF358045 AF358050 AF358053 AF358054AF358055 AF368358 AF368373 AJ278727 AJ299947AJ299948 AJ299951 AJ299955 AJ299957 AJ299963AJ299964 AJ459006 AY080942 AY080950 AY080955AY236074 AY236075 AY236076 AY236077 AY236078AY236079 AY236080 AY236081 AY236082 AY236083AY236084 AY236085 AY236086 AY236087 AY236517AY236518 U31650 U31654 U72670 U81596 U89302U89304 and U94337 M2 + pmoA sequences have beendeposited with GenBank (accession number AY195653-AY195672)

Acknowledgements

The authors thank Ann Auman Svetlana Dedysh Peter Dun-field Werner Liesack Ian McDonald Samantha Morris andStephen Nold for unpublished sequences pmoA clones andmethanotroph isolates We thank Saacutendor Bottka JoacutezsefCsontos Fodor Szilvia Andrew J Holmes and many activemembers of the Yahoo microarray discussion group (httpwwwgroupsyahoocomgroupmicroarray) for their help andsuggestions concerning various phases of the work pre-sented here Helpful comments and suggestions by twoanonymous reviewers are gratefully acknowledged Work atARC was supported by the Fonds zur Foumlrderung der wissen-schaftlichen Forschung Austria (Project number P15044)and funding through the EU 6th framework quality of life andmanagement of living resources grant QLK-3 CT-2000-01528

References

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Behr T Koob C Schedl M Mehlen A Meier H KnoppD et al (2000) A nested array of rRNA targeted probesfor the detection and identification of enterococci byreverse hybridization Syst Appl Microbiol 23 563ndash572

Bourne DG McDonald IR and Murrell JC (2001) Com-parison of pmoA PCR primer sets as tools for investigatingmethanotroph diversity in three Danish soils Appl EnvironMicrobiol 67 3802ndash3809

Breslauer KJ Frank R Blocker H and Marky LA(1986) Predicting DNA duplex stability from the basesequence Proc Natl Acad Sci USA 83 3746ndash3750

Brown TJ and Anthony RM (2000) The addition of lownumbers of 3cent thymine bases can be used to improve thehybridization signal of oligonucleotides for use withinarrays on nylon supports J Microbiol Meth 42 203ndash207

Chizhikov V Wagner M Ivshina A Hoshino Y KapikianAZ and Chumakov K (2002) Detection and genotyping



of human group a rotaviruses by oligonucleotide microar-ray hybridization J Clin Microbiol 40 2398ndash2407

Cho JC and Tiedje JM (2001) Bacterial species determi-nation from DNA-DNA hybridization by using genome frag-ments and DNA microarrays Appl Environ Microbiol 673677ndash3682

Cho JC and Tiedje JM (2002) Quantitative detection ofmicrobial genes by using DNA microarrays Appl EnvironMicrobiol 68 1425ndash1430

Dedysh N Liesack W Khmelenina VN Suzina NETrotsenko YA Semrau JD et al (2000) Methylocellapalustris General nov sp nov a new methane-oxidizingacidophilic bacterium from peat bogs representing a novelsubtype of serine-pathway methanotrophs Int J Syst EvolMicrobiol 50 955ndash969

Dunfield PF Yimga MT Dedysh SN Berger U Lie-sack W and Heyer J (2002) Isolation of a Methylocystisstrain containing a novel pmoA-like gene FEMS MicrobiolEcol 41 17ndash26

Fotin AV Drobyshev AL Proudnikov DY Perov ANand Mirzabekov AD (1998) Parallel thermodynamic anal-ysis of duplexes on oligodeoxyribonucleotide microchipsNucleic Acids Res 26 1515ndash1521

Gulledge J Ahmad A Steudler PA Pomerantz WJand Cavanaugh CM (2001) Genus- and family-level 16SrRNA-targeted oligonucleotide probes for ecological stud-ies on methanotrophic bacteria Appl Environ Microbiol 674726ndash4733

Guo Z Guilfoyle RA Thiel AJ Wang R and SmithLM (1994) Direct fluorescence analysis of genetic poly-morphisms by hybridization with oligonucleotide arrays onglass supports Nucleic Acids Res 22 5456ndash5465

Hamels S Gala JL Dufour S Vannuffel P ZammatteoN and Remacle J (2001) Consensus PCR and microar-ray for diagnosis of the genus Staphylococcus speciesand methicillin resistance Biotechniques 31 1364ndash1362

Hanson RS and Hanson TE (1996) Methanotrophic bac-teria Microbiol Rev 60 439ndash471

Henckel T Jackel U Schnell S and Conrad R (2000a)Molecular analyses of novel methanotrophic communitiesin forest soil that oxidize atmospheric methane Appl Envi-ron Microbiol 66 1801ndash1808

Henckel T Roslev P and Conrad R (2000b) Effects ofO2 and CH4 on presence and activity of the indigenousmethanotrophic community in rice field soil Environ Micro-biol 2 666ndash679

Henckel T Jackel U and Conrad R (2001) Vertical dis-tribution of the methanotrophic community after drainageof rice field soil FEMS Microbiol Ecol 34 279ndash291

Holmes AJ Roslev P McDonald IR Iversen N Hen-riksen K and Murrell JC (1999) Characterization ofmethanotrophic bacterial populations in soils showingatmospheric methane uptake Appl Environ Microbiol 653312ndash3318

Hughes TR Mao M Jones AR Burchard J MartonMJ Shannon KW et al (2001) Expression profilingusing microarrays fabricated by an ink-jet oligonucleotidesynthesizer Nat Biotechnol 19 342ndash347

Hung SH Gray QYu DM and Ratliff RL (1994) Evi-dence from CD spectra that d (purine) r (pyrimidine) and r

(purine) d (pyrimidine) hybrids are in different structuralclasses Nucleic Acids Res 22 4326ndash4334

Ishii K and Fukui M (2001) Optimization of annealingtemperature to reduce bias caused by a primer mismatchin multi-template PCR Appl Environ Microbiol 67 3753ndash3755

Koizumi Y Kelly JJ Nakagawa T Urakawa H El Fan-troussi S Al Muzaini S et al (2002) Parallel character-ization of anaerobic toluene- and ethylbenzene-degradingmicrobial consortia by PCR-denaturing gradient gel elec-trophoresis RNA-DNA membrane hybridization and DNAmicroarray technology Appl Environ Microbiol 68 3215ndash3225

Liu WT Mirzabekov AD and Stahl DA (2001) Optimi-zation of an oligonucleotide microchip for microbial identi-fication studies a non-equilibrium dissociation approachEnviron Microbiol 3 619ndash629

Lovell CR Friez MJ Longshore JW and Bagwell CE(2001) Recovery and phylogenetic analysis of nifHsequences from diazotrophic bacteria associated withdead aboveground biomass of Spartina alterniflora ApplEnviron Microbiol 67 5308ndash5314

Loy A Lehner A Lee N Adamczyk J Meier H ErnstJ et al (2002) Oligonucleotide microarray for 16S rRNAgene-based detection of all recognized lineages of sulfate-reducing prokaryotes in the environment Appl EnvironMicrobiol 68 5064ndash5508

Lueders T Chin KJ Conrad R and Friedrich M (2001)Molecular analyses of methyl-coenzyme M reductasealpha-subunit (mcrA) genes in rice field soil and enrich-ment cultures reveal the methanogenic phenotype of anovel archaeal lineage Environ Microbiol 3 194ndash204

McDonald IR and Murrell JC (1997) The particulatemethane monooxygenase gene pmoA and its use as afunctional gene probe for methanotrophs FEMS MicrobiolLett 156 205ndash210

McTavish H Fuchs JA and Hooper AB (1993) Sequenceof the gene coding for ammonia monooxygenase inNitrosomonas europaea J Bacteriol 175 2436ndash2444

Meroueh M and Chow CS (1999) Thermodynamics ofRNA hairpins containing single internal mismatchesNucleic Acids Res 27 1118ndash1125

Mikhailovich V Lapa S Gryadunov D Sobolev AStrizhkov B Chernyh N et al (2001) Identification ofrifampin-resistant Mycobacterium tuberculosis strains byhybridization PCR and ligase detection reaction on oligo-nucleotide microchips J Clin Microbiol 39 2531ndash2540

Polz MF and Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR Appl Environ Micro-biol 64 3724ndash3730

Purkhold U Pommerening-Roser A Juretschko SSchmid MC Koops HP and Wagner M (2000) Phy-logeny of all recognized species of ammonia oxidizersbased on comparative 16S rRNA and amoA sequenceanalysis implications for molecular diversity surveys ApplEnviron Microbiol 66 5368ndash5382

Radajewski S Ineson P Parekh NR and Murrell JC(2000) Stable-isotope probing as a tool in microbial ecol-ogy Nature 403 646ndash649

Radajewski S Webster G Reay DS Morris SA Ine-son P Nedwell DB et al (2002) Identification of active



methylotroph populations in an acidic forest soil by stable-isotope probing Microbiology 148 2331ndash2342

Raskin L Stromley JM Rittmann BE and Stahl DA(1994) Group-specific 16S rRNA hybridization probes todescribe natural communities of methanogens Appl Envi-ron Microbiol 60 1232ndash1240

Reay DS Radajewski S Murrell JC McNamara N andNedwell DB (2001) Effects of land-use on the activity anddiversity of methane oxidizing bacteria in forest soils SoilBiol Biochem 33 1613ndash1623

Reysenbach AL Giver LJ Wickham GS and PaceNR (1992) Differential amplification of rRNA genes bypolymerase chain reaction Appl Environ Microbiol 583417ndash3418

Rudi K Flateland SL Hanssen JF Bengtsson G andNissen H (2002) Development and evaluation of a 16Sribosomal DNA array-based approach for describing com-plex microbial communities in ready-to-eat vegetable sal-ads packed in a modified atmosphere Appl EnvironMicrobiol 68 1146ndash1156

Rudi K Skulberg OM Skulberg R and Jakobsen KS(2000) Application of sequence-specific labeled 16S rRNAgene oligonucleotide probes for genetic profiling of cyano-bacterial abundance and diversity by array hybridizationAppl Environ Microbiol 66 4004ndash4011

Sambrook J Fritsch EF and Maniatis T (1989) Molecu-lar Cloning a Laboratory Manual 2nd edn Cold SpringHarbor New York Cold Spring Harbor Laboratory Press

Schena M Shalon D Heller R Chai A Brown POand Davis RW (1996) Parallel human genome analysismicroarray-based expression monitoring of 1000 genesProc Natl Acad Sci USA 93 10614ndash10619

Semrau JD Chistoserdov A Lebron J Costello A Dav-agnino J Kenna E et al (1995) Particulate methanemonooxygenase genes in methanotrophs J Bacteriol 1773071ndash3079

Sessitsch A Weilherter A Gerzabek MH Kirchmann Hand Kandeler E (2001) Microbial population structures in

soil particle size fractions of a long-term fertilizer fieldexperiment Appl Environ Microbiol 67 4215ndash4224

Shchepinov MS Case-Green SC and Southern EM(1997) Steric factors influencing hybridization of nucleicacids to oligonucleotide arrays Nucleic Acids Res 251155ndash1161

Small J Call DR Brockman FJ Straub TM and Chan-dler DP (2001) Direct detection of 16S rRNA in soilextracts by using oligonucleotide microarrays Appl EnvironMicrobiol 67 4708ndash4716

Spiro A Lowe M and Brown D (2000) A bead-basedmethod for multiplexed identification and quantitation ofDNA sequences using flow cytometry Appl Environ Micro-biol 66 4258ndash4265

Strunk O Gross O Reichel B May M Hermann SStuckman N et al (2000) ARB A software environmentfor for sequence data [WWW document] URL httpwwwmikrobiologietu-muenchende

Sugimoto N Nakano M and Nakano S (2000) Thermo-dynamics-structure relationship of single mismatches inRNADNA duplexes Biochemistry 39 11270ndash11281

Tao H Bausch C Richmond C Blattner FR and Con-way T (1999) Functional genomics expression analysisof Escherichia coli growing on minimal and rich media JBacteriol 181 6425ndash6440

Wilson KH Wilson WJ Radosevich JL DeSantis TZViswanathan VS Kuczmarski TA and Andersen GL(2002) High-density microarray of small-subunit ribosomalDNA probes Appl Environ Microbiol 68 2535ndash2541

Witzingerode F Gobel UB and Stackebrandt E (1997)Determination of microbial diversity in environmental sam-ples pitfalls of PCR-based rRNA analysis FEMS Rev 21213ndash229

Wood WI Gitschier J Lasky LA and Lawn RM (1985)Base composition-independent hybridization in tetrameth-ylammonium chloride a method for oligonucleotidescreening of highly complex gene libraries Proc Natl AcadSci USA 82 1585ndash1588


567



5

566ndash582

genes Originally developed for whole genome geneexpression analyses (Schena

et al

1996 Tao

et al

1999) DNA microarrays have very strong applicationpotential in many areas of microbiology Upon availabilityof corresponding probe sets they enable the detection ofup to several thousand microbial strains species generaor higher clades (depending on the design of the probe)in a single assay In clinical veterinary and plant microbi-ology food and water quality control this means that asingle test can be developed to detect all pathogenicbeneficialcontaminating bacteria which might be presentin the sample being investigated The potential for envi-ronmental microbiology is even stronger By applyingnested sets of oligonucleotide probes (Behr

et al

2000)which target genes reflecting the phylogeny of the targetorganism it becomes possible to assess the wholeprokaryotic diversity of an environment The most obvioustarget for such studies are the 16S and 23S rRNA genesOther (often referred to as lsquofunctionalrsquo ie encoding forrelated enzymes carrying out a defined function) possibletarget genes can also be used at least for a physiologi-cally restricted group of microorganisms The applicationof functional genes narrows down the analysis to a func-tionally (sometimes also phylogenetically) defined groupof microbes The main advantage of this approach is thatit also enables the detection and analysis of uncultivatedmembers of the microbial groups being investigated(Raskin

et al

1994 McDonald and Murrell 1997Purkhold

et al

2000 Radajewski

et al

2000 Lovell

etal

2001 Lueders

et al

2001) In contrast to 16S rRNAenvironmental sequences belonging to a novel but func-tionally related phylum can easily be recognised andincluded in the analysis

Oligonucleotide probe sets spotted onto nylon or nitro-cellulose membranes (lsquomacroarraysrsquo) have been used forthe diagnosis of bacteraemia (Anthony

et al

2000) food-contamination (Rudi

et al

2002) detection of enterococci(Behr

et al

2000) or cyanobacteria (Rudi

et al

2000) Aspecial microarray format consisting of individual poly-acrylamide gel micropads with immobilized oligonucle-otides (Liu

et al

2001) was used for the characterizationof aromatic hydrocarbon degrading consortia (Koizumi

et al

2002) and identification of rifampicin resistantstrains of

Mycobacterium tuberculosis

(Mikhailovich

et al

2001) A lsquotraditionalrsquo glass microarray consisting ofseven probes was developed for

Staphylococcus

diagno-sis (Hamels

et al

2001) Different genotypes of rotavi-ruses were specifically detected by an oligonucleotidemicroarray (Chizhikov

et al

2002) Cho and Tiedje (2001)applied whole genome DNAndashDNA hybridization for thedetection and community analysis of microorganisms Ahigh-density Affymetrix GeneChip containing over 30 00016S rRNA targeting oligo probes was used to identifyculture collection species and subsequently to character-

ize populations of airborne bacteria at the level of higherphylogenetic taxa (Wilson

et al

2002) A 16S rRNA genebased oligonucleotide microarray targeting and coveringthe entire known diversity of sulphate reducers was devel-oped and successfully validated with environmental sam-ples (Loy

et al

2002)In most cases the target consists of labelled large gene

fragments (several hundred nucleotides long) increasingthe potential for the accumulation of background signalarising from a low rate of non-specific hybridization Alter-native labelling approaches such as terminal transferaselabelling of specific oligonucleotides (Rudi

et al

2002)and the application of end labelled specific stackingprobes in conjunction with immobilized capture probes(Small

et al

2001) may improve the detection limit ofdiagnostic microbial microarrays by decreasing the abovementioned non-specific background hybridization

Microbial community structures can be assessed by thequantitative analysis of micro or macroarray results Asimple and elegant method to quantify specific microbialgenes from a sample has recently been published (Choand Tiedje 2002) In this method gene fragments (500ndash900 bp in length) were applied as probes Each spot con-sisted of a mixture of an individual probe and a commonreference gene fragment Hybridization was done with aCy3-labelled environmental mixture and a Cy5-labelledreference DNA Quantification was based on the Cy3Cy5ratios Unfortunately the same principle cannot be appliedto the quantification of oligonucleotide chip results becauseof the inherent differences in hybridization efficienciesbetween oligonucleotide probes In practice a differentreference oligo for each probe should be designed

Even though high expectations exist for diagnosticmicrobial microarrays applications utilizing their fullpotential (high throughput detection of large numbers ofmicrobes and quantitative assessment of their communitystructures) are still very limited (Loy

et al

2002) Part ofthe reason for this is that a technical platform of reason-able cost based on readily available consumables andequipment with demonstrated potential in high-through-put quantitative microbial diagnostics is missing

Here we describe the development and validation of adiagnostic microbial microarray for the high-throughputdetection and community structure analysis of methan-otrophic and functionally related bacteria A set of tech-niques based on well established and widely appliedtechnologies and commercially available consumablesand equipment is reported

Results and discussion

Protocol optimization

One of the main problems when designing oligonucleotidemicroarrays is to achieve nearly identical melting temper-

568

L Bodrossy

et al



5

566ndash582


et al

1985 Spiro

et al



et al

1994 Shchepinov

et al


cent



2


yen



et al



in vitro


m

l

-

1


et al





pmoA

amoA


pmoA

682 can amplify

pmoA

amoA


pmoA


pmoA


et al


pmoA



569



5

566ndash582




pmoA

amoA



Mc capsulatus

(Bath)

pmoA



pmoA

amoA


Table 1


Name

a


cent

AElig

3

cent

b

Length T

m

Selected

c

MM

d

Mb460

Methylobacter


24 609

+

Mb478

Methylobacter

TGGTATGGC

A

TGGGGTCTGT

20 597

+

TMb271

Methylobacter

TTGTGGTGGCGTTACCGT

18 580

+

511ndash436

Methylobacter


20 555

+

BB51-299





at theends













Table 1 cont
























Fig

2

Ran

ge o

f st

rain

cov

erag

e fo

r ol

igon

ucle

otid

e pr

obes

tar

getin

g pm

oAa

moA

gen

es o

f m

etha

notr

ophs

A

OB

s an

d ba

cter

ia c

arry

ing

pmoA

am

oA h

omol

ogue

s A

sim

ilar

tabl

e w

ith a

ll 51

4 se

quen

ces

cons

ider

ed (

with

out

hybr

idiz

atio

n re

sults

) is

ava

ilabl

e fr

om t

he a

utho

rs u

pon

requ

est

Und

er lsquoP

robe

srsquo b

lack

fill

indi

cate

s ex

pect

ed p

ositi

ve r

esul

ts

grey

fill

indi

cate

s po

sitiv

e re

sults

not

pr

edic

ted

and

thic

k bl

ack

fram

ing

indi

cate

s ne

gativ

e re

sults

whe

re h

ybrid

izat

ion

was

pre

dict

ed W

hite

num

bers

insi

de b

lack

and

gre

y bo

xes

indi

cate

the

num

ber

of lsquom

ism

atch

equ

ival

ents

rsquo as

desc

ribed

in t

he r

elev

ant

sect

ion

of R

esul

ts L

ette

rs in

gre

y bo

xes

indi

cate

pro

bes

of u

nrel

iabl

e hy

brid

izat

ion

beha

viou

r lsquoA

cent a

shor

t pr

obe

with

sig

nific

ant

seco

ndar

y st

ruct

ure

lsquoBcent a

sho

rt p

robe

N

ames

of

envi

ronm

enta

l clo

nes

are

prec

eded

by

an in

dica

tion

of t

heir

pred

icte

d im

med

iate

phy

loge

netic

rel

atio

nshi

p H

ighe

st a

nd lo

wes

t si

gnal

val

ues

( o

f th

at o

f m

trof

173

) ob

tain

ed w

ith f

ull

mat

ch ta

rget

s ar

e in

dica

ted

(lsquomax

val

rsquo an

d lsquom

inv

alrsquo)

Gre

y bo

xes

indi

cate

dat

a w

hich

are

not

rep

rese

ntat

ive

(Jpn

284

Est

514

Nc_

oce4

26 g

p23ndash

454

and

MR

1-34

8 n

o fu

ll m

atch

ref

eren

ce ta

rget

av

aila

ble

II5

10 a

nd I

I630

sho

rt p

robe

s of

unr

elia

ble

hybr

idiz

atio

n be

havi

our

B2r

el26

0 an

d N

sNV

207

dat

a fr

om s

ingl

e m

ism

atch

tar

gets

mtr

of 1

73 r

efer

ence

pro

be m

trof

661

tar

getin

g th

e re

vers

e pr

imer

app

lied

in m

ost

ampl

ifica

tions

) A

pm

oAa

moA

DN

A n

eigh

bour

tre

e is

add

ed t

o th

e le

ft pr

ovid

ing

furt

her

insi

ght

into

the

phy

loge

netic

rel

atio

nshi

ps o

f th

e re

fere

nce

set

The

sca

le

bar

indi

cate

s th

e es

timat

ed n

umbe

r of

bas

e ch

ange

s pe

r nu

cleo

tide

sequ

ence

pos

ition

Str

ain

C

lon

eP

r o

b e

s

Mm275

PS80-291

Mb460

Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

Mcy409

Peat264

Msi520

Msi_tri309

Msi232

II510

II630

RA14-598

B2rel260

B2-400

B2all343

pmoAMO3-400

xb6-539

LP21-232

NmNc533

Nsm_eut381

Pl6-306

PS5-226

NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

6261

6160

6058

6061

6261

6362

5959

6160

5960

5759

5862

5859

6162

6161

6062

5860

62

Min

val

130

26

389

353

14

16

50

NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

NA

192

87

189

91

373

112

12

335

49

103

29

88

111

17

116

NA

NA

22

34

58

100

84

NA

Max

val

180

26

389

353

72

16

80

NA

108

NA

31

NA

166

41

50

NA

41

175

312

10

47

158

371

194

38

75

34

46

167

39

29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

29

88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

e M

90-P

12+

33

1M

eth

ylo

mo

nas

clo

ne

mv1

9pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv2

1pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv9

pa

+2

++

1M

eth

ylo

mo

nas

met

han

ica

clo

ne

D1

+2

++

2++

clo

ne

PS

-80

2+

33

Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

b2

+3

1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

3+

3M

eth

ylo

bac

ter

clo

ne

5hm

-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

+M

eth

ylo

bac

ter

clo

ne

RB

-100

1+

33

2+

2+

Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

+M

eth

ylo

cyst

is c

lon

e F

12

+1

1A

B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

1A

B1

3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

1A

B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

+A

B3

1M

eth

ylo

sin

us

tric

ho

spo

riu

m O

b3b

3+

+A

B3

+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+


















82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References






























































568

L Bodrossy

et al



5

566ndash582


et al

1985 Spiro

et al



et al

1994 Shchepinov

et al


cent



2


yen



et al



in vitro


m

l

-

1


et al





pmoA

amoA


pmoA

682 can amplify

pmoA

amoA


pmoA


pmoA


et al


pmoA



569



5

566ndash582




pmoA

amoA



Mc capsulatus

(Bath)

pmoA



pmoA

amoA


Table 1


Name

a


cent

AElig

3

cent

b

Length T

m

Selected

c

MM

d

Mb460

Methylobacter


24 609

+

Mb478

Methylobacter

TGGTATGGC

A

TGGGGTCTGT

20 597

+

TMb271

Methylobacter

TTGTGGTGGCGTTACCGT

18 580

+

511ndash436

Methylobacter


20 555

+

BB51-299





at theends













Table 1 cont
























Fig

2

Ran

ge o

f st

rain

cov

erag

e fo

r ol

igon

ucle

otid

e pr

obes

tar

getin

g pm

oAa

moA

gen

es o

f m

etha

notr

ophs

A

OB

s an

d ba

cter

ia c

arry

ing

pmoA

am

oA h

omol

ogue

s A

sim

ilar

tabl

e w

ith a

ll 51

4 se

quen

ces

cons

ider

ed (

with

out

hybr

idiz

atio

n re

sults

) is

ava

ilabl

e fr

om t

he a

utho

rs u

pon

requ

est

Und

er lsquoP

robe

srsquo b

lack

fill

indi

cate

s ex

pect

ed p

ositi

ve r

esul

ts

grey

fill

indi

cate

s po

sitiv

e re

sults

not

pr

edic

ted

and

thic

k bl

ack

fram

ing

indi

cate

s ne

gativ

e re

sults

whe

re h

ybrid

izat

ion

was

pre

dict

ed W

hite

num

bers

insi

de b

lack

and

gre

y bo

xes

indi

cate

the

num

ber

of lsquom

ism

atch

equ

ival

ents

rsquo as

desc

ribed

in t

he r

elev

ant

sect

ion

of R

esul

ts L

ette

rs in

gre

y bo

xes

indi

cate

pro

bes

of u

nrel

iabl

e hy

brid

izat

ion

beha

viou

r lsquoA

cent a

shor

t pr

obe

with

sig

nific

ant

seco

ndar

y st

ruct

ure

lsquoBcent a

sho

rt p

robe

N

ames

of

envi

ronm

enta

l clo

nes

are

prec

eded

by

an in

dica

tion

of t

heir

pred

icte

d im

med

iate

phy

loge

netic

rel

atio

nshi

p H

ighe

st a

nd lo

wes

t si

gnal

val

ues

( o

f th

at o

f m

trof

173

) ob

tain

ed w

ith f

ull

mat

ch ta

rget

s ar

e in

dica

ted

(lsquomax

val

rsquo an

d lsquom

inv

alrsquo)

Gre

y bo

xes

indi

cate

dat

a w

hich

are

not

rep

rese

ntat

ive

(Jpn

284

Est

514

Nc_

oce4

26 g

p23ndash

454

and

MR

1-34

8 n

o fu

ll m

atch

ref

eren

ce ta

rget

av

aila

ble

II5

10 a

nd I

I630

sho

rt p

robe

s of

unr

elia

ble

hybr

idiz

atio

n be

havi

our

B2r

el26

0 an

d N

sNV

207

dat

a fr

om s

ingl

e m

ism

atch

tar

gets

mtr

of 1

73 r

efer

ence

pro

be m

trof

661

tar

getin

g th

e re

vers

e pr

imer

app

lied

in m

ost

ampl

ifica

tions

) A

pm

oAa

moA

DN

A n

eigh

bour

tre

e is

add

ed t

o th

e le

ft pr

ovid

ing

furt

her

insi

ght

into

the

phy

loge

netic

rel

atio

nshi

ps o

f th

e re

fere

nce

set

The

sca

le

bar

indi

cate

s th

e es

timat

ed n

umbe

r of

bas

e ch

ange

s pe

r nu

cleo

tide

sequ

ence

pos

ition

Str

ain

C

lon

eP

r o

b e

s

Mm275

PS80-291

Mb460

Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

Mcy409

Peat264

Msi520

Msi_tri309

Msi232

II510

II630

RA14-598

B2rel260

B2-400

B2all343

pmoAMO3-400

xb6-539

LP21-232

NmNc533

Nsm_eut381

Pl6-306

PS5-226

NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

6261

6160

6058

6061

6261

6362

5959

6160

5960

5759

5862

5859

6162

6161

6062

5860

62

Min

val

130

26

389

353

14

16

50

NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

NA

192

87

189

91

373

112

12

335

49

103

29

88

111

17

116

NA

NA

22

34

58

100

84

NA

Max

val

180

26

389

353

72

16

80

NA

108

NA

31

NA

166

41

50

NA

41

175

312

10

47

158

371

194

38

75

34

46

167

39

29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

29

88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

e M

90-P

12+

33

1M

eth

ylo

mo

nas

clo

ne

mv1

9pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv2

1pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv9

pa

+2

++

1M

eth

ylo

mo

nas

met

han

ica

clo

ne

D1

+2

++

2++

clo

ne

PS

-80

2+

33

Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

b2

+3

1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

3+

3M

eth

ylo

bac

ter

clo

ne

5hm

-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

+M

eth

ylo

bac

ter

clo

ne

RB

-100

1+

33

2+

2+

Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

+M

eth

ylo

cyst

is c

lon

e F

12

+1

1A

B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

1A

B1

3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

1A

B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

+A

B3

1M

eth

ylo

sin

us

tric

ho

spo

riu

m O

b3b

3+

+A

B3

+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+


















82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References































































569



5

566ndash582




pmoA

amoA



Mc capsulatus

(Bath)

pmoA



pmoA

amoA


Table 1


Name

a


cent

AElig

3

cent

b

Length T

m

Selected

c

MM

d

Mb460

Methylobacter


24 609

+

Mb478

Methylobacter

TGGTATGGC

A

TGGGGTCTGT

20 597

+

TMb271

Methylobacter

TTGTGGTGGCGTTACCGT

18 580

+

511ndash436

Methylobacter


20 555

+

BB51-299





at theends













Table 1 cont
























Fig

2

Ran

ge o

f st

rain

cov

erag

e fo

r ol

igon

ucle

otid

e pr

obes

tar

getin

g pm

oAa

moA

gen

es o

f m

etha

notr

ophs

A

OB

s an

d ba

cter

ia c

arry

ing

pmoA

am

oA h

omol

ogue

s A

sim

ilar

tabl

e w

ith a

ll 51

4 se

quen

ces

cons

ider

ed (

with

out

hybr

idiz

atio

n re

sults

) is

ava

ilabl

e fr

om t

he a

utho

rs u

pon

requ

est

Und

er lsquoP

robe

srsquo b

lack

fill

indi

cate

s ex

pect

ed p

ositi

ve r

esul

ts

grey

fill

indi

cate

s po

sitiv

e re

sults

not

pr

edic

ted

and

thic

k bl

ack

fram

ing

indi

cate

s ne

gativ

e re

sults

whe

re h

ybrid

izat

ion

was

pre

dict

ed W

hite

num

bers

insi

de b

lack

and

gre

y bo

xes

indi

cate

the

num

ber

of lsquom

ism

atch

equ

ival

ents

rsquo as

desc

ribed

in t

he r

elev

ant

sect

ion

of R

esul

ts L

ette

rs in

gre

y bo

xes

indi

cate

pro

bes

of u

nrel

iabl

e hy

brid

izat

ion

beha

viou

r lsquoA

cent a

shor

t pr

obe

with

sig

nific

ant

seco

ndar

y st

ruct

ure

lsquoBcent a

sho

rt p

robe

N

ames

of

envi

ronm

enta

l clo

nes

are

prec

eded

by

an in

dica

tion

of t

heir

pred

icte

d im

med

iate

phy

loge

netic

rel

atio

nshi

p H

ighe

st a

nd lo

wes

t si

gnal

val

ues

( o

f th

at o

f m

trof

173

) ob

tain

ed w

ith f

ull

mat

ch ta

rget

s ar

e in

dica

ted

(lsquomax

val

rsquo an

d lsquom

inv

alrsquo)

Gre

y bo

xes

indi

cate

dat

a w

hich

are

not

rep

rese

ntat

ive

(Jpn

284

Est

514

Nc_

oce4

26 g

p23ndash

454

and

MR

1-34

8 n

o fu

ll m

atch

ref

eren

ce ta

rget

av

aila

ble

II5

10 a

nd I

I630

sho

rt p

robe

s of

unr

elia

ble

hybr

idiz

atio

n be

havi

our

B2r

el26

0 an

d N

sNV

207

dat

a fr

om s

ingl

e m

ism

atch

tar

gets

mtr

of 1

73 r

efer

ence

pro

be m

trof

661

tar

getin

g th

e re

vers

e pr

imer

app

lied

in m

ost

ampl

ifica

tions

) A

pm

oAa

moA

DN

A n

eigh

bour

tre

e is

add

ed t

o th

e le

ft pr

ovid

ing

furt

her

insi

ght

into

the

phy

loge

netic

rel

atio

nshi

ps o

f th

e re

fere

nce

set

The

sca

le

bar

indi

cate

s th

e es

timat

ed n

umbe

r of

bas

e ch

ange

s pe

r nu

cleo

tide

sequ

ence

pos

ition

Str

ain

C

lon

eP

r o

b e

s

Mm275

PS80-291

Mb460

Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

Mcy409

Peat264

Msi520

Msi_tri309

Msi232

II510

II630

RA14-598

B2rel260

B2-400

B2all343

pmoAMO3-400

xb6-539

LP21-232

NmNc533

Nsm_eut381

Pl6-306

PS5-226

NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

6261

6160

6058

6061

6261

6362

5959

6160

5960

5759

5862

5859

6162

6161

6062

5860

62

Min

val

130

26

389

353

14

16

50

NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

NA

192

87

189

91

373

112

12

335

49

103

29

88

111

17

116

NA

NA

22

34

58

100

84

NA

Max

val

180

26

389

353

72

16

80

NA

108

NA

31

NA

166

41

50

NA

41

175

312

10

47

158

371

194

38

75

34

46

167

39

29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

29

88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

e M

90-P

12+

33

1M

eth

ylo

mo

nas

clo

ne

mv1

9pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv2

1pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv9

pa

+2

++

1M

eth

ylo

mo

nas

met

han

ica

clo

ne

D1

+2

++

2++

clo

ne

PS

-80

2+

33

Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

b2

+3

1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

3+

3M

eth

ylo

bac

ter

clo

ne

5hm

-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

+M

eth

ylo

bac

ter

clo

ne

RB

-100

1+

33

2+

2+

Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

+M

eth

ylo

cyst

is c

lon

e F

12

+1

1A

B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

1A

B1

3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

1A

B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

+A

B3

1M

eth

ylo

sin

us

tric

ho

spo

riu

m O

b3b

3+

+A

B3

+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+


















82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References









































































Table 1 cont
























Fig

2

Ran

ge o

f st

rain

cov

erag

e fo

r ol

igon

ucle

otid

e pr

obes

tar

getin

g pm

oAa

moA

gen

es o

f m

etha

notr

ophs

A

OB

s an

d ba

cter

ia c

arry

ing

pmoA

am

oA h

omol

ogue

s A

sim

ilar

tabl

e w

ith a

ll 51

4 se

quen

ces

cons

ider

ed (

with

out

hybr

idiz

atio

n re

sults

) is

ava

ilabl

e fr

om t

he a

utho

rs u

pon

requ

est

Und

er lsquoP

robe

srsquo b

lack

fill

indi

cate

s ex

pect

ed p

ositi

ve r

esul

ts

grey

fill

indi

cate

s po

sitiv

e re

sults

not

pr

edic

ted

and

thic

k bl

ack

fram

ing

indi

cate

s ne

gativ

e re

sults

whe

re h

ybrid

izat

ion

was

pre

dict

ed W

hite

num

bers

insi

de b

lack

and

gre

y bo

xes

indi

cate

the

num

ber

of lsquom

ism

atch

equ

ival

ents

rsquo as

desc

ribed

in t

he r

elev

ant

sect

ion

of R

esul

ts L

ette

rs in

gre

y bo

xes

indi

cate

pro

bes

of u

nrel

iabl

e hy

brid

izat

ion

beha

viou

r lsquoA

cent a

shor

t pr

obe

with

sig

nific

ant

seco

ndar

y st

ruct

ure

lsquoBcent a

sho

rt p

robe

N

ames

of

envi

ronm

enta

l clo

nes

are

prec

eded

by

an in

dica

tion

of t

heir

pred

icte

d im

med

iate

phy

loge

netic

rel

atio

nshi

p H

ighe

st a

nd lo

wes

t si

gnal

val

ues

( o

f th

at o

f m

trof

173

) ob

tain

ed w

ith f

ull

mat

ch ta

rget

s ar

e in

dica

ted

(lsquomax

val

rsquo an

d lsquom

inv

alrsquo)

Gre

y bo

xes

indi

cate

dat

a w

hich

are

not

rep

rese

ntat

ive

(Jpn

284

Est

514

Nc_

oce4

26 g

p23ndash

454

and

MR

1-34

8 n

o fu

ll m

atch

ref

eren

ce ta

rget

av

aila

ble

II5

10 a

nd I

I630

sho

rt p

robe

s of

unr

elia

ble

hybr

idiz

atio

n be

havi

our

B2r

el26

0 an

d N

sNV

207

dat

a fr

om s

ingl

e m

ism

atch

tar

gets

mtr

of 1

73 r

efer

ence

pro

be m

trof

661

tar

getin

g th

e re

vers

e pr

imer

app

lied

in m

ost

ampl

ifica

tions

) A

pm

oAa

moA

DN

A n

eigh

bour

tre

e is

add

ed t

o th

e le

ft pr

ovid

ing

furt

her

insi

ght

into

the

phy

loge

netic

rel

atio

nshi

ps o

f th

e re

fere

nce

set

The

sca

le

bar

indi

cate

s th

e es

timat

ed n

umbe

r of

bas

e ch

ange

s pe

r nu

cleo

tide

sequ

ence

pos

ition

Str

ain

C

lon

eP

r o

b e

s

Mm275

PS80-291

Mb460

Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

Mcy409

Peat264

Msi520

Msi_tri309

Msi232

II510

II630

RA14-598

B2rel260

B2-400

B2all343

pmoAMO3-400

xb6-539

LP21-232

NmNc533

Nsm_eut381

Pl6-306

PS5-226

NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

6261

6160

6058

6061

6261

6362

5959

6160

5960

5759

5862

5859

6162

6161

6062

5860

62

Min

val

130

26

389

353

14

16

50

NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

NA

192

87

189

91

373

112

12

335

49

103

29

88

111

17

116

NA

NA

22

34

58

100

84

NA

Max

val

180

26

389

353

72

16

80

NA

108

NA

31

NA

166

41

50

NA

41

175

312

10

47

158

371

194

38

75

34

46

167

39

29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

29

88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

e M

90-P

12+

33

1M

eth

ylo

mo

nas

clo

ne

mv1

9pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv2

1pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv9

pa

+2

++

1M

eth

ylo

mo

nas

met

han

ica

clo

ne

D1

+2

++

2++

clo

ne

PS

-80

2+

33

Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

b2

+3

1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

3+

3M

eth

ylo

bac

ter

clo

ne

5hm

-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

+M

eth

ylo

bac

ter

clo

ne

RB

-100

1+

33

2+

2+

Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

+M

eth

ylo

cyst

is c

lon

e F

12

+1

1A

B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

1A

B1

3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

1A

B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

+A

B3

1M

eth

ylo

sin

us

tric

ho

spo

riu

m O

b3b

3+

+A

B3

+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+


















82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References





















































































Fig

2

Ran

ge o

f st

rain

cov

erag

e fo

r ol

igon

ucle

otid

e pr

obes

tar

getin

g pm

oAa

moA

gen

es o

f m

etha

notr

ophs

A

OB

s an

d ba

cter

ia c

arry

ing

pmoA

am

oA h

omol

ogue

s A

sim

ilar

tabl

e w

ith a

ll 51

4 se

quen

ces

cons

ider

ed (

with

out

hybr

idiz

atio

n re

sults

) is

ava

ilabl

e fr

om t

he a

utho

rs u

pon

requ

est

Und

er lsquoP

robe

srsquo b

lack

fill

indi

cate

s ex

pect

ed p

ositi

ve r

esul

ts

grey

fill

indi

cate

s po

sitiv

e re

sults

not

pr

edic

ted

and

thic

k bl

ack

fram

ing

indi

cate

s ne

gativ

e re

sults

whe

re h

ybrid

izat

ion

was

pre

dict

ed W

hite

num

bers

insi

de b

lack

and

gre

y bo

xes

indi

cate

the

num

ber

of lsquom

ism

atch

equ

ival

ents

rsquo as

desc

ribed

in t

he r

elev

ant

sect

ion

of R

esul

ts L

ette

rs in

gre

y bo

xes

indi

cate

pro

bes

of u

nrel

iabl

e hy

brid

izat

ion

beha

viou

r lsquoA

cent a

shor

t pr

obe

with

sig

nific

ant

seco

ndar

y st

ruct

ure

lsquoBcent a

sho

rt p

robe

N

ames

of

envi

ronm

enta

l clo

nes

are

prec

eded

by

an in

dica

tion

of t

heir

pred

icte

d im

med

iate

phy

loge

netic

rel

atio

nshi

p H

ighe

st a

nd lo

wes

t si

gnal

val

ues

( o

f th

at o

f m

trof

173

) ob

tain

ed w

ith f

ull

mat

ch ta

rget

s ar

e in

dica

ted

(lsquomax

val

rsquo an

d lsquom

inv

alrsquo)

Gre

y bo

xes

indi

cate

dat

a w

hich

are

not

rep

rese

ntat

ive

(Jpn

284

Est

514

Nc_

oce4

26 g

p23ndash

454

and

MR

1-34

8 n

o fu

ll m

atch

ref

eren

ce ta

rget

av

aila

ble

II5

10 a

nd I

I630

sho

rt p

robe

s of

unr

elia

ble

hybr

idiz

atio

n be

havi

our

B2r

el26

0 an

d N

sNV

207

dat

a fr

om s

ingl

e m

ism

atch

tar

gets

mtr

of 1

73 r

efer

ence

pro

be m

trof

661

tar

getin

g th

e re

vers

e pr

imer

app

lied

in m

ost

ampl

ifica

tions

) A

pm

oAa

moA

DN

A n

eigh

bour

tre

e is

add

ed t

o th

e le

ft pr

ovid

ing

furt

her

insi

ght

into

the

phy

loge

netic

rel

atio

nshi

ps o

f th

e re

fere

nce

set

The

sca

le

bar

indi

cate

s th

e es

timat

ed n

umbe

r of

bas

e ch

ange

s pe

r nu

cleo

tide

sequ

ence

pos

ition

Str

ain

C

lon

eP

r o

b e

s

Mm275

PS80-291

Mb460

Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

Mcy409

Peat264

Msi520

Msi_tri309

Msi232

II510

II630

RA14-598

B2rel260

B2-400

B2all343

pmoAMO3-400

xb6-539

LP21-232

NmNc533

Nsm_eut381

Pl6-306

PS5-226

NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

6261

6160

6058

6061

6261

6362

5959

6160

5960

5759

5862

5859

6162

6161

6062

5860

62

Min

val

130

26

389

353

14

16

50

NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

NA

192

87

189

91

373

112

12

335

49

103

29

88

111

17

116

NA

NA

22

34

58

100

84

NA

Max

val

180

26

389

353

72

16

80

NA

108

NA

31

NA

166

41

50

NA

41

175

312

10

47

158

371

194

38

75

34

46

167

39

29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

29

88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

e M

90-P

12+

33

1M

eth

ylo

mo

nas

clo

ne

mv1

9pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv2

1pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv9

pa

+2

++

1M

eth

ylo

mo

nas

met

han

ica

clo

ne

D1

+2

++

2++

clo

ne

PS

-80

2+

33

Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

b2

+3

1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

3+

3M

eth

ylo

bac

ter

clo

ne

5hm

-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

+M

eth

ylo

bac

ter

clo

ne

RB

-100

1+

33

2+

2+

Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

+M

eth

ylo

cyst

is c

lon

e F

12

+1

1A

B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

1A

B1

3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

1A

B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

+A

B3

1M

eth

ylo

sin

us

tric

ho

spo

riu

m O

b3b

3+

+A

B3

+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+


















82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References







































































Fig

2

Ran

ge o

f st

rain

cov

erag

e fo

r ol

igon

ucle

otid

e pr

obes

tar

getin

g pm

oAa

moA

gen

es o

f m

etha

notr

ophs

A

OB

s an

d ba

cter

ia c

arry

ing

pmoA

am

oA h

omol

ogue

s A

sim

ilar

tabl

e w

ith a

ll 51

4 se

quen

ces

cons

ider

ed (

with

out

hybr

idiz

atio

n re

sults

) is

ava

ilabl

e fr

om t

he a

utho

rs u

pon

requ

est

Und

er lsquoP

robe

srsquo b

lack

fill

indi

cate

s ex

pect

ed p

ositi

ve r

esul

ts

grey

fill

indi

cate

s po

sitiv

e re

sults

not

pr

edic

ted

and

thic

k bl

ack

fram

ing

indi

cate

s ne

gativ

e re

sults

whe

re h

ybrid

izat

ion

was

pre

dict

ed W

hite

num

bers

insi

de b

lack

and

gre

y bo

xes

indi

cate

the

num

ber

of lsquom

ism

atch

equ

ival

ents

rsquo as

desc

ribed

in t

he r

elev

ant

sect

ion

of R

esul

ts L

ette

rs in

gre

y bo

xes

indi

cate

pro

bes

of u

nrel

iabl

e hy

brid

izat

ion

beha

viou

r lsquoA

cent a

shor

t pr

obe

with

sig

nific

ant

seco

ndar

y st

ruct

ure

lsquoBcent a

sho

rt p

robe

N

ames

of

envi

ronm

enta

l clo

nes

are

prec

eded

by

an in

dica

tion

of t

heir

pred

icte

d im

med

iate

phy

loge

netic

rel

atio

nshi

p H

ighe

st a

nd lo

wes

t si

gnal

val

ues

( o

f th

at o

f m

trof

173

) ob

tain

ed w

ith f

ull

mat

ch ta

rget

s ar

e in

dica

ted

(lsquomax

val

rsquo an

d lsquom

inv

alrsquo)

Gre

y bo

xes

indi

cate

dat

a w

hich

are

not

rep

rese

ntat

ive

(Jpn

284

Est

514

Nc_

oce4

26 g

p23ndash

454

and

MR

1-34

8 n

o fu

ll m

atch

ref

eren

ce ta

rget

av

aila

ble

II5

10 a

nd I

I630

sho

rt p

robe

s of

unr

elia

ble

hybr

idiz

atio

n be

havi

our

B2r

el26

0 an

d N

sNV

207

dat

a fr

om s

ingl

e m

ism

atch

tar

gets

mtr

of 1

73 r

efer

ence

pro

be m

trof

661

tar

getin

g th

e re

vers

e pr

imer

app

lied

in m

ost

ampl

ifica

tions

) A

pm

oAa

moA

DN

A n

eigh

bour

tre

e is

add

ed t

o th

e le

ft pr

ovid

ing

furt

her

insi

ght

into

the

phy

loge

netic

rel

atio

nshi

ps o

f th

e re

fere

nce

set

The

sca

le

bar

indi

cate

s th

e es

timat

ed n

umbe

r of

bas

e ch

ange

s pe

r nu

cleo

tide

sequ

ence

pos

ition

Str

ain

C

lon

eP

r o

b e

s

Mm275

PS80-291

Mb460

Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

Mcy409

Peat264

Msi520

Msi_tri309

Msi232

II510

II630

RA14-598

B2rel260

B2-400

B2all343

pmoAMO3-400

xb6-539

LP21-232

NmNc533

Nsm_eut381

Pl6-306

PS5-226

NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

6261

6160

6058

6061

6261

6362

5959

6160

5960

5759

5862

5859

6162

6161

6062

5860

62

Min

val

130

26

389

353

14

16

50

NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

NA

192

87

189

91

373

112

12

335

49

103

29

88

111

17

116

NA

NA

22

34

58

100

84

NA

Max

val

180

26

389

353

72

16

80

NA

108

NA

31

NA

166

41

50

NA

41

175

312

10

47

158

371

194

38

75

34

46

167

39

29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

29

88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

e M

90-P

12+

33

1M

eth

ylo

mo

nas

clo

ne

mv1

9pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv2

1pa

+2

++

1M

eth

ylo

mo

nas

clo

ne

mv9

pa

+2

++

1M

eth

ylo

mo

nas

met

han

ica

clo

ne

D1

+2

++

2++

clo

ne

PS

-80

2+

33

Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

b2

+3

1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

3+

3M

eth

ylo

bac

ter

clo

ne

5hm

-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

+M

eth

ylo

bac

ter

clo

ne

RB

-100

1+

33

2+

2+

Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

+M

eth

ylo

cyst

is c

lon

e F

12

+1

1A

B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

1A

B1

3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

1A

B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

+A

B3

1M

eth

ylo

sin

us

tric

ho

spo

riu

m O

b3b

3+

+A

B3

+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+


















82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References
































































Fig

2

Ran

ge o

f st

rain

cov

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ucle

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ing

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ant

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izat

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pred

icte

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( o

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) ob

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Gre

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are

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imer

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Mb478

Mb271

511-436

Mb292

peat1-3-287

Mb_SL-299

Jpn284

Mm_pel467

Est514

LP20-644

Mmb303

Ia577

Nc_oce426

Mth413

Mc396

fw1-639

M90-201

Mcl408

Ib453

Ib559

Mcy262

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Msi520

Msi_tri309

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pmoAMO3-400

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NmNc533

Nsm_eut381

Pl6-306

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NsNv207

NsNv363

Nit_rel223

Nit_rel351

Nit_rel470

Nit_rel304

M84P105-451

WC306_54-385

M84P22-514

gp23-454

MR1-348

gp391

gp2-581

RA21-466

mtrof173

mtrof362-I

mtrof661

L21

2224

2018

2020

2025

1823

2023

2021

2025

1719

2019

2022

2019

2317

1918

1716

2025

1923

1917

2024

2626

2317

2020

2020

1920

2017

1820

1823

1818

1919

Tm

5958

6160

5856

5959

6158

6260

5860

5960

5862

6262

5862

6161

6160

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6160

6058

6061

6261

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6160

5960

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5862

5859

6162

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6062

5860

62

Min

val

130

26

389

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14

16

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NA

77

NA

31

NA

152

41

22

NA

41

175

312

10

47

50

256

194

20

75

32

46

54

11

10

513

22

64

78

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192

87

189

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112

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88

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17

116

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26

389

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72

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108

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31

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166

41

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41

175

312

10

47

158

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194

38

75

34

46

167

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29

513

22

64

89

NA

192

278

189

91

373

112

15

335

49

166

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88

111

17

116

NA

NA

22

34

58

100

270

NA

Met

hyl

om

on

as c

lon

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90-P

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1M

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nas

clo

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9pa

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nas

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ne

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pa

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++

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ylo

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nas

met

han

ica

clo

ne

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2++

clo

ne

PS

-80

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Met

hyl

ob

acte

r cl

on

e S

L-5

102

2+

++

32

33

+3

Met

hyl

ob

acte

r cl

on

e m

v6p

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1+

2+ +

Met

hyl

ob

acte

r cl

on

e m

v16p

a+

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ylo

bac

ter

clo

ne

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-81

+3

32

+3

Un

iden

tifi

ed s

trai

n L

K5

1+

Met

hyl

ob

acte

rcl

on

e 5h

m-2

21

22

+3

1+

3M

eth

ylo

bac

ter

clo

ne

RB

-16

12

+3

2+

1M

eth

ylo

bac

ter

clo

ne

SL

-41

+3

3+

2+

Met

hyl

ob

acte

r c

lon

e 5h

m-2

31

32

+2

+M

eth

ylo

bac

ter

clo

ne

RB

-100

1+

33

2+

2+

Met

hyl

ob

acte

r c

lon

e L

OP

B 1

35

33

1+

31

+3

+L

P 2

0 g

rou

p c

lon

e M

90-P

503

++

1+

LP

20

gro

up

clo

ne

M90

-P24

3+

+1

+M

eth

ylo

mic

rob

ium

alb

um

BG

83

++

+2

+ ++

Met

hyl

osa

rcin

a fi

bri

ata

AM

L-C

10+

23

2+

+2

+

++2

+M

eth

ylo

ther

mu

sst

rain

HB

2+

1+

Met

hyl

oco

ccu

s ca

psu

latu

s (

Bat

h)

33

+2

++

Met

hyl

oco

ccu

s ca

psu

latu

s B

L4

33

12

++

++

501

gro

up

clo

ne

FW

-18

+1

+1

+ F

W-1

gro

up

clo

ne

pA

MC

512

33

+2

++

2M

eth

ylo

cald

um

rel

ated

clo

ne

M84

-P11

32

++

13

3+

+M

eth

ylo

cald

um

rela

ted

clo

ne

M90

-P75

33

2+

+1

33

++

Met

hyl

oca

ldu

m t

epid

um

LK

6+

+1

++

1M

eth

ylo

cyst

is c

lon

e S

L-5

70

+1

1A

B+

2M

eth

ylo

cyst

is c

lon

e L

OP

A 1

35

22

31

AB

1+

+M

eth

ylo

cyst

is c

lon

e F

12

+1

1A

B1

+1

Met

hyl

ocy

stis

str

ain

M2

+1

1A

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3+

+M

eth

ylo

cyst

is p

arvu

s O

BB

P2

+1

1A

B2

3gt7

+1

Pea

t cl

on

e P

129

E+

31

AB

+2

Met

hyl

osi

nu

scl

on

e rb

p46

3+

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1M

eth

ylo

sin

us

tric

ho

spo

riu

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b3b

3+

+A

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+1

Met

hyl

osi

nu

s c

lon

e L

OP

B 1

33

33

3+

AB

13

+1

Met

hyl

osi

nu

s sp

ori

um

SE

23

++

AB

1+

+U

nid

enti

fied

str

ain

Y3

+A

B1

++

RA

14 g

rou

p c

lon

e R

A14

1+

+2

Met

hyl

oca

psa

rel

ated

clo

ne

JY-6

48

32

11

+3

Met

hyl

oca

psa

rel

ated

clo

ne

LO

PB

13

43

21

++

2M

eth

ylo

cap

sa a

cid

op

hila

B2

B1

++

gt7+

2L

P21

gro

up

clo

ne

P13

63

23

++

+1

LP

21 g

rou

p c

lon

e m

v12p

a+

1L

P21

gro

up

clo

ne

LO

PA

12

8B

3B

32

+2

++

1N

itro

som

on

as c

lon

e g

p1a

+1

1+

Nit

roso

mo

nas

eu

tro

ph

a1

+2

2+

Nit

roso

mo

nas

rela

ted

clo

ne

pl6

3+

21

2+

Nit

roso

mo

nas

rel

ated

clo

ne

PS

-5+

12

+N

itro

sosp

ira

clo

ne

LO

PA

12

32

++

AO

B r

elat

ed c

lon

e P

124

++

+A

OB

rel

ated

clo

ne

LO

PA

12

4+

1+

AO

B r

elat

ed c

lon

e g

p22

3+

+cl

on

e M

84-P

105

3+

clo

ne

WC

306-

54+

++

+ +cl

on

e M

84-P

222

1cl

on

e R

A21

3

+

+

+

+

01

1

+

+

+ +

+


















82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References















































































82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References



































































82

67

Soda lake strain 5b

15



31

66



M2 105 (1 clone)

47

88


88

55





80

66

80


M2 37 (1 clone)

88

66


M2 69 (1 clone)

80

M2 66 (1 clone)

99

56


88

M2 26 (3 clones)

66

Clone M90-P4

99

Clone FW-1

54


CloneFW-18

78

43

36

46



88


80


39

Methylocystis sp M


80


88

99


23

37

25

Clone peat1-2

69


Clone SL 5101


41

92

94


Clone RA 14

96

89

86





42

Clone K1

99

94

97

Clone M84-P22

49

Clone RA 21

77

010

Mb478

Mb478

Mcl408Ib453

Ib559

Mcy262

Mcy409

Msi 232II510II630

Mtrof661

Mtrof173

Mmb303

A

B








82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References





































































82

67

Soda lake strain 5b

15


31




47

55



Clone FW-18


Clone M90-P4

Clone FW-1

54

43

78

36

46




39


Methylocystis sp M


23

37

25

Clone peat1-2


69

29


Clone SL 5101


41

92

94


Clone RA 14

96


Clone ferm_xb6

54

Clone LP 21

66

86

61

89

88




11


43



42

Clone K1

99

94

97

Clone M84-P22

49

Clone gp5

77

Clone gp2


98

90

Clone RA 21

010

Xb6-539

Peat264

Msi 232II510II630

Mtrof661

Mtrof173

LP21-232


Gp2-581 Gp391

A

B








Conclusions



















Target preparation






Hybridization

















Acknowledgements


References





































































Conclusions



















Target preparation






Hybridization

















Acknowledgements


References

































































Conclusions



















Target preparation






Hybridization

















Acknowledgements


References



































































Target preparation






Hybridization

















Acknowledgements


References












































































Acknowledgements


References











































































































































Documents

Development and validation of a diagnostic microbial microarray … · 2017-08-12 · of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase