Dumont et al. 2006 - The Department of Ecology and Evolutionary ...

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SIP and metagenomic analysis 1241Amarker12C-DNA13C-DNABFig. 1. A. The CsCl-ethidium bromide gradientcontaining 12 C-DNA and 13 C-DNA from theDNA-SIP experiment with forest soil that consumed13 CH 4 . The DNA was visible and photographedin ambient light and the position of the12C-DNA and 13 C-DNA bands is indicated by thearrows.B. Denaturing gradient gel electrophoresisanalysis of the 12 C-DNA and 13 C-DNA recoveredfrom the gradient. The marker containeda mixture of 16S rRNA gene products from purebacterial cultures. The predominant DGGEbands from the 13 C-DNA were sequenced anda genus name is indicated where the per centidentity of the sequence was greater than 98%to that of a cultivated representative. TheGS_band_3 sequence belongs to an uncultivatedGammaproteobacteria that was alsoPCR amplified in a previous DNA-SIP analysisof this soil (Radajewski et al., 2002).GS_band_1 possesses similarity toBacteroidetes 16S rRNA sequences.12C-DNA13C-DNAGS_band_1; uncultured bacteriumGS_band_2; uncultured Methylobacter sp.GS_band_3; uncultured Gammaproteobacteria sp.GS_band_4; uncultured Methylocella sp.GS_band_5; uncultured Methylocystis sp.considerable interest in the possibility of combiningDNA-SIP with metagenomic studies to solve thisdilemma (Schloss and Handelsman, 2003; Wellingtonet al., 2003; Dumont and Murrell, 2005a). In a metagenomiclibrary constructed from 13 C-DNA from a DNA-SIPexperiment, the cloned DNA would originate fromgenomes of organisms that have obtained the majority oftheir carbon, either directly or indirectly via crossfeeding,from the 13 C-substrate. This limits the library togenome fragments of organisms involved in a specificmetabolic process of interest and increases the likelihoodof obtaining and sequencing genes that are integralto the process.In this study, a metagenomic library was constructedusing 13 C-DNA from a DNA-SIP experiment with 13 CH 4 .The objective was to perform an experiment to determinethe feasibility of cloning larger fragments of the 13 C-DNAand identifying a clone containing methylotrophy genes. Aclone library was made from 13 C-DNA using a bacterialartificial chromosome (BAC) plasmid and a clone containinga complete pmoCAB operon, encoding the particulatemethane monooxygenase (pMMO) enzyme, was identifiedand sequenced. The pmoA gene was similar to thatof an uncultivated Methylocystis sp. previously identifiedin this soil by DNA-SIP and PCR analysis. A very briefpreliminary report of these data was published in a reviewof DNA-SIP (Dumont and Murrell, 2005a).ResultsStable isotope probing with 13 CH 4 and constructionof a metagenomic libraryThe SIP experiment was performed in a manner similarto that described previously (Radajewski et al., 2002) andwith sample taken from the same forest soil site. Soil wascollected from the acidic oak forest soil and incubated with13CH 4 until a total of 50 ml had been consumed. The DNAwas extracted from the 13 CH 4 -labelled soil and the DNAwas visible in ambient light after isopycnic centrifugationin CsCl (Fig. 1A). Therefore, the 13 C-DNA band could becollected without exposure to DNA-damaging UV irradiation.The 13 C-DNA was analysed by pulsed-field gel electrophoresis(PFGE) and the majority of fragments werebetween ∼15 kb and ∼100 kb in length (results notshown).Restriction enzyme activity is sensitive to inhibition bysoil organics which are co-extracted with DNA (Zhouet al., 1996). Although several rounds of CsCl gradientcentrifugation had been used to purify the DNA, the gradient-purifiedDNA was not sufficiently pure to be digestedwith restriction enzyme BamHI (results not shown). Therefore,further purification was achieved by electrophoresisof the 13 C-DNA through a 1% (w/v) agarose gel. Theagarose gel electrophoresis had two convenient purposes:first, it eliminated contaminants from the DNA sothat it could be digested with restriction enzymes, andsecond, the 13 C-DNA formed a tight band that could beexcised in an agarose plug in which restriction enzymedigestion of the 13 C-DNA could be performed. This minimizedadditional DNA shearing that could potentiallyoccur when pipetting the DNA solution.Partially digested 13 C-DNA was resolved on a standardagarose gel (20 cm × 20 cm) rather than a PFGE system,because standard electrophoresis resulted in a more tightdistribution of the desired size fragments within the geland easier elution from the gel. As described in the Experimentalprocedures section, extreme care was taken toavoid exposure of the DNA to UV irradiation. The partiallyBamHI-digested and size-selected DNA was recoveredfrom the agarose and ligated into plasmid pCC1BACand a library of 2300 clones was generated using the© 2006 The AuthorsJournal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 1240–1250
1242 M. G. Dumont et al.orf2154 dnaJ gloBtrkA mptG folC folK pmoC pmoA pmoB bolA cobS cobT orf5501 moxFFig. 2. Genetic map of ORFs identified on clone GSC357. The fragment is 15.2 kb in length. The assigned names represent the closest knowngenes (refer to Table 1).1kbE. coli TransforMax EC300 cells provided with the vector(Epicentre).Analysis of the SIP labelling and the metagenomic libraryBacterial 16S rRNA genes were amplified from the 12 C-DNA and 13 C-DNA by PCR and analysed by denaturinggradient gel electrophoresis (DGGE) (Fig. 1B). The DGGEpattern for the 12 C-DNA was complex, as expected for asoil bacterial community. The DGGE pattern for the 13 C-DNA showed less than 10 predominant bands. Sequencingof the dominant 13 C-DNA DGGE bands indicated thepresence of the methanotrophic genera Methylobacter(GS_band_2), Methylocella (GS_band_4) and Methylocystis(GS_band_5) (Fig. 1B). The Methylocystis 16SrRNA sequence was very similar to the UP4, UP5, UP6and UP7 16S rRNA sequences (AY080911, AY080917,AY080912, AY080914 respectively) that together constituted96% of the 16S rRNA gene sequences from the 13 C-DNA in the original 13 CH 4 SIP analysis of this forest soil(Radajewski et al., 2002). In addition to the methanotroph16S rRNA gene sequences, a predominant sequencetype (GS_band_1) was present with similarity to theBacteroidetes and a second type (GS_band_3) with similarityto a Gammaproteobacteria; the latter was alsoretrieved in the previous SIP study of this forest soil(Radajewski et al., 2002).After construction of the metagenomic library from 13 C-DNA, the plasmids were isolated from 48 clones chosenat random and analysed by digestion with BamHI followedby agarose gel electrophoresis. Most clones containedinserts between 10 and 30 kb and two of the 48 plasmidscontained no visible insert DNA. Also, nearly half of theclones analysed had additional BamHI sites within theinsert fragment (results not shown), which indicated thatthe cloned 13 C-DNA had been incompletely digested asintended, or alternatively had been partially protectedfrom cutting by methylation. The sequencing primers T7and RP2 annealed adjacent to the cloning site ofpCC1BAC and were used to sequence the ends ofseveral of the cloned inserts. The sequence data from theclones were compared with the GenBank database byBLAST analysis (Altschul et al., 1990) and were differentfrom each other, with the exception of two clones thatappeared to be identical. None of the genes identified byend sequencing were of obvious relevance to methylotrophyand these clones were not sequenced further.The metagenomic library was screened by colonyhybridization for pmoA, mmoX and mxaF, which encodesubunits of the pMMO, soluble methane monooxygenaseand methanol dehydrogenase respectively. Polymerasechain reaction primers were used to amplify the probeusing the 13 C-DNA as template. As the probes were amplifiedfrom 13 C-DNA, there was a high probability that thehomologous probe would be present for a cloned gene.The three gene hybridizations were performed separatelyand a total of four membranes each with approximately575 clones were hybridized with 32 P-GTP random primedPCR probes.The mmoX and mxaF probes did not hybridize with anyof the clones in the library. Two clones, GSC357 (GisburnSoil Clone) and GSC1346, hybridized strongly to thepmoA probe and the pmoA gene could be amplified fromthese clones by PCR (results not shown). The restrictionfragment length polymorphism (RFLP) patterns ofGSC357 and GSC1346 digested with NotI were identical(results not shown). GSC357 was completely sequencedusing a shotgun approach. The complete sequence was15 230 bp and a summary of the genes contained on thisplasmid and their arrangement is given in Fig. 2 andTable 1. GSC1346 was partially sequenced and found tobe very similar to the sequence of GSC357. It is likely thatGSC357 and GSC1346 were from different strains of thesame species.Analysis of the pmoCAB operon on clone GSC357GSC357 contained a complete pmoCAB operon, withpmoC, pmoA and pmoB of 765 bp, 759 bp and 1257 bprespectively. The intergenic region between pmoC andpmoA was 269 bp and between pmoA and pmoB was227 bp. The sequences of pmoCAB operons and derivedpolypeptide sequences demonstrated considerable similarityto those of Methylocystis sp. strain M (Gilbert et al.,2000), Methylosinus trichosporium OB3b (Gilbert et al.,2000), Methylocystis sp. strain SC2 (Ricke et al., 2004)and Methylococcus capsulatus (Bath) (Semrau et al.,1995; Stolyar et al., 1999) (Table 2). Putative ribosomebinding sites were present upstream (5′) of all three pmogenes on the clone. The pmoCAB operon in Methylocystis© 2006 The AuthorsJournal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 1240–1250
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