Environmental Science and Engineering, M/C 138-78, California Institute of Technology, Pasadena, CA 91125, USA
Correspondence
Jared R. Leadbetter
jleadbetter{at}caltech.edu
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ABSTRACT |
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The nucleotide and deduced amino acid sequences for the FTHFS genes recovered from pure cultures and environmental samples during the course of this study can be found in GenBank under accession numbers AY162294AY162314, AY162316 and AY254548.
Present address: Faculty for Biological Sciences, BS 344, California State University, Los Angeles, Los Angeles, CA 90032-4226, USA.
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INTRODUCTION |
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In many termite species, bacterial homoacetogens outprocess methanoarchaea for reductant, most notably H2 (Brauman et al., 1992). As a result, termites emit less CH4 than might be expected if their fermentation pattern were similar to that observed in ruminants, and the basis for this is not well understood. Termites are as intriguing for the CH4 that they do not emit, as much as for the CH4 that they do.
Because of the influence of acetogenesis on termite nutrition and the production of greenhouse gases, there has been considerable interest in learning more about the gut microbiota accounting for this activity (Breznak, 1994). Four homoacetogenic, spore-forming Firmicutes have previously been isolated from four different termite species and described as novel taxa (Boga et al., 2003
; Breznak et al., 1988
; Kane & Breznak, 1991
; Kane et al., 1991
). Two homoacetogenic spirochaetes have been isolated from a fifth termite species (Leadbetter et al., 1999
). The latter strains, Treponema ZAS-1 and Treponema ZAS-2, represented the first examples of spirochaetes isolated from any termite-gut environment, as well as the first demonstration of lithotrophic metabolism (much less CO2-reductive acetogenesis) in this phylum-level group of bacteria. The isolates exhibited enzymic activities associated with the WoodLjungdahl pathway used by homoacetogenic Firmicutes (Leadbetter et al., 1999
), a pathway also used in the oxidative energy metabolism and autotrophic anabolism of diverse archaea and sulfate-reducing bacteria (Drake et al., 1997
; Ragsdale, 1997
; Wood, 1991
). However, the evolutionary history of this pathway in spirochaetes, and its relationships to those from better-studied organisms, is not currently known. The actual contribution to gut acetogenesis by any of the aforementioned isolates and the full diversity of homoacetogens present and active in any single termite species are also poorly understood. It has proven tempting to speculate that spirochaete species may dominate acetogenesis in the guts of termites (Leadbetter et al., 1999
), especially since spirochaetes are among the most numerous microbiota present in the guts of virtually all termite species, representing as many as 50 % of all prokaryotic cells (Breznak & Leadbetter, 2002
; Lilburn et al., 1999
; Paster et al., 1996
). However, at least 50 % of the prokaryotic cells found in termite guts are not spirochaetes and not all Zootermopsis-gut spirochaetes are CO2-reducing acetogens. One strain isolated from this environment, Treponema ZAS-9, does not display this mode of metabolism (Breznak & Leadbetter, 2002
; Lilburn et al., 2001
).
We have adopted the use of a functional-gene approach employing degenerate, oligonucleotide primers developed to amplify portions of genes encoding a key enzyme of the CO2-reductive acetogenesis pathway: formyltetrahydrofolate synthetase (FTHFS) (Leaphart & Lovell, 2001; Leaphart et al., 2003
). These primers were successfully applied to three termite-gut spirochaete isolates. The sequences for FTHFS partial genes for two of these spirochaetes have also been recently reported by others (Leaphart et al., 2003
). Here, we detail our analysis of a sizeable collection of FTHFS genes derived from spirochaetal isolates and the gut community of the termite Zootermopsis, as well as two putative acetogenesis-related genes flanking the FTHFS gene in Treponema strain ZAS-2.
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METHODS |
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DNA extraction.
Z. angusticollis-gut community DNA was purified using a modification of a previously described technique (Purdy et al., 1996). DNA was extracted from the entire, intact hindgut of nine uniformly sized worker larvae. Three termite hindguts were combined into each of three 2 ml microcentrifuge tubes containing 0·2 g of 0·1 mm Biospec zirconium/silica beads, 700 µl of 1 % (w/v) polyvinylpolypyrrolidone in 120 mM sodium phosphate, pH 8·0, 50 µl of 20 % (w/v) SDS and 500 µl TRIS-equilibrated phenol, pH 8·0. The samples were disrupted using a Biospec MiniBeadbeater-8 as described previously (Purdy et al., 1996
). The reaction mixtures were pooled and applied to hydroxyapatite-containing microcentrifuge spin columns (Bio-Rad). Sample application, washing and elution steps were performed by gently spinning the column for <10 s in a fixed-angle bench-top microcentrifuge. DNA was extracted from the treponemal isolates using the DNeasy extraction kit (Qiagen). DNA samples were stored at -20 °C until used.
PCR amplification, cloning and RFLP analysis.
FTHFS homologues were amplified from DNA templates prepared from cultivated spirochaete strains and termite-gut contents using the primers and PCR conditions exactly as described by others previously (Leaphart & Lovell, 2001), except that Roche Taq polymerase and reagents were used. Reactions yielded the expected
1·1 kb gene fragments as products. When community DNA was used as template, reaction products of various other sizes were also observed. PCR products generated from genomic templates from isolated strains were cloned directly using the Topo TA cloning kit (Invitrogen); products amplified from gut template were similarly cloned after purification of the desired-size band using the QIAEX II Gel Extraction Kit (Qiagen). Using an adaptation of a previously described method (Ng et al., 1996
), the community-derived clone library was characterized via RFLP analyses after double digestion with MspI and HinP1I following the manufacturer's recommendations (New England Biolabs). RFLPs were visualized via gel electrophoresis using 2·5 % (w/v) low-melting-point agarose and analysed manually.
Inverse PCR generation of Treponema ZAS-2 genomic fragment.
A synopsis of this technique has been described previously (Raponi et al., 2000). Genomic DNA was digested with NcoI according to the manufacturer's guidelines (New England Biolabs). The digested-DNA fragments were circularized using T4 DNA ligase. The ligation mixture was purified using the QIAEX II Gel Extraction Kit (Qiagen). Five microlitres of the purified ligation mixture was used as template in 20 µl reactions using the Failsafe PCR System and PCR PreMix Selection Kit (Epicenter). The internal FTHFS out primers employed were 5'-GGCCGAAGAAGTCGTAAAAC-3', designed to amplify the downstream gene sequence, and 5'-CCAGGTTCGGTTTCAGGGC-3', designed to amplify the upstream gene sequence. The amplification conditions used were initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min and extension at 72 °C for 6 min, with a final extension at 72 °C for 10 min. Products were RFLP-sorted and cloned as outlined above.
Nucleotide sequencing and analysis.
Sequencing was performed at the DNA Sequencing Core Facility at the Beckman Institute of Caltech using the dideoxy chain termination method, Sequenase (United States Biochemical) and a Perkin-Elmer ABI 373A automated sequencer. Sequence reads were assembled and edited using SEQUENCHER software for Windows (Genecodes). Multiple sequence alignments, translations and phylogenetic analyses were performed using the ARB freeware package (http://www.arb-home.de/) running within the Linux environment. FTHFS trees were constructed using PUZZLE-MAP 5.0 maximum-likelihood analyses (Schmidt et al., 2002). For analyses of aligned protein sequences, 10 000 puzzling-steps were employed using the WAG model for substitution-rate frequencies and uniform models for rate heterogeneity to construct the input matrices (Goldman & Whelan, 2000
). For analysis of FTHFS clones at the DNA sequence level, the Schöniger & von Haeseler substitution and uniform rate heterogeneity models were employed (Schöniger & von Haeseler, 1999
). Transversion parameters and doublet frequencies were estimated from the dataset by the software. Phylogenetic-tree layout-editing was performed using TREEVIEW 1.6.6 for Windows (Page, 1996
). Collector's curve analyses were used to estimate the completeness of the clone inventory and were performed using ANALYTIC RAREFACTION 1.3, written and made freely available by Steven M. Holland (http://www.uga.edu/
strata/software/), and ESTIMATES 6.01b1, written and made freely available by Robert K. Colwell (http://viceroy.eeb.uconn.edu/EstimateS).
Nucleotide accession numbers.
The GenBank accession numbers for the sequences generated in this study and other FTHFS sequences used in the construction of Fig. 2 are: Moorella thermoacetica, J02911; Horse Manure Clone H 1, AF295711; Horse Manure Clone H 2, AF295715; Horse Manure Clone H 4, AF295714; Horse Manure Clone H 5, AF295713; Spartina Clone SR 10, AF295723; Acetobacterium woodii, AF295701; Clostridium aceticum, AF295705; Clostridium magnum, AF295703; Eubacterium limosum, AF295706; Proteus vulgaris, AF295710; Ruminococcus productus, AF295707; Sporomusa ovata, AF295708; Sporomusa termitida, AF295709; Thermoanaerobacter kivui, AF295704; Za-gut Clone A, AY162294; Za-gut Clone C, AY162295; Za-gut Clone E, AY162296; Za-gut Clone E2, AY162297; Za-gut Clone F, AY162298; Za-gut Clone F2, AY162299; Za-gut Clone G, AY162300; Za-gut Clone G2, AY162301; Za-gut Clone H, AY162302; Za-gut Clone I, AY162303; Za-gut Clone L, AY162304; Za-gut Clone M, AY162305; Za-gut Clone N, AY162306; Za-gut Clone P, AY162307; Za-gut Clone R, AY162308; Za-gut Clone T, AY162309; Za-gut Clone U, AY162310; Za-gut Clone Y, AY162311; Za-gut Clone Z, AY162312; Treponema strain ZAS-1A, AY162313; Treponema strain ZAS-1B, AY162314; Treponema strain ZAS-2, AY254548; Treponema strain ZAS-9, AY162316. An FTHFS gene was identified in the unpublished genome of Treponema denticola (http://www.hgsc.bcm.tmc.edu/microbial/Tdenticola/).
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RESULTS |
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Amplification and cloning of diverse FTHFS homologues from the termite-gut community
DNA was purified from the gut contents of robust specimens. Amplification reactions using the FTHFS primer set and gut DNA as template successfully yielded products of the anticipated 1·1 kb size. These were ligated into a cloning vehicle and transformed into Escherichia coli. From this, a library of 91 insert-containing plasmid clones was collected. After sorting of the library by comparing RFLP patterns, 31 clones were targeted for sequencing and further analyses. BLAST analyses confirmed that all of the clones encoded FTHFS homologues. When the 31 sequences were aligned and compared with each other, several were found to share near identity (greater than 99·5 % identity at the DNA level) and were thereafter considered as being representative of the same genotype. From these data, 19 unique clone types were established. Each was assigned a Za-gut epithet (for Zootermopsis angusticollis). After in silico translation of FTHFS genes, the deduced amino acid sequences for the 19 Za-gut clone types were aligned and compared. Despite clear differences at the DNA level, many of these were nearly identical in their amino acid sequences. Using 98 % amino acid identity as a cut-off, the clone inventory was collapsed into eight distinct amino acid groups. The rank abundances of the DNA and amino acid types recovered in the 91-clone collection are presented in Fig. 1
. At both levels, the recovery of unique clones in the collection appeared to be close to saturation: rarefaction and collector's curve analyses indicated that the FTHFS inventory was essentially complete using this particular template DNA and primer set. With infinite sampling, rarefaction analysis suggested that no more than 23 DNA and nine amino acid types would have been recovered. Chou and ACE estimators (available within the ESTIMATES program) predicted that no more than 22 DNA and the observed eight amino acid types would have been recovered.
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Over 85 % of the FTHFS homologues recovered from termite-gut contents grouped within the Lovell cluster, and most of these comprised a unique, distinct and well-supported branch together with the homologues obtained from the treponemal isolates (Fig. 2a). All clone types comprising this termite treponeme cluster shared greater than 80 % amino acid identity with those derived from the treponemal isolates. Of particular note, members of Za-gut Clone P group and Za-gut Clone N shared 96·3 and 94·3 % sequence identity, respectively, with the FTHFS homologue identified from Treponema strain ZAS-2. The nine clone types comprising the H group accounted for 43 % of the clones recovered from gut-community DNA, and shared
91 % amino acid identity with the FTHFS homologue from strain ZAS-2. Despite close similarities with each other at the amino acid level (>98 % identity), clone types comprising the Za-gut Clone H group were far from identical at the DNA level, thus they appear to represent different microbial strains or species (Fig. 2b
). For example, Za-gut Clone F2 and Za-gut Clone R shared only 88 % DNA sequence identity despite sharing 99·3 % amino acid identity.
Two Lovell-cluster clone types, Za-gut Clone F and Za-gut Clone Y, showed no affiliation with the termite treponemes (Fig. 2a). These, together with a clone recovered from horse manure DNA, formed a unique radiation and were not highly similar to homologues identified in known homoacetogens. They shared less than 75 % amino acid identity with the FTHFS from R. productus. Thus they may represent a novel, not-yet-cultivated microbial lineage. None of the recovered clones evidenced any affiliation with the FTHFS homologue identified in S. termitida, the spore-forming homoacetogen isolated from the gut of the Caribbean wood-feeding, higher termite, Nasutitermes nigriceps. Approximately 15 % of the recovered clones (i.e. Za-gut Clones T and E) did not affiliate with the Lovell cluster and thus may not represent homoacetogens. When our analyses of termite-gut-derived sequences were expanded to include a recently reported, larger collection of FTHFS homologues (Leaphart et al., 2003
), none of these aforementioned relationships were meaningfully changed or further resolved. Za-gut Clone E, for example, remained quite remote from any available FTHFS sequences.
Character analysis of FTHFS sequences
With a few minor exceptions, all FTHFS genes retrieved during the course of this study contained all of the conserved FTHFS polypeptide hallmarks that have previously been noted (Leaphart & Lovell, 2001; Leaphart et al., 2003
). The majority of the FTHFS genes comprising the termite treponeme cluster contained a distinguishing polypeptide character not observed in other FTHFS genes analysed. As many as eight amino acid residues, instead of the more typically encountered two, were found inserted into a region corresponding to Moorella residues Tyr231 and Lys234 (Fig. 3
). Of all other FTHFS homologues analysed, only R. productus contained additional residues within this region. This unique region was filtered from alignments used for making phylogenetic comparisons, thus serves as an independent character or signpost providing additional support for the coherency of the termite treponeme radiation.
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Downstream from the FTHFS-encoding gene was an obvious ORF. The deduced 209 residue protein shared 50 % amino acid sequence identity with a putative methenyltetrahydrofolate cyclohydrolase (MTHFC) from the unpublished genome of Desulfitobacterium hafniense, a spore-forming member of the class Clostridia. MTHFCs catalyse the enzymic step in acetogenesis following that exerted by FTHFS and are also involved in other THF-dependent metabolisms. Because the gene encoding this enzyme was not available from any known homoacetogen, the homologue from strain ZAS-2 was compared with those identified homologues from the D. hafniense and 20 other, mostly unpublished, genome sequences. It clustered within a well-supported branch along with the homologues from D. hafniense, Clostridium botulinum, Clostridium difficile and Carboxydothermus hydrogenoformans, all of which are Firmicutes belonging to the class Clostridia (Fig. 4a). This radiation was distinct from the branch which contained a MTHFC homologue identified from the unpublished genome of the non-acetogenic, human oral treponeme, T. denticola (Fig. 4a
). As was the case for FTHFS-encoding genes, the published genomes of two other spirochaetes, T. pallidum and B. burgdorferi, did not contain any obvious MTHFC homologues.
Downstream from the putative MTHFC homologue from Treponema strain ZAS-2 was a partial gene interrupted by the terminus of the iPCR-generated amplification product. The first 102 amino acids of the deduced polypeptide were homologous to the catalytic domains of FolD-type methylenetetrahydrofolate dehydrogenases (MTHFDs), and shared 40 % amino acid identity with that from the unpublished genome of D. hafniense. MTHFD catalyses the enzymic step in acetogenesis that follows those exerted by FTHFS and MTHFC and is also involved in other THF-dependent metabolisms in other organisms. As was the case with MTHFC, the gene encoding a MTHFD was not yet available from any known homoacetogen. Accordingly, the ZAS-2 homologue was used in a phylogenetic comparison with those identified in the D. hafniense and 10 other, mostly unpublished, genome sequences. The homologue from strain ZAS-2 was the deepest branch on a well-supported cluster comprising the enzyme homologues from D. hafniense, Clostridium botulinum, Clostridium difficile and Carboxydothermus hydrogenoformans (Fig. 4b). FolD homologues identified in the unpublished genome sequences of the non-homoacetogenic oral treponeme T. denticola, and published genome of the syphilis spirochaete T. pallidum, did not cluster phylogenetically with Treponema strain ZAS-2 or the four clostridia (Fig. 4b
). As was the case for genes encoding FTHFS and MTHFC, the published genome of the Lyme disease spirochaete B. burgdorferi did not contain an obvious MTHFD homologue. The branching topology did not change significantly with the addition of more than 50 other demonstrated or deduced MTHFDs to the analysis (not shown).
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DISCUSSION |
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Members of the genus Treponema, a genus within the phylum-level assemblage Spirochaetes, are only distantly related to the classical homoacetogens, most of which belong to the phylum Firmicutes. Treponemes are also only distantly related to other homoacetogens, such as Desulfotignum phosphitoxidans, a -proteobacterium, and Holophaga foetida, one of the few cultivated representatives of the phylum Acidobacteria (Liesack et al., 1994
; Schink et al., 2002
). It was somewhat surprising that primers designed to amplify FTHFS genes from clostridia functioned in doing so from termite-gut spirochaetes, an observation that has also been recently reported by others (Leaphart et al., 2003
). More surprising, FTHFS homologues from termite-gut Treponema strains clustered phylogenetically with those from homoacetogenic clostridia and not, for example, with that from the non-homoacetogenic oral spirochaete T. denticola (Fig. 2a
). This pattern was re-encountered during the analysis of each of two other genes likely to encode THF-dependent enzymes identified from Treponema ZAS-2 (Fig. 4a, b
). This suggests that THF-dependent enzymes from closely related termite-gut and human-associated treponemes may have distinctly different evolutionary origins. Thus, it can be postulated that the termite-gut spirochaete pathway may have been acquired via lateral gene transfer from a species closely related to R. productus. If so, was the event before or after spirochaetes had first become established within the symbiotic, termite-gut communities? The identification of a homoacetogen-like FTHFS gene from the H2-producing isolate Treponema ZAS-9 raises similar intrigue, as this strain is not a homoacetogen (Breznak & Leadbetter, 2002
; Lilburn et al., 2001
). Of all the FTHFS genes that comprise Lovell's homoacetogen cluster, to date this is the only example representing a known non-homoacetogen. Might this strain represent a snapshot of a species in evolutionary transition either into or from having CO2-reductive capacities?
Our initial concerns that the FTHFS primer set might be biased towards the overamplification of genes from the homoacetogenic clostridia (for which they had been designed) did not play out. Three-quarters of the clones recovered clustered closely with the spirochaete-derived sequences, several sharing remarkable amino acid similarities with the isolates, for example, 96·3 % similarity shared between Za-gut Clone P and Treponema ZAS-2 (Fig. 2a). The results of this study establish that there could be as many as 20 strains representing at least two homoacetogenic phyla present in Zootermopsis guts. We are now interested in examining the nature and physical location of the cells encoding these genes, and in determining which types actually dominate FTHFS expression in situ. Are the organisms representing Clone P and Clone H truly spirochaetes, are they as abundant and active as the inventory results might suggest, and are they actually homoacetogens? Are the cells representing Clone F and Clone Y actually spore-forming clostridia? The termite-gut community is now recognized as being highly structured (Brune & Friedrich, 2000
). Are all homoacetogen species found localized to the same temporal and spatial niches within the gut? The results of the current study should help facilitate the design of quantitative mRNA, cell-localization, community fingerprinting, and other approaches to address all such questions.
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ACKNOWLEDGEMENTS |
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Received 14 March 2003;
revised 13 May 2003;
accepted 28 May 2003.
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