Anaerobic Microbial Metabolism of Dichloroacetate.

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising "Candidatus Dichloromethanomonas elyunquensis" strain RM utilizes DCA as an energy source, and the transient formation of formate, H2, and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated "Candidatus Dichloromethanomonas elyunquensis" strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg-1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.IMPORTANCE Dichloroacetate (DCA) is ubiquitous in the environment due to natural formation via biological and abiotic chlorination processes and the turnover of chlorinated organic materials (e.g., humic substances). Additional sources include DCA usage as a chemical feedstock and cancer drug and its unintentional formation during drinking water disinfection by chlorination. Despite the ubiquitous presence of DCA, its fate under anoxic conditions has remained obscure. We discovered an anaerobic bacterium capable of metabolizing DCA, identified the enzyme responsible for DCA dehalogenation, and elucidated a novel DCA fermentation pathway. The findings have implications for the turnover of DCA and the carbon and electron flow in electron acceptor-depleted environments and the human gastrointestinal tract.

Recoding of the selenocysteine UGA codon by cysteine in the presence of a non-canonical tRNACys and elongation factor SelB.

In many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNASec is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNASec-like tRNA genes distributed across Bacteria that also encode a canonical tRNASec. These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNASec with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNASec-like tRNACys, and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNACys as 'tRNAReC' (Recoding with Cys). We performed a comprehensive survey of tRNAReC genes to establish their phylogenetic distribution, and found that, in a particular lineage of clostridial Pelotomaculum, the Cys identity elements of tRNAReC had mutated. This novel tRNA, which contains a UCA anticodon, is capable of Sec incorporation in E. coli, albeit with lower efficiency relative to Pelotomaculum tRNASec. We renamed this unusual tRNASec derived from tRNAReC as 'tRNAReU' (Recoding with Sec). Together, our results suggest that tRNAReC and tRNAReU may serve as safeguards in the production of selenoproteins and - to our knowledge - they provide the first example of programmed codon-anticodon mispairing in bacteria.

A benzene-degrading nitrate-reducing microbial consortium displays aerobic and anaerobic benzene degradation pathways.

In this study, we report transcription of genes involved in aerobic and anaerobic benzene degradation pathways in a benzene-degrading denitrifying continuous culture. Transcripts associated with the family Peptococcaceae dominated all samples (21-36% relative abundance) indicating their key role in the community. We found a highly transcribed gene cluster encoding a presumed anaerobic benzene carboxylase (AbcA and AbcD) and a benzoate-coenzyme A ligase (BzlA). Predicted gene products showed >96% amino acid identity and similar gene order to the corresponding benzene degradation gene cluster described previously, providing further evidence for anaerobic benzene activation via carboxylation. For subsequent benzoyl-CoA dearomatization, bam-like genes analogous to the ones found in other strict anaerobes were transcribed, whereas gene transcripts involved in downstream benzoyl-CoA degradation were mostly analogous to the ones described in facultative anaerobes. The concurrent transcription of genes encoding enzymes involved in oxygenase-mediated aerobic benzene degradation suggested oxygen presence in the culture, possibly formed via a recently identified nitric oxide dismutase (Nod). Although we were unable to detect transcription of Nod-encoding genes, addition of nitrite and formate to the continuous culture showed indication for oxygen production. Such an oxygen production would enable aerobic microbes to thrive in oxygen-depleted and nitrate-containing subsurface environments contaminated with hydrocarbons.

Benzene degradation in a denitrifying biofilm reactor: activity and microbial community composition.

Benzene is an aromatic compound and harmful for the environment. Biodegradation of benzene can reduce the toxicological risk after accidental or controlled release of this chemical in the environment. In this study, we further characterized an anaerobic continuous biofilm culture grown for more than 14 years on benzene with nitrate as electron acceptor. We determined steady state degradation rates, microbial community composition dynamics in the biofilm, and the initial anaerobic benzene degradation reactions. Benzene was degraded at a rate of 0.15 µmol/mg protein/day and a first-order rate constant of 3.04/day which was fourfold higher than rates reported previously. Bacteria belonging to the Peptococcaceae were found to play an important role in this anaerobic benzene-degrading biofilm culture, but also members of the Anaerolineaceae were predicted to be involved in benzene degradation or benzene metabolite degradation based on Illumina MiSeq analysis of 16S ribosomal RNA genes. Biomass retention in the reactor using a filtration finger resulted in reduction of benzene degradation capacity. Detection of the benzene carboxylase encoding gene, abcA, and benzoic acid in the culture vessel indicated that benzene degradation proceeds through an initial carboxylation step.

Isolation of a sulfide-producing bacterial consortium from cooling-tower water: Evaluation of corrosive effects on galvanized steel.

Sulfidogenic Clostridia and sulfate reducing bacteria (SRB) often cohabit in nature. The presence of these microorganisms can cause microbially influenced corrosion (MIC) of materials in different ways. To investigate this aspect, bacteria were isolated from cooling tower water and used in corrosion tests of galvanized steel. The identity of the isolates was determined by comparative sequence analysis of PCR-amplified 16S rDNA gene fragments, separated by denaturing gradient gel electrophoresis (DGGE). This analysis showed that, in spite of the isolation process, colonies were not pure and consisted of a mixture of bacteria affiliated with Desulfosporosinus meridiei and Clostridium sp. To evaluate the corrosive effect, galvanized steel coupons were incubated with a mixed culture for 4, 8, 24, 72, 96, 168, 360 and 744 h, along with a control set in sterile culture medium only. The corrosion rate was determined by weight loss, and biofilm formation and corroded surfaces were observed by scanning electron microscopy (SEM). Although the sulfide-producing bacterial consortium led to a slight increase in the corrosion of galvanized steel coupons, when compared to the previous studies it can be said that Clostridium sp. can reduce the corrosive effect of the Desulfosporosinus sp. strain.

Genomic insights into a new acidophilic, copper-resistant Desulfosporosinus isolate from the oxidized tailings area of an abandoned gold mine.

Microbial sulfate reduction in acid mine drainage is still considered to be confined to anoxic conditions, although several reports have shown that sulfate-reducing bacteria occur under microaerophilic or aerobic conditions. We have measured sulfate reduction rates of up to 60 nmol S cm(-3) day(-1) in oxidized layers of gold mine tailings in Kuzbass (SW Siberia). A novel, acidophilic, copper-tolerant Desulfosporosinus sp. I2 was isolated from the same sample and its genome was sequenced. The genomic analysis and physiological data indicate the involvement of transporters and additional mechanisms to tolerate metals, such as sequestration by polyphosphates. Desulfosporinus sp. I2 encodes systems for a metabolically versatile life style. The genome possessed a complete Embden-Meyerhof pathway for glycolysis and gluconeogenesis. Complete oxidation of organic substrates could be enabled by the complete TCA cycle. Genomic analysis found all major components of the electron transfer chain necessary for energy generation via oxidative phosphorylation. Autotrophic CO2 fixation could be performed through the Wood-Ljungdahl pathway. Multiple oxygen detoxification systems were identified in the genome. Taking into account the metabolic activity and genomic analysis, the traits of the novel isolate broaden our understanding of active sulfate reduction and associated metabolism beyond strictly anaerobic niches.

Long-Term Incubation Reveals Methanogenic Biodegradation of C5 and C6 iso-Alkanes in Oil Sands Tailings.

iso-Alkanes are major components of petroleum and have been considered recalcitrant to biodegradation under methanogenic conditions. However, indigenous microbes in oil sands tailings ponds exposed to solvents rich in 2-methylbutane, 2-methylpentane, 3-methylpentane, n-pentane, and n-hexane produce methane in situ. We incubated defined mixtures of iso- or n-alkanes with mature fine tailings from two tailings ponds of different ages historically exposed to different solvents: one, ~10 years old, receiving C5-C6 paraffins and the other, ~35 years old, receiving naphtha. A lengthy incubation (>6 years) revealed iso-alkane biodegradation after lag phases of 900-1800 and ~280 days, respectively, before the onset of methanogenesis, although lag phases were shorter with n-alkanes (~650-1675 and ~170 days, respectively). 2-Methylpentane and both n-alkanes were completely depleted during ~2400 days of incubation, whereas 2-methylbutane and 3-methylpentane were partially depleted only during active degradation of 2-methylpentane, suggesting co-metabolism. In both cases, pyrotag sequencing of 16S rRNA genes showed codominance of Peptococcaceae with acetoclastic (Methanosaeta) and hydrogenotrophic (Methanoregula and Methanolinea) methanogens. These observations are important for predicting long-term greenhouse-gas emissions from oil sands tailings ponds and extend the known range of hydrocarbons susceptible to methanogenic biodegradation in petroleum-impacted anaerobic environments.

Anaerobic alkane biodegradation by cultures enriched from oil sands tailings ponds involves multiple species capable of fumarate addition.

A methanogenic short-chain alkane-degrading culture (SCADC) was enriched from oil sands tailings and transferred several times with a mixture of C6, C7, C8 and C10 n-alkanes as the predominant organic carbon source, plus 2-methylpentane, 3-methylpentane and methylcyclopentane as minor components. Cultures produced ∼40% of the maximum theoretical methane during 18 months incubation while depleting the n-alkanes, 2-methylpentane and methylcyclopentane. Substrate depletion correlated with detection of metabolites characteristic of fumarate activation of 2-methylpentane and methylcyclopentane, but not n-alkane metabolites. During active methanogenesis with the mixed alkanes, reverse-transcription PCR confirmed the expression of functional genes (assA and bssA) associated with hydrocarbon addition to fumarate. Pyrosequencing of 16S rRNA genes amplified during active alkane degradation revealed enrichment of Clostridia (particularly Peptococcaceae) and methanogenic Archaea (Methanosaetaceae and Methanomicrobiaceae). Methanogenic cultures transferred into medium containing sulphate produced sulphide, depleted n-alkanes and produced the corresponding succinylated alkane metabolites, but were slow to degrade 2-methylpentane and methylcyclopentane; these cultures were enriched in Deltaproteobacteria rather than Clostridia. 3-Methylpentane was not degraded by any cultures. Thus, nominally methanogenic oil sands tailings harbour dynamic and versatile hydrocarbon-degrading fermentative syntrophs and sulphate reducers capable of degrading n-, iso- and cyclo-alkanes by addition to fumarate.

DNA stable-isotope probing of oil sands tailings pond enrichment cultures reveals different key players for toluene degradation under methanogenic and sulfidogenic conditions.

Oil sands tailings ponds are anaerobic repositories of fluid wastes produced by extraction of bitumen from oil sands ores. Diverse indigenous microbiota biodegrade hydrocarbons (including toluene) in situ, producing methane, carbon dioxide and/or hydrogen sulfide, depending on electron acceptor availability. Stable-isotope probing of cultures enriched from tailings associated specific taxa and functional genes to (13)C6- and (12)C7-toluene degradation under methanogenic and sulfate-reducing conditions. Total DNA was subjected to isopycnic ultracentrifugation followed by gradient fraction analysis using terminal restriction fragment length polymorphism (T-RFLP) and construction of 16S rRNA, benzylsuccinate synthase (bssA) and dissimilatory sulfite reductase (dsrB) gene clone libraries. T-RFLP analysis plus sequencing and in silico digestion of cloned taxonomic and functional genes revealed that Clostridiales, particularly Desulfosporosinus (136 bp T-RF) contained bssA genes and were key toluene degraders during methanogenesis dominated by Methanosaeta. Deltaproteobacterial Desulfobulbaceae (157 bp T-RF) became dominant under sulfidogenic conditions, likely because the Desulfosporosinus T-RF 136 apparently lacks dsrB and therefore, unlike its close relatives, is presumed incapable of dissimilatory sulfate reduction. We infer incomplete oxidation of toluene by Desulfosporosinus in syntrophic association with Methanosaeta under methanogenic conditions, and complete toluene oxidation by Desulfobulbaceae during sulfate reduction.

Conductive iron oxide minerals accelerate syntrophic cooperation in methanogenic benzoate degradation.

Recent studies have suggested that conductive iron oxide minerals can facilitate syntrophic metabolism of the methanogenic degradation of organic matter, such as ethanol, propionate and butyrate, in natural and engineered microbial ecosystems. This enhanced syntrophy involves direct interspecies electron transfer (DIET) powered by microorganisms exchanging metabolic electrons through electrically conductive minerals. Here, we evaluated the possibility that conductive iron oxides (hematite and magnetite) can stimulate the methanogenic degradation of benzoate, which is a common intermediate in the anaerobic metabolism of aromatic compounds. The results showed that 89-94% of the electrons released from benzoate oxidation were recovered in CH4 production, and acetate was identified as the only carbon-bearing intermediate during benzoate degradation. Compared with the iron-free controls, the rates of methanogenic benzoate degradation were enhanced by 25% and 53% in the presence of hematite and magnetite, respectively. This stimulatory effect probably resulted from DIET-mediated methanogenesis in which electrons transfer between syntrophic partners via conductive iron minerals. Phylogenetic analyses revealed that Bacillaceae, Peptococcaceae, and Methanobacterium are potentially involved in the functioning of syntrophic DIET. Considering the ubiquitous presence of iron minerals within soils and sediments, the findings of this study will increase the current understanding of the natural biological attenuation of aromatic hydrocarbons in anaerobic environments.

Diet shapes the gut microbiota of the omnivorous cockroach Blattella germanica.

The gut microbiota of insects contributes positively to the physiology of its host mainly by participating in food digestion, protecting against pathogens, or provisioning vitamins or amino acids, but the dynamics of this complex ecosystem is not well understood so far. In this study, we have characterized the gut microbiota of the omnivorous cockroach Blattella germanica by pyrosequencing the hypervariable regions V1-V3 of the 16S rRNA gene of the whole bacterial community. Three diets differing in the protein content (0, 24 and 50%) were tested at two time points in lab-reared individuals. In addition, the gut microbiota of wild adult cockroaches was also analyzed. In contrast to the high microbial richness described on the studied samples, only few species are shared by wild and lab-reared cockroaches, constituting the bacterial core in the gut of B. germanica. Overall, we found that the gut microbiota of B. germanica is highly dynamic as the bacterial composition was reassembled in a diet-specific manner over a short time span, with no-protein diet promoting high diversity, although the highest diversity was found in the wild cockroaches analyzed. We discuss how the flexibility of the gut microbiota is probably due to its omnivorous life style and varied diets.

Inhibition of sulfate reducing bacteria in aquifer sediment by iron nanoparticles.

Batch microcosms were setup to determine the impact of different sized zero valent iron (Fe(0)) particles on microbial sulfate reduction during the in situ bio-precipitation of metals. The microcosms were constructed with aquifer sediment and groundwater from a low pH (3.1), heavy-metal contaminated aquifer. Nano (nFe(0)), micro (mFe(0)) and granular (gFe(0)) sized Fe(0) particles were added to separate microcosms. Additionally, selected microcosms were also amended with glycerol as a C-source for sulfate-reducing bacteria. In addition to metal removal, Fe(0) in microcosms also raised the pH from 3.1 to 6.5, and decreased the oxidation redox potential from initial values of 249 to -226 mV, providing more favorable conditions for microbial sulfate reduction. mFe(0) and gFe(0) in combination with glycerol were found to enhance microbial sulfate reduction. However, no sulfate reduction occurred in the controls without Fe(0) or in the microcosm amended with nFe(0). A separate dose test confirmed the inhibition for sulfate reduction in presence of nFe(0). Hydrogen produced by Fe(0) was not capable of supporting microbial sulfate reduction as a lone electron donor in this study. Microbial analysis revealed that the addition of Fe(0) and glycerol shifted the microbial community towards Desulfosporosinus sp. from a population initially dominated by low pH and metal-resisting Acidithiobacillus ferrooxidans.

Microbial community response to addition of polylactate compounds to stimulate hexavalent chromium reduction in groundwater.

To evaluate the efficacy of bioimmobilization of Cr(VI) in groundwater at the Department of Energy Hanford site, we conducted a series of microcosm experiments using a range of commercial electron donors with varying degrees of lactate polymerization (polylactate). These experiments were conducted using Hanford Formation sediments (coarse sand and gravel) immersed in Hanford groundwater, which were amended with Cr(VI) and several types of lactate-based electron donors (Hydrogen Release Compound, HRC; primer-HRC, pHRC; extended release HRC) and the polylactate-cysteine form (Metal Remediation Compound, MRC). The results showed that polylactate compounds stimulated an increase in bacterial biomass and activity to a greater extent than sodium lactate when applied at equivalent carbon concentrations. At the same time, concentrations of headspace hydrogen and methane increased and correlated with changes in the microbial community structure. Enrichment of Pseudomonas spp. occurred with all lactate additions, and enrichment of sulfate-reducing Desulfosporosinus spp. occurred with almost complete sulfate reduction. The results of these experiments demonstrate that amendment with the pHRC and MRC forms result in effective removal of Cr(VI) from solution most likely by both direct (enzymatic) and indirect (microbially generated reductant) mechanisms.

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment.

Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with (13)C(7)-toluene, the production of both sulfide and (13)CO(2) was clearly coupled to the (13)C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only ∼50%, pointing toward high ratios of heterotrophic CO(2)-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

Functional characterization of an anaerobic benzene-degrading enrichment culture by DNA stable isotope probing.

The flow of carbon under sulfate-reducing conditions within a benzene-mineralizing enrichment culture was analysed using fully labelled [13C6]-benzene. Over 180 days of incubation, 95% of added 13C-benzene was released as 13C-carbon dioxide. DNA extracted from cultures that had degraded different amounts of unlabelled or 13C-labelled benzene was centrifuged in CsCl density gradients to identify 13C-benzene-assimilating organisms by density-resolved terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA gene fragments. Two phylotypes showed significantly increased relative abundance of their terminal restriction fragments in 'heavy' fractions of 13C-benzene-incubated microcosms compared with a 12C-benzene-incubated control: a member of the Cryptanaerobacter/Pelotomaculum group within the Peptococcaceae, and a phylotype belonging to the Epsilonproteobacteria. The Cryptanaerobacter/Pelotomaculum phylotype was the most frequent sequence type. A small amount of 13C-methane was aceticlastically produced, as concluded from the linear relationship between methane production and benzene degradation and the detection of Methanosaetaceae as the only methanogens present. Other phylotypes detected but not 13C-labelled belong to several genera of sulfate-reducing bacteria, that may act as hydrogen scavengers for benzene oxidation. Our results strongly support the hypothesis that benzene is mineralized by a consortium consisting of syntrophs, hydrogenotrophic sulfate reducers and to a minor extent of aceticlastic methanogens.

The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation.

Here, we present a detailed functional and phylogenetic characterization of an iron-reducing enrichment culture maintained in our lab with benzene as sole carbon and energy source. We used DNA-stable isotope probing to identify microbes within the enrichment most active in the assimilation of (13)C-label. When (12)C(6)- and (13)C(6)-benzene were added as comparative substrates, marked differences in the quantitative buoyant density distribution became apparent especially for uncultured microbes within the Gram-positive Peptococcaceae, closely related to environmental clones retrieved from contaminated aquifers world wide and only distantly related to cultured representatives of the genus Thermincola. Prominent among the other constituents of the enrichment were uncultured Deltaproteobacteria, as well as members of the Actinobacteria. Although their presence within the enrichment seems to be stable they did not assimilate (13)C-label as significantly as the Clostridia within the time course of our experiment. We hypothesize that benzene degradation in our enrichment involves an unusual syntrophy, where members of the Clostridia primarily oxidize benzene. Electrons from the contaminant are both directly transferred to ferric iron by the primary oxidizers, but also partially shared with the Desulfobulbaceae as syntrophic partners. Alternatively, electrons may also be quantitatively transferred to the partners, which then reduce the ferric iron. Thus our results provide evidence for the importance of a novel clade of Gram-positive iron-reducers in anaerobic benzene degradation, and a role of syntrophic interactions in this process. These findings shed a totally new light on the factors controlling benzene degradation in anaerobic contaminated environments.

Non-sulfate-reducing, syntrophic bacteria affiliated with desulfotomaculum cluster I are widely distributed in methanogenic environments.

The classical perception of members of the gram-positive Desulfotomaculum cluster I as sulfate-reducing bacteria was recently challenged by the isolation of new representatives lacking the ability for anaerobic sulfate respiration. For example, the two described syntrophic propionate-oxidizing species of the genus Pelotomaculum form the novel Desulfotomaculum subcluster Ih. In the present study, we applied a polyphasic approach by using cultivation-independent and culturing techniques in order to further characterize the occurrence, abundance, and physiological properties of subcluster Ih bacteria in low-sulfate, methanogenic environments. 16S rRNA (gene)-based cloning, quantitative fluorescence in situ hybridization, and real-time PCR analyses showed that the subcluster Ih population composed a considerable part of the Desulfotomaculum cluster I community in almost all samples examined. Additionally, five propionate-degrading syntrophic enrichments of subcluster Ih bacteria were successfully established, from one of which the new strain MGP was isolated in coculture with a hydrogenotrophic methanogen. None of the cultures analyzed, including previously described Pelotomaculum species and strain MGP, consumed sulfite, sulfate, or organosulfonates. In accordance with these phenotypic observations, a PCR-based screening for dsrAB (key genes of the sulfate respiration pathway encoding the alpha and beta subunits of the dissimilatory sulfite reductase) of all enrichments/(co)cultures was negative with one exception. Surprisingly, strain MGP contained dsrAB, which were transcribed in the presence and absence of sulfate. Based on these and previous findings, we hypothesize that members of Desulfotomaculum subcluster Ih have recently adopted a syntrophic lifestyle to thrive in low-sulfate, methanogenic environments and thus have lost their ancestral ability for dissimilatory sulfate/sulfite reduction.

Nucleotide sequence and transcriptional analysis of the celD beta-glucanase gene from Ruminococcus flavefaciens FD-1.

The nucleotide sequence of the celD gene, which encodes endoglucanase and xylanase activity, from Ruminococcus flavefaciens FD-1 was determined. The DNA sequence of celD contains an open reading frame of 1215 nucleotides that encodes a polypeptide of 405 amino acids with a molecular mass of 44,631 Da. The primary amino acid sequence of CelD was screened against the GenBank data base for similar polypeptide sequences and the analysis indicated that CelD has common features with endoglucanases from the family E cellulases. Both hydrophobic cluster and BESTFIT (Genetics Computer Group (University of Wisconsin) package) analyses confirmed this relationship. Pairwise alignments using BESTFIT revealed that CelD was most closely related to endE4 from Thermomonospora fusca over a 160 amino acid window. The histidine, aspartate, and glutamate residues identified as being essential for catalytic activity in family E cellulases are conserved in CelD. A Shine-Dalgarno-like sequence was present 5 base pairs (bp) upstream of the translation start site. Primer extension analysis indicated that different transcription initiation sites are used to initiate transcription of celD in Escherichia coli and R. flavefaciens. In the case of R. flavefaciens the transcription initiation site is at a T residue (nucleotide 273) 16 bp upstream from the translational start site. A region resembling a sigma 70-like-10 promoter sequence is present upstream from the transcription initiation site but there is no apparent-35 region. In contrast, transcription in E. coli is initiated at a C residue 258 bp upstream from the translational start site and a sequence resembling a omega 70-like-10 region is present 5 bp upstream of this residue.(ABSTRACT TRUNCATED AT 250 WORDS)