The Wolinella succinogenes mcc gene cluster encodes an unconventional respiratory sulphite reduction system

Assimilatory and dissimilatory sulphite reductions are key reactions in the biogeochemical sulphur cycle and several distinct sirohaem-containing sulphite reductases have been characterized. Here, we describe that the Epsilonproteobacterium Wolinella succinogenes is able to grow by sulphite respiration (yielding sulphide) with formate as electron donor. Sulphite is reduced by MccA, a prototypical member of an emerging new class of periplasmic cytochrome c sulphite reductases that, phylogenetically, belongs to a multihaem cytochrome c superfamily whose members play crucial roles in the global sulphur and nitrogen cycles. Within this family, MccA represents an unconventional octahaem cytochrome c containing a special haem c group that is bound via two cysteine residues arranged in a unique CX(15)CH haem c binding motif. The phenotypes of numerous W.succinogenes mutants producing MccA variants underlined the structural importance of this motif. Several open reading frames of the mcc gene cluster were individually inactivated and characterization of the corresponding mutants indicated that the predicted iron-sulphur protein MccC, the putative quinol dehydrogenase MccD (a member of the NrfD/PsrC family) as well as a peptidyl-prolyl cis-trans isomerase, MccB, are essential for sulphite respiration. MccA synthesis in W. succinogenes was found to be induced by sulphite (but not by thiosulphate or sulphide) and repressed in the presence of fumarate or nitrate. Based on the results, a sophisticated model of respiratory sulphite reduction by the Mcc system is presented.     

Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK₆ was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK₆, 8-MMK₆ and 7,8-DMMK₆ were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.     

Hydrogen sulfide-producing kinetics of Shewanella oneidensis in sulfite and thiosulfate respiration

Shewanella oneidensis is a model species for aquatic ecosystems and plays an important role in bioremediation, biofuel cell manufacturing and biogeochemical cycling. S. oneidensis MR-1 is able to generate hydrogen sulfide from various sulfur species; however, its catalytic kinetics have not been determined. In this study, five in-frame deletion mutants of S. oneidensis were constructed and their H₂S-producing activities were analyzed. SirA and PsrA were the two major contributors to H₂S generation under anoxic cultivation, and the optimum SO₃²⁻ concentration for sulfite respiration was approximately 0.8 mM, while the optimum S₂O₃²⁻ concentration for thiosulfate respiration was approximately 0.4 mM. Sulfite and thiosulfate were observed to interfere with each other during respiration, and a high concentration of sulfite or thiosulfate chelated extracellular free-iron but did not repress the expression of sirA or psrA. Nitrite and nitrate were two preferred electron acceptors during anaerobic respiration; however, under energy-insufficient conditions, S. oneidensis could utilize multiple electron acceptors simultaneously. Elucidiating the stoichiometry of H₂S production in S. oneidensis would be helpful for the application of this species in bioremediation and biofuel cell manufacturing, and would help to characterize the ecophysiology of sulfur cycling.     

Natranaeroarchaeum sulfidigenes gen. nov., sp. nov., carbohydrate-utilizing sulfur-respiring haloarchaeon from hypersaline soda lakes,a member of a new family Natronoarchaeaceae fam. nov. in the order Halobacteriales

A pure culture of alkaliphilic haloarchaeon strain AArc-S^T capable of anaerobic growth by carbohydrate-dependent sulfur respiration was obtained from hypersaline lakes in southwestern Siberia. According to phylogenetic analysis, AArc-S^T formed a new genus level branch most related to the genus Natronoarchaeum in the order Halobacteriales. The strain is facultatively anaerobic with strictly respiratory metabolism growing either by anaerobic respiration with elemental sulfur and thiosulfate as the electron acceptors or by aerobic respiration at microoxic conditions. Thiosulfate is reduced partially to sulfide and sulfite. It is a first sulfur-reducing alkaliphilic haloarchaeon utilizing sugars, starch and glycerol as substrates for anaerobic growth. It is extremely halophilic (optimum at 3.5 M total Na⁺) and obligately alkaliphilic (optimum at pH 9.5). The dominant polar lipids include PG and PGP-Me with the archaeol (C₂₀-C₂₀) or extended archaeol (C₂₀-C₂₅) cores. The dominant respiratory lipoquinone is MK-8:8. On the basis of unique physiological properties and results of phylogenetic analysis, the soda lake isolate is suggested to be classified into a novel genus and species Natranaeroarchaeum sulfidigenes gen. nov., sp. nov. (=JCM 34033^T = UNIQEM U1000^T). Furthermore, on the bases of phylogenomic reconstruction, a new family Natronoarchaeaceae fam. nov. is proposed within the order Halobacteriales incorporating Natranaeroarchaeum and three related genera: Natronoarchaeum, Salinarchaeum and Halostella.     

Halapricum desulfuricans sp. nov., carbohydrate-utilizing,sulfur-respiring haloarchaea from hypersaline lakes

Nine pure cultures of neutrophilic haloaloarchaea capable of anaerobic growth by carbohydrate-dependent sulfur respiration were isolated from hypersaline lakes in southwestern Siberia and southern Russia. According to phylogenomic analysis the isolates were closely related to each other and formed a new species within the genus Halapricum (family Haloarculaceae). They have three types of catabolism: fermentative, resulting in H₂ formation; anaerobic respiration using sulfur compounds as e-acceptors and aerobic respiration. Apart from elemental sulfur, all isolates can also use three different sulfoxides as acceptors and the type strain also grows with thiosulfate, reducing it partially to sulfide and sulfite. All strains utilized sugars and glycerol as the e-donors and C source for anaerobic growth and some can also grow with alpha-glucans, such as starch and dextrins. The major respiratory menaquinones are MK-8:8 and MK-8:7, but 5–19% consists of “thermoplasmata” quinones (MMK-8:8 and MMK-8:7), whose occurrence in haloarchaea is unprecedented. On the basis of their unique physiological properties and results of phylogenomic analysis, the isolates are suggested to be classified into a novel species Halapricum desulfuricans sp. nov. (type strain HSR12-2^T = JCM 34032^T = UNIQEM U1001^T).     

Regulation of anaerobic respiratory pathways in Wolinella succinogenes by the presence of electron acceptors

In Wolinella succinogenes ATP synthesis and consequently bacterial growth can be driven by the reduction of either nitrate (E₀=+0.42 V), nitrite (E₀=+0.36 V), fumarate (E₀=+0.03 V) or sulphur (E₀=-0.27 V) with formate as the electron donor. Bacteria growing in the presence of nitrate and fumarate were found to reduce both acceptors simultaneously, while the reduction of both nitrate and fumarate is blocked during growth with sulphur. These observations were paralleled by the presence and absence of the corresponding bacterial reductase activities. Using a specific antiserum, fumarate reductase was shown to be present in bacteria grown with fumarate and nitrate, and to be nearly absent from bacteria grown in the presence of sulphur. The contents of polysulphide reductase, too, corresponded to the enzyme activities found in the bacteria. This suggests that the activities of anaerobic respiration are regulated at the biosynthetic level in W. succinogenes. Thus nitrate and fumarate reduction are repressed by the most electronegative acceptor of anacrobic respiration, sulphur. By contrast, in Escherichia coli a similar effect is exerted by the most electropositive acceptor, O₂. W. succinogenes also differs from E. coli in that fumarate reductase is not repressed by nitrate.Abbreviations BV benzyl viologen - DMN 2,3-dimethyl-1,4-naphthoquinone - DMSO dimethylsulfoxide - TMAO trimethylamine-N-oxide     

Properties of a Wolinella succinogenes mutant lacking periplasmic sulfide dehydrogenase (Sud)

A Δsud deletion mutant of Wolinella succinogenes that lacked the periplasmic sulfide dehydrogenase (Sud) was constructed using homologous recombination. The mutant grew with sulfide and fumarate, indicating that Sud was not a component of the electron transport chain that catalyzed fumarate respiration with sulfide as an electron donor. Likewise, growth with formate and either polysulfide or sulfur was not affected by the deletion. Removal of Sud from wild-type W. succinogenes by spheroplast formation did not decrease the activity of electron transport to polysulfide. The Δpsr deletion mutant that lacks polysulfide reductase (Psr) grew by fumarate respiration with sulfide as an electron donor, indicating that Psr is not required for this activity. Received: 31 August 1995 / Accepted: 25 October 1995     

Sulfite oxidation by the quinone-reducing molybdenum sulfite dehydrogenase SoeABC from the bacterium Aquifex aeolicus

The microaerophilic bacterium Aquifex aeolicus is a chemolitoautotroph that uses sulfur compounds as electron sources. The model of oxidation of the energetic sulfur compounds in this bacterium predicts that sulfite would probably be a metabolic intermediate released in the cytoplasm. In this work, we purified and characterized a membrane-bound sulfite dehydrogenase, identified as an SoeABC enzyme, that was previously described as a sulfur reductase. It is a member of the DMSO-reductase family of molybdenum enzymes. This type of enzyme was identified a few years ago but never purified, and biochemical data and kinetic properties were completely lacking. An enzyme catalyzing sulfite oxidation using Nitro-blue tetrazolium as artificial electron acceptor was extracted from the membrane fraction of Aquifex aeolicus. The purified enzyme is a dimer of trimer (αβγ)₂ of about 390 kDa. The K_M for sulfite and k_cat values were 34 μM and 567 s⁻¹ respectively, at pH 8.3 and 55 °C. We furthermore showed that SoeABC reduces a UQ₁₀ analogue, the decyl-ubiquinone, as well, with a K_M of 2.6 μM and a k_cat of 52.9 s⁻¹. It seems to specifically oxidize sulfite but can work in the reverse direction, reduction of sulfur or tetrathionate, using reduced methyl viologen as electron donor. The close phylogenetic relationship of Soe with sulfur and tetrathionate reductases that we established, perfectly explains this enzymatic ability, although its bidirectionality in vivo still needs to be clarified. Oxygen-consumption measurements confirmed that electrons generated by sulfite oxidation in the cytoplasm enter the respiratory chain at the level of quinones.     

DMSO respiration by the anaerobic rumen bacterium Wolinella succinogenes

The anaerobic rumen bacterium Wolinella succinogenes was able to grow by respiration with dimethylsulphoxide (DMSO) as electron acceptor and formate or H₂ as electron donors. The growth yield amounted to 6.7 g and 6.4 g dry cells/mol DMSO with formate or H₂ as the donors, respectively. This suggested an ATP yield of about 0.7 mol ATP/mol DMSO. Cell homogenates and the membrane fraction contained DMSO reductase activity with a high K _m (43 mM) for DMSO. The electron transport from H₂ to DMSO in the membranes was inhibited by 2-(heptyl)-4-hydroxyquinoline N-oxide, indicating the participation of menaquinone. Formation of DMSO reductase activity occurred only during growth on DMSO, presence of other electron acceptors (fumarate, nitrate, nitrite, N₂O, and sulphur) repressed the DMSO reductase activity. DMSO can therefore be used by W. succinogenes as an acceptor for phosphorylative electron transport, but other electron acceptors are used preferentially.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - DMS Dimethylsulphide (CH₃)₂S - DMSO Dimethylsulphoxide (CH₃)₂SO - HQNO 2-(Heptyl)-4-hydroxyquinoline-N-oxide - TMAO Trimethylamine-N-oxide - Y _s Growth yield for substrate S     

Oxidation of H2, organic compounds and inorganic sulfur compounds coupled to reduction of O2 or nitrate by sulfate-reducing bacteria

All of fourteen sulfate-reducing bacteria tested were able to carry out aerobic respiration with at least one of the following electron donors: H₂, lactate, pyruvate, formate, acetate, butyrate, ethanol, sulfide, thiosulfate, sulfite. Generally, we did not obtain growth with O₂ as electron acceptor. The bacteria were microaerophilic, since the respiration rates increased with decreasing O₂ concentrations or ceased after repeated O₂ additions. The amounts of O₂ consumed indicated that the organic substrates were oxidized incompletely to acetate; only Desulfobacter postgatei oxidized acetate with O₂ completely to CO₂. Many of the strains oxidized sulfite (completely to sulfate) or sulfide (incompletely, except Desulfobulbus propionicus); thiosulfate was oxidized only by strains of Desulfovibrio desulfuricans; trithionate and tetrathionate were not oxidized by any of the strains. With Desulfovibrio desulfuricans CSN and Desulfobulbus propionicus the oxidation of inorganic sulfur compounds was characterized in detail. D. desulfuricans formed sulfate during oxidation of sulfite, thiosulfate or elemental sulfur prepared from polysulfide. D. propionicus oxidized sulfite and sulfide to sulfate, and elemental sulfur mainly to thiosulfate. A novel pathway that couples the sulfur and nitrogen cycles was detected: D. desulfuricans and (only with nitrite) D. propionicus were able to completely oxidize sulfide coupled to the reduction of nitrate or nitrite to ammonia. Cell-free extracts of both strains did not oxidize sulfide or thiosulfate, but formed ATP during oxidation of sulfite (37 nmol per 100 nmol sulfite). This, and the effects of AMP, pyrophosphate and molybdate on sulfite oxidation, suggested that sulfate is formed via the (reversed) sulfate activation pathway (involving APS reductase and ATP sulfurylase). Thiosulfate oxidation with O₂ probably required a reductive first step, since it was obtained only with energized intact cells.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - APS adenosine phosphosulfate or adenylyl sulfate     

Ability of Selenomonas ruminantium,Veillonella parvula,and Wolinella succinogenes to Reduce Nitrate and Nitrite with Special Reference to the Suppression of Ruminal Methanogenesis

When mixed ruminal microbes were grown in a medium containing ground hay and concentrate, cell numbers of nitrate-reducing bacteria, Veillonella parvula and Wolinella succinogenes, drastically decreased as estimated by competitive PCR. However, decrease in their numbers was prevented by the addition of nitrate, suggesting that energy acquisition by electron transport phosphorylation (ETP) coupled with nitrate and/or nitrite reduction is important for the survival of these bacteria in the rumen. On the other hand, the number of Selenomonas ruminantium increased, and addition of nitrate did not affect the number, suggesting that the numbers of nitrate-reducing strains of S. ruminantium are low. A nitrate-reducing strain of S. ruminantium subsp. lactilytica was found to have the ETP system coupled with both nitrate and nitrite reduction. V. parvula, W. succinogenes, S. ruminantium, and Streptococcus bovis were more tolerant to nitrite toxicity than other representative ruminal bacteria. Methanogens were particularly sensitive to nitrite. W. succinogenes reduced nitrate and nitrite at higher rates than V. parvula and the subsp.lactilytica , but growth of W. succinogenes on nitrate and H₂ was slower than the growth of V. parvula and the subsp. lactilytica. Methane production by unidentified methanogen mixture was markedly reduced by the coexistence of W. succinogenes, V. parvula or the subsp. lactilytica in the presence of nitrate and H₂. W. succinogenes was shown to be most effective to augment nitrate and nitrite reduction, and to reduce methanogenesis.     

Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria

Recent phylogenetic analyses have established that the Epsilonproteobacteria form a globally ubiquitous group of ecologically significant organisms that comprises a diverse range of free-living bacteria as well as host-associated organisms like Wolinella succinogenes and pathogenic Campylobacter and Helicobacter species. Many Epsilonproteobacteria reduce nitrate and nitrite and perform either respiratory nitrate ammonification or denitrification. The inventory of epsilonproteobacterial genomes from 21 different species was analysed with respect to key enzymes involved in respiratory nitrogen metabolism. Most ammonifying Epsilonproteobacteria employ two enzymic electron transport systems named Nap (periplasmic nitrate reductase) and Nrf (periplasmic cytochrome c nitrite reductase). The current knowledge on the architecture and function of the corresponding proton motive force-generating respiratory chains using low-potential electron donors are reviewed in this article and the role of membrane-bound quinone/quinol-reactive proteins (NapH and NrfH) that are representative of widespread bacterial electron transport modules is highlighted. Notably, all Epsilonproteobacteria lack a napC gene in their nap gene clusters. Possible roles of the Nap and Nrf systems in anabolism and nitrosative stress defence are also discussed. Free-living denitrifying Epsilonproteobacteria lack the Nrf system but encode cytochrome cd₁ nitrite reductase, at least one nitric oxide reductase and a characteristic cytochrome c nitrous oxide reductase system (cNosZ). Interestingly, cNosZ is also found in some ammonifying Epsilonproteobacteria and enables nitrous oxide respiration in W. succinogenes.     

How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic

The 39‐kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a ‘Trojan horse’ antibiotic. The approximately 260‐residue C‐terminal region of MccB is homologous to ubiquitin‐like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB‐catalysed C‐terminal MccA‐acyl‐adenylation is reminiscent of the E1‐catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C‐terminal di‐glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C‐terminal Asn. Furthermore, after an intramolecular rearrangement of MccA‐acyl‐adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate‐linked analogue of acyl‐adenylated aspartic acid. We report six‐crystal structures of MccB in apo, substrate‐, intermediate‐, and inhibitor‐bound forms. Structural and kinetic analyses reveal a novel‐peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic‐active site for catalysing dual adenosine triphosphate‐consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two‐step activation for generating the peptidyl‐antibiotic MccC7.     

Phorphorylative electron transport chains lacking a cytochromebc 1 complex

Electron transport-coupled phosphorylation with fumarate as terminal acceptor inWolinella succinogenes yields less than 1 ATP/2 electrons. The generated by the electron transport is 0.18V and the H⁺/electron ratio is 1. The electron transport chain is made up of two dehydrogenases (hydrogenase and formate dehydrogenase) that catalyze the reduction of menaquinone, and fumarate reductase which catalyzes the oxidation of menaquinol.C-type cytochromes are not involved. The phosphorylative electron transport with sulfur as terminal acceptor inW. succinogenes orDesulfuromonas acetoxidans does not involve known quinones. The ATP yields should be even smaller than those with fumarate. Succinate oxidation by sulfur, which is a catabolic reaction inD. acetoxidans, is accomplished by reversed electron transport.     

A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a ΔfccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The ΔfccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor. Received: 26 September 1997 / Accepted: 8 December     

Inhibition of colicin synthesis by the antibiotic globomycin

Wolinella succinogenes can grow by anaerobic respiration with fumarate or polysulfide as the terminal electron acceptor, and H₂ or formate as the electron donor. A ΔhydABC mutant lacking the hydrogenase structural genes did not grow with H₂ and either fumarate or polysulfide. In contrast to the wild-type strain, the mutant grown with fumarate and with formate instead of H₂ did not catalyze the reduction of fumarate, polysulfide, dimethylnaphthoquinone, or benzyl viologen by H₂. Growth and enzymic activities were restored upon integration of a plasmid carrying hydABC into the genome of the ΔhydABC mutant. The ΔhydABC mutant was complemented with hydABC operons modified by artificial stop codons in hydA (StopA) or at the 5′-end of hydC (StopC). The StopC mutant lacked HydC, and the hydrophobic C-terminus of HydA was missing in the hydrogenase of the StopA mutant. The two mutants catalyzed benzyl viologen reduction by H₂. The enzyme activity was located in the membrane of the mutants. A mutant with both modifications (StopAC) contained the activity in the periplasm. The three mutants did not grow with H₂ and either fumarate or polysulfide, and did not catalyze dimethylnaphthoquinone reduction by H₂. We conclude that the same hydrogenase serves in the anaerobic respiration with fumarate and with polysulfide. HydC and the C-terminus of HydA appear to be required for both routes of electron transport and for dimethylnaphthoquinone reduction by H₂. The hydrogenase is anchored in the membrane by HydC and by the C-terminus of HydA. The catalytic subunit HydB is oriented towards the periplasmic side of the membrane. Received: 29 December 1997 / Accepted: 6 March 1998     

Isolation of the sulphur reductase and reconstitution of the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes can grow at the expense of sulphur reduction by formate. The enzymes involved in the catalysis of this catabolic reaction have been investigated. From the results the following conclusions are drawn: 1. The enzyme isolated as a sulphide dehydrogenase from the cytoplasmic membrane of W. succinogenes is the functional sulphur reductase that operates in the electron transport from formate to sulphur. 2. The enzyme (M_r 200,000) consists essentially of one type of subunit with the M_r 85,000 and contains equal amounts of free iron and sulphide (120 mol/g protein), but no heme. It represents the first functional sulphur reductase ever isolated. 3. The electron transport chain catalyzing sulphur reduction by formate consists merely of formate dehydrogenase and sulphur reductase. A lipophilic quinone which mediates the transfer of electrons between enzymes in other chains, is apparently not involved. This is the first known example of a phosphorylative electron transport chain that operates without a quinone. 4. The same formate dehydrogenase appears to operate in the electron transport both with sulphur and with fumarate as the terminal electron acceptor in W. succinogenes.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DTT dithiothreitol - MK menaquinone (vitamin K₂) - PMSF phenylmethane sulfonylfluoride - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine - Tea triethanolamine - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonate Dedicated to Professor F. Schneider (Philipps-Universität Marburg) on the occasion of his 60th birthday     

Genomic Differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, Identified by Suppression Subtractive Hybridization

Fibrobacter is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in Fibrobacter succinogenes S85 but absent from F. intestinalis DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to F. intestinalis were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the F. succinogenes genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of F. succinogenes genomic DNA, were identified that lack similarity to those of F. intestinalis chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to F. succinogenes. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in F. succinogenes S85 was either missing from or not functional in F. intestinalis DR7, which explains the requirement of vitamin B₁₂ for the growth of the F. intestinalis species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by Fibrobacter genera appear to have an important role in energy metabolism.     

16S rRNA analysis and the phylogenetic position of Wolinella succinogenes

The 16S ribosomal RNA of Wolinella succinogenes ATCC29543 was analyzed by the RNase T1 oligonucleotide cataloguing approach. In contrast to its present classification, W. succinogenes is related neither to members of the genus Bacteroides nor to any other genus of the family Bacteroidaceae. As derived from the similarity coefficients (S_AB values) calculated on the basis of more than 350 eubacterial species, W. succinogenes appears to be a distantly related member of the division of purple photosynthetic bacteria and their relatives; however, S_AB values do not indicate that this species is preferentially related to any representative of the 4 subdivisions.