A flavodiiron protein and high molecular weight rubredoxin from Moorella thermoacetica with nitric oxide reductase activity

A five-gene "oxidative stress protection" cluster has recently been described from the strictly anaerobic, acetogenic bacterium, Moorella thermoacetica [Das, A., et al. (2001) J. Bacteriol. 183, 1560-1567]. Within this cluster are two cotranscribed genes, fprA (for A-type flavoprotein) and hrb (for high molecular weight rubredoxin) whose encoded proteins have no known functions. Here we show that FprA and Hrb are expressed in M. thermoacetica under normal anaerobic growth conditions and report characterizations of the recombinant FprA and Hrb. FprA contains flavin mononucleotide (FMN) and a non-heme diiron site. M?ssbauer spectroscopy shows that the irons of the diferric site are antiferromagnetically coupled, implying a single-atom, presumably solvent, bridge between the irons. Hrb contains FMN and a rubredoxin-like [Fe(SCys)4] site. NADH does not directly reduce either the FMN or the diiron site in FprA, whereas Hrb functions as an efficient NADH:FprA oxidoreductase. Substitution of zinc for iron in Hrb completely abolished this activity. The observation that homologues of FprA from other organisms show O2 and/or anaerobic NO consumption activity prompted an examination of these activities for M. thermoacetica FprA. The Hrb/FprA combination does indeed have both NADH:O2 and NADH:NO oxidoreductase activities. The NO reductase activity, however, was significantly more efficient due to a lower Km for NO (4 M) and to progressive and irreversible inactivation of FprA during O2 reductase turnover but retention of activity during NO reductase turnover. Substitution of zinc for iron in FprA completely abolished these reductase activities. The stoichiometry of 1 mol of NADH oxidized:2 mol of NO consumed implies reduction to N2O. Fits of an appropriate rate law to the kinetics data are consistent with a mechanism in which 2NO's react at each FprA active site in the committed step. Expression of FprA in an Escherichia coli strain deficient in NO reductase restored the anaerobic growth phenotype of cultures exposed to otherwise toxic levels of exogenous NO. The accumulated results indicate that Hrb/FprA is fully capable of functioning in nitrosative stress protection in M. thermoacetica.     

Susceptibility to Heavy Metals and Cadmium Accumulation in Aerobic and Anaerobic Thermophilic Microorganisms Isolated from Deep-Sea Hydrothermal Vents

Thirty thermophilic strains isolated from heavy metal-rich hydrothermal vent sites at Lau Basin were tested for their susceptibility to cadmium, zinc, cobalt, and nickel. The 14 aerobic spore formers belonging to the genus Bacillus, 6 anaerobic fermenters from the order Thermotogales, and 10 anaerobic sulfur reducers from the order Thermococcales could be clearly distinguished according to their metal susceptibilities. The Thermococcales were found to exhibit the highest resistance to cadmium and zinc, whereas Thermotogales were highly sensitive to these metals. In contrast, the Thermotogales displayed the highest resistance to cobalt ions. No clear distinction could be established between the metal susceptibilities of these strains and seven reference organisms used for comparative studies. Cadmium resistance, slightly inducible in some cadmium-resistant bacilli, was not plasmid mediated. The amount of cadmium immobilized by the Thermotogales was related to their level of resistance to this metal. Received: 24 February 2000 / Accepted: 28 March 2000     

Acquisition of a bacterial RumA-type tRNA(uracil-54, C5)-methyltransferase by Archaea through an ancient horizontal gene transfer

The Pyrococcus abyssi genome displays two genes possibly coding for S-adenosyl-l-methionine-dependent RNA(uracil, C5)-methyltransferases (PAB0719 and PAB0760). Their amino acid sequences are more closely related to Escherichia coli RumA catalysing the formation of 5-methyluridine (m(5)U)-1939 in 23S rRNA than to E. coli TrmA (tRNA methyltransferase A) methylating uridine-54 in tRNA. Comparative genomic and phylogenetic analyses show that homologues of PAB0719 and PAB0760 occur only in a few Archaea, these genes having been acquired via a single horizontal gene transfer from a bacterial donor to the common ancestor of Thermococcales and Nanoarchaea. This transfer event was followed by a duplication event in Thermococcales leading to two closely related genes. None of the gene products of the two P. abyssi paralogues catalyses in vitro the formation of m(5)U in a P. abyssi rRNA fragment homologous to the bacterial RumA substrate. Instead, PAB0719 enzyme (renamed (Pab)TrmU54) displays an identical specificity to TrmA, as it catalyses the in vitro formation of m(5)U-54 in tRNA. Thus, during evolution, at least one of the two P. abyssi RumA-type enzymes has changed of target specificity. This functional shift probably occurred in an ancestor of all Thermococcales. This study also provides new evidence in favour of a close relationship between Thermococcales and Nanoarchaea.     

Genome sequence of Thermotoga sp. strain RQ2, a hyperthermophilic bacterium isolated from a geothermally heated region of the seafloor near Ribeira Quente, the Azores

Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.     

Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales

From a hot spring of the Azores islands a novel thermophilic bacterium belonging to the Thermotogales order was isolated. This strain, which grows optimally at 70(deg)C and pH 6.5, is the first known extreme thermophile that is able to degrade native feathers at high temperatures. The enzyme system converts feather meal to amino acids and peptides. On the basis of physiological, morphological, and 16S rDNA studies the new isolate was found to be a member of the Thermotogales order and was identified as Fervidobacterium pennavorans. The strain was highly related to Fervidobacterium islandicum and Fervidobacterium pullulanolyticum. The cell-bound keratinolytic enzyme system was purified 32-fold by detergent treatment with CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was characterized as a serine protease with a molecular mass of 130 kDa and an isoelectric point of 3.8. Optimal activity was measured at 80(deg)C and pH 10.0. Furthermore, 19 anaerobic thermophilic archaea and bacteria belonging to the orders Thermococcales, Thermoproteales, Thermotogales, and Clostridiales (growth temperatures between 60 and 105(deg)C) were tested for their abilities to grow on feathers and produce heat-stable keratinolytic enzymes. None of the tested extremophilic microorganisms was able to attack the substrate in a native form.     

Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum

Two flavo-diiron proteins (FDPs), FprA1 and FprA2, are up-regulated when the strictly anaerobic solvent producer, Clostridium acetobutylicum, is exposed to dioxygen. These two FDPs were purified following heterologous overexpression in Escherichia coli as N-terminal Strep-tag fusion proteins. The recombinant FprA1 and FprA2 were found to be homodimeric and homotetrameric, respectively, and both FDPs functioned as terminal components of NADH oxidases (NADH:O₂ oxidoreductases) when using C. acetobutylicum NADH:rubredoxin oxidoreductase (NROR) and rubredoxin (Rd) as electron transport intermediaries. Both FDPs catalyzed the four-electron reduction of molecular oxygen to water with similar specific activities. The results are consistent with these FDPs functioning as efficient scavengers of intracellular dioxygen under aerobic or microoxic growth conditions.     

A flavo-diiron protein from Desulfovibrio vulgaris with oxidase and nitric oxide reductase activities. Evidence for an in vivo nitric oxide scavenging function

A few members of a widespread class of bacterial and archaeal flavo-diiron proteins, dubbed FprAs, have been shown to function as either oxidases (dioxygen reductases) or scavenging nitric oxide reductases, but the questions of which of these functions dominates in vivo for a given FprA and whether all FprAs function as oxidases or nitric oxide reductases remain to be clarified. To address these questions, an FprA has been characterized from the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris. The gene encoding this D. vulgaris FprA lies downstream of an operon encoding superoxide reductase and rubredoxin, consistent with an O(2)-scavenging oxidase function for this FprA. The recombinant D. vulgaris FprA can indeed serve as the terminal component of an NADH oxidase. However, this oxidase turnover results in irreversible inactivation of the enzyme. On the other hand, the recombinant D. vulgaris FprA shows robust anaerobic nitric oxide reductase activity in vitro and also protects a nitric oxide-sensitive Escherichia coli strain against exposure to exogenous nitric oxide. It is, therefore, proposed that this D. vulgaris FprA functions as a scavenging nitric oxide reductase in vivo and that this activity protects D. vulgaris against anaerobic exposure to nitric oxide. The location of a gene encoding a second FprA homologue in the D. vulgaris genome also suggests its involvement in nitrogen oxide metabolism.     

Distribution of folates and modified folates in extremely thermophilic bacteria.

Analyses were made of the structures and levels of folates and modified folates present in extremely thermophilic bacteria. These procedures involved the chemical analysis of products resulting from the oxidative cleavage of the 6-substituted, folatelike tetrahydropterins present in the cells. Air-oxidized cell extracts of extreme thermophiles from two members of the archaebacterial order Thermococcales, Thermococcus celer and Pyrococcus furiosus, contained only 7-methylpterin, indicating that these cells contain a modified folate with a methylated pterin. Cell extracts also contained 6-acetyl-7-methyl-7,8-dihydropterin, another product derived from the oxidative cleavage of a dimethylated folate, demonstrating that both the C-7 and C-9 carbons of the pterin were methylated. Extracts, however, contained neither p-aminobenzoylpolyglutamates nor methaniline, the oxidative cleavage products of folates and methanopterin, respectively, indicating that they contain a previously undescribed C1 carrier(s). On the basis of the level of the 7-methylpterin isolated, the levels of modified folate were 2 to 10 times higher than those typically found in mesophilic bacteria and 10 to 100 times less than the level of methanopterin found in the methanogenic bacteria. Oxidized cell extracts of Sulfolobus spp. of the archaebacterial order Sulfolobales contained only pterin, and, like members of the order Thermococcales, they contained neither-p-aminobenzoylpolyglutamates nor methaniline. Oxidized cell extracts of the extreme thermophiles Pyrobaculum sp. strain H10 and Pyrodictium occultum, from the archaebacterial orders Thermoproteales and Pyrodictiales, respectively, and Thermotoga maritima from the eubacterial order Thermotogales, contained pterin and p-aminobenzoylpolyglutamates, indicating that these cells contained unmodified folates. The levels of p-aminobenzoylpolyglutamates in these archaebacterial cell extracts indicate that the folates were present in the cells at levels 4 to 10 times higher than generally found in those mesophilic eubacteria which do not folates in energy metabolism. The levels and chain lengths of the of p-aminobenzoylpolyglutamates present in Thermotoga maritima were typical of those found in mesophilic eubacteria.     

Chromosome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences

In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.     

Thiosulfate Reduction, an Important Physiological Feature Shared by Members of the Order Thermotogales

Several members of the order Thermotogales in the domain Bacteria, viz., Thermotoga neapolitana, Thermotoga maritima, Thermosipho africanus, Fervidobacterium islandicum, and Thermotoga strain SEBR 2665, an isolate from an oil well, reduced thiosulfate to sulfide. This reductive process enhanced cellular yields and growth rates of all the members but was more significant with the two hyperthermophiles T. neapolitana and T. maritima. This is the first report of such an occurrence in this group of thermophilic and hyperthermophilic anaerobic bacteria. The results suggest that thiosulfate reduction is important in the geochemical cycling of sulfur in anaerobic thermal environments such as the slightly acidic and neutral-pH volcanic hot springs and oil reservoirs.     

Genes for the major structural components of Thermotogales species' togas revealed by proteomic and evolutionary analyses of OmpA and OmpB homologs

The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath.     

O2 and Reactive Oxygen Species Detoxification Complex, Composed of O2-Responsive NADH:Rubredoxin Oxidoreductase-Flavoprotein A2-Desulfoferrodoxin Operon Enzymes, Rubperoxin, and Rubredoxin, in Clostridium acetobutylicum

Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O₂-flow culture conditions, where the cells consume O₂ proficiently. An O₂-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacterized flavoprotein A2 (FprA2), and the predicted superoxide reductase desulfoferrodoxin (Dsr), has been proposed to participate in defense against O₂ stress. To functionally characterize these proteins, native NROR from C. acetobutylicum, recombinant NROR (rNROR), FprA2, Dsr, and rubredoxin (Rd) expressed in Escherichia coli were purified. Purified native NROR and rNROR both exhibited weak H₂O₂-forming NADH oxidase activity that was slightly activated by Rd. A mixture of NROR, Rd, and FprA2 functions as an efficient H₂O-forming NADH oxidase with a high affinity for O₂ (the K_m for O₂ is 2.9 ± 0.4 μM). A mixture of NROR, Rd, and Dsr functions as an NADH-dependent O₂⁻ reductase. A mixture of NROR, Rd, and rubperoxin (Rpr, a rubrerythrin homologue) functions as an inefficient H₂O-forming NADH oxidase but an efficient NADH peroxidase with a low affinity for O₂ and a high affinity for H₂O₂ (the K_ms for O₂ and H₂O₂ are 303 ± 39 μM and ≤1 μM, respectively). A gene encoding Rd is dicistronically transcribed with a gene encoding a glutaredoxin (Gd) homologue, and the expression levels of the genes encoding Gd and Rd were highly upregulated upon exposure to O₂. Therefore, nror operon enzymes, together with Rpr, efficiently function to scavenge O₂, O₂⁻, and H₂O₂ by using an O₂-responsive rubredoxin as a common electron carrier protein.     

Crystal structure of rubredoxin from Desulfovibrio gigas to ultra-high 0.68 A resolution

Rubredoxin (D.g. Rd) is a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas. The protein is generally purified from anaerobic bacteria in which it is thought to be involved in electron transfer or exchange processes. Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin:oxygen oxidoreductase (ROO) in D.g. The crystal structure of D.g. Rd has been determined by means of both a Fe single-wavelength anomalous dispersion (SAD) signal and the direct method, and refined to an ultra-high 0.68 A resolution, using X-ray from a synchrotron. Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. Hydrophobic and pi-pi interactions maintain the internal Rd folding. Multiple conformations of the iron-sulfur cluster and amino acid residues are observed and indicate its unique mechanism of electron transfer. Several hydrogen bonds, including N-H...SG of the iron-sulfur, are revealed clearly in maps of electron density. Abundant waters bound to C-O peptides of residues Val8, Cys9, Gly10, Ala38, and Gly43, which may be involved in electron transfer. This ultrahigh-resolution structure allows us to study in great detail the relationship between structure and function of rubredoxin, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron-sulfur cluster, and distributions of electron density among activity sites. For the first time, this information will provide a clear role for this protein in a strict anaerobic bacterium.     

Acquired tetracycline and/or macrolide-lincosamides-streptogramin resistance in anaerobes

In general bacterial antibiotic resistance is acquired on mobile elements such as plasmids, transposons and/or conjugative transposons. This is also true for many antibiotic resistant anaerobic species described in the literature. Of the 23 different tetracycline resistant efflux genes identified, tet(B), tet(K), tet(L), and tetA(P) have been found in anaerobic species and six of the ten tetracycline resistant genes coding for ribosomal protection proteins, tet(M), tet(O), tetB(P), tet(Q), tet(W), and tet(32), have been identified in anaerobes. There are now three enzymes which inactivate tetracycline, of which the tet(X) has been identified in Bacteroides though is not functional under anaerobic growth conditions. A similar situation exists with the genes conferring macrolide-lincosamide-streptogramin (MLS) resistance. Of the 26 rRNA methylase MLS resistant genes characterized, five genes; erm(B), erm(C), erm(F), erm(G), and erm(Q), have been identified in anaerobes. In contrast, no genes coding for MLS resistant efflux proteins or inactivating enzymes have been described in anaerobic species. This mini-review will summarize what is known about tetracycline and MLS resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes.     

Phylogenetic analyses of two "archaeal" genes in thermotoga maritima reveal multiple transfers between archaea and bacteria

The genome sequence of Thermotoga maritima revealed that 24% of its open reading frames (ORFs) showed the highest similarity scores to archaeal genes in BLAST analyses. Here we screened 16 strains from the genus Thermotoga and other related Thermotogales for the occurrence of two of these "archaeal" genes: the gene encoding the large subunit of glutamate synthase (gltB) and the myo-inositol 1P synthase gene (ino1). Both genes were restricted to the Thermotoga species within the Thermotogales. The distribution of the two genes, along with results from phylogenetic analyses, showed that they were acquired from Archaea during the divergence of the Thermotogales. Database searches revealed that three other bacteria-Dehalococcoides ethenogenes, Sinorhizobium meliloti, and Clostridium difficile-possess archaeal-type gltBs, and the phylogenetic analyses confirmed at least two lateral gene transfer (LGT) events between Bacteria and Archaea. These LGT events were also strongly supported by gene structure data, as the three domains in bacterial-type gltB are homologous to three independent ORFs in Archaea and Bacteria with archaeal-type gltBs. The ino1 gene has a scattered distribution among Bacteria, and apart from the Thermotoga strains it is found only in Aquifex aeolicus, D. ethenogenes, and some high-G+C Gram-positive bacteria. Phylogenetic analysis of the ino1 sequences revealed three highly supported prokaryotic clades, all containing a mixture of archaeal and bacterial sequences, and suggested that all bacterial ino1 genes had been recruited from archaeal donors. The Thermotoga strains and A. aeolicus acquired this gene independently from different archaeal species. Although transfer of genes from hyperthermophilic Archaea may have facilitated the evolution of bacterial hyperthermophily, between-domain transfers also affect mesophilic species. For hyperthermophiles, we hypothesize that LGT may be as much a consequence as the cause of adaptation to hyperthermophily.     

Gene transfer and genome plasticity in Thermotoga maritima, a model hyperthermophilic species

The genome sequence of the hyperthermophilic bacterium Thermotoga maritima MSB8 presents evidence for lateral gene transfer events between bacterial and archaeal species. To estimate the extent of genomic diversity across the order Thermotogales, a comparative genomic hybridization study was initiated to compare nine Thermotoga strains to the sequenced T. maritima MSB8. Many differences could be associated with substrate utilization patterns, which are most likely a reflection of the environmental niche that these individual species occupy. A detailed analysis of some of the predicted variable regions demonstrates many examples of the deletion/insertion of complete cassettes of genes and of gene rearrangements and insertions of DNA within genes, with the C or N terminus being retained. Although the mechanism for gene transfer in this lineage remains to be elucidated, this analysis suggests possible associations with repetitive elements and highlights the possible benefits of rampant genetic exchange to these species.     

Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica

The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.     

Evidence for existence of "mesotogas," members of the order Thermotogales adapted to low-temperature environments

All cultivated isolates of the bacterial order Thermotogales are either thermophiles or hyperthermophiles, but Thermotogales 16S rRNA gene sequences have been detected in many mesophilic anaerobic and microaerophilic environments, particularly within communities involved in the remediation of pollutants. Here we provide metagenomic evidence for the existence of Thermotogales lineages, which we informally call "mesotoga," that are adapted to growth at lower temperatures. Two fosmid clones containing mesotoga DNA, originating from a low-temperature enrichment culture that degrades a polychlorinated biphenyl congener, were sequenced. Phylogenetic analysis clearly puts this bacterial lineage within the Thermotogales order, with the rRNA gene trees and 21 of 58 open reading frames strongly supporting this relationship. An analysis of protein sequence composition showed that mesotoga proteins are adapted to function at lower temperatures than are their identifiable homologs from thermophilic and hyperthermophilic members of the order Thermotogales, supporting the notion that this bacterium lives and grows optimally at lower temperatures. The phylogenetic analysis suggests that the mesotoga lineage from which our fosmids derive has used both the acquisition of genes from its neighbors and the modification of existing thermophilic sequences to adapt to a mesophilic lifestyle.     

A new member of the endonuclease III family of DNA repair enzymes that removes methylated purines from DNA.

DNA is constantly exposed to endogenous andexogenous alkylating agents that can modify its bases,resulting in mutagenesis in the absence of DNA repair [1,2]. Alkylation damage is removed by the action of DNA glycosylases, which initiate the base excision repair pathway and protect the sequence information of the genome [3-5]. We have identified a new class of methylpurine DNA glycosylase, designated MpgII, that is a member of the endonuclease III family of DNA repair enzymes. We expressed and purified MpgII from Thermotoga maritima and found that the enzyme releases both 7-methylguanine and 3-methyladenine from DNA. We cloned the MpgII genes from T. maritima and from Aquifex aeolicus and found that both genes could restore methylmethanesulfonate (MMS) resistance to Escherichia coli alkA tagA double mutants, which are deficient in the repair of alkylated bases. Analogous genes are found in other Bacteria and Archaea and appear to be the only genes coding for methylpurine DNA glycosylase activity in these organisms. MpgII is the fifth member of the endonuclease III family of DNA repair enzymes, suggesting that the endonuclease III protein scaffold has been modified during evolution to recognize and repair a variety of DNA damage.