Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.     

Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium.

Dissimilatory arsenate-reducing bacteria have been implicated in the mobilization of arsenic from arsenic-enriched sediments. An As(V)-reducing bacterium, designated strain GBFH, was isolated from arsenic-contaminated sediments of Lake Coeur d'Alene, Idaho. Strain GBFH couples the oxidation of formate to the reduction of As(V) when formate is supplied as the sole carbon source and electron donor. Additionally, strain GBFH is capable of reducing As(V), Fe(III), Se(VI), Mn(IV) and a variety of oxidized sulfur species. 16S ribosomal DNA sequence comparisons reveal that strain GBFH is closely related to Desulfitobacterium hafniense DCB-2(T) and Desulfitobacterium frappieri PCP-1(T). Comparative physiology demonstrates that D. hafniense and D. frappieri, known for reductively dechlorinating chlorophenols, are also capable of toxic metal or metalloid respiration. DNA-DNA hybridization and comparative physiological studies suggest that D. hafniense, D. frappieri, and strain GBFH should be united into one species. The isolation of an Fe(III)- and As(V)-reducing bacterium from Lake Coeur d'Alene suggests a mechanism for arsenic mobilization in these contaminated sediments while the discovery of metal or metalloid respiration in the genus Desulfitobacterium has implications for environments cocontaminated with arsenious and chlorophenolic compounds.     

Energy yield of respiration on chloroaromatic compounds in Desulfitobacterium dehalogenans

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with 13C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO2. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.     

Desulfacinum subterraneum sp.nov.--a new thermophilic sulfate-reducing bacterium isolated from a high temperature oil field

A new thermophilic sulfate-reducing bacterium isolated from the high-temperature White Tiger oil field (Vietnam) is described. Cells of the bacterium are oval (0.4-0.6 by 0.6-1.8 microns), nonmotile, non-spore-forming, and gram-negative. Growth occurs at 45 to 65 degrees C (with an optimum at 60 degrees C) at NaCl concentrations of 0 to 50 g/l. In the course of sulfate reduction, the organism can utilize lactate, pyruvate, malate, fumarate, ethanol, salts of fatty acids (formate, acetate, propionate, butyrate, caproate, palmitate), yeast extract, alanine, serine, cysteine, and H2 + CO2 (autotrophically). In addition to sulfate, the bacterium can use sulfite, thiosulfate, and elemental sulfur as electron acceptors. In the absence of electron acceptors, the bacterium can ferment pyruvate and yeast extract (a yet unrecognized capacity of sulfate reducers) with the formation of acetate and H2. The G + C content of DNA is 60.8 mol %. The level of DNA-DNA hybridization of the isolate (strain 101T) and Desulfacinum infernum (strain B alpha G1T) is as low as 34%. Analysis of the nucleotide sequence of 16S rDNA places strain 101T in the phylogenetic cluster of the Desulfacinum species within the sulfate reducer subdivision of the delta subclass of Proteobacteria. All these results allowed the bacterium studied to be described as a new species, Desulfacinum subterraneum sp. nov., with strain 101 as the type strain.     

Energy Yield of Respiration on Chloroaromatic Compounds in Desulfitobacterium dehalogenans

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with ¹³C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO₂. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.     

Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate.

Strain Co23, an anaerobic spore-forming microorganism, was enriched and isolated from a compost soil on the basis of its ability to grow with 2,3-dichlorophenol (DCP) as its electron acceptor, ortho chlorines were removed from polysubstituted phenols but not from monohalophenols. Growth by chlororespiration was indicated by a growth yield of 3.24 g of cells per mol of reducing equivalents (as 2[H]) from lactate oxidation to acetate in the presence of 3-chloro-4-hydroxybenzoate but no growth in the absence of the halogenated electron acceptor. Other indicators of chlororespiration were the fraction of electrons from the electron donor used for dechlorination (0.67) and the H2 threshold concentration of < 1.0 ppm. Additional electron donors utilized for reductive dehalogenation were pyruvate, formate, butyrate, crotonate, and H2. Pyruvate supported homoacetogenic growth in the absence of an electron acceptor. Strain Co23 also used sulfite, thiosulfate, and sulfur as electron acceptors for growth, but it did not use sulfate, nitrate or fumarate. The temperature optimum for growth was 37 degrees C; however, the rates of dechlorination were optimum at 45 degrees C and activity persisted to temperatures as high as 55 degrees C. The 16S rRNA sequence was determined, and strain Co23 was found to be related to Desulfitobacterium dehalogenans JW/IU DC1 and Desulfitobacterium strain PCE1, with sequence similarities of 97.2 and 96.8%, respectively. The phylogenetic and physiological properties exhibited by strain Co23 place it into a new species designated Desulfitobacterium chlororespirans.     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6 could grow at a NaCl concentration in the medium of 30-230 g/l, with an optimum at 80-100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H2 + CO2 were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H2 + CO2 or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37 degrees C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the subclass Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.     

An aerobic formate-utilizing bacterium,Cupriavidus sp., isolated from activated sludge of wastewater treatment

An aerobic formate-assimilating bacterium, denoted as strain FAB, was newly isolated from activated sludge of wastewater treatment. Phylogenetic analysis based on 16S rDNA sequence assigned the isolate to the genus Cupriavidus. Scanning electron micrography revealed that this bacterium has a coccal morphology, and from some physiological assays, the bacterium was characterized to be Gram-negative, nitrate-reduction-positive and catalase-positive. In addition to formate, strain FAB was able to utilize fructose, acetate or pyruvate as a preferred carbon source. Compared with a close relative, Cupriavidus necator, our isolate exhibited a greater growth rate on formate under an aerobic condition.     

Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulfate and tetrachloroethene

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis -dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.     

Desulfacinum subterraneumsp. nov., a New Thermophilic Sulfate-Reducing Bacterium Isolated from a High-Temperature Oil Field

A new thermophilic sulfate-reducing bacterium isolated from the high-temperature White Tiger oil field (Vietnam) is described. Cells of the bacterium are oval (0.4–0.6 by 0.6–1.8 m), nonmotile, non-spore-forming, and gram-negative. Growth occurs at 45 to 65°C (with an optimum at 60°C) at NaCl concentrations of 0 to 50 g/l. In the course of sulfate reduction, the organism can utilize lactate, pyruvate, malate, fumarate, ethanol, salts of fatty acids (formate, acetate, propionate, butyrate, caproate, palmitate), yeast extract, alanine, serine, cysteine, and H₂+ CO₂(autotrophically). In addition to sulfate, the bacterium can use sulfite, thiosulfate, and elemental sulfur as electron acceptors. In the absence of electron acceptors, the bacterium can ferment pyruvate and yeast extract (a yet unrecognized capacity of sulfate reducers) with the formation of acetate and H₂. The G+C content of DNA is 60.8 mol %. The level of DNA–DNA hybridization of the isolate (strain 101^T) and Desulfacinum infernum(strain BG1^T) is as low as 34%. Analysis of the nucleotide sequence of 16S rDNA places strain 101^Tin the phylogenetic cluster of the Desulfacinumspecies within the sulfate reducer subdivision of the delta subclass of Proteobacteria. All these results allowed the bacterium studied to be described as a new species, Desulfacinum subterraneumsp. nov., with strain 101 as the type strain.     

Desulfitobacterium sp. strain PCE1, an anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene or ortho-chlorinated phenols

A strictly anaerobic bacterium, strain PCE1, was isolated from a tetrachloroethene-dechlorinating enrichment culture. Cells of the bacterium were motile curved rods, with approximately four lateral flagella. They possessed a gram-positive type of cell wall and contained cytochrome c. Optimum growth occurred at pH 7.2–7.8 and 34–38° C. The organism grew with l-lactate, pyruvate, butyrate, formate, succinate, or ethanol as electron donors, using either tetrachloroethene, 2-chlorophenol, 2,4,6-trichlorophenol, 3-chloro-4-hydroxy-phenylacetate, sulfite, thiosulfate, or fumarate as electron acceptors. Strain PCE1 also grew fermentatively with pyruvate as the sole substrate. l-Lactate and pyruvate were oxidized to acetate. Tetrachloroethene was reductively dechlorinated to trichloroethene and small amounts (< 5%) of cis-1,2-dichloroethene and trans-1,2-dichloroethene. Chlorinated phenolic compounds were dechlorinated specifically at the ortho-position. On the basis of 16S rRNA sequence analysis, the organism was identified as a species within the genus Desulfitobacterium, which until now only contained the chlorophenol-dechlorinating bacterium, Desulfitobacterium dehalogenans. Received: 31 August 1995 / Accepted: 14 November 1995     

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A

An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6, could grow at a NaCl concentration in the medium of 30–230 g/l, with an optimum at 80–100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H₂/CO₂ were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H₂/CO₂ or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37°C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the class Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6^T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.     

Complete oxidation of catechol by the strictly anaerobic sulfate-reducing Desulfobacterium catecholicum sp. nov.

From an anaerobic enrichment culture with vanillate as substrate, a catechol-degrading lemon-shaped nonsporing sulfate-reducing bacterium, strain NZva20, was isolated in pure culture. Growth occurred in defined, bicarbonate-buffered, sulfide-reduced freshwater medium with catechol as sole electron donor and carbon source. Catechol was completely oxidized to CO₂ with an average growth yield of 31 g cell dry mass per mol of catechol, corresponding to 9.5 g cell dry mass per mol of sulfate reduced. Further substrates utilized as electron donors and carbon sources were resorcinol, hydroquinone, benzoate and several other aromatic compounds, hydrogen plus carbon dioxide, formate, lactate, pyruvate, alcohols including methanol, dicarboxylic acids, acetate, propionate and higher fatty acids up to 18 carbon atoms. Instead of sulfate, sulfite, thiosulfate, dithionite or nitrate served as electron acceptors. Nitrate was reduced to ammonium. Strain NZva20 is the first bacterium in which the complete oxidation of organic substrates is linked to the ammonification of nitrate. Elemental sulfur was not utilized as electron acceptor. In the absence of an electron acceptor slow growth occurred on pyruvate or fumarate. The G+C content of the DNA of strain NZva20 was 52.4 mol%. Cytochromes were present. Desulfoviridin could not be detected. Strain NZva20 is described as type strain of a new species, Desulfobacterium catecholicum sp. nov.Affectionately dedicated to Professor Ralph S. Wolfe on the occassion of his 65th birthday     

Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.     

Halonatronum saccharophilum gen. nov. sp. nov--a new haloalkalophilic bacteria from the order Haloanaerobiales from Lake Magadi

A new alkaliphilic and moderately halophilic chemoorganotrophic anaerobic bacterium (strain Z-7986), which is spore-forming, rod-shaped, and has a gram-negative cell wall pattern, was isolated from the coastal lagoon mud of the highly mineralized Lake Magadi (Kenya). The organism is an obligatorily carbonate- and sodium chloride-dependent. It is a motile peritrichously flagellated rod that has developed within 3-17% NaCl concentration (with an optimum at 7-12% NaCl) and within a pH range of 7.7-10.3 (with an optimum at pH values of 8-8.5). It is a moderate thermophile with a broad temperature optimum from 36-55 degrees C and a growth maximum at 60 degrees C. The bacterium catabolizes glucose, fructose, sucrose, maltose, starch, glycogen, N-acetyl-D-glucosamine, and, to a slight degree, peptone and yeast extract. Its anabolism requires yeast extract or casamino acids. Glucose fermentation yields formate, acetate, ethanol, H2, and CO2. The bacterium is sulfidetolerant and capable of the nonspecific reduction of S0 to H2S. The G + C content of the DNA is 34.4 mol %. The analysis of the 16S rRNA sequence revealed that strain Z-7986 belongs to the order Haloanaerobiales and represents a new genus in the family Halobacteroidaceae. We suggest calling the organism Halonatronum saccharophilum gen. nov. sp. nov. The type strain of this species is Z-7986T (= DSM13868, = Uniqem 211).     

Characterization of Desulfomicrobium escambium sp. nov. and proposal to assign Desulfovibrio desulfuricans strain Norway 4 to the genus Desulfomicrobium

A sulfate-reducing bacterium, designated strain ESC1, was isolated and found to be a new species. Strain ESC1 is a strictly anaerobic, gram-negative, non-sporeforming, motile, short, round-ended rod often occurring in pairs. Of 31 fermentative substrates tested, only pyruvate was utilized. Sulfate enhanced growth with pyruvate and allowed growth with ethanol, lactate, formate and hydrogen. Both sulfate and thiosulfate were reduced. Lactate was incompletely oxidized to acetate and CO₂. The strain was desulfoviridin negative. The G+C content is 59.9%. These data suggested placement of strain ESC1 in the genus Desulfomicrobium. Comparative 16S rRNA analysis showed that strain ESC1 shares 98% rRNA sequence similarity with Desulfomicrobium baculatum and Desulfovibrio desulfuricans strain Norway 4. The latter two strains shared greater than 99% 16S rRNA sequence similarity. Strain ESC1 has been designated as the new species Desulfomicrobium escambium. We also recommend that D. desulfuricans strain Norway 4 be considered for reclassification as a Desulfomicrobium species.     

Use of Ferric and Ferrous Iron Containing Minerals for Respiration by Desulfitobacterium frappieri

The potential of Desulfitobacterium frappieri strain G2, which was isolated from subsurface smectite bedding, to participate in iron redox reactions was investigated. Strain G2 can use poorly crystalline Fe(III) oxide, soluble forms of Fe(III) and Fe(III) in the structure of phyllosilicate minerals as electron acceptors. It can also oxidize Fe(II)-NTA or Fe(II) in the structure of phyllosilicate minerals with nitrate as the electron acceptor. These results suggest for the first time that strains of Desulfitobacterium frappieri may play an important role in iron cycling in sedimentary environments.     

Isolation and characterization of <Emphasis Type="Italic">Sulfurospirillum carboxydovorans</Emphasis> sp. nov., a new microaerophilic carbon monoxide oxidizing epsilon Proteobacterium

A new microaerophilic, Gram-negative, motile, 2–3 m long and 0.3 m wide, vibrioid to spirillum-shaped, CO oxidizing bacterium, designated strain MV, isolated from marine sediment (The North Sea) is described. Strain MV was able to couple the oxidation of CO to the reduction of elemental sulphur, DMSO and thiosulphate. Growth occurred with up to 100% (v/v) CO in the headspace. Acetate was needed as carbon source. No growth on CO was observed with nitrate and selenate as electron acceptor. Sulphite, elemental sulphur, DMSO, thiosulphate, nitrate, nitrite, perchloroethylene, arsenate and selenate were used as electron acceptors with pyruvate as energy and carbon source. Microaerophilic growth was observed. In non-agitated cultures growth occurred at atmospheric oxygen concentrations in the headspace. Hydrogen (with acetate as carbon source), formate (with acetate as carbon source), pyruvate, lactate, succinate, fumarate, malate -ketoglutaric acid, aspartate and yeast extract (1% (w/v)) supported growth with nitrate as electron acceptor. Fumarate and malate were fermented. Vitamins were not required for growth. The strain was cytochrome C oxidase and catalase positive. The DNA mol G+C content was 30.5%. 16S rRNA gene sequence comparison showed that strain MV grouped within the genus Sulfurospirillum with Sulfurospirillum arcachonense (sequence similarity 98.3%) as closest relative. The relative DNA–DNA relatedness between strain MV and S. arcachonense was 33.1%. Based on a detailed phenotypic and phylogenetic analysis, inclusion of strain MV in the genus Sulfurospirillum as a well separated new species is proposed. As species name we propose Sulfurospirillum carboxydovorans. The type strain is strain MV (ATCC BAA-937 = DSM 16295, GenBank accession number: AY740528).     

Occurrence of several genes encoding putative reductive dehalogenases in Desulfitobacterium hafniense/frappieri and Dehalococcoides ethenogenes

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].