RESUMEN

Artificial climbing walls represent a unique indoor environment in which humans interact closely with a variety of surface types. Climbing wall holds may mediate transmission of organisms between individuals, and yet there are no studies that identify microorganisms present on these surfaces. In the current study, the microorganisms found on climbing wall holds were characterized by analysis of amplified SSU rRNA gene sequences. In contrast to many other studies of built environments, the majority of microorganisms on holds were most closely related to microbes annotated as being recovered from environmental sources, such as soil, with human skin also representing an important source. Regional patterns were evident as rRNA gene sequences from the marine cyanobacterium Prochlorococcus were abundant in gyms found within 16 km of the ocean. Enterobacteriaceae were present on 100 % of holds surveyed, and the members detected are commonly associated with fecal matter.

Asunto(s)

Bacterias/clasificación , Bacterias/genética , Biota , Microbiología Ambiental , Análisis por Conglomerados , ADN Ribosómico/química , ADN Ribosómico/genética , Humanos , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Deportes

RESUMEN

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.

Asunto(s)

Chloroflexi/clasificación , Chloroflexi/enzimología , Oxidorreductasas/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteómica , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Chloroflexi/crecimiento & desarrollo , Cromatografía Liquida , Medios de Cultivo , Formiato Deshidrogenasas/química , Formiato Deshidrogenasas/metabolismo , Datos de Secuencia Molecular , Oxidorreductasas/química , Filogenia , Especificidad de la Especie , Relación Estructura-Actividad , Espectrometría de Masas en Tándem

RESUMEN

A combination of microcosm studies, polymerase chain reaction (PCR) analysis, and site data was used to assess the indigenous reductive dechlorinating potential in a trichloroethene (TCE)-contaminated aquifer at Cape Canaveral Air Station, Florida. Sediment and groundwater were obtained from two distinct locations approximately 10 m apart. Microcosm studies were performed to assess dechlorinating activity under a variety of nutrient and electron donor amendment conditions. Most live microcosms constructed using material from the first location, near well 9 (W09), were negative for dechlorination. All live microcosms constructed using material from the second location (W06) exhibited dechlorination of TCE to vinyl chloride (VC) and ethene (ETH). DNA encoding 16S ribosomal RNA (rDNA) with a sequence nearly identical with that from Dehalococcoides ethenogenes strain 195 was detected in the active microcosms and in the sediment from W06 with polymerase chain reaction (PCR) using primers targeted to unique regions of Dehalococcoides 16S rDNA. Dehalococcoides was not detected in the autoclaved microcosms from W06, nor in sediment and most microcosms from W09. The results of the microcosm studies and PCR analysis were supported by field data, which indicated significant accumulation of cis-1,2-dichloroethene (cisDCE) and VC at W06, but not at W09. The different microcosm results obtained for the two locations and the spatial variation of positive PCR results indicates heterogeneous distribution of dechlorinating activity and a specific dechlorinating organism, Dehalococcoides, at the site. As both Dehalococcoides and dechlorination activity were similarly, heterogeneously distributed, this suggests that molecular-probing (which could and should be extended in the future to include virtually all known dechlorinators and/or dehalogenases) can provide a relatively quick and facile method for investigating spatial distributions of dechlorinators on-site.

Asunto(s)

Tricloroetileno/metabolismo , Contaminantes Químicos del Agua/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Secuencia de Bases , Biodegradación Ambiental , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , Ecosistema , Monitoreo del Ambiente , Florida , Sedimentos Geológicos/microbiología , Oxidación-Reducción , Filogenia , Reacción en Cadena de la Polimerasa , Microbiología del Agua

RESUMEN

cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination. "Dehalococcoides ethenogenes" strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE. Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization. To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp. culture. The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory. Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform. Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE. VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination. However, high concentrations of PCE or TCE inhibited VC dechlorination. Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC. At contaminated sites in which "D. ethenogenes" or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination.

Asunto(s)

Bacterias Anaerobias/fisiología , Dicloruros de Etileno/metabolismo , Contaminantes del Suelo/metabolismo , Cloruro de Vinilo/metabolismo , Compuestos de Cloro/química , Cloroformo/química , Oxidación-Reducción , Microbiología del Suelo

RESUMEN

Methanosarcina barkeri 227 possesses two clusters of genes potentially encoding nitrogenases. We have previously demonstrated that one cluster, called nif2, is expressed under molybdenum (Mo)-sufficient conditions, and the deduced amino acid sequences for nitrogenase structural genes in that cluster most closely resemble those for the Mo nitrogenase of the gram-positive eubacterium Clostridium pasteurianum. The previously cloned nifH1 from M. barkeri shows phylogenetic relationships with genes encoding components of eubacterial Mo-independent eubacterial alternative nitrogenases and other methanogen nitrogenases. In this study, we cloned and sequenced nifD1 and part of nifK1 from M. barkeri 227. The deduced amino acid sequence encoded by nifD1 from M. barkeri showed great similarity with vnfD gene products from vanadium (V) nitrogenases, with an 80% identity at the amino acid level with the vnfD gene product from Anabaena variabilis. Moreover, there was a small open reading frame located between nifD1 and nifK1 with clear homology to vnfG, a hallmark of eubacterial alternative nitrogenases. Stimulation of diazotrophic growth of M. barkeri 227 by V in the absence of Mo was demonstrated. The unusual complement of nif genes in M. barkeri 227, with one cluster resembling that from a gram-positive eubacterium and the other resembling a eubacterial V nitrogenase gene cluster, suggests horizontal genetic transfer of those genes.

Asunto(s)

Proteínas Bacterianas , Genes Arqueales , Methanosarcina barkeri/genética , Fijación del Nitrógeno/genética , Nitrogenasa/genética , Vanadio , Methanosarcina barkeri/enzimología , Datos de Secuencia Molecular , Molibdeno , Familia de Multigenes , Nitrogenasa/clasificación , Nitrogenasa/efectos de los fármacos , Oxidorreductasas/genética , Filogenia , Compuestos de Tungsteno/farmacología

RESUMEN

"Dehalococcoides ethenogenes" 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.

Asunto(s)

Bacterias Anaerobias/metabolismo , Dicloruros de Etileno/metabolismo , Hidrocarburos Clorados/metabolismo , Bacterias Anaerobias/crecimiento & desarrollo , Biodegradación Ambiental , Medios de Cultivo , Oxidación-Reducción , Tetracloroetileno/metabolismo , Tricloroetileno/metabolismo , Cloruro de Vinilo/metabolismo

RESUMEN

Methane-oxidizing activity in natural samples is typically measured by amending 14CH4 to the sample and then following the accumulation of 14CO2. Current biological techniques to synthesize 14CH4 yield significant quantities of 14CO that when oxidized to 14CO2 would artificially inflate the measured methane-oxidizing activity of a sample. We present here a new method to biologically produce highly-pure 14CH4 using Methanothrix sp. Strain CALS-1 which produces very little CO. Using this method, 14CH4 was produced at nearly 100% efficiency and at a high specific activity (2.2 GBq.mmol-1) equal to the parent compound, [2-14C] sodium acetate. Furthermore, only trace quantities of H2 and CO were produced with only one molecule of CO produced for every 17,000 molecules of CH4. When compared to the standard CH4 generation method, this technique produced 97% purer CH4.

Asunto(s)

Metano/metabolismo , Methanosarcinaceae/metabolismo , Radioisótopos de Carbono , Methanosarcinaceae/crecimiento & desarrollo , Acetato de Sodio/metabolismo

RESUMEN

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.

Asunto(s)

Clostridium/clasificación , Escherichia coli/clasificación , Taninos Hidrolizables/metabolismo , Filogenia , ARN Ribosómico 16S/genética , Rumen/microbiología , Streptococcus/clasificación , Yersinia/clasificación , Animales , Clostridium/genética , Clostridium/aislamiento & purificación , Colombia , ADN Ribosómico/genética , Ciervos , Erwinia/clasificación , Erwinia/genética , Erwinia/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Eubacterium/clasificación , Eubacterium/genética , Eubacterium/aislamiento & purificación , Cabras , Honduras , Taninos Hidrolizables/farmacología , New York , Oregon , Peptostreptococcus/clasificación , Peptostreptococcus/genética , Peptostreptococcus/aislamiento & purificación , Fenotipo , Ovinos , Streptococcus/genética , Streptococcus/aislamiento & purificación , Yersinia/genética , Yersinia/aislamiento & purificación

RESUMEN

Transcription initiation in Archaea (archaebacteria) resembles the eucaryotic process, having been shown to involve TATA box-like promoter regions as well as TATA-binding protein and TFIIB homologs. However, little is known about transcription regulation in archaea. We have previously demonstrated that transcripts of nifHDK2 genes, encoding Methanosarcina barkeri nitrogenase, are present in N2-grown cells but not in ammonium-grown cells, indicating that nif transcription is regulated by the nitrogen source. In this study, we detected proteins in M. barkeri cell extracts that bind specifically to DNA containing the putative promoter region of nifHDK2. No binding was found when the promoter region was deleted from the DNA. A competition assay showed that the methyl coenzyme M reductase (mcr) promoter region DNA and the nifH2 promoter region DNA competed for a common factor(s). There was no binding to the nifH2 promoter region by extracts of ammonium-grown cells, but there was binding by these extracts to promoter regions for mcr genes, which are presumably constitutively expressed. Interestingly, extracts of ammonium-grown cells inhibited binding to the nif promoter region by extracts of N2-grown cells. Fractionation of extracts of ammonium-grown cells with a heparin-Sepharose column resolved them into a fraction eluting at 0 M NaCl, which inhibited binding by extracts of N2-grown cells, and a fraction eluting at 0.5 to 0.75 M NaCl, which showed binding to the promoter region. These results are congruent with a model for regulation of nif gene expression in M. barkeri in which a substance present in ammonium-grown cells inhibits DNA binding by a transcription-associated protein or proteins.

Asunto(s)

Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Arqueales/genética , Methanosarcina barkeri/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Methanosarcina barkeri/enzimología , Datos de Secuencia Molecular , Nitrogenasa/genética

RESUMEN

Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium (III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 micromol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE, cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 micromol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.

Asunto(s)

Bacterias Gramnegativas/enzimología , Hidrolasas/metabolismo , Oxidorreductasas/metabolismo , Contaminantes Ambientales/metabolismo , Hidrolasas/química , Hidrolasas/aislamiento & purificación , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Tetracloroetileno/metabolismo , Tricloroetileno/metabolismo

RESUMEN

Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene. Strain 195, a coccoid bacterium that dechlorinates tetrachloroethene to ethene, was isolated and characterized. Growth of strain 195 with H2 and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures. Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea. Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups.

Asunto(s)

Carcinógenos/metabolismo , Etilenos/metabolismo , Bacterias Gramnegativas/metabolismo , Tetracloroetileno/metabolismo , Biodegradación Ambiental , Medios de Cultivo , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Gramnegativas/ultraestructura , Filogenia , ARN Ribosómico 16S/clasificación , Tricloroetileno/metabolismo , Contaminantes Químicos del Agua/metabolismo

RESUMEN

Determination of the nucleotide sequence of the nitrogenase structural genes (nifHDK2) from Methanosarcina barkeri 227 was completed in this study by cloning and sequencing a 2.7-kb BamHI fragment containing the 3' end of nifK2 and 1,390 bp of the nifE2-homologous genes. Open reading frame nifK2 is 1,371 bp long including the stop codon TAA and encodes a polypeptide of 456 amino acids. Phylogenetic analysis of the deduced amino acid sequences of the nifK2 and nifE2 gene products from M. barkeri showed that both genes cluster most closely with the corresponding nif-1 gene products from Clostridium pasteurianum, consistent with our previous analyses of nifH2 and nifD2. The nifE gene product is known to be homologous to that of nifD, and our analysis shows that the branching pattern for the nifE proteins resembles that for the nifD product (with the exception of vnfE from Azotobacter vinelandii), suggesting that a gene duplication occurred before the divergence of nitrogenases. Primer extension showed that nifH2 had a single transcription start site located 34 nucleotides upstream of the ATG translation start site for nifH2, and a sequence resembling the archaeal consensus promoter sequence [TTTA(A/T)ATA] was found 32 nucleotides upstream from that transcription start site. A tract of four T's, previously identified as a transcription termination site in archaea, was found immediately downstream of the nifK2 gene, and a potential promoter was located upstream of the nifE2 gene. Hybridization with nifH2 and nifDK2 probes with M. barkeri RNA revealed a 4.6-kb transcript from N2-grown cells, large enough to harbor nifHDK genes and their internal open reading frames, while no transcript was detected from NH4(+)-grown cells. These results support a model in which the nitrogenase structural genes in M. barkeri are cotranscribed in a single NH4(+)-repressed operon.

Asunto(s)

Genes Bacterianos/genética , Methanosarcina barkeri/genética , Nitrogenasa/genética , Oxidorreductasas , Filogenia , Secuencia de Bases , Clonación Molecular , Methanosarcina barkeri/enzimología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN Bacteriano/genética , ARN Mensajero/genética , Análisis de Secuencia de ADN , Transcripción Genética/genética

RESUMEN

We have been studying an anaerobic enrichment culture which, by using methanol as an electron donor, dechlorinates tetrachloroethene (PCE) to vinyl chloride and ethene. Our previous results indicated that H2 was the direct electron donor for rductive dechlorination of PCE by the methanol-PCE culture. Most-probable-number counts performed on this culture indicated low numbers (< or equal to 10(4)/ml)) of methanogens and PCE dechlorinators using methanol and high numbers (> or equal to 10(6)/ml)) of sulfidogens, methanol-utilizing acetogens, fermentative heterotrophs, and PCE dechlorinators using H2. An anaerobic H2-PCE enrichment culture was derived from a 10(-6) dilution of the methanol-PCE culture. This H2-PCE culture used PCE at increasing rates over time when transferred to fresh medium and could be transferred indefinitely with H2 as the electron donor for the PCE dechlorination, indicating that H2-PCE can serve as an electron donor-acceptor pair for energy conservation and growth. Sustained PCE dechlorination by this culture was supported by supplementation with 0.05 mg of vitamin B12 per liter, 25% (vol/vol) anaerobic digestor sludge supernatant, and 2 mM acetate, which presumably served as a carbon source. Neither methanol nor acetate could serve as an electron donor for dechlorination by the H2-PCE culture, and it did not produce CH4 or acetate from H2-CO2 or methanol, indicating the absence of methanogenic and acetogenic bacteria. Microscopic observatios of the pruified H2-PCE culture showed only two major morphotypes: irregular cocci and small rods.

Asunto(s)

Bacterias Anaerobias/metabolismo , Tetracloroetileno/metabolismo , Acetatos/metabolismo , Ácido Acético , Biodegradación Ambiental , Contaminantes Ambientales/metabolismo , Etilenos/metabolismo , Hidrógeno/metabolismo , Metanol/metabolismo , Solventes/metabolismo , Cloruro de Vinilo/metabolismo

RESUMEN

Tetrachloroethene (PCE) and other chloroethenes are major contaminants in groundwater, and PCE is particularly resistant to attack by aerobes. We have developed an anaerobic enrichment culture that carries out reductive dechlorination of chloroethenes to ethene at high rates, thereby detoxifying them. Although the electron donor added to the culture is methanol, our evidence indicates that H2 is the electron donor used directly for dechlorination. We have recently obtained a culture from 10(-6) dilution of the original methanol/PCE culture that uses H2 as an electron donor for PCE dechlorination. Because the culture can be transferred indefinitely and the rate of PCE dechlorination increases after inoculation, we suggest that dechlorinating organisms in the culture use the carbon-chlorine bonds in chloroethenes as electron acceptors for energy conservation.

Asunto(s)

Bacterias Anaerobias/metabolismo , Cloro/metabolismo , Tetracloroetileno/metabolismo , Biodegradación Ambiental , Catálisis , Tetracloroetileno/química

RESUMEN

L. Sibold, M. Henriquet, O. Possot, and J.-P. Aubert (Res. Microbiol. 142:5-12, 1991) cloned and sequenced two nifH-homologous open reading frames (ORFs) from Methanosarcina barkeri 227. Phylogenetic analysis of the deduced amino acid sequences of the nifH ORFs from M. barkeri showed that nifH1 clusters with nifH genes from alternative nitrogenases, while nifH2 clusters with nifH1 from the gram-positive eubacterium Clostridium pasteurianum. The N-terminal sequence of the purified nitrogenase component 2 (the nifH gene product) from M. barkeri was identical with that predicted for nifH2, and dot blot analysis of RNA transcripts indicated that nifH2 (and nifDK2) was expressed in M. barkeri when grown diazotrophically in Mo-containing medium. To obtain nifD2 from M. barkeri, a 4.7-kbp BamHI fragment of M. barkeri DNA was cloned which contained at least five ORFs, including nifH2, ORF105, and ORF125 (previously described by Sibold et al.), as well as nifD2 and part of nifK2. ORFnifD2 is 1,596 bp long and encodes 532 amino acid residues, while the nifK2 fragment is 135 bp long. The deduced amino acid sequences for nifD2 and the nifK2 fragment from M. barkeri cluster most closely with the corresponding nifDK1 gene products from C. pasteurianum. The predicted M. barkeri nifD2 product contains a 50-amino acid insert near the C terminus which has previously been found only in the clostridial nifD1 product. Previous biochemical and sequencing evidence indicates that the C. pasteurianum nitrogenase is the most divergent of known eubacterial Mo-nitrogenases, most likely representing a distinct nif gene family, which now also contains M. barkeri as a member. The similarity between the methanogen and clostridial nif sequences is especially intriguing in light of the recent findings of sequence similarities between gene products from archaea and from low-G+C gram-positive eubacteria for glutamate dehydrogenase, glutamine synthetase I, and heat shock protein 70. It is not clear whether this similarity is due to horizontal gene transfer or to the resemblance of the M. barkeri and C. pasteurianum nitrogenase sequences to an ancestral nitrogenase.

Asunto(s)

Genes Bacterianos/genética , Methanosarcina barkeri/genética , Nitrogenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Clostridium/genética , Medios de Cultivo , Methanosarcina barkeri/crecimiento & desarrollo , Datos de Secuencia Molecular , Molibdeno/metabolismo , Fijación del Nitrógeno/genética , Nitrogenasa/aislamiento & purificación , Sistemas de Lectura Abierta/genética , Filogenia , ARN Mensajero/genética , Análisis de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.

Asunto(s)

Bacterias/metabolismo , Hidrógeno/metabolismo , Tetracloroetileno/metabolismo , Adaptación Biológica , Anaerobiosis , Bacterias/efectos de los fármacos , Biodegradación Ambiental , Etilenos/biosíntesis , Euryarchaeota/metabolismo , Metanol/metabolismo , Oxidación-Reducción , Vancomicina/farmacología , Cloruro de Vinilo/metabolismo

RESUMEN

CO and H(2) have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism of H(2), CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H(2) to partial pressures of 40 to 70 Pa (1 Pa = 0.987 x 10 atm), as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H(2) to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa (some CO came from the rubber stopper) during the middle of methanogenesis; this was followed by a decrease in CO partial pressures to less than 0.01 Pa by the end of methanogenesis. Accumulation or consumption of CO by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N(2)-CO(2), accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial pressures of H(2) (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrix sp. strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp. strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp. strain CALS-1 were 9.1, 0.045, and 5.8 mumol of viologen reduced min mg of protein. These results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H(2) in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate.

RESUMEN

Tetrachloroethene, also known as perchloroethylene (PCE), is a common groundwater contaminant throughout the United States. The incomplete reductive dechlorination of PCE--resulting in accumulations of trichloroethene, dichloroethene isomers, and/or vinyl chloride--has been observed by many investigators in a wide variety of methanogenic environments. Previous mixed-culture studies have demonstrated that complete dechlorination to ethene is possible, although the final dechlorination step from vinyl chloride to ethene is rate limiting, with significant levels of vinyl chloride typically persisting. In this study, anaerobic methanol-PCE enrichment cultures which proved capable of dechlorinating high concentrations PCE to ethene were developed. Added concentrations of PCE as high as 550 microM (91-mg/liter nominal concentration; approximately 55-mg/liter actual aqueous concentration) were routinely dechlorinated to 80% ethene and 20% vinyl chloride within 2 days at 35 degrees C. The methanol level used was approximately twice that needed for complete dechlorination of PCE to ethene. The observed transformations occurred in the absence of methanogenesis, which was apparently inhibited by the high concentrations of PCE. When incubation was allowed to proceed for as long as 4 days, virtually complete conversion of PCE to ethene resulted, with less than 1% persisting as vinyl chloride. An electron balance demonstrated that methanol consumption was completely accounted for by dechlorination (31%) and acetate production (69%). The high volumetric rates of PCE dechlorination (up to 275 mumol/liter/day) and the relatively large fraction (ca. one-third) of the supplied electron donor used for dechlorination suggest that reductive dechlorination could be exploited for bioremediation of PCE-contaminated sites.

Asunto(s)

Cloro/metabolismo , Etilenos/metabolismo , Metano/metabolismo , Tetracloroetileno/metabolismo , Anaerobiosis , Electrones , Hongos/metabolismo , Oxidación-Reducción , Transformación Bacteriana , Cloruro de Vinilo/metabolismo

RESUMEN

The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20- to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. The component 1 analog had a molecular size of approximately 250 kDa, as estimated by gel filtration, and sodium dodecyl sulfate-polyacrylamide gels revealed two predominant bands with molecular sizes near 57 and 62 kDa, consistent with an alpha 2 beta 2 tetramer as in eubacterial component 1 proteins. For the component 2 analog, a molecular size of approximately 120 kDa was estimated by gel filtration, with a subunit molecular size near 31 kDa, indicating that the component 2 protein is a tetramer, in contrast to eubacterial component 2 proteins, which are dimers. Rates of C2H2 reduction by the nearly pure subunits were 1,000 nmol h-1 mg of protein-1, considerably lower than those for conventional Mo nitrogenases but similar to that of the non-Mo non-V nitrogenase from Azotobacter vinelandii. Strain 227 nitrogenase reduced N2 at a higher rate per electron than it reduced C2H2, also resembling the non-Mo non-V nitrogenase of A. vinelandii. Ethane was not produced from C2H2. NH4+ concentrations as low as 10 microM caused a transient inhibition of C2H2 reduction by strain 227 cells. Antiserum against component 2 Rhodospirillum rubrum nitrogenase was found to cross-react with component 2 from strain 227, and Western immunoblots using this antiserum showed no evidence for covalent modification of component 2. Also, extracts of strain 227 cells prepared before and after switch-off had virtually the same level of nitrogenase activity. In conclusion, the nitrogenase from strain 227 is similar in overall structure to the eubacterial nitrogenases and shows greatest similarity to alternative nitrogenases.

Asunto(s)

Euryarchaeota/enzimología , Nitrogenasa/metabolismo , Acetileno/metabolismo , Amoníaco/metabolismo , Western Blotting , Etilenos/metabolismo , Euryarchaeota/metabolismo , Sustancias Macromoleculares , Peso Molecular , Nitrógeno/metabolismo , Fijación del Nitrógeno , Nitrogenasa/química , Nitrogenasa/aislamiento & purificación , Oxidación-Reducción