Search

In oxidative phosphorylation, respiratory complex I serves as an entry point in the electron transport chain for electrons generated in catabolic processes in the form of NADH. An ancestral version of the complex, lacking the NADH-oxidising module, is encoded in a significant number of bacterial genomes. Amongst them is Desulfitobacterium hafniense, a strict anaerobe capable of conserving energy via organohalide respiration. This study investigates the role of the complex I-like enzyme in D. hafniense energy metabolism using rotenone as a specific complex I inhibitor under different growth conditions. The investigation revealed that the complex I-like enzyme was essential for growth with lactate and pyruvate but not in conditions involving H2 as an electron donor. In addition, a previously published proteomic dataset of strain DCB-2 was analysed to reveal the predominance of the complex under different growth conditions and to identify potential redox partners. This approach revealed seven candidates with expression patterns similar to Nuo homologues, suggesting the use of diverse electron sources. Based on these results, we propose a model where the complex I-like enzyme serves as an electron entry point into the respiratory chain for substrates delivering electrons within the cytoplasm, such as lactate or pyruvate, with ferredoxins shuttling electrons to the complex. [ABSTRACT FROM AUTHOR]

In oxidative phosphorylation, respiratory complex I serves as an entry point in the electron transport chain for electrons generated in catabolic processes in the form of NADH. An ancestral version of the complex, lacking the NADH-oxidising module, is encoded in a significant number of bacterial genomes. Amongst them is Desulfitobacterium hafniense, a strict anaerobe capable of conserving energy via organohalide respiration. This study investigates the role of the complex I-like enzyme in D. hafniense energy metabolism using rotenone as a specific complex I inhibitor under different growth conditions. The investigation revealed that the complex I-like enzyme was essential for growth with lactate and pyruvate but not in conditions involving H2 as an electron donor. In addition, a previously published proteomic dataset of strain DCB-2 was analysed to reveal the predominance of the complex under different growth conditions and to identify potential redox partners. This approach revealed seven candidates with expression patterns similar to Nuo homologues, suggesting the use of diverse electron sources. Based on these results, we propose a model where the complex I-like enzyme serves as an electron entry point into the respiratory chain for substrates delivering electrons within the cytoplasm, such as lactate or pyruvate, with ferredoxins shuttling electrons to the complex.

The coupling of thermal remediation with microbial reductive dechlorination (MRD) has shown promising potential for the cleanup of chlorinated solvent contaminated sites. In this study, thermal treatment and bioaugmentation were applied in series, where prior higher thermal remediation temperature led to improved TCE dechlorination performance with both better organohalide-respiring bacteria (OHRB) colonization and electron donor availability. The 60 °C was found to be a key temperature point where the promotion effect became obvious. Amplicon sequencing and co-occurrence network analysis demonstrated that temperature was a more dominating factor than bioaugmentation that impacted microbial community structure. Higher temperature of prior thermal treatment resulted in the decrease of richness, diversity of indigenous microbial communities, and simplified the network structure, which benefited the build-up of newcoming microorganisms during bioaugmentation. Thus, the abundance of Desulfitobacterium increased from 0.11 % (25 °C) to 3.10 % (90 °C). Meanwhile, released volatile fatty acids (VFAs) during thermal remediation functioned as electron donors and boosted MRD. Our results provided temperature-specific information on synergistic effect of sequential thermal remediation and bioaugmentation, which contributed to better implementation of the coupled technologies in chloroethene-impacted sites. [Display omitted] • Microbial reductive dechlorination (MRD) benefited from prior thermal treatment. • Higher temperature promoted following bioaugmented MRD performance. • Desulfitobacterium abundance increased from 0.11 % (25 °C) to 3.10 % (90 °C). • Higher temperature facilitated positive interspecies interactions. • Indigenous released VFAs at 90 °C was 7.70 times of that at 75 °C after heating. [ABSTRACT FROM AUTHOR]

In acid drainage environments, biosulfidogenesis by sulfate-reducing bacteria (SRB) attenuates the extreme conditions by enabling the precipitation of metals as their sulfides, and the neutralization of acidity through proton consumption. So far, only a handful of moderately acidophilic SRB species have been described, most of which are merely acidotolerant. Here, a novel species within a novel genus of moderately acidophilic SRB is described, Acididesulfobacillus acetoxydans gen. nov. sp. nov. strain INE, able to grow at pH 3.8. Bioreactor studies with strain INE at optimum (5.0) and low (3.9) pH for growth showed that strain INE alkalinized its environment, and that this was more pronounced at lower pH. These studies also showed the capacity of strain INE to completely oxidize organic acids to CO2, which is uncommon among acidophilic SRB. Since organic acids are mainly in their protonated form at low pH, which increases their toxicity, their complete oxidation may be an acid stress resistance mechanism. Comparative proteogenomic and membrane lipid analysis further indicated that the presence of saturated ether-bound lipids in the membrane, and their relative increase at lower pH, was a protection mechanism against acid stress. Interestingly, other canonical acid stress resistance mechanisms, such as a Donnan potential and increased active charge transport, did not appear to be active. [ABSTRACT FROM AUTHOR]

In acid drainage environments, biosulfidogenesis by sulfate-reducing bacteria (SRB) attenuates the extreme conditions by enabling the precipitation of metals as their sulfides, and the neutralization of acidity through proton consumption. So far, only a handful of moderately acidophilic SRB species have been described, most of which are merely acidotolerant. Here, a novel species within a novel genus of moderately acidophilic SRB is described, Acididesulfobacillus acetoxydans gen. nov. sp. nov. strain INE, able to grow at pH 3.8. Bioreactor studies with strain INE at optimum (5.0) and low (3.9) pH for growth showed that strain INE alkalinized its environment, and that this was more pronounced at lower pH. These studies also showed the capacity of strain INE to completely oxidize organic acids to CO2, which is uncommon among acidophilic SRB. Since organic acids are mainly in their protonated form at low pH, which increases their toxicity, their complete oxidation may be an acid stress resistance mechanism. Comparative proteogenomic and membrane lipid analysis further indicated that the presence of saturated ether-bound lipids in the membrane, and their relative increase at lower pH, was a protection mechanism against acid stress. Interestingly, other canonical acid stress resistance mechanisms, such as a Donnan potential and increased active charge transport, did not appear to be active.

This study demonstrated the potential for reductive dechlorination of aryl halides in a sedimentary environment by naturally occurring bacteria. A laboratory sediment microcosm and highly enriched, stable dechlorinating cultures were established using hydrogen as electron donor and 2,3-dichlorophenol (2,3DCP), monochlorophenol (MCP) and dichlorobenzene (DCB) as electron acceptors. 16S rRNA gene sequencing of dominant DGGE bands assigned detected phylotypes in chlorophenols (CPs) amended sediment microcosms to the genera Anaerospora, Pseudomonas, Desulfitobacterium, Clostridium, Mycobacterium, and Peptoclostridium, beside uncultured marine Bacterioplankton, Halochromatium and Sedimentibacter. Chlorobenzene (CB) amended sediments showed the same community in addition to Bacillus sp. Major operational taxonomic units (OTUs) in CPs enrichment cultures were assigned to Anaerospora hongkongensis, Pseudomonas stutzeri, Pseudomonas pseudoalcaligenes, Clostridium sp., Desulfitobacterium dichloroeliminans, beside unidentified marine bacterioplankton. Enrichment on DCB resulted in predominance of the same populations in addition to Peptoclostridium sp. and Dehalococcoides mccartyi. Dechlorination in enrichment cultures was mainly assigned to Desulfitobacterium and Dehalococcoides, which are both known for their ability to couple dechlorination to growth in a halorespiration way. Enrichment cultures containing Desulfitobacterium showed ortho dechlorination activity of 2,3DCP to 2 chlorophenol (2CP) and 3 chlorophenol (3CP). These results provided compelling evidence that sedimentary environments in the vicinity of oil refineries do harbor dehalorespiring bacteria capable of reductive dechlorination of various aryl halides pollutants.

Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.

The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01 Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation.

The bioremediation of persistent organohalide molecules under anoxic conditions mostly relies on the bacterial process called organohalide respiration (OHR). Organohalide-respiring bacteria (OHRB) are phylogenetically diverse anaerobic bacteria that share the capacity to use organohalides as terminal electron acceptors in an energy-conserving process. The reductive dehalogenase (rdh) gene clusters encode for proteins specialized in the respiration of one or a limited number of organohalides. One particular OHRB may harbor up to several dozens of rdh gene clusters suggesting a wide potential for bioremediation. To avoid wasting energy in producing unnecessary proteins, rdh gene clusters often include a transcriptional regulator. In organohalide-respiring Firmicutes , RdhK is a dedicated transcriptional regulator of OHR and represents a subfamily of proteins among the CRP/FNR superfamily of regulators. RdhK proteins are composed of an effector-binding domain (EBD) which recognizes a given organohalide and subsequently controls the interaction of its C-terminal DNA-binding domain (DBD) with a DNA motif (referred to as dehalobox, or DB) located in the promoter region of the target rdh genes. The two binding partners (i.e. an organohalide molecule and a DB sequence) of RdhK proteins are interdependent which impairs the exploration of OHR regulatory networks. Here, we propose a strategy relying on hybrid proteins to efficiently screen the DNA target of a single RdhK protein without prior knowledge on its effector. To demonstrate the potential of the method, two hybrids with alternative fusion points were designed based on RdhK6 EBD and RdhK1 DBD from Desulfitobacterium hafniense. Electrophoretic mobility shift assay was performed with purified hybrids along with the parental proteins and their binding properties were further tested in vivo through a β-galactosidase reporter assay. Along with revealing new RdhK6 features, we show that both hybrids resulted in active regulatory proteins with distinct binding patterns. While Hybrid A was less specific for the DNA motif, Hybrid B successfully mimicked the binding behavior of the parental proteins and thus represents a promising template for the design of new RdhK hybrids to screen yet uncharacterized RdhK proteins and also possibly other members of the CRP/FNR superfamily. [ABSTRACT FROM AUTHOR]

Abstract The study was conducted to demonstrate the influence of extracellular secretions from Microbacterium on the reductive dechlorination of tetrachloroethene (PCE). A series of mixed cultures were established from a paddy soil sample. In the mixed cultures amended with extracellular secretions from Microbacterium , PCE was rapidly and completely converted into cis -1,2-dichloroethene (cis -DCE) and trans -1,2-dichloroethene (trans -DCE) within 40 days. The unamended mixed cultures showed weak signs of dechlorination after a pronounced lag phase, and trichloroethene (TCE) was accumulated as a major end product. This result means that amendment with extracellular secretions from Microbacterium shortened the lag phase, increased the dechlorination velocity and promoted the production of less-chlorinated chloroethene. The results were corroborated by defined subculture experiments, which proved that microorganisms from unamended mixed cultures could also be stimulated by extracellular secretions from Microbacterium. Desulfitobacterium was identified as the main dechlorinating population in all mixed cultures by direct PCR. Additionally, the 16S rRNA gene copies of Desulfitobacterium increased by one or two orders of magnitude with PCE dechlorination, which provided corroborative evidence for the identification result. The volatile fatty acids were monitored, and most interestingly, a close association between propionate oxidation and dechlorination was found, which has rarely been mentioned before. It was assumed that the oxidation of propionate provided hydrogen for dechlorination, while dechlorination facilitated the shift of the reaction toward propionate oxidation by reducing the partial pressure of hydrogen. Graphical abstract Image Highlights • PCE dechlorination was accelerated by extracellular secretions from Microbacterium. • Desulfitobacterium was identified to be the dechlorinator in the mixed cultures. • Propionate oxidization was associated with the PCE dechlorination in the cultures. The perchloroethylene dechlorination was accelerated by extracellular secretions from Microbacterium in a mixed culture containing Desulfitobacterium. [ABSTRACT FROM AUTHOR]

This study demonstrated the potential for reductive dechlorination of aryl halides in a sedimentary environment by naturally occurring bacteria. A laboratory sediment microcosm and highly enriched, stable dechlorinating cultures were established using hydrogen as electron donor and 2,3-dichlorophenol (2,3DCP), monochlorophenol (MCP) and dichlorobenzene (DCB) as electron acceptors. 16S rRNA gene sequencing of dominant DGGE bands assigned detected phylotypes in chlorophenols (CPs) amended sediment microcosms to the genera Anaerospora, Pseudomonas, Desulfitobacterium, Clostridium, Mycobacterium, and Peptoclostridium, beside uncultured marine Bacterioplankton, Halochromatium and Sedimentibacter. Chlorobenzene (CB) amended sediments showed the same community in addition to Bacillus sp. Major operational taxonomic units (OTUs) in CPs enrichment cultures were assigned to Anaerospora hongkongensis, Pseudomonas stutzeri, Pseudomonas pseudoalcaligenes, Clostridium sp., Desulfitobacterium dichloroeliminans, beside unidentified marine bacterioplankton. Enrichment on DCB resulted in predominance of the same populations in addition to Peptoclostridium sp. and Dehalococcoides mccartyi. Dechlorination in enrichment cultures was mainly assigned to Desulfitobacterium and Dehalococcoides, which are both known for their ability to couple dechlorination to growth in a halorespiration way. Enrichment cultures containing Desulfitobacterium showed ortho dechlorination activity of 2,3DCP to 2 chlorophenol (2CP) and 3 chlorophenol (3CP). These results provided compelling evidence that sedimentary environments in the vicinity of oil refineries do harbor dehalorespiring bacteria capable of reductive dechlorination of various aryl halides pollutants. [ABSTRACT FROM AUTHOR]

Trichothecenes are the most common mycotoxins contaminating small grain cereals worldwide. The C12,13 epoxide group in the trichothecenes was identified as a toxic group posing harm to humans, farm animals, and plants. Aerobic biological de-epoxidation is considered the ideal method of controlling these types of mycotoxins. In this study, we isolated a novel trichothecene mycotoxin-de-epoxidating bacterium, Desulfitobacterium sp. PGC-3-9, from a consortium obtained from the soil of a wheat field known for the occurrence of frequent Fusarium head blight epidemics under aerobic conditions. Along with MMYPF media, a combination of two antibiotics (sulfadiazine and trimethoprim) substantially increased the relative abundance of Desulfitobacterium species from 1.55% (aerobic) to 29.11% (aerobic) and 28.63% (anaerobic). A single colony purified strain, PGC-3-9, was isolated and a 16S rRNA sequencing analysis determined that it was Desulfitobacterium. The PGC-3-9 strain completely de-epoxidated HT-2, deoxynivalenol (DON), nivalenol and 15-acetyl deoxynivalenol, and efficiently eliminated DON in wheat grains under aerobic and anaerobic conditions. The strain PGC-3-9 exhibited high DON de-epoxidation activity at a wide range of pH (6–10) and temperature (15–50 °C) values under both conditions. This strain may be used for the development of detoxification agents in the agriculture and feed industries and the isolation of de-epoxidation enzymes.

Iron artifacts are common among the findings of archaeological excavations. The corrosion layer formed on these objects requires stabilization after their recovery, without which the destruction of the item due to physicochemical damage is likely. Current technologies for stabilizing the corrosion layer are lengthy and generate hazardous waste products. Therefore, there is a pressing need for an alternative method for stabilizing the corrosion layer on iron objects. The aim of this study was to evaluate an alternative conservation-restoration method using bacteria. For this, anaerobic iron reduction leading to the formation of stable iron minerals in the presence of chlorine was investigated for two strains of Desulfitobacterium hafniense (strains TCE1 and LBE). Iron reduction was observed for soluble Fe(III) phases as well as for akaganeite, the most troublesome iron compound in the corrosion layer of archaeological iron objects. In terms of biogenic mineral production, differential efficiencies were observed in assays performed on corroded iron coupons. Strain TCE1 produced a homogeneous layer of vivianite covering 80% of the corroded surface, while on the coupons treated with strain LBE, only 10% of the surface was covered by the same mineral. Finally, an attempt to reduce iron on archaeological objects was performed with strain TCE1, which led to the formation of both biogenic vivianite and magnetite on the surface of the artifacts. These results demonstrate the potential of this biological treatment for stabilizing archaeological iron as a promising alternative to traditional conservation-restoration methods. IMPORTANCE Since the Iron Age, iron has been a fundamental material for the building of objects used in everyday life. However, due to its reactivity, iron can be easily corroded, and the physical stability of the object built is at risk. This is particularly true for archaeological objects on which a potentially unstable corrosion layer is formed during the time the object is buried. After excavation, changes in environmental conditions (e.g., higher oxygen concentration or lower humidity) alter the stability of the corrosion layer and can lead to the total destruction of the object. In this study, we demonstrate the feasibility of an innovative treatment based on bacterial iron reduction and biogenic mineral formation to stabilize the corrosion layer and protect these objects. [ABSTRACT FROM AUTHOR]

Microbial growth coupled to O-demethylation of phenyl methyl ethers, which are lignin decomposition products, was described for acetogenic bacteria and recently also for two species belonging to the nonacetogenic genus Desulfitobacterium. To elucidate the potential role of desulfitobacteria in the O-demethylation of phenyl methyl ethers in the environment, we cultivated Desulfitobacterium chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense with phenyl methyl ethers as sole electron donors. With the exception of D. metallireducens, all species and strains tested were able to demethylate at least three of the four phenyl methyl ethers applied with fumarate, nitrate, or thiosulfate as electron acceptor. Furthermore, a high number of operons putatively encoding demethylase systems were identified in the genomes of Desulfitobacterium spp., although discrimination between O-, S-, N- and, Cl-demethylases was not possible. These findings provide evidence for the importance of the methylotrophic metabolism for desulfitobacteria and point to their involvement in the O-demethylation of phenyl methyl ethers in the environment. [ABSTRACT FROM AUTHOR]

We have developed a novel approach to identifying and quantifying closely related organohalide-respiring bacteria. Our approach made use of the unique genomic associations of specific reductive dehalogenase subunit A encoding genes ( rdhA) that exist in known strains of Dehalococcoides mccartyi and Desulfitobacterium and the distinguishing covariance pattern of observed rdhA genes to assign genes to unknown strains. To test this approach, we operated five anaerobic reductively dechlorinating chemostats for 3-4 years with tetrachloroethene and trichloroethene as terminal electron acceptors and lactate/formate as electron donors. The presence and abundance of rdhA genes were determined comprehensively at the community level using a custom-developed Reductive Dehalogenase Chip ( RDH Chip) DNA microarray and used to define putative strains of Dehalococcoides mccartyi and Desulfitobacterium sp. This monitoring revealed that stable chemical performance of chemostats was reflected by a stable community of reductively dechlorinating bacteria. However, perturbations introduced by, for example, electron donor limitation or addition of the competing electron acceptor sulfate led to overall changes in the chemostat performance, including incomplete reduction in the chloroethene substrates, and in the population composition of reductively dehalogenating bacteria. Interestingly, there was a high diversity of operationally defined D. mccartyi strains between the chemostats with almost all strains unique to their specific chemostats in spite of similar selective pressure and similar inocula shared between chemostats. [ABSTRACT FROM AUTHOR]

Organohalide compounds such as chloroethenes, chloroethanes and polychlorinated benzenes are among the most significant pollutants in the world. These compounds are often found in contamination plumes with other pollutants such as solvents, pesticides and petroleum derivatives. Microbial bioremediation of contaminated sites, has become commonplace whereby key processes involved in bioremediation include anaerobic degradation and transformation of these organohalides by organohalide respiring bacteria and also via hydrolytic, oxygenic and reductive mechanisms by aerobic bacteria. Microbial ecogenomics has enabled us to not only study the microbiology involved in these complex processes but also develop tools to better monitor and assess these sites during bioremediation. Microbial ecogenomics have capitalized on recent advances in high-throughput and -output genomics technologies in combination with microbial physiology studies to address these complex bioremediation problems at a system level. Advances in environmental metagenomics, transcriptomics and proteomics have provided insights into key genes and their regulation in the environment. They have also given us clues into microbial community structures, dynamics and functions at contaminated sites. These techniques have not only aided us in understanding the lifestyles of common organohalide respirers, for example Dehalococcoides, Dehalobacter and Desulfitobacterium, but also provided insights into novel and yet uncultured microorganisms found in organohalide respiring consortia. In this paper we look at how ecogenomic studies have aided us to understand the microbial structures and functions in response to environmental stimuli such as the presence of chlorinated pollutants.

Across the U. S. Upper Midwest, a natural geographical sulfate gradient exists in lakes. Sediment grab samples and cores were taken to explore whether this sulfur gradient impacted organohalide-respiring Chloroflexi in lake sediments. Putative organohalide-respiring Chloroflexi were detected in 67 of 68 samples by quantitative polymerase chain reaction. Their quantities ranged from 3.5 × 104 to 8.4 × 1010 copies 16 S r RNA genes g−1 dry sediment and increased in number from west to east, whereas lake sulfate concentrations decreased along this west-to-east transect. A terminal restriction fragment length polymorphism ( TRFLP) method was used to corroborate this inverse relationship, with sediment samples from lower sulfate lakes containing both a higher number of terminal restriction fragments ( TRFs) belonging to the organohalide-respiring Dehalococcoidetes, and a greater percentage of the TRFLP amplification made up by Dehalococcoidetes members. Statistical analyses showed that dissolved sulfur in the porewater, measured as sulfate after oxidation, appeared to have a negative impact on the total number of putative organohalide-respiring Chloroflexi, the number of Dehalococcoidetes TRFs, and the percentage of the TRFLP amplification made up by Dehalococcoidetes. These findings point to dissolved sulfur, presumably present as reduced sulfur species, as a potentially controlling factor in the natural cycling of chlorine, and perhaps as a result, the natural cycling of some carbon as well. [ABSTRACT FROM AUTHOR]

The rhodanese protein domain is common throughout all kingdoms of life and is characterized by an active site cysteine residue that is able to bind sulfane sulfur and catalyse sulfur transfer. No unique function has been attributed to rhodanese-domain-containing proteins, most probably because of their diversity at both the level of sequence and protein domain architecture. In this study, we investigated the biochemical properties of an unusual rhodanese protein, PhsE, from Desulfitobacterium hafniense strain TCE1 which we have previously shown to be massively expressed under anaerobic respiration with tetrachloroethene. The peculiarity of the PhsE protein is its domain architecture which is constituted of two rhodanese domains each with an active site cysteine. The N-terminal rhodanese domain is preceded by a lipoprotein signal peptide anchoring PhsE on the outside of the cytoplasmic membrane. In vitro sulfur-transferase activity of recombinant PhsE variants was measured for both domains contrasting with other tandem-domain rhodaneses in which usually only the C-terminal domain has been found to be active. The genetic context of phsE shows that it is part of a six-gene operon displaying homology with gene clusters encoding respiratory molybdoenzymes of the PhsA/PsrA family, possibly involved in the reduction of sulfur compounds. Our data suggest, however, that the presence of sulfide in the medium is responsible for the high expression of PhsE in Desulfitobacterium, where it could play a role in the sulfur homeostasis of the cell. [ABSTRACT FROM AUTHOR]

The biodegradation of 2,4,6-trichlorophenol (246TCP) was studied using expanded granular sludge bed (EGSB) reactors and a fluidized bed biofilm reactor (FBBR) filled with activated carbon. One of the EGSB reactor and the FBBR were bioaugmented with Desulfitobacterium strains. 246TCP loading rate was gradually incremented from 10 to 250 mg L-1 day-1. The main pathway of dechlorination was in ortho-position, generating 4-chlorophenol and 2,4-dichlorophenol. The maintenance of both COD degradation efficiency (higher than 80%) and methanogenic efficiency (between 0.3 and 0.6 g CH4-COD g-1 COD consumed) in EGSB reactor implies a great stability of the process. Through isotherm studies in FBBR, it could be deduced that around 52% of 246TCP was completely dechlorinated, whereas the adsorption involved around 16%. By means of FISH studies it was proved that the methanogenic Archaea community was maintained in the bioaugmented EGSB reactor, whereas in the FBBR this community was gradually developed until reaching stability. Desulfitobacterium community was also maintained in the reactors, although D. chlororespirans proportion rise in the FBBR at the higher 246TCP loading rates, implying that this species can withstand the 246TCP toxicity better than D. hafniense. [ABSTRACT FROM AUTHOR]

Microbial dehalogenation of tetrachloroethene (PCE) and cis-dichloroethene ( cis-DCE) was studied in cultures from a continuous stirred tank reactor initially inoculated with aquifer material from a PCE-contaminated site. Cultures amended with hydrogen and acetate readily dechlorinated PCE and cis-DCE; however, this transformation was incomplete and resulted in the accumulation of chlorinated intermediates and only small amounts of ethene within 60 days of incubation. Conversely, microbial PCE and cis-DCE dechlorination in cultures with benzoate and acetate resulted in the complete transformation to ethene within 30 days. Community fingerprinting by denaturing gradient gel electrophoresis (DGGE) revealed the predominance of phylotypes closely affiliated with Desulfitobacterium, Dehalococcoides, and Syntrophus species. The Dehalococcoides culture VZ, obtained from small whitish colonies in cis-DCE dechlorinating agarose cultures, revealed an irregular cell diameter between 200 and 500 nm, and a spherical or biconcave disk-shaped morphology. These organisms were identified as responsible for the dechlorination of cis-DCE to ethene in the PCE-dechlorinating consortia, operating together with the Desulfitobacterium as PCE-to- cis-DCE dehalogenating bacterium and with a Syntrophus species as potential hydrogen-producing partner in cultures with benzoate. [ABSTRACT FROM AUTHOR]

The in situ degradation of chlorinated ethenes was assessed in an anaerobic aquifer using stable isotope fractionation approaches, microcosm studies and taxon specific detection of specific dehalogenating groups of bacteria. The aquifer in the Bitterfeld/Wolfen region in Germany contained all chlorinated ethenes, benzene and toluene as contaminants. The concentrations and isotope composition of the chlorinated ethenes indicated biodegradation of the contaminants. Microcosm studies confirmed the presence of in situ microbial communities capable of the complete dechlorination of tetrachloroethene. Taxon specific investigation of the microbial communities indicated the presence of various potential dechlorinating organisms including Dehalococcoides, Desulfuromonas, Desulfitobacterium and Dehalobacter. The integrated approach, using metabolite spectra, molecular marker analysis and isotope studies, provided several lines of evidence for natural attenuation of the chlorinated ethenes. [Copyright &y& Elsevier]

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts. [ABSTRACT FROM AUTHOR]

Desulfitobacterium spp. are strictly anaerobic bacteria that were first isolated from environments contaminated by halogenated organic compounds. They are very versatile microorganisms that can use a wide variety of electron acceptors, such as nitrate, sulfite, metals, humic acids, and man-made or naturally occurring halogenated organic compounds. Most of the Desulfitobacterium strains can dehalogenate halogenated organic compounds by mechanisms of reductive dehalogenation, although the substrate spectrum of halogenated organic compounds varies substantially from one strain to another, even with strains belonging to the same species. A number of reductive dehalogenases and their corresponding gene loci have been isolated from these strains. Some of these loci are flanked by transposition sequences, suggesting that they can be transmitted by horizontal transfer via a catabolic transposon. Desulfitobacterium spp. can use H2 as electron donor below the threshold concentration that would allow sulfate reduction and methanogenesis. Furthermore, there is some evidence that syntrophic relationships occur between Desulfitobacterium spp. and sulfate-reducing bacteria, from which the Desulfitobacterium cells acquire their electrons by interspecies hydrogen transfer, and it is believed that this relationship also occurs in a methanogenic consortium. Because of their versatility, desulfitobacteria can be excellent candidates for the development of anaerobic bioremediation processes. The release of the complete genome of Desulfitobacterium hafniense strain Y51 and information from the partial genome sequence of D. hafniense strain DCB-2 will certainly help in predicting how desulfitobacteria interact with their environments and other microorganisms, and the mechanisms of actions related to reductive dehalogenation. [ABSTRACT FROM AUTHOR]

Desulfitobacterium hafniense PCP-1 (formerly frappieri PCP-1) has two reductive dehalogenases (RDases) that have been characterized. One is a membrane-associated 2,4,6-trichlorophenol RDase, which is encoded by crdA, and the other is a 3,5-dichlorophenol RDase encoded by cprA5. In this report, we determined the occurrence of these two RDase genes in seven other Desulfitobacterium strains. The presence or absence of these two RDases may explain the differences in the spectrum of halogenated compounds by these Desulfitobacterium strains. crdA gene sequences were found in all of the tested strains. It was expressed in strain PCP-1 regardless of the absence or presence of chlorophenols in the culture medium. crdA was also expressed in D. hafniense strains DCB-2 and TCE-1. cprA5 was detected only in D. hafniense strains PCP-1, TCP-A, and DCB-2. In these strains, cprA5 transcripts were detected only in the presence of chlorophenols. We also examined the expression of putative cprA RDases (cprA2, cprA3, and cprA4) that were shown to exist in the D. hafniense DCB-2 genome. RT-PCR experiments showed that cprA2, cprA3, and cprA4 were expressed in D. hafniense strains PCP-1, DCB-2, and TCP-A in the presence of chlorophenols. However, contrary to cprA5, these three genes were also expressed in the absence of halogenated compounds in the culture medium. [ABSTRACT FROM AUTHOR]

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene ( cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min−1 mg protein−1 . The apparent K m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we report on phylogenetic and physiological characterization of an anaerobic culture capable of reductive dehalogenation of tetrachloroethene (PCE) obtained from a PCE-contaminated site. The culture was enriched using different combinations of electron donors (hydrogen and acetate) and electron acceptors (PCE, cis-1,2-dichloroethene (cDCE) and controls without chlorinated ethenes). The resulting subcultures were analyzed using three different approaches: chemical analysis to document conversion of chlorinated ethenes; polymerase chain reaction (PCR) of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE) to compare community compositions; fluorescence in situ hybridization (FISH) to quantify specific groups of microorganisms using oligonucleotide probes previously designed or newly designed based on the sequences retrieved from sequence analysis of specific DGGE bands. Members of two genera which contain bacteria capable of reductive dehalogenation were detected in the culture: Dehalococcoides and Desulfitobacterium. The combined analyses suggested that Dehalococcoides-like bacteria are associated with complete dehalogenation of chlorinated ethenes to ethene with hydrogen as electron donor; and Desulfitobacterium-like bacteria, in contrast, are associated with incomplete PCE dehalogenation to cDCE and appear to be able to use acetate as electron donor. In addition, Sporomusa-like bacteria were identified, which most likely act as homoacetogens. The results demonstrated that combination of culture enrichment with different substrates, DGGE, and FISH allowed a detailed qualitative and quantitative characterization of the dominant microorganisms associated with reductive dehalogenation. [Copyright &y& Elsevier]

An anaerobic bacterium, strain DP7, was isolated from human feces in mineral medium with formate and 0.02% yeast extract as energy and carbon source. This rod-shaped motile bacterium used pyruvate, lactate, formate, hydrogen, butyrate, and ethanol as electron donor for sulfite reduction. Other electron acceptors such as thiosulfate, nitrate and fumarate stimulated growth in the presence of 0.02% yeast extract and formate. Acetate was the only product during fermentative growth on pyruvate. Six mol of pyruvate were fermented to 7 mol of acetate. 13C-NMR labeling experiments showed homoacetogenic 13C-CO2 incorporation into acetate. The pH and temperature optimum of fermentative growth on pyruvate was 7.4 and 37 °C, respectively. The growth rate under these conditions was approximately 0.10 h–1. Strain DP7 was identified as a new strain of Desulfitobacterium frappieri on the basis of 16S rRNA sequence analysis (99% similarity) and DNA-DNA hybridization (reassociation value of 83%) with Desulfitobacterium frappieri TCE1. In contrast to described Desulfitobacterium strains, the newly isolated strain has not been isolated from a polluted environment and did not use chloroethenes or chlorophenols as electron acceptor. [ABSTRACT FROM AUTHOR]