Role of humic-bound iron as an electron transfer agent in dissimilatory Fe(III) reduction.
The dissimilatory Fe(III) reducer Geobacter metallireducens reduced Fe(III) bound in humic substances, but the concentrations of Fe(III) in a wide range of highly purified humic substances were too low to account for a significant portion of the electron-accepting capacities of the humic substances. Furthermore, once reduced, the iron in humic substances could not transfer electrons to Fe(III) oxide. These results suggest that other electron-accepting moieties in humic substances, such as quinones, are the important electron-accepting and shuttling agents under Fe(III)-reducing conditions. (+info)
Unexpected population distribution in a microbial mat community: sulfate-reducing bacteria localized to the highly oxic chemocline in contrast to a eukaryotic preference for anoxia.
The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-microm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families "Desulfovibrionaceae" and "Desulfobacteriaceae," Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia. (+info)
Diversity of sulfate-reducing bacteria in oxic and anoxic regions of a microbial mat characterized by comparative analysis of dissimilatory sulfite reductase genes.
Sequence analysis of genes encoding dissimilatory sulfite reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the Desulfonema-like organisms within the delta-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating Desulfonema-like organisms with oxic habitats. (+info)
Cloning, sequencing and heterologous expression of pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici.
A genomic lambda-library of Pelobacter acidigallici has been established. Proteolytic digestion of homogeneous pyrogallol-phloroglucinol transhydroxylase from the same microorganism afforded polypeptide fragments whose N-terminal sequences allowed the generation of oligonucleotide primers. Together with primers deduced from the known N-terminal sequences of the two intact subunits these were used in PCR experiments to obtain three amplificates. Screening the lambda-library with the three amplificates led eventually to clones containing the whole gene coding for the transhydroxylase. Sequencing the gene revealed two open reading frames coding for 875 and 275 amino acids which correspond to the alpha- and beta-subunits of THL, respectively. The two subunits are separated by a 48-bp noncoding region. Comparison of the sequence with those of other molybdopterin cofactor (MoCo)-enzymes places THL in the dimethylsulfoxide reductase family. Possible contact sites to the MoCo and to the iron-sulphur clusters were spotted. Using the expression vectors pQE 30 and pT 7-7 three constructs harbouring the THL gene were created. One of them carried a His6-tag at the N-terminus of the alpha-subunit, another at the C-terminus of the beta-subunit. Immunoblot analysis showed high expression of THL, but the inclusion bodies could not be refolded to active enzyme. (+info)
Extracellular metal-binding activity of the sulphate-reducing bacterium Desulfococcus multivorans.
Polarography was used to measure the copper-binding ability of culture filtrates from a range of sulphate-reducing bacteria (SRB), including pure cultures and environmental isolates. Of those tested, Desulfococcus multivorans was shown to have the greatest copper-binding capacity and this organism was used for further experiments. Extracellular copper- and zinc-binding activities of Dc. multivorans culture filtrates from batch cultures increased over time and reached a maximum after 10 d growth. The culture filtrate was shown to bind copper reversibly and zinc irreversibly. Twelve-day-old Dc. multivorans culture filtrates were shown to have a copper-binding capacity of 3.64 +/- 0.33 micromol ml(-1) with a stability constant, log10K, of 5.68 +/- 0.64 (n=4). The metal-binding compound was partially purified from culture growth media by dichloromethane extraction followed by HPLC using an acetonitrile gradient. (+info)
Psychrophilic sulfate-reducing bacteria isolated from permanently cold arctic marine sediments: description of Desulfofrigus oceanense gen. nov., sp. nov., Desulfofrigus fragile sp. nov., Desulfofaba gelida gen. nov., sp. nov., Desulfotalea psychrophila gen. nov., sp. nov. and Desulfotalea arctica sp. nov.
Five psychrophilic, Gram-negative, sulfate-reducing bacteria were isolated from marine sediments off the coast of Svalbard. All isolates grew at the in situ temperature of -1.7 degrees C. In batch cultures, strain PSv29T had the highest growth rate at 7 degrees C, strains ASv26T and LSv54T had the highest growth rate at 10 degrees C, and strains LSv21T and LSv514T had the highest growth rate at 18 degrees C. The new isolates used the most common fermentation products in marine sediments, such as acetate, propionate, butyrate, lactate and hydrogen, but only strain ASv26T was able to oxidize fatty acids completely to CO2. The new strains had growth optima at neutral pH and marine salt concentration, except for LSv54T which grew fastest with 1% NaCl. Sulfite and thiosulfate were used as electron acceptors by strains ASv26T, PSv29T and LSv54T, and all strains except PSv29T grew with Fe3+ (ferric citrate) as electron acceptor. Chemotaxonomy based on cellular fatty acid patterns and menaquinones showed good agreement with the phylogeny based on 16S rRNA sequences. All strains belonged to the delta subclass of Proteobacteria but had at least 9% evolutionary distance from known sulfate reducers. Due to the phylogenetic and phenotypic differences between the new isolates and their closest relatives, establishment of the new genera Desulfotalea gen. nov., Desulfofaba gen. nov. and Desulfofrigus gen. nov. is proposed, with strain ASv26T as the type strain of the type species Desulfofrigus oceanense sp. nov., LSv21T as the type strain of Desulfofrigus fragile sp. nov., PSv29T as the type strain of the type species Desulfofaba gelida sp. nov., LSv54T as the type strain of the type species Desulfotalea psychrophila sp. nov. and LSv514T as the type strain of Desulfotalea arctica sp. nov. (+info)
Random transposition by Tn916 in Desulfitobacterium dehalogenans allows for isolation and characterization of halorespiration-deficient mutants.
To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted. (+info)
A proton-NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans. Comparison between the reduced and the oxidized forms.
The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant. (+info)