Hydrogenase activity of mineral-associated and suspended populations of Desulfovibrio desulfuricans Essex 6.

The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate electron transfer to metals and may contribute to the formation and speciation of ferrous sulfides formed at the cell-mineral interface. In the present study, we compared the whole cell hydrogenase activity of Desulfovibrio desulfuricans strain Essex 6 growing as biofilms on hematite (hematite-associated) or as suspended populations using different metabolic pathways. Hematite-associated cells exhibited significantly greater hydrogenase activity than suspended populations during sulfate respiration but not during pyruvate fermentation. The enhanced activity of the hematite-associated, sulfate-grown cells appears to be dependent on iron availability rather than a general response to surface attachment since the activity of glass-associated cells did not differ from that of suspended populations. Hydrogenase activity of pyruvate-fermenting cells was stimulated by addition of iron as soluble Fe(II)Cl2 and, in the absence of added iron, both sulfate-reducing and pyruvate-fermenting cells displayed similar rates of hydrogenase activity. These data suggest that iron exerts a stronger influence on whole cell hydrogenase activity than either metabolic pathway or mode of growth. The location of hydrogenase to the cell envelope and the enhanced activity at the hematite surface in sulfate-reducing cells may influence the redox conditions that control the species of iron sulfides on the mineral surface.

Combining in situ reverse transcriptase polymerase chain reaction, optical microscopy, and X-ray photoelectron spectroscopy to investigate mineral surface-associated microbial activities.

A study was undertaken to investigate expression of a gene encoding a c-type cytochrome in cells of the dissimilatory metal reducing bacterium (DMRB) Geobacter sulfurreducens during association with poorly crystalline and crystalline solid-phase Fe(III)-oxides. The gene encoding OmcC (outer membrane c-type cytochrome) was used as a target for PCR-based molecular detection and visualization of omcC gene expression by individual cells and aggregates of cells of G. sulfurreducens associated with ferrihydrite and hematite mineral particles. Expression of omcC was demonstrated in individual bacterial cells associated with these Fe-oxide surfaces by in situ RT-PCR (IS-RT PCR) and epifluorescence microscopy. Epifluorescence microscopy also permitted visualization of total DAPI-stained cells in the same field of view to assess the fraction of the cell population expressing omcC. By combining reflected differential interference contrast (DIC) microscopy and epifluorescence microscopy, it was possible to determine the spatial relationship between cells expressing omcC and the mineral surface. Introduction of the fluorescently labeled lectin concanavalin A revealed extracellular polymeric substances (EPS) extending between aggregations of bacterial cells and the mineral surface. The results indicate that EPS mediates an association between cells of G. sulfurreducens and ferrihydrite particles, but that direct cell contact with the mineral surface is not required for expression of omcC. XPS analysis revealed forms of reduced Fe associated with areas of the mineral surface where EPS-mediated bacterial associations occurred. The results demonstrate that by combining molecular biology, reflectance microscopy, and XPS, chemical transformations at a mineral surface can be related to the expression of specific genes by individual bacterial cells and cell aggregates associated with the mineral surface. The approach should be useful in establishing involvement of specific gene products in a wide variety of surface chemical processes.

Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens.

Geobacter sulfurreducens is capable of anaerobic respiration with Fe(III) as a terminal electron acceptor via a membrane-bound Fe(III) reductase activity associated with a large molecular mass cytochrome c. This cytochrome was purified by detergent extraction of the membrane fraction, Q-Sepharose ion-exchange chromatography, preparative electrophoresis, and MonoQ ion-exchange chromatography. Spectrophotometric analysis of the purified cytochrome reveals a c-type haem, with no evidence of haem a, haem b or sirohaem. The cytochrome has an M(r) of 89000 as determined by denaturing PAGE, and has an isoelectric point of 5.2 as determined by analytical isoelectric focusing. Dithionite-reduced cytochrome can donate electrons to Fe(III)-nitrilotriacetic acid and synthetic ferrihydrite, thus demonstrating that the cytochrome has redox and thermodynamic properties required for reduction of Fe(III). Analysis using cyclic voltammetry confirmed that the reduced cytochrome can catalytically transfer electrons to ferrihydrite, further demonstrating its ability to be an electron transport mediator in anaerobic Fe(III) respiration. Sequence analysis of a cloned chromosomal DNA fragment revealed a 2307 bp open reading frame (ferA) encoding a 768 amino acid protein corresponding to the 89 kDa cytochrome. The deduced amino acid sequence (FerA) translated from the open reading frame contained 12 putative haem-binding motifs, as well as a hydrophobic N-terminal membrane anchor sequence, a lipid-attachment site and an ATP/GTP-binding site. FerA displayed 20% or less identity with amino acid sequences of other known cytochromes, although it does share some features with characterized polyhaem cytochromes c.

Isolation and analysis of three peroxide sensor regulatory gene homologs ahpC, ahpX and oxyR in Streptomyces viridosporus T7A--a lignocellulose degrading actinomycete.

Increased lignolytic peroxidase activity has been demonstrated with the addition of sublethal doses of toxic H2O2 in Streptomyces viridosporus T7A. Until now, the effect of H2O2 at the molecular level has not been well characterized. Here, for the first time we report the isolation and analysis of three peroxide-induced gene homologs from S. viridosporus T7A; ahpC and ahpX (encoding alkyl hydroxyperoxidase subunits) and oxyR (encoding oxygen stress regulatory protein). The genome organization of these stress related genes were found to be divergently adjacent to each other. The protein sequence analysis of the oxyR homolog revealed a helix-turn-helix DNA-binding motif characteristic to the LysR of regulatory proteins induced by H2O2. The nucleotide sequence analysis of the intergenic region between ahpC and oxyR revealed that they shared a core T-n11-A, a signature protein-binding region of LysR family members. Based on similarities in sequence analysis, genetic organization, and the induction of lignin peroxidase activity upon exposure to hydrogen peroxide, we hypothesize a peroxide induction mechanism for the regulation of oxidative lignin biodegradation by S. viridosporus, possibly via use of OxyR which is also involved in regulating the peroxide stress response in this actinomycete.

Characterization of a membrane-bound NADH-dependent Fe(3+) reductase from the dissimilatory Fe(3+)-reducing bacterium Geobacter sulfurreducens.

Geobacter sulfurreducens produces a single, membrane-associated Fe(3+) reductase activity when grown on fumarate or Fe(3+). The activity was initially isolated by solubilization of membranes with the non-ionic detergent dodecyl-beta-D-maltoside, and partially purified by a combination of ion exchange chromatography and preparative non-denaturing gel electrophoresis. Molecular mass of the reductase, as determined by gel filtration chromatography, was approximately 300 kDa. Cofactor analysis of the purified reductase demonstrates that it contains a hemoprotein and flavin adenine dinucleotide. Kinetic and inhibitor studies show that the reductase is specific for NADH as electron donor, and confirm that the reductase enzymatically reduces Fe(3+). The cytochrome associated with the complex undergoes a reoxidation upon addition of Fe(3+) compounds, indicating an ability to pass reducing equivalents to Fe(3+). This is the first description of a purified NADH-dependent Fe(3+) reductase from a microorganism capable of coupling Fe(3+) reduction to growth.

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.

Immunologic relatedness of extracellular ligninases from the actinomycetes Streptomyces viridosporus T7A and Streptomyces badius 252.

Four isoforms of the extracellular lignin peroxidase of the ligninolytic actinomycete Streptomyces viridosporus T7A (ALip-P1, P2, P3, and P4) were individually purified by ultrafiltration and ammonium sulfate precipitation, followed by electro-elution using polyacrylamide gel electrophoresis. Three of the purified peroxidases were compared for their immunologic relatedness by Western blot analysis using a polyclonal antibody preparation produced in rabbits against pure isoform P3. The anti-P3 antibody was also tested for its reactivity towards a lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and another ligninolytic actinomycete Streptomyces badius 252. Results showed that peroxidases ALip-P1 through ALip-P3 are immunologically related to one another. The peroxidases of S. badius, but not the peroxidase of P. chrysosporium, also reacted with the antibody, thus indicating that the lignin peroxidases of S. viridosporus and S. badius are immunologically related. Based upon its specific affinity, lignin peroxidase isoform ALip-P3 of S. viridosporus was readily purified using an anti-P3 antibody affinity column.