Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion.

A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.

Hydrogenase I of Clostridium pasteurianum functions as a novel selenite reductase.

Clostridium pasteurianum's hydrogenase I, an important constitutive metabolic enzyme, has been shown to function as a 'novel selenite reductase'. Selenite reductase activity was found to co-purify with hydrogenase I activity; the fold purification and specific activities for these two activities paralleled each other throughout the purification steps. The highly purified hydrogenase I apparent K(m) for the selenite substrate was 0.2 mM. The stoichiometry for the enzymatic reduction of SeO3(2-) to Se(0) via H2 oxidation, was determined to be 2.3:1 (H2:Se(0)), very close to the theoretical ratio of 2:1 for this reduction reaction. Known electron carriers required for hydrogenase I activity were also found to couple its selenite reductase activity, the most efficient one being ferredoxin. The purified hydrogenase I not only reduced selenite but also tellurite, and its selenite activity was completely inhibited by O2 and CuSO4, potent inhibitors of hydrogenase I activity.

Regulation of the Periplasmic [Fe] Hydrogenase by Ferrous Iron in Desulfovibrio vulgaris (Hildenborough).

The periplasmic [Fe] hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) DSM 8303 was found to be regulated by ferrous iron availability. During growth with 5 ppm of iron, the enzyme derepressed and the specific activity increased approximately fourfold, whereas the presence of 100 ppm of ferrous iron repressed the enzyme. The repression-derepression phenomenon with ferrous iron was found to be operative when the cells were cultured under either hydrogen or nitrogen gas. This is the first reported case showing that the hydrogenase enzyme is regulated by iron, and the implications of this finding relative to the corrosion industry are discussed.

Effect of hydrogenase and mixed sulfate-reducing bacterial populations on the corrosion of steel.

The importance of hydrogenase activity to corrosion of steel was assessed by using mixed populations of sulfate-reducing bacteria isolated from corroded and noncorroded oil pipelines. Biofilms which developed on the steel studs contained detectable numbers of sulfate-reducing bacteria (10 increasing to 10/0.5 cm). However, the biofilm with active hydrogenase activity (i.e., corrosion pipeline organisms), as measured by a semiquantitative commercial kit, was associated with a significantly higher corrosion rate (7.79 mm/year) relative to noncorrosive biofilm (0.48 mm/year) with 10 sulfate-reducing bacteria per 0.5 cm but no measurable hydrogenase activity. The importance of hydrogenase and the microbial sulfate-reducing bacterial population making up the biofilm are discussed relative to biocorrosion.

Physiological effects of metronidazole on Clostridium pasteurianum.

The physiological effects of metronidazole on the growth, viability, fermentation end-product production and cellular morphology of Clostridium pasteurianum cells growing logarithmically were studied. Metronidazole (a 5-nitroimidazole) was found to be the most potent of the nitroimidazole compounds tested against C. pasteurianum. It inhibited the growth rate of C. pasteurianum cultures by varying degrees over a range of drug concentrations (2.5-10 mg/L). Metronidazole had an immediate bactericidal effect at a concentration of 10 mg/L, killing 99.9% of cells within 5 min of drug addition. The same concentration caused an immediate cessation of fermentation end-product (acetate and butyrate) production in these cultures. These observations may be relevant to a proposed cell lysing mechanism which may form an additional mode of action of this important antibiotic.

Reduction of 2-, 4- and 5-nitroimidazole drugs by hydrogenase 1 in Clostridium pasteurianum.

Highly-purified bidirectional hydrogenase (hydrogenase 1) of Clostridium pasteurianum could rapidly reduce several 2-, 4- and 5-nitroimidazole compounds via an electron carrier-coupled mechanism. Hydrogenase 1 was also shown to reduce a 2-nitroimidazole (misonidazole) and a 4-nitroimidazole in the presence of its required electron carriers including ferredoxin, the flavin coenzymes FAD and FMN, and the low potential electron carrier dyes methyl- and benzyl-viologen. No drug reduction by hydrogenase 1 occurred when any one of these electron carriers was replaced by nicotinamide electron carriers (NAD and NADP), or was omitted from the reaction mixture. The rates of reduction of the nitroimidazole compounds correlated with their one electron reduction potentials at pH 7(E7(1)); the higher the drug's E7(1), the faster its rate of reduction by the enzyme. Reduction rates for the drugs did not correlate with the antibacterial activity of these compounds against C. pasteurianum, suggesting that other factors are also important in determining the antimicrobial potencies of nitroimidazoles.

Evidence for proton motive force dependent transport of selenite by Clostridium pasteurianum.

The proton motive force mediated the transport of selenite (SeO3(2-)) in Clostridium pasteurianum cells by proton symport. The proton conductor, carbonyl cyanide m-chlorophenylhydrazone, inhibited SeO3(2-) uptake while N,N'-dicyclohexylcarbodiimide prevented SeO3(2-) uptake by presumably inhibiting the unidirectional ATPase. Acid pulse studies and antibiotic experiments with valinomycin suggest that the transmembrane delta pH component of the proton motive force mediated the transport of SeO3(2-) into the cells. The SeO3(2-) porter system in C. pasteurianum was found to be dependent upon energy source, temperature, and medium pH.

Role of hydrogenase 1 of Clostridium pasteurianum in the reduction of metronidazole.

Competition studies between the phosphoroclastic reaction and the metronidazole reduction reaction using dialyzed crude cell-free extracts of Clostridium pasteurianum which were essentially devoid of Hydrogenase 1 activity demonstrated that this enzyme plays an important role in the reduction of metronidazole. To determine further the exact function for Hydrogenase 1 in the reduction of the drug, this enzyme was highly purified from C. pasteurianum. Metronidazole reduction activity copurified with Hydrogenase 1 specific activity throughout the purification procedure. Drug reduction required the presence of an electron carrier and could not be accomplished by the enzyme alone. Ferredoxin, and also the low potential electron carrier dyes, methyl and benzyl viologen, and the flavin coenzymes, FAD and flavin mononucleotide (FMN), could couple the reduction of metronidazole. Hydrogenase 1 activity and its metronidazole reduction activity were inactivated irreversibly in the presence of oxygen. Metronidazole could be reduced only by an electron carrier-Hydrogenase 1 mechanism or directly by sodium dithionite.

Role of the phosphoroclastic reaction of Clostridium pasteurianum in the reduction of metronidazole.

To demonstrate the importance of electron siphoning by the metronidazole reductase system from reduced ferredoxin to the mechanism of action of the drug in Clostridium pasteurianum, the effects of the reduction of metronidazole on the phosphoroclastic reaction were studied. Metronidazole concentrations between 0.5 and 5 mM caused a significant increase in acetyl phosphate production by the phosphoroclastic reaction compared to the control system without metronidazole. When this enzymatic reaction was assayed by standard manometric techniques under nitrogen gas, two simultaneous effects of electron siphoning were demonstrated: (i) the electrons from reduced ferredoxin were initially consumed for the reduction of metronidazole instead of being evolved as H2 via the ferredoxin-linked hydrogenase and (ii) phosphoroclastic activity was stimulated, with augmented production of CO2 and acetyl phosphate. This work further supports the notion of preferential scavenging of electrons away from ferredoxin-linked enzymatic reactions by metronidazole reductase(s) in C. pasteurianum.

Ferredoxin-linked reduction of metronidazole in Clostridium pasteurianum.

Clostridium pasteurianum cell-free extracts enzymatically reduced metronidazole when coupled by hydrogenase via reduced ferredoxin. A 5 mM concentration of methyl viologen, flavin adenine dinucleotide, or flavin mononucleotide could completely replace ferredoxin (0.05 mM) in the in vitro reduction assay system, whereas 5 mM benzyl viologen was less effective. However, when these electron carriers were used at a concentration of 0.05 mM, there was a drastic loss in their abilities to couple the metronidazole reduction system compared with the comparable concentration of ferredoxin. It is not understood why these flavin coenzymes participate in this enzymatic reaction. NAD and NADP had no activity when substituted for ferredoxin in the enzyme system. Two reduced ferredoxin-linked pathways, "metronidazole reductase" and the inducible dissimilatory sulfite reductase system, when combined in a single in vitro competition experiment demonstrated a preferential flow of electrons to metronidazole away from sulfite. A proposed bactericidal mechanism for metronidazole against C. pasteurianum incorporating the above findings is discussed.

Sulfur isotope fractionation during SO3(2-) reduction by different clostridial species.

Sulfur isotope composition patterns for sulfide evolved from cultures supplemented with 1 mM Na2SO3, suggested that an inducible dissimilatory type SO3(2-) reduction pathway, as previously found in C. pasteurianum, probably exists in many clostridial species. Data are presented for five additional species which include pathogens and nonpathogens.

Purification and characterization of an inducible dissimilatory type sulfite reductase from Clostridium pasteurianum.

An inducible sulfite reductase was purified from Clostridium pasteurianum. The pH optimum of the enzyme is 7.5 in phosphate buffer. The molecular weight of the reductase was determined to be 83,600 from sodium dodecyl sulfate gel electrophoresis with a proposed molecular structure: alpha 2 beta 2. Its absorption spectrum showed a maximum at 275 nm, a broad shoulder at 370 nm and a very small absorption maximum at 585 nm. No siroheme chromophore was isolated from this reductase. The enzyme could reduce the following substrates in preferential order: NH2OH greater than SeO3 (2-) greater than NO(2-) 2 at rates 50% or less of its preferred substrate SO3(2-). The proposed dissimilatory intermediates, S3O6 (2-) or S2O3(2-), were not utilized by this reductase while KCN inhibited its activity. Varying the substrate concentration [SO3(2-)] from 1 to 2.5 mumol affected the stoichiometry of the enzyme reaction by alteration of the ratio of H2 uptake to S2- formed from 2.5:1 to 3.1:1. The inducible sulfite reductase was found to be linked to ferredoxin which could be completely replaced by methyl viologen or partially by benzyl viologen. Some of the above-mentioned enzyme properties and physiological considerations indicated that it was a dissimilatory type sulfite reductase.

The role of Thiobacillus albertis glycocalyx in the adhesion of cells to elemental sulfur.

Thiobacillus albertis, a newly characterized acidophilic Thiobacillus sp., was found not to be dependent on physiological conditions such as pH, cellular energy, or peripheral cell envelope sulfhydryl groups for attachment to elemental sulfur (S0). Heat-killed cells or those pretreated with sulfhydryl reagents (iodoacetate or iodoacetamide) were able to adhere to S0 in comparable numbers as assayed by epifluorescence microscopy. In addition, iodoacetate and iodoacetamide were found to be bactericidal, the former more potent than the latter. Sodium lauryl sulfate was found to cause nearly complete detachment of T. albertis cells from glass slides implicating its glycocalyx for this cell-glass attachment. In addition scanning electron microscopy visually revealed T. albertis cellular adhesion to S0 was due to the organism's threadlike glycocalyx material interacting with the sulfur surface. It was concluded that T. albertis glycocalyx plays an important role in the attachment to solid surfaces (glass or S0). In addition T. albertis was shown to colonize S0 surfaces by microcolonies.

Stable isotope fractionation by Clostridium pasteurianum. 4. Sulfur isotope fractionation during enzymatic S3O6(2-), S2O3(2-), and SO3(2-) reductions.

Cell-free extracts from Clostridium pasteurianum grown on SO3(2-) utilize H2 to reduce S3O6(2-), S2O3(2-), SO3(2-) to H2S at a much faster rate than extracts from SO4(2-)-grown cells. This further supports the concept of an inducible dissimilatory type SO3(2-) reductive pathway in this organism. 35S dilution experiments further support the concept that S3O6(2-) and S2O3(2-) are pathway intermediates. The inducible SO3(2-) reductase is ferredoxin linked and the kinetics of the reduction and the sulfur isotope fractionation of the product can be altered by altering the growth conditions. The attending sulfur isotope fractionations are similar to those observed during the chemical decomposition of these compounds. In the case of S2O3(2-), 35S labelling experiments verified the conclusions derived from the stable isotope fractionation data concerning the relative reduction rates of the sulfane and sulfonate sulfurs. The reduction rates were also affected by enzyme concentration. The integrity of the whole cell is a necessary requirement for the large inverse isotope effects previously reported.

Physiological and ultrastructural characterization of a new acidophilic Thiobacillus species (T. kabobis).

A new autotrophic acidophilic Thiobacillus sp. was isolated from acidic soil adjacent to a natural gas processing plant's sulfur stockpile. This isolate metabolized S2O3(2-) to S4O6(2-) during growth and could not reoxidize this product; instead, the remaining S2O3(2-) substrate was oxidized to SO4(2-) in the stationary phase which represented a significant metabolic change as compared with other acidophilic thiobacilli. In contrast the isolate oxidized S0 to H2SO4 during the log-phase growth with no S4O6(2-) being formed. Ultrastructural studies revealed that the isolate's cytoplasm contained unidentified membrane-bound granules, volutin type bodies, and carboxysomes. Cleavage planes within the cell wall revealed a pattern of highly ordered subunits in the outer leaflet of the outer membrane and these ordered subunits were also discerned through the eutectic at the cell surface. This regular structure was not observed at the surface of cells of other acidophilic thiobacilli. The isolate bears a single pilus and one unusually long polar flagellum. The physiological and ultrastructural data are discussed in relation to other known thiobacilli and show this isolate to be a new Thiobacillus sp. named T. kabobis.

Stable isotope fractionation by Clostridium pasteurianum. 3. Effect of SeO32- on the physiology and associated sulfur isotope fractionation during SO32- and SO42- reductions.

Increased SeO32- concentration reduced H2S evolution from SO32- during whole cell and cell-free extract reductions by Clostridium pasteurianum. H2S production from SO42- was completely inhibited by SeO32- in stationary phase cells. Generation times increased with greater SeO32- concentration, the increase with 1 mM SeO32- being a factor of 2.5 for 1 mM SO32-, and over 3 for 1 mM SO42- reductions. In vitro and in vivo experiments with proposed intermediates of the SO32- reduction pathway show that SeO32- inhibited both the S3O62- to S2O32- and S2O32- to S2- reaction sequences with the latter being more pronounced in growth experiments. Both extracts and whole cells reduced SeO32- to Se0 but Se0 granules were not found in the cell's cytoplasm. The formation of S2O32- by an extracellular chemical mechanism appears not to have occurred in these experiments. Increased SeO32- concentration had the effect of compressing the isotopic release pattern for H2S along the H2S production axis and did not significantly alter the maximum and minimum values of delta 34S. Thus, inhibition by SeO32- limited the conversions of sulfur species without altering the isotopic selectivity of rate-controlling steps in the pathway.

Stable isotope fractionation by Clostridium pasteurianum. 2. Regulation of sulfite reductases by sulfur amino acids and their influence on sulfur isotope fractionation during SO32- and SO42- reduction.

In addition to an assimilatory sulfite reductase, studies of cultures of Clostridium pasteurianum supplemented with methionine, cysteine, and 35SO42- provides evidence for another reductase which is induced by SO32-. This inducible reductase appears to be dissimaltory because of the copious sulfide production arising when the cells are grown on SO32-. Cysteine can repress the assimilatory sulfite reductase but does not affect the inducible reductase. During late logarithmic growth on 1 mM SO42- + 10mM cysteine, depression of the inducible reductase occurred along with increased sulfide production. The presence of 1 mM cysteine and (or) 1 mM cysteine and (or) 1 mM methionine does not affect the inverse sulfur isotope effect for evolved H2S. However, 5 and 10 mM cysteine reduce the maximum delta34S value for released H2S from +40 to 10%. A small conversion of cysteine to H2S by C. pasteurianum occurs, but only in the stationary phase.

The use of stable sulfur isotope labelling to elucidate sulfur metabolism by Clostridium pasteurianum.

An unique isotope labelling experiment was conducted whereby mixtures of sulfate and sulfite of different isotopic compositions were metabolized by Clostridium pasteurianum. The results showed during reduction of 1 mM SO3= plus 1 mM SO4=, essentially all evolved H2S arose from the sulfite whereas in the case of cellular sulfur, 85% was derived from sulfite and the remainder from sulfate.

Morphological modifications of cells of Clostridium pasteurianum caused by growth on sulfite.

The morphology of Clostridium pasteurianum cells grown on 10(-2) M SO32- showed significant alteration in cell shape and the absence of the electron translucent reserve polysaccharide (amylopectin) when compared to sulfate-grown cells. At the lower sulfite concentrations (10(-3) and 10(-4)M SO32-) the cells showed the cytoplasmic changes noted above but the cell shapes were not modified.