Guy Fauque

Biochemistry, physiology and biotechnology of sulfate-reducing bacteria.

Chemolithotrophic bacteria that use sulfate as terminal electron acceptor (sulfate-reducing bacteria) constitute a unique physiological group of microorganisms that couple anaerobic electron transport to ATP synthesis. These bacteria (220 species of 60 genera) can use a large variety of compounds as electron donors and to mediate electron flow they have a vast array of proteins with redox active metal groups. This chapter deals with the distribution in the environment and the major physiological and metabolic characteristics of sulfate-reducing bacteria (SRB). This chapter presents our current knowledge of soluble electron transfer proteins and transmembrane redox complexes that are playing an essential role in the dissimilatory sulfate reduction pathway of SRB of the genus Desulfovibrio. Environmentally important activities displayed by SRB are a consequence of the unique electron transport components or the production of high levels of H(2)S. The capability of SRB to utilize hydrocarbons in pure cultures and consortia has resulted in using these bacteria for bioremediation of BTEX (benzene, toluene, ethylbenzene and xylene) compounds in contaminated soils. Specific strains of SRB are capable of reducing 3-chlorobenzoate, chloroethenes, or nitroaromatic compounds and this has resulted in proposals to use SRB for bioremediation of environments containing trinitrotoluene and polychloroethenes. Since SRB have displayed dissimilatory reduction of U(VI) and Cr(VI), several biotechnology procedures have been proposed for using SRB in bioremediation of toxic metals. Additional non-specific metal reductase activity has resulted in using SRB for recovery of precious metals (e.g. platinum, palladium and gold) from waste streams. Since bacterially produced sulfide contributes to the souring of oil fields, corrosion of concrete, and discoloration of stonework is a serious problem, there is considerable interest in controlling the sulfidogenic activity of the SRB. The production of biosulfide by SRB has led to immobilization of toxic metals and reduction of textile dyes, although the process remains unresolved, SRB play a role in anaerobic methane oxidation which not only contributes to carbon cycle activities but also depletes an important industrial energy reserve.

Structure-function relationship in hemoproteins: The role of cytochrome c3 in the reduction of colloidal sulfur by sulfate-reducing bacteria

Cytochromes c3 of different strains of sulfatereducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c3 is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c3. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c3 is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c3 (or another sulfur reductase) than lactate-sulfate-grown cells.

Macellibacteroides fermentans gen. nov., sp. nov., a member of the family Porphyromonadaceae isolated from an upflow anaerobic filter treating abattoir wastewaters

A novel obligately anaerobic, non-spore-forming, rod-shaped mesophilic bacterium, which stained Gram-positive but showed the typical cell wall structure of Gram-negative bacteria, was isolated from an upflow anaerobic filter treating abattoir wastewaters in Tunisia. The strain, designated LIND7H(T), grew at 20-45 °C (optimum 35-40 °C) and at pH 5.0-8.5 (optimum pH 6.5-7.5). It did not require NaCl for growth, but was able to grow in the presence of up to 2 % NaCl. Sulfate, thiosulfate, elemental sulfur, sulfite, nitrate and nitrite were not used as terminal electron acceptors. Strain LIND7H(T) used cellobiose, glucose, lactose, mannose, maltose, peptone, rhamnose, raffinose, sucrose and xylose as electron donors. The main fermentation products from glucose metabolism were lactate, acetate, butyrate and isobutyrate. The predominant cellular fatty acids were anteiso-C(15 : 0), C(15 : 0), C(17 : 0) 2-OH and a summed feature consisting of C(18 : 2)ω6,9c and/or anteiso-C(18 : 0), and the major menaquinones were MK-9, MK-9(H(2)) and MK-10. The G+C content of the genomic DNA was 41.4 mol%. Although the closest phylogenetic relatives of strain LIND7H(T) were Parabacteroides merdae, Parabacteroides goldsteinii and Parabacteroides gordonii, analysis of the hsp60 gene sequence showed that strain LIND7H(T) was not a member of the genus Parabacteroides. On the basis of phylogenetic inference and phenotypic properties, strain LIND7H(T) ( = CCUG 60892(T) = DSM 23697(T) = JCM 16313(T)) is proposed as the type strain of a novel species in a new genus within the family Porphyromonadaceae, Macellibacteroides fermentans gen. nov., sp. nov.

Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were embedded and ultrathin sections were incubated with antibodies and subsequently labeled with protein A-gold. The bisulfite reductase in all three strains and APS reductase in d. gigas and D. vulgaris were found in the cytoplasm. The labeling of d. thermophilus with APS reductase antibodies resulted in a distribution of gold particles over the cytoplasmic membrane region. The localization of the two enzymes is discussed with respect to the mechanism and energetics of dissimilatory sulfate reduction.

Activation, reduction and proton-deuterium exchange reaction of the periplasmic hydrogenase from Desulfovibrio gigas in relation with the role of cytochrome c3.

The enzyme hydrogenase catalyzes the reversible oxidoreduction of the dihydrogen molecule, according to the reaction: Ha * 2H’ t 2e[ 11. Depending on the microorganisms and on the environmental conditions [2] the overall balance of the reaction can be directed towards either the production or the consumption of hydrogen gas. With the purified enzyme it is always possible with an adequate electron donor or acceptor to let the reversible reaction go in one or the other direction but the activity exhibited strongly depends upon the nature of the redox agent, especially its potential, and of the medium conditions [3]. The H+-D2 (or D’-HZ) exchange reaction is part of the reversible activity of hydrogenase [4] and provides an intrinsic measure of this activity. Since the overall balance is nil the exchange should proceed in the absence of any electron donor or redox substance. Yet, whereas the reaction readily takes place with the living cells [S], crude extracts [6] or even partly purified hydrogenases [7], the isolated enzyme in contrast has to be first activated for the exchange to proceed. This is generally achieved by addition of dithionite [6] or, in the case of Desulfovibrio, of the specific cytochrome c3 [8]; however both agents may interfere in several ways with the exchange reaction. The problem arises from the involvement in the loss and recovery of hydrogenase activity of different processes such as oxidation and reduction, oxygenation and deoxygenation. Dithionite for instance scavenges oxygen from the medium and from the enzyme centers and at the same time reduces hydrogenase [6]. Moreover, its action is altered according to the pH conditions [9]. As for cytochrome c3 it can act once reduced as a simple oxygen scavenger or, as it had been reported for Desuffovibrio vulgaris Miyazaki [8,10], it can accelerate both Hz evolution and the exchange reaction. On the contrary such a stimulation of Hz evolution has not been observed with Desulfovibrio gigas hydrogenase after addition of cytochrome c3 from that same organism [ Ill. It was therefore of interest to check whether in the case of the latter species cytochrome c3 had an effect upon the exchange reaction. This work shows that with the purified periplasmic hydrogenase from D. gigas the addition of an electron carrier or of a reducing agent was unnecessary either for the exchange reaction or for the activation of the enzyme. In the latter process, two successive steps were observed: (1) Requiring that oxygen be excluded from the enzyme centers but also probably implying a modification in these centers; this step could be achieved by physical or chemical means and was only accelerated in the presence of dithionite or cytochrome c3. (2) Consisting in a spontaneous reduction of the enzyme under molecular hydrogen and following the same kinetics whether cytochrome c3 was present or not.

Inhibition studies of three classes of Desulfovibrio hydrogenase: application to the further characterization of the multiple hydrogenases found in Desulfovibrio vulgaris Hildenborough

The three types of hydrogenase hitherto characterized in genus Desulfovibrio exhibit distinctive inhibition patterns of their proton-deuterium exchange activity by CO, NO and NO2-. The (Fe) and (NiFeSe) hydrogenases are the most sensitive to all three inhibitors while the (NiFe) enzymes, relatively little inhibited by CO, are still very sensitive to NO but unaffected by NO2-. These differences together with some specific catalytic properties, in particular the pH profile and the H2 to HD ratio in the exchange reaction, constitute a simple means of characterizing multiple hydrogenases present in one or different species.

The pH dependence of proton-deuterium exchange, hydrogen production and uptake catalyzed by hydrogenases from sulfate-reducing bacteria

Different patterns have been found in the pH dependence of hydrogenase activity with enzymes purified from different species of Desulfovibrio. With the cytoplasmic hydrogenase from Desulfovibrio baculatus strain 9974, the pH optima in H2 production and uptake were respectively 4.0 and 7.5 with a higher activity in production than in uptake. The highest D2-H+ exchange activity was found also at pH 4.0 but the optima differed for the HD and the H2 components. Both similarly rose when the pH decreased from 9.0 to 4.5, but the rate of H2 evolution slowed whereas the HD evolution continued rising till pH values around 3.0 were reached. The H2 to HD ratio at pH above 4.5 was higher than one. With the periplasmic hydrogenase from Desulfovibrio vulgaris Hildenborough, the highest exchange activity was near pH 5.5, the same value as in hydrogen production. The periplasmic hydrogenase from Desulfovibrio gigas had in contrast the same pH optimum in the exchange (7.5-8.0) as in the H2 uptake. The ratio of H2 to HD was below one for both enzymes. These different patterns may be related to functional and structural differences in the three hydrogenases so far studied, particularly in the composition of their catalytic centers.

Thermodesulfovibrio hydrogeniphilus sp. nov., a new thermophilic sulphate-reducing bacterium isolated from a Tunisian hot spring

A new thermophilic sulphate-reducing bacterium (strain Hbr5T) was enriched and isolated from a terrestrial Tunisian hot spring. It was a non-spore-forming Gram-negative curved or vibrio-shaped bacterium. It appeared singly or in long chains and was actively motile by a polar flagellum. It possessed c-type cytochromes and desulfofuscidin. Growth occurred between 50 and 70 degrees C, with an optimum of 65 degrees C at pH 7.1. In the presence of sulphate as a terminal electron acceptor, this strain readily used H2 but formate only poorly. It could use sulphate, thiosulphate, sulphite or arsenate as electron acceptors. Its DNA G+C content was 36.1 mol%. Based on phenotypic, genomic, and phylogenetic characteristics, strain Hbr5T (=DSM 18151T, =JCM 13991T) is proposed to be assigned to a novel species of genus Thermodesulfovibrio, T. hydrogeniphilus sp. nov.

ESR studies of cytochrome c3 from Desulfovibrio desulfuricans strain Norway 4: Midpoint potentials of the four haems, and interactions with ferredoxin and colloidal sulphur

Abstract ESR spectra were recorded of cytochrome c 3 from Desulfovibrio desulfuricans , strain Norway 4, after poising at different redox potentials in the presence of dye mediators. The spectra of the four different haems were resolved by their different g values and redox potentials. Estimation of the midpoint potentials indicated that there are two higher-potential haems (both −125 ± 15 mV) and two lower-potential haems (−305 and −325 ± 15 mV). The ESR spectrum of the oxidized cytochrome was slightly shifted by interaction with ferredoxin I from D. desulfuricans , Norway strain. Addition of the oxidizing substrate, colloidal sulphur, to the cytochrome c 3 reduced in the presence of hydrogen + hdrogenase, caused a partial reoxidation of the cytochrome, as judged by ESR and spectrophotometry. If redox titrations were carried out in the presence of colloidal sulphur, only the two higher-potential haems could be reduced, with potentials − 110 ± 15 mV. A mechanism of sulphur reduction by an exposed, low-potential haem is discussed. The reaction may involve insoluble S 8 molecules as reaction intermediates.

Comparative studies of two ferredoxins from Desulfovibrio desulfuricans Norway

Two ferredoxins isolated from Desulfovibrio desulfuricans Norway have been purified and characterized. The less acidic, designated as ferredoxin I, contains four iron atoms, four acid-labile sulfur groups and six cysteine residues per molecule. Ferredoxin II is more acidic and abundant than ferredoxin I, but is very unstable to O2. Ferredoxin I and ferredoxin II differ according to amino acid composition but are homologous with respect to their N-terminal amino acid sequence. The absorption spectra of the two ferredoxins are similar to those of other Desulfovibrio species. Both proteins appear to be dimers of identical 6000-dalton subunits. Their activity was tested in two types of reaction in the electron transfer chain (phosphoroclastic reaction and sulfite reductase activity). The isolation of two different ferredoxins from the same organism, Desulfovibrio, has been reported in Desulfovibrio africanus but the significance of two ferredoxins functioning in the same electron transfer chain is not yet understood.