Alain Dolla

Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains

BackgroundDue to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions.ResultsThe protein composition of the secretome produced by industrial T. reesei (strain CL847) grown on a medium promoting the production of both cellulases and hemicellulases was explored using two-dimensional electrophoresis and MALDI-TOF or LC-MS/MS protein identification. A total of 22 protein species were identified. As expected, most of them are potentially involved in biomass degradation. The 2D map obtained was then used to compare the secretomes produced by CL847 and another efficient cellulolytic T. reesei strain, Rut-C30, the reference cellulase-overproducing strain using lactose as carbon source and inducer of cellulases.ConclusionThis study provides the most complete mapping of the proteins secreted by T. reesei to date. We report on the first use of proteomics to compare secretome composition between two cellulase-overproducing strains Rut-C30 and CL847 grown under similar conditions. Comparison of protein patterns in both strains highlighted many unexpected differences between cellulase cocktails. The results demonstrate that 2D electrophoresis is a promising tool for studying cellulase production profiles, whether for industrial characterization of an entire secretome or for a more fundamental study on cellulase expression at genome-wide scale.

Function of oxygen resistance proteins in the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough.

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.

Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H(2) as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H(2) was also present, growth in lactate- and sulfate-containing media lacking H(2) was less efficient. The hyd mutant produced, transiently, significant amounts of H(2) under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688-3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.

A New Function of the Desulfovibrio vulgaris Hildenborough [Fe] Hydrogenase in the Protection against Oxidative Stress

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, have developed a set of reactions allowing them to survive in oxic environments and even to reduce molecular oxygen to water. D. vulgaris contains a cytoplasmic superoxide reductase (SOR) and a periplasmic superoxide dismutase (SOD) involved in the elimination of superoxide anions. To assign the function of SOD, the periplasmic [Fe] hydrogenase activity was followed in both wild-type and sod deletant strains. This activity was lower in the strain lacking the SOD than in the wild-type when the cells were exposed to oxygen for a short time. The periplasmic SOD is thus involved in the protection of sensitive iron-sulfur-containing enzyme against superoxide-induced damages. Surprisingly, production of the periplasmic [Fe] hydrogenase was higher in the cells exposed to oxygen than in those kept in anaerobic conditions. A similar increase in the amount of [Fe] hydrogenase was observed when an increase in the redox potential was induced by addition of chromate. Viability of the strain lacking the gene encoding [Fe] hydrogenase after exposure to oxygen for 1 h was lower than that of the wild-type. These data reveal for the first time that production of the periplasmic [Fe] hydrogenase is up-regulated in response to an oxidative stress. A new function of the periplasmic [Fe] hydrogenase in the protective mechanisms of D. vulgaris Hildenborough toward an oxidative stress is proposed.

A sequential electron transfer from hydrogenases to cytochromes in sulfate-reducing bacteria.

A central step in the energy metabolism of sulfate-reducing bacteria is the oxidation of molecular hydrogen, catalyzed by a periplasmic hydrogenase. The resulting electrons are then transferred to various electron transport chains and used for cytoplasmic sulfate reduction. The complex formation between [NiFeSe] hydrogenase and the soluble periplasmic polyheme cytochromes from Desulfomicrobium norvegicum was characterized by cross-linking experiments, BIAcore and kinetics analysis. Analysis of electron transfer between [NiFeSe] hydrogenase and octaheme cytochrome c(3) (M(r) 26¿ omitted¿000) pointed out that this cytochrome is reduced faster in the presence of catalytic amounts of tetraheme cytochrome c(3) (M(r) 13¿ omitted¿000) isolated from the same organism. The activation of the hydrogenase-dependent reduction of polyheme cytochromes by cytochrome c(3) (M(r) 13¿ omitted¿000), which is now described in both Desulfovibrio and Desulfomicrobium, is proposed as a general mechanism. During this process, cytochrome c(3) (M(r) 13¿ omitted¿000) would act as an electron shuttle in between hydrogenase and the polyheme cytochromes and its conductivity appears to be an important factor.

Anaerobic oxidation of long-chain n -alkanes by the hyperthermophilic sulfate-reducing archaeon, Archaeoglobus fulgidus

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n-alkenes, was shown to oxidize saturated hydrocarbons (n-alkanes in the range C10–C21) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n-hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n-alkane (C16) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.

The First Genomic and Proteomic Characterization of a Deep-Sea Sulfate Reducer: Insights into the Piezophilic Lifestyle of Desulfovibrio piezophilus

Desulfovibrio piezophilus strain C1TLV30T is a piezophilic anaerobe that was isolated from wood falls in the Mediterranean deep-sea. D. piezophilus represents a unique model for studying the adaptation of sulfate-reducing bacteria to hydrostatic pressure. Here, we report the 3.6 Mbp genome sequence of this piezophilic bacterium. An analysis of the genome revealed the presence of seven genomic islands as well as gene clusters that are most likely linked to life at a high hydrostatic pressure. Comparative genomics and differential proteomics identified the transport of solutes and amino acids as well as amino acid metabolism as major cellular processes for the adaptation of this bacterium to hydrostatic pressure. In addition, the proteome profiles showed that the abundance of key enzymes that are involved in sulfate reduction was dependent on hydrostatic pressure. A comparative analysis of orthologs from the non-piezophilic marine bacterium D. salexigens and D. piezophilus identified aspartic acid, glutamic acid, lysine, asparagine, serine and tyrosine as the amino acids preferentially replaced by arginine, histidine, alanine and threonine in the piezophilic strain. This work reveals the adaptation strategies developed by a sulfate reducer to a deep-sea lifestyle.

Anaerobic Oxidation of Fatty Acids and Alkenes by the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus

ABSTRACT Archaeoglobus fulgidus oxidizes fatty acids (C4 to C18) and n-alk-1-enes (C12:1 to C21:1) in the presence of thiosulfate as a terminal electron acceptor. End products of metabolism were CO2 and sulfide. Growth on perdeuterated hexadecene yielded C15- to C17-labeled fatty acids as metabolites, thus confirming the ability of A. fulgidus to oxidize alkyl chains.

The protein moiety modulates the redox potential in cytochromes c

Cytochrome c is one of the most thoroughly documented oxidoreduction proteins. Its electron transfer activity, which involves an association between the heme group and the polypeptidic chain, is correlated with the redox potential value of the heme group. The redox potential covers a wide range up to 0.8 V, an extreme case being observed in the low-potential cytochromes c from sulfate reducing bacteria. On of the main roles of the polypeptidic moiety consists of modulating the redox potential value of the heme group. In this paper, some structural factors that seem likely to be involved in maintaining the redox potential value are described.

Escherichia coli is able to produce heterologous tetraheme cytochrome c3 when the ccm genes are co-expressed

The production of Desulfovibrio vulgaris Hildenborough cytochrome c(3) (M(r) 13000), which is a tetraheme cytochrome, in Escherichia coli was examined. This cytochrome was successfully produced in an E. coli strain co-expressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. The apocytochrome c(3) was matured in either anaerobic or aerobic conditions, but aerobic growth in the presence of delta-aminolevulinic acid was found to be best for cytochrome c(3) production. Site-directed mutagenesis was performed to investigate the effect of the presence of four amino acids in between the two cysteines of the heme binding sites 2 and 4 on the maturation of holocytochrome c(3) in E. coli.