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Archives of Microbiology (v.181, #3)
Protein phosphorylation on tyrosine in bacteria by Alain J. Cozzone; Christophe Grangeasse; Patricia Doublet; Bertrand Duclos (pp. 171-181).
Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts. The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight. Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type. In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome. In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides. Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors. Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity.
Keywords: Bacterial protein phosphorylation; Tyrosine kinase; Tyrosine phosphatase; Phosphotyrosine; Exopolysaccharides; Capsular polysaccharides; Signal transduction; Bacterial pathogenicity
Genes involved in the anaerobic degradation of toluene in a denitrifying bacterium, strain EbN1 by Michael Kube; Johann Heider; Judith Amann; Peter Hufnagel; Simon Kühner; Alfred Beck; Richard Reinhardt; Ralf Rabus (pp. 182-194).
The organization of all genes required for the anaerobic conversion of toluene to benzoyl-CoA was investigated in denitrifying Azoarcus-like strain EbN1. All of these genes are clustered within 25.3 kb of contiguous DNA sequence, which includes only a few intervening sequences. The toluene-catabolic genes are organized in two apparent operons. One contains the genes (bssCAB) for the three subunits of benzylsuccinate synthase, which initiates anaerobic toluene degradation by converting toluene to (R)-benzylsuccinate. The BssCAB proteins of strain EbN1 are most similar to those of Thauera aromatica strain K172. The bssCAB genes are part of a larger putative operon (bssDCABEFGH), which contains the gene bssD, encoding the activase for benzylsuccinate synthase, and four genes (bssEFGH) encoding proteins of unknown function. RT-PCR experiments showing continuation of transcription over the three largest intergenic regions of the bss operon support the assumed structure. Moreover, BssG was identified as toluene-induced protein. Downstream of the bss genes, another large putative operon (bbsA–H) was identified that contains all genes required for β-oxidation of benzylsuccinate to benzoyl-CoA, e.g. bbsEF, encoding succinyl-CoA:(R)-benzylsuccinate CoA-transferase. Immediately upstream of the bss operon, genes for a two-component regulatory system were identified; their products may sense toluene and induce the expression of both catabolic operons. The order and sequences of the bss and bbs genes are highly similar among toluene-degrading denitrifiers. The bss and bbs genes of the FeIII-reducing Geobacter metallireducens display less sequence similarity and are organized differently. The genes between the bss and bbs operons and in the flanking regions differ between strain EbN1 and the other strains.
Keywords: Toluene; Denitrifying bacterium; Anaerobic degradation; Gene prediction; Gene annotation; Genomics; Proteomics
Chorismate mutase of Thermus thermophilus is a monofunctional AroH class enzyme inhibited by tyrosine by Kerstin Helmstaedt; Gabriele Heinrich; Rainer Merkl; Gerhard H. Braus (pp. 195-203).
aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-α/β barrel enzyme that is organized as a trimer. Comparison of the enzyme’s structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the protein’s surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase. The kinetic data show Michaelis-Menten substrate saturation with a K m of 290 μM, and a k cat/K m value of 180 s−1 mM−1. TtCM was inhibited by tyrosine with a K i =34 μM, possibly in a competitive manner.
Keywords: Chorismate mutase; aroG ; TtCM; BsCM; AroH; AroQ
Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of Aspergillus parasiticus by Li-Wei Lee; Ching-Hsun Chiou; Karen L. Klomparens; Jeffrey W. Cary; John E. Linz (pp. 204-214).
The biosynthesis of aflatoxin in Aspergillus parasiticus is a complex process that involves the activities of at least 18 pathway enzymes. The distribution of these enzymes within fungal colonies and fungal cells is not clearly understood. The objective of this study was to investigate the distribution and subcellular location of Nor-1, Ver-1, and OmtA, which represent early, middle, and late enzymatic activities, respectively, in the aflatoxin biosynthetic pathway. The distribution of these three enzymes within A. parasiticus SU-1 was analyzed in time-fractionated, 72-h fungal colonies (fraction 1, 48–72 h; fraction 2, 24–48 h; fraction 3, 0–24 h). Western blot analysis and immunofluorescence microscopy demonstrated the highest abundance of Nor-1, Ver-1, and OmtA in colony fraction 2. Fungal tissues in this fraction were analyzed by immunoelectron microscopy. Nor-1 and Ver-1 were primarily localized to the cytoplasm, suggesting that they are cytosolic enzymes. OmtA was also detected in the cytoplasm. However, in cells located near the basal (substrate) surface of the colony, OmtA was predominantly detected in organelles tentatively identified as vacuoles. The role of this organelle in toxin biosynthesis is unclear. The relative distribution of OmtA to the cytoplasm or to vacuole-like organelles may depend on the age and/or physiological condition of the fungal cells.
Keywords: Aspergillus parasiticus ; OmtA; Nor-1; Ver-1; Vacuoles; Immunoelectron microscopy; Immunofluorescence microscopy; Microbodies; Woronin bodies
Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in Propionibacterium freudenreichii by Pauline Leverrier; Johannes P. C. Vissers; Annette Rouault; Patrick Boyaval; Gwénaël Jan (pp. 215-230).
Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.
Keywords: Propionibacterium ; Probiotic; Stress; Acid; Bile salts; Cross-protection; Proteomics; Quadrupole/time-of-flight mass spectrometry; Nanoscale LC-MS/MS; De novo sequencing
In Saccharomyces cerevisiae, the effect of H2O2 on ATP, but not on glyceraldehyde-3-phosphate dehydrogenase, depends on the glucose concentration by Hugo Osório; Pedro Moradas-Ferreira; María A. Günther Sillero; Antonio Sillero (pp. 231-236).
As has been previously shown, Saccharomyces cerevisiae grown in 2% or 0.025% glucose uses this carbohydrate by the fermentative or oxidative pathways, respectively. Depending on the glucose concentration in the medium, the effect of the addition of H2O2 on the level of ATP and on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity differed. In the presence of 2% glucose, ATP and GAPDH decreased sharply during the first few minutes of treatment, whereas in the presence of 0.025% glucose, GAPDH activity decreased similarly, but the ATP level remained practically unchanged. The addition of 3 mM glutathione to the culture media prevented the depletion of ATP levels and GAPDH activity in the presence of H2O2. Catalase and superoxide dismutase activities did not vary significantly when yeast cells were grown either in 2% or in 0.025% glucose.
Keywords: Saccharomyces cerevisiae ; ATP; Glyceraldehyde-3-phosphate dehydrogenase; Inosine; H2O2 ; Oxidative stress
Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum by Shih-Kuang Hsu; Long-Liu Lin; Hsueh-Hsia Lo; Wen-Hwei Hsu (pp. 237-244).
Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 μM phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K m and k cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K m and k cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.
Keywords: Prephenate dehydratase; Site-directed mutagenesis; Feedback inhibition; Catalytic activity; Corynebacterium glutamicum
Phenyl methyl ethers: novel electron donors for respiratory growth of Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S by Anke Neumann; Tina Engelmann; Roland Schmitz; Yvonne Greiser; Adelheid Orthaus; Gabriele Diekert (pp. 245-249).
Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor. The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds. O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO2 as electron acceptor. One mol phenyl methyl ether R-O-CH3 was O-demethylated to R-OH per 3 mol fumarate reduced to succinate. The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted. D. hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers. Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate. Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation. This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.
Keywords: Desulfitobacterium hafniense ; Desulfitobacterium sp. strain PCE-S; Reductive dehalogenation; O-demethylation; O-demethylase; Phenyl methyl ether; Vanillate; Syringate; Fumarate reduction; Dehalorespiration
Specificity grouping of the accessory gene regulator quorum-sensing system of Staphylococcus epidermidis is linked to infection by A. B. Carmody; Michael Otto (pp. 250-253).
Staphylococcus epidermidis represents the most frequent pathogen involved in nosocomial infections and infections of indwelling medical devices. The strain-to-strain variation of the gene encoding the quorum-sensing pheromone of S. epidermidis as well as the correlation between specificity groups and origin from infection were determined. The pro-pheromone gene was highly conserved and showed infrequent, non-synonymous, single-nucleotide polymorphisms that led to conservative amino acid exchanges only. Importantly, one specificity group was significantly more frequent among strains isolated from infection. The finding that quorum-sensing specificity groups are linked to infection demonstrates the relevance of quorum-sensing for virulence in this critical human pathogen and contributes to the scientific basis needed for the development of quorum-sensing-targeting drugs.
Keywords: Staphylococcus epidermidis ; Infection; Quorum-sensing