Identification and partial characterisation of an extracellular activator of fatty acid modifying enzyme (FAME) expression in Staphylococcus epidermidis.
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Fatty acid modifying enzyme (FAME) is an extracellular enzyme that inactivates bactericidal fatty acids by esterifying them to cholesterol. Inactivation of these fatty acids may allow Staphylococcus epidermidis to live for long periods of time on the skin. This study describes the identification and partial characterisation of an extracellular activator of FAME production. Addition of FAME-free concentrated culture filtrate (activator) to S. epidermidis cultures (OD600 = 0.05) caused a 3-5-fold increase in FAME activity. Addition of the activator did not increase the amount of exopolysaccharide produced by S. epidermidis. The mol. wt of this activator was <3000 kDa and it was quite resistant to boiling. Treatment of the activator with proteinase K did not destroy its ability to induce FAME expression. Addition of S. aureus activator to S. epidermidis cultures also increased FAME expression. However, when S. epidermidis activator was added to S. aureus cultures no increase or inhibition in FAME production was observed. (+info)
Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.
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Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined. (+info)
A pyruvated mannose-specific xanthan lyase involved in xanthan degradation by Paenibacillus alginolyticus XL-1.
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The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55 degrees C. (+info)
Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1.
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An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp. strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765-3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide. (+info)
Enhancement of solubilization and biodegradation of polyaromatic hydrocarbons by the bioemulsifier alasan.
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Alasan, a high-molecular-weight bioemulsifier complex of an anionic polysaccharide and proteins that is produced by Acinetobacter radioresistens KA53 (S. Navon-Venezia, Z. Zosim, A. Gottlieb, R. Legmann, S. Carmeli, E. Z. Ron, and E. Rosenberg, Appl. Environ. Microbiol. 61:3240-3244, 1995), enhanced the aqueous solubility and biodegradation rates of polyaromatic hydrocarbons (PAHs). In the presence of 500 microg of alasan ml-1, the apparent aqueous solubilities of phenanthrene, fluoranthene, and pyrene were increased 6.6-, 25.7-, and 19.8-fold, respectively. Physicochemical characterization of the solubilization activity suggested that alasan solubilizes PAHs by a physical interaction, most likely of a hydrophobic nature, and that this interaction is slowly reversible. Moreover, the increase in apparent aqueous solubility of PAHs does not depend on the conformation of alasan and is not affected by the formation of multimolecular aggregates of alasan above its saturation concentration. The presence of alasan more than doubled the rate of [14C]fluoranthene mineralization and significantly increased the rate of [14C]phenanthrene mineralization by Sphingomonas paucimobilis EPA505. The results suggest that alasan-enhanced solubility of hydrophobic compounds has potential applications in bioremediation. (+info)
Broadly protective vaccine for Staphylococcus aureus based on an in vivo-expressed antigen.
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Vaccines based on preferential expression of bacterial antigens during human infection have not been described. Staphylococcus aureus synthesized poly-N-succinyl beta-1-6 glucosamine (PNSG) as a surface polysaccharide during human and animal infection, but few strains expressed PNSG in vitro. All S. aureus strains examined carried genes for PNSG synthesis. Immunization protected mice against kidney infections and death from strains that produced little PNSG in vitro. Nonimmune infected animals made antibody to PNSG, but serial in vitro cultures of kidney isolates yielded mostly cells that did not produce PNSG. PNSG is a candidate for use in a vaccine to protect against S. aureus infection. (+info)
A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.
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The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase. (+info)
Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide.
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In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics. (+info)