Chitin catabolism in the marine bacterium Vibrio furnissii. Identification, molecular cloning, and characterization of A N, N'-diacetylchitobiose phosphorylase.
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The major product of bacterial chitinases is N,N'-diacetylchitobiose or (GlcNAc)(2). We have previously demonstrated that (GlcNAc)(2) is taken up unchanged by a specific permease in Vibrio furnissii (unlike Escherichia coli). It is generally held that marine Vibrios further metabolize cytoplasmic (GlcNAc)(2) by hydrolyzing it to two GlcNAcs (i.e. a "chitobiase "). Here we report instead that V. furnissii expresses a novel phosphorylase. The gene, chbP, was cloned into E. coli; the enzyme, ChbP, was purified to apparent homogeneity, and characterized kinetically. The DNA sequence indicates that chbP encodes an 89-kDa protein. The enzymatic reaction was characterized as follows. (GlcNAc)(2)+P(i) GlcNAc-alpha-1-P+GlcNAc K'(cq)=1.0+/-0.2 Reaction 1 The K(m) values for the four substrates were in the range 0.3-1 mm. p-Nitrophenyl-(GlcNAc)(2) was cleaved at 8.5% the rate of (GlcNAc)(2), and p-nitrophenyl (PNP)-GlcNAc was 36% as active as GlcNAc in the reverse direction. All other compounds tested displayed +info)
Differentiation of chitinase-active and non-chitinase-active subpopulations of a marine bacterium during chitin degradation.
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The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97-N-acetyl-beta-D-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase. (+info)
Spatial and temporal variations in chitinolytic gene expression and bacterial biomass production during chitin degradation.
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Growth of the chitin-degrading marine bacterium S91 on solid surfaces under oligotrophic conditions was accompanied by the displacement of a large fraction of the surface-derived bacterial production into the flowing bulk aqueous phase, irrespective of the value of the surface as a nutrient source. Over a 200-h period of surface colonization, 97 and 75% of the bacterial biomass generated on biodegradable chitin and a nonnutritional silicon surface, respectively, detached to become part of the free-living population in the bulk aqueous phase. Specific surface-associated growth rates that included the cells that subsequently detached from the substrata varied depending on the nutritional value of the substratum and during the period of surface colonization. Specific growth rates of 3.79 and 2.83 day(-1) were obtained when cells first began to proliferate on a pure chitin film and a silicon surface, respectively. Later, when cell densities on the surface and detached cells as CFU in the bulk aqueous phase achieved a quasi-steady state, specific growth rates decreased to 1.08 and 0.79 day(-1) on the chitin and silicon surfaces, respectively. Virtually all of the cells that detached from either the chitin or the silicon surfaces and the majority of cells associated with the chitin surface over the 200-h period of surface colonization displayed no detectable expression of the chitin-degrading genes chiA and chiB. Cells displaying high levels of chiA-chiB expression were detected only on the chitin surface and then only clustered in discrete areas of the surface. Surface-associated, differential gene expression and displacement of bacterial production from surfaces represent adaptations at the population level that promote efficient utilization of limited resources and dispersal of progeny to maximize access to new sources of energy and maintenance of the population. (+info)
Suramin inhibits initiation of defense signaling by systemin, chitosan, and a beta-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells.
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Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and beta-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog (125)I-Tyr-2, Ala-15-systemin to the systemin receptor with an IC(50) of 160 microM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptor-mediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspension-cultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemin-induced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding. (+info)
Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1 mutant of Saccharomyces cerevisiae.
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Deletion of the 1,3-beta-D-glucan synthase gene FKS1 in Saccharomyces cerevisiae induces a compensatory mechanism that is reflected in a significant increase in chitin synthase III (CSIII) activity, leading to high rates of chitin synthesis. Deregulation of CSIII activity is mainly due to the intracellular delocalization of Chs3p and Chs4p, the two main components of the CSIII active complex. (+info)
Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba.
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An antifungal peptide from leaves of Ginkgo biloba, designated GAFP, has been isolated. Its molecular mass of 4244.0 Da was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation. GAFP exhibited antifungal activity towards Pellicularia sasakii Ito, Alternaria alternata (Fries) Keissler, Fusarium graminearum Schw. and Fusarium moniliforme. Its activities differed among various fungi. GAFP could also cause increased hyphal membrane permeabilization and a rapid alkalization of the medium when applied at 100 microgram/ml to Pellicularia sasakii Ito hyphae. The amino acid sequence of GAFP shows characteristics of the cysteine/glycine-rich chitin binding domain of many chitin binding proteins. The cysteine residues are well conserved. (+info)
Overproduction of an inducible extracellular serine protease improves biological control of Pythium ultimum by Stenotrophomonas maltophilia strain W81.
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Stenotrophomonas maltophilia W81 can protect sugar beet against PYTHIUM:-mediated damping-off disease through the production of an extracellular protease. Here, the proteolytic enzyme of W81 was purified by anion-exchange chromatography and characterized as a serine protease. The purified enzyme was fungicidal against PYTHIUM: ultimum in vitro. Its synthesis was inducible by casein in W81, and mutagenesis of this strain using the luciferase (luxAB) reporter transposon Tn5-764cd resulted in the isolation of two mutant derivatives (W81M3 and W81M4) capable of producing significantly increased levels of extracellular protease in the presence of casein. Strain W81M4 also exhibited increased chitinolytic activity. The luxAB fusions in strains W81M3 and W81M4 were highly expressed in the absence of casein but not in its presence, suggesting that the corresponding loci were involved in down-regulating extracellular protease production. Extracellular protease production in the W81 wild-type strain and protease overproduction in mutants W81M3 and W81M4 were also induced in the presence of the autoclaved fungal mycelium. In soil microcosms naturally infested by PYTHIUM: spp., inoculation of sugar beet seeds with W81M3 or W81M4 resulted in improved biocontrol of PYTHIUM:-mediated damping-off disease compared with W81, and the level of protection achieved was equivalent to that conferred by chemical fungicides. The wild-type W81 and its mutant derivatives did not differ in rhizosphere colonization. Therefore, the improved biocontrol ability of W81M3 and W81M4 resulted from their capacity to overproduce extracellular serine protease. (+info)
Chitin synthesis in a gas1 mutant of Saccharomyces cerevisiae.
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The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1 delta. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene. However, deletion of the CHS1 gene did not affect the phenotype of the gas1 delta mutant and only slightly reduced the chitin content. Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity. CHS3 expression was not affected in the gas1 mutant. Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin. (+info)