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The respiratory system of Chromobacterium violaceum grown under conditions of high and low cyanide evolution.

The particulate fraction of disrupted Chromobacterium violaceum grown under cyanide-evolving conditions was unable to oxidize ascorbate plus N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD), but oxidized NADH and succinate by a linear respiratory pathway which was very resistant to inhibition by cyanide. When the bacteria were grown under conditions where little cyanide evolution occurred, particulate fractions developed the ability to oxidize ascorbate-TMPD by a pathway highly sensitive to cyanide inhibition; respiratory activity with NADH and succinate proceeded via both the cyanide-sensitive and-resistant pathways. Studies with respiratory inhibitors, and the cytochrome compositions of the fractions derived from cultures grown under both conditions, are presented. A soluble, carbon monoxide-binding cytochrome c was found, and this appears similar to those found recently in Beneckea natiegens, methylotrophic bacteria and the marine pseudomonad B16.

Antimycin A↗

Production and properties of the native Chromobacterium violaceum fucose-binding lectin (CV-IIL) compared to homologous lectins of Pseudomonas aeruginosa (PA-IIL) and Ralstonia solanacearum (RS-IIL).

Chromobacterium violaceum is a versatile, violet pigment (violacein)-producing beta-proteobacterium, confined to tropical and subtropical regions, dwelling in soil and water, like Pseudomonas aeruginosa and Ralstonia solanacearum. These three bacteria are saprophytes that occasionally become aggressive opportunistic pathogens virulently attacking animals (the first two) and plants (the third). The recent availability of their genome sequences enabled identification in the C. violaceum genome of an ORF (locus no. 1744) that is similar to those of P. aeruginosa and R. solanacearum lectins, PA-IIL and RS-IIL, respectively. A recombinant protein, CV-IIL, encoded by that ORF exhibited fucose>mannose-specific lectin activity resembling PA-IIL. This paper describes production and properties of the native CV-IIL, which, like PA-IIL and RS-IIL, is probably also a quorum-sensing-driven secondary metabolite, appearing concomitantly with violacein. Its formation is repressed in the CV026 mutant of C. violaceum, which lacks endogenous N-acylhomoserine lactone. The upstream extragenic sequence of its ORF contains a 20 bp sequence (5'-101-120) with partial similarities to the luxI-box and the related P. aeruginosa and R. solanacearum promoter boxes of quorum-sensing-controlled genes. The lectin level is augmented by addition of trehalose to the medium. The subunit size of CV-IIL (around 11.86 kDa) is similar to those of PA-IIL (11.73 kDa) and RS-IIL (11.60 kDa). Like PA-IIL, in the tetrameric form CV-IIL preferentially agglutinates alpha1-2 fucosylated H-positive human erythrocytes (regardless of their A, B or O type), as opposed to the O(h) Bombay type, but differs from it in having no interaction with rabbit erythrocytes and in displaying stronger affinity to l-galactose than to l-fucose. The greater similarity of CV-IIL to PA-IIL than to RS-IIL might be related to the selective adaptation of both C. violaceum and P. aeruginosa to animal tissues versus the preferential homing of R. solanacearum to plants.

Bacterial Proteins↗

The chemical structure of the lipid A component of lipopolysaccharides from Chromobacterium violaceum NCTC 9694.

The chemical structure of the lipid A component of lipopolysaccharides from Chromobacterium violaceum NCTC9694 was studied. Sequential treatment of lipopolysaccharide with alkali, acid, sodium borohydride and hydrazine allowed the isolation of a reduced glucosamine disaccharide. According to methylation studies and enzymic analysis with beta-N-acetylglucosaminidase the D-glucosamine residues are beta(1 leads to 6) linked. The disaccharide carries two phosphate groups, one being linked glycosidically, the other being linked as an ester to the non-reducing glucosamine. Application of a different degradation pathway shows that the ester-bound phosphate group is substituted by a 4-aminoarabinosyl residue and that the glycosidically linked phosphate group is substituted by a glucosaminyl residue. Neither the amino nor the hydroxyl groups of both these substituents are acylated. This backbone structure is shown in the following formula: (formula: see text). The amino groups of the central glucosamine disaccharide are substituted by D-3-hydroxy-dodecanoic acid, the hydroxyl groups by dodecanoic, L-2-hydroxydodecanoic and D-3-hydroxy-decanoic acid.

Acetylglucosamine↗

Genetic characterization of Chromobacterium isolates from black water environments in the Brazilian Amazon.

AIMS: To isolate and to characterize the diversity of Chromobacterium violaceum from the Brazilian Amazon region. METHODS AND RESULTS: Twenty-two isolates were obtained from the waters and banks of the river Negro, in the Brazilian Amazon. All isolates were able to grow in vitro at 44 degrees C and pH 4.0, but were adversely affected by temperatures below 15 degrees C, and unable to survive at 4 degrees C, properties that may be related to the adaptation to the ecosystem. The isolates were joined at a final level of similarity of only 13% in the rep-PCR analysis. The analysis of 16S rRNA genes resulted in three main groups clustered at a final level of similarity of 97% and only three isolates were clustered with the type strain. Similar data were obtained for the 23S rRNA gene. CONCLUSIONS: A high level of genetic diversity was verified with indications that the Brazilian isolates would fit into at least two new clusters besides C. violaceum species. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show remarkable bacterial adaptability and genetic diversity of C. violaceum in the Amazon region.

Brazil↗

The Serratia-type hemolysin of Chromobacterium violaceum.

Chromobacterium violaceum is a Gram-negative opportunistic human pathogen and an inhabitant of tropical soils and waterways. Although known primarily for the synthesis of the pigment violacein, and more recently as a reporter strain for quorum sensing, clinical reports of chromobacteriosis comprise the largest block of published literature on this organism. Genome sequencing has revealed many potential virulence factors in this microorganism, and this paper establishes the presence in C. violaceum of a Serratia type-hemolysin (ChlA) and transporter (ChlB). We also show that the hemolysin operon includes a third gene (chlC) that is predicted to encode a phosphorylation domain similar to the receiver domain of response regulators in bacterial signal transduction systems.

Animals↗

Cloning, molecular analysis, and expression of the polyhydroxyalkanoic acid synthase (phaC) gene from Chromobacterium violaceum.

The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaC(Cv)) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaC(Cv) and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaA(Cv)). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes and Ralstonia eutropha (formerly Alcaligenes eutrophus), harboring phaC(Cv), accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. coli nor recombinant Pseudomonas putida harboring phaC(Cv) accumulated significant levels of PHA. Sequence analysis of the phaC(Cv) product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no. AAD10274).

Acyltransferases↗

Effect of cold temperatures on the viability of Chromobacterium violaceum.

The effect of low, nonfreezing temperatures on the viability of five strains of Chromobacterium violaceum was studied. The viability of cultures grown at 30 C was determined after exposure to various diluents held at 0 to 2 C. A culture diluted at its growth temperature served as the control. Cells of strain N were most sensitive in the early part of the exponential phase of growth. Cells of strains 252 and 341 were most sensitive in the late exponential, early stationary phase of growth. Cells of strain 9 showed greatest loss of viability during the maximal stationary phase. Strain 69 was completely resistant throughout its growth cycle to cold injury. Cell viability was much greater in buffered salts solution than in distilled water, broth, or physiological saline, whether cultures were diluted at room temperature or in the cold. The proportion of cells surviving after exposure to cold, however, was the same regardless of the composition of the diluent. Loss of viability was progressive at 0 to 2 C and reached a maximum after 2 hr. There was no loss of viability of cells exposed to 20 C, but there was some loss at 12 C. Strain 341 cultivated at 15 C was much less sensitive to 0 to 2 C than when it was cultivated at 30 C. The composition of the growth medium seemed to have no effect on the survival of cells exposed to cold. The polyamines, spermine and trimethylenediamine, as well as erythritol and sucrose, exerted some protective action against the effects of cold but not uniformly for all strains studied.

Chromobacterium↗

Mouse and human cell activation by N-dodecanoyl-DL-homoserine lactone, a Chromobacterium violaceum autoinducer.

Chromobacterium violaceum produces autoinducers, including homoserine lactones (HSLs), for genetic regulation. Among the seven HSLs derived from C. violaceum we evaluated, only C(12)-HSL stimulated the production of inflammatory cytokines in mammalian monocytic cell lines through the activation of the NF-kappaB signaling pathway besides their quorum-sensing role, like 3-oxo-C(12)-HSL from Pseudomonas aeruginosa.

4-Butyrolactone↗

Phagocyte NADPH oxidase, but not inducible nitric oxide synthase, is essential for early control of Burkholderia cepacia and chromobacterium violaceum infection in mice.

Reactive oxygen and nitrogen intermediates have critical, partially overlapping roles in host defense against a variety of pathogens. Using mice deficient in generating phagocyte superoxide (p47(phox)(-/-)) and mice deficient in generating inducible nitric oxide synthase (iNOS(-/-)), we examined the roles of these reactive species in host defense against Burkholderia cepacia and Chromobacterium violaceum, organisms known to have unusual virulence in chronic granulomatous disease. Intraperitoneal B. cepacia challenge (4.0 x 10(3) to 4.0 x 10(5) organisms/mouse) resulted in mortality in all p47(phox)(-/-) mice, with the survival interval being inversely proportionate to the amount of inoculum. Pretreatment with gamma interferon did not affect survival. C. violaceum was strikingly virulent in p47(phox)(-/-) mice (the 50% lethal dose [LD(50)] was <13 organisms). iNOS(-/-) and wild-type mice were resistant to B. cepacia challenges of at least 10(6) organisms per mouse, and the LD(50) of C. violaceum was between 10(6) and 10(7) organisms per mouse. Consistent with the survival data, numbers of organisms in cultures of B. cepacia from multiple sites were higher for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice at day 4 after challenge, but numbers of organisms for different B. cepacia strains varied. The recovery of C. violaceum was strikingly greater at 18 h after challenge for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice, in which the organism burdens were virtually nil. In vitro, both B. cepacia and C. violaceum were sensitive to H(2)O(2) and to reactive nitrogen intermediates but the sensitivities of different strains varied significantly. Host defense against B. cepacia and C. violaceum is critically dependent in vivo on reactive oxygen intermediates, and these species are model organisms to further dissect host and pathogen interactions related to the generation and scavenging of microbicidal reactive intermediates.

Animals↗

Mesosome structure in Chromobacterium violaceum.

Exponentially growing cells of the gram-negative bacterium Chromobacterium violaceum demonstrate invaginations of the cytoplasmic membrane with a high frequency. These invaginations conform to the ultrastructural appearance of mesosomes of gram-positive bacteria. As many as four mesosomes are observed per cell, each of which may increase the total membrane surface of the cell by 30%. Washing of cells in dilute tris(hydroxymethyl)aminomethane buffer effects a distension of the mesosome "neck" and/or cytoplasmic membrane clarifying the association of the mesosome to the cytoplasmic membrane. Plasmolysis effects an eversion of the mesosome into the plasmolysis vacuole.

Buffers↗

Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing.

Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N-hexanoyl-L-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 microM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.

Acetylglucosaminidase↗

Tryptophan synthetic pathway and its regulation in Chromobacterium violaceum.

Extracts of Chromobacterium violaceum catalyzed all of the reactions involved in synthesizing tryptophan from chorismic acid. Tryptophan auxotrophs which had lost any of these activities did not produce the characteristic purple pigment, violacein, when grown on a medium in which tryptophan was limiting. Gel filtration of extracts allowed us to estimate molecular weights for the tryptophan enzymes. All of the enzymes appeared to have molecular weights below 100,000. No enzymes were observed to occur in aggregates. The specific activities of the enzymes of the tryptophan pathway did not change when mutants were grown under conditions of limiting or excess tryptophan. The first enzyme in the pathway, anthranilate synthetase, was subject to feedback control by the end product, tryptophan. Tryptophan acted as a noncompetitive inhibitor with respect to glutamine, one of the substrates for anthranilate synthetase, and as a competitive inhibitor of the reaction when chorismate, the other substrate, was varied. The nonlinearity observed in the Lineweaver-Burk plot in the latter case suggests that there may be more than one chorismate-binding site on anthranilate synthetase.

Cell-Free System↗

Beta-cyanoalanine formation by Chromobacterium violaceum.

Nonproliferating cells of Chromobacterium violaceum incubated with glycine, methionine, and succinate as substrates accumulated beta-cyanoalanine in the culture fluid. Tracer experiments showed that carbons-2, -3, and -4 of beta-cyanoalanine are derived from the 2-carbon of glycine. When methionine-methyl-(14)C, succinate-1,4-(14)C, or succinate-2,3-(14)C was used as substrate, beta-cyanoalanine did not become labeled. If K(14)CN and serine were used as substrates, the cyano group of beta-cyanoalanine was labeled. Radioactive beta-cyanoalanine, labeled in the 3-carbon, was formed when glycine and H(14)CHO were used as substrates. (14)C-formic acid did not replace formaldehyde. Asparagine also accumulated in the incubated mixture and was found to be labeled in the amide carbon. Incubation of cells with beta-cyanoalanine-4-(14)C produced labeled aspartic acid in cell hydrolysates.

Alanine↗

Effect of enzymes on the composition and structure of Chromobacterium violaceum cell envelopes.

Cell envelopes of Chromobacterium violaceum were isolated and treated under controlled conditions with trypsin, Pronase, lipase, phospholipase C, lysozyme, and a mixture of enzymes produced by a bacteriolytic Pseudomonas sp. After each enzyme treatment, losses in dry weight, protein, lipid, carbohydrate, 2,6-diaminopimelic acid, and total phosphorus were determined. Electron-microscopic examination of the enzyme-treated envelopes indicated complete or partial loss of envelope rigidity or some envelope fragmentation, or both. Each enzyme hydrolyzed at least one envelope component and liberated several others into the supernatant fluid, where they appeared as nondialyzable particulate components, identified by means of electron microscopy. Unlike the other enzymes, the Pseudomonas sp. enzyme mixture partially liberated all major envelope components except phosphorus, heptose, and 2-keto-3-deoxy octonic acid. In spite of these large losses, the envelopes preserved some features of their integrity and elongated shape.

Bacterial Proteins↗