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PubMed1994

Regioselectivity and fatty acid specificity of Chromobacterium viscosum lipase.

The fatty acid specificity of Chromobacterium viscosum lipase was studied by comparing the pseudo-first-order rate constants for the transesterification of different fatty acid methyl esters with 1-propanol in dry acetonitrile as solvent. It was found that this enzyme shows a significant preference towards long chain fatty acids and, for chains with the same length, towards saturated ones. The same enzyme was used to study the esterification of sorbitol and decanoic acid. A mixture of mono-, di-, tri- and tetraesters was obtained. The concentration of esters was strongly increased upon raising the temperature from 35 to 70 degrees C. The structures of the di-, tri- and tetraesters were determined using 13C NMR spectrometry. The diester appeared to be sorbitol 1,6-didecanoate, the triester was sorbitol 1,5,6-tridecanoate and the tetraester was the 1,2,5,6-tetradecanoate, which indicates that the C. viscosum lipase acylates sorbitol in a regioselective manner.

Chromobacterium↗
PubMed2006

Alginate-chaperoned facile refolding of Chromobacterium viscosum lipase.

Urea denatured lipase from Chromobacterium viscosum lipase could be refolded by addition of alginate with high guluronic acid content. The refolded molecule could be recovered by affinity precipitation. This approach resulted in recovery of 80% (of original activity) as compared to classical dilution method which gave only 21% activity recovery. Dynamic light scattering showed that binding required about 45 min and activity data obtained from affinity precipitation experiments indicated that refolding was almost instantaneous after binding. Circular dichroism (CD) and fluorescence data showed that refolded molecule was identical to the native molecule. It also showed that refolding takes place at the binding stage and not at the precipitation stage. Preliminary studies showed that the refolding strategy worked equally well with lipases from wheat germ and porcine pancreas.

Alginates↗
PubMed2004

YM-254890 analogues, novel cyclic depsipeptides with Galpha(q/11) inhibitory activity from Chromobacterium sp. QS3666.

The structure elucidation and biological activity of novel YM-254890 (1) analogues and semi-synthetic derivatives are described. Three natural analogues, YM-254891 (2), YM-254892 (3), and YM-280193 (4), were isolated from the fermentation broth of Chromobacterium sp. QS3666, and two hydrogenated derivatives, YM-385780 (5) and YM-385781 (6), were synthesized from YM-254890. Their structures were determined by one- and two-dimensional NMR studies and mass spectrometry. Among these compounds, two natural analogues 2-3 which possessed acyl groups at beta-HyLeu-1 and one derivative 6 whose conformation was similar to that of 1 showed comparable Galpha(q/11) inhibitory activity to that of 1. This indicates that the acyl beta-HyLeu residue plays an important role in activity and also that the alpha,beta-unsaturated carbonyl group of the N-MeDha residue is not critical to activity. The other hydrogenated derivative 5 had significantly less activity, which could be attributed to conformational differences.

Adenosine Diphosphate↗
PubMed2005

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis.

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.

Bacterial Typing Techniques↗
PubMed2004

Flow microcalorimetric studies of phenol and its chlorinated derivatives and a theoretical evaluation of their possible inhibition mode on Chromobacterium violaceum respiration.

The general belief that chemical structure determines the biological effect of drugs has led to several techniques to establish structure-activity relationships (SAR) that is useful in the development of more active compounds. Predicting toxic effects based on SAR, one can obtain toxicological data with a low cost-benefit ratio. Chlorophenols that represent a class of toxic agents frequently used in industrial processes are not satisfactorily described in the literature in relation to their toxicity. The main objective of this work is to relate the microbial activities of phenol, anisole and their chlorinated derivatives on Chromobacterium violaceum respiration with their physicochemical properties. Anisole and its chlorinated derivatives were used to evaluate the influence of phenol acidity on biological activity. The calculations were carried out at the semi-empirical AM1 and ab initio DFT levels employing the basis sets CEP-31G, CEP-31+Ge CEP-31G** that were parameterized using the continuum-solvation model COSMO for solvent contribution. Both empirical and theoretical properties were evaluated by chemometric analyses (hierarchical cluster analysis (HCA) and principal component analysis (PCA)), to correlate the physicochemical properties of the phenol, anisole and their chlorinated derivatives with their biological activities. The results obtained for the current work indicate that the biological activities of these compounds increase as the n-octanol/water (logP) partition coefficients, ionization energies (IP), melting points (mp) and dissociation constants increase and the solvent effects (SE), enthalpies of formation (DeltafH degrees ) and proton affinities (PA) decrease.

Anisoles↗
PubMed2006

Ecthyma gangrenosum and septic shock syndrome secondary to Chromobacterium violaceum.

Chromobacterium violaceum is a gram-negative bacterium of soil and water in tropical and subtropical environments. Typically, it is considered a bacterium of low virulence although, uncommonly, it causes human infection, particularly in persons with defects in host defenses. Infection generally follows exposure of broken skin to contaminated water and soil, and is often characterized by pustules, lymphadenitis, fever, and vomiting, as well as rapid dissemination and a high mortality rate. Unfortunately, because C violaceum is ubiquitous, it is often dismissed as a contaminant when cultured. Because rapid diagnosis (by taking appropriate specimens) and treatment are vital to a good prognosis, it is imperative that physicians be aware of this organism. In addition, patients with chromobacterial infections should have an immunologic workup because infections in immunocompetent individuals are rare. Here we report an aggressive yet nonfatal case of C violaceum septicemia in an adolescent male, diagnosed through a punch biopsy of a skin lesion, and resulting in a new diagnosis of chronic granulomatous disease.

Adolescent↗
PubMed2004

Metal solubilization from metal-containing solid materials by cyanogenic Chromobacterium violaceum.

Different cyanogenic bacterial strains (Chromobacterium violaceum, Pseudomonas fluorescens, Bacillus megaterium) were cultivated under cyanide-forming conditions in the presence of metal-containing solids such as nickel powder or electronic scrap. All microorganisms were able to form water-soluble metal cyanides, however, with different efficiencies. C. violaceum was able to mobilize nickel as tetracyanonickelate [Ni(CN)4(2-)] from fine-grained nickel powder. Gold was microbially solubilized as dicyanaoaurate [Au(CN)2-] from electronic waste. Additionally, cyanide-complexed copper was detected during biological treatment of shredded printed circuit boards scrap. Regarding the formation of tetracyanonickelate, C. violaceum was more effective than P. fluorescens or B. megaterium. Besides a few previous reports on gold solubilization from gold-containing ores or native gold by C. violaceum, the findings demonstrate for the first time the microbial mobilization of metals other than gold from solid materials and represent a novel type of microbial metal mobilization based on the ability of certain microbes to form HCN. The results might have the potential for industrial applications (biorecovery, bioremediation) regarding the treatment of metal-containing solids since metal cyanides can easily be separated by chromatographic means and be recovered by sorption onto activated carbon.

Biodegradation, Environmental↗
PubMed2005

Detection of Chromobacterium violaceum by multiplex PCR targeting the prgI, spaO, invG, and sipB genes.

Based on the recently completed genomic sequence of Chromobacterium violaceum American Type Culture Collection (ATCC) 12472 a multiplex PCR assay targeting the prgI, spaO, invG, and sipB genes of the Salmonella SPI-1 homologue type-III secretion system was developed. PCR products of 255bp (prgI), 749bp (spaO), 1685bp (invG), and 1752bp (sipB) were successfully amplified simultaneously in a single reaction with all Chr. violaceum strains investigated whereas other bacteria tested negative. The detection limit for pure cultures in multiplex PCR analysis was 100CFU. The developed assay significantly improves rapid identification of Chr. violaceum and allows its differentiation from closely related organisms.

Animals↗
PubMed2004

An analysis of type-III secretion gene clusters in Chromobacterium violaceum.

Chromobacterium violaceum is an environmental Gram-negative bacterium that is common in soil and water in tropical and sub-tropical regions. It is also a model organism for studying quorum-sensing and is a rare but deadly human pathogen. Recent completion of the genome sequence of C. violaceum strain ATCC 12472 revealed the presence of genes associated with type-III secretion systems (TTSSs). One of these systems resembles the Spi-1 system found in Salmonella enterica, whereas another is similar to the Spi-2 system from the same organism. Here, we present a detailed analysis and a fresh annotation of the two gene clusters. Moreover, we highlight the presence of several genes encoding putative type-III effector proteins that lead us to predict that this organism can manipulate vesicular trafficking, the actin cytoskeleton and apoptotic pathways within mammalian cells to its own advantage.

Bacterial Proteins↗
PubMed1984

Recurrent infection with Chromobacterium violaceum: first case report from South America.

A case of chromobacteriosis in a young Brazilian with toxaemia and multiple skin abscesses is described. The infection responded to treatment with chloramphenicol and cotrimoxazole but recurred 18 months later following insect bites received while fishing in a river. Chromobacterium violaceum was subsequently isolated from the river water. This is the first case of this kind to be reported from South America.

Abscess↗
PubMed2000

Extraction of violacein from Chromobacterium violaceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers.

Fatty acyl homoserine lactones (AHLs) are used as extracellular quorum sensing signals by a variety of gram-negative bacteria. By activating proteins belonging to the LuxR family of transcriptional regulators, these signal metabolites allow population density-dependent gene regulation within a species, as well as interspecies communication among different bacteria. The experimental detection of AHLs is important in the identification of quorum sensing capabilities in bacteria. Chromobacterium violaceum is a gram-negative bacterium that produces the purple pigment violacein in response to the presence of the AHL N-hexanoyl homoserine lactone (C6HSL). The mini-Tn5 mutant strain C. violaceum CV0blu is deficient in the production of this signal molecule but retains the ability to synthesize violacein in response to the presence of C6HSL and a variety of other short-chain AHLs. We have developed a quantitative bioassay that measures the amount of violacein produced by this strain in response to the presence of different concentrations of various AHL molecules. This new assay provides a means of quantifying the amount of a given AHL present in a bacterial culture and can be used to measure differences in AHL production among different strains or different batch cultures of a given species.

4-Butyrolactone↗
PubMed2000

A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase.

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.

Chemical Phenomena↗
PubMed2001

Expression and partial characterization of an elastase from Chromobacterium violaceum.

Chromobacterium violaceum was recovered at necropsy from the lungs, liver, spleen, and an interscapular abscess of a Chinese red panda (strain 98-9187) [J. Vet. Diagn. Invest. 12 (2000) 177]. As the lungs exhibited extensive, necrotizing lesions harboring bacterial aggregates, we sought to determine whether C. violaceum produced an elastase that might in part account for these lesions. The C. violaceum type strain (ATCC 12472(T)) and strain 98-9187 were shown to exhibit elastolytic activity by elastin Congo red and elastin nutrient agar assays. The activity was isolated from the periplasmic fraction and was present throughout the growth cycle. Activity increased markedly in late logarithmic phase growth. In elastin-limiting medium, activity rapidly decreased in early stationary phase indicating a tight regulation of yield. The activity was optimal at neutral pH and was sensitive to the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. Activity was restored upon addition of zinc indicating the enzyme is a zinc metalloproteinase. A band corresponding to purified elastase activity was present at approximately 30kDa in a denaturing polyacrylamide gel.

Animals↗
PubMed1995

Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing a tetrahydrobiopterin cofactor. Several key mechanistic questions have yet to be resolved, specifically the identity of the hydroxylating species and the role of the non-heme iron which is present in all of the mammalian PAHs. Recently, we have demonstrated that a bacterial PAH from Chromobacterium violaceum does not require any redox active metal for activity [Carr, R. T., & Benkovic, S. J. (1993) Biochemistry 32, 14132-14138]. To identify the function of iron in the mammalian PAH's, we have undertaken a series of experiments to compare the mechanisms of this metal-independent PAH with the iron-dependent PAH from rat liver. Using [4-2H]phenylalanine as a substrate gave a kinetic isotope effect on hydroxylation of unity for CVPAH which is in agreement with previous values reported for RLPAH. The [4-2H]phenylalanine underwent an NIH shift upon hydroxylation by CVPAH. The extent of deuterium retention at the 3-position of the tyrosine product was identical within experimental error for both RLPAH and CVPAH using [4-2H]phenylalanine and [2,3,5,6-2H]phenylalanine as substrates. This suggests that PAH from either source probably does not directly mediate the NIH shift mechanism. No uncoupled pterin turnover was observed for CVPAH with either L-tyrosine or p-chloro-L-phenylalanine as substrate or tetrahydropterin as cofactor, each of which causes uncoupled turnover with RLPAH. CVPAH readily accepts 4-methylphenylalanine as a substrate giving 4-(hydroxymethyl)phenylalanine as the major product and 3-methyltyrosine as the only other minor product.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

X-ray absorption studies of the Cu-dependent phenylalanine hydroxylase from Chromobacterium violaceum. Comparison of the copper coordination in oxidized and dithionite-reduced enzymes.

The coordination chemistry of the Cu sites of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum has been studied by X-ray absorption spectroscopy (XAS). The EXAFS of the Cu(II) form of the enzyme resembles that of other non-blue copper proteins such as plasma amine oxidases and dopamine-beta-hydroxylase and is characteristic of a mixed N/O coordination shell containing histidine ligation. Detailed simulations of the raw EXAFS data have been carried out using full curved-wave restrained refinement methodologies which allow imidazole ligands to be treated as structural units. The results suggest a Cu(II) coordination of two histidines and two additional O/N-donor groups. A reasonable fit to both data sets can be obtained by assuming that the non-imidazole first-shell donor atoms are derived from solvent (H2O or OH-). The EXAFS of the reduced enzyme shows major differences. The amplitude of the first shell in the Fourier transform is only 50% of that of the oxidized enzyme, indicative of a substantial reduction in coordination number. In addition, the first shell of the transform is split into two components. Simulations of the reduced data can be obtained by either two histidines at a long distance of 2.08 A and an O ligand at a short distance of 1.88 A or two histidines at a short distance of 1.90 A and one second-row scatterer such as S or Cl at 2.20 A. Comparison of absorption edge data on the reduced enzyme with data from Cu(I) bis- and tris(1,2-dimethylimidazole) complexes suggests a pseudo-three-coordinate structure.

Chromobacterium↗
PubMed1994

Identification of metal ligands in Cu(II)-inhibited Chromobacterium violaceum phenylalanine hydroxylase by electron spin echo envelope modulation analysis of histidine to serine mutations.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) is known to bind an equivalent of divalent copper. The "metal-free" form of the protein is fully active, and Cu(II) is now shown to be an inhibitor of CVPAH rather than an activator of the enzyme [Carr, R. T., & Benkovic, S. J. (1994) Biochemistry 32, 14132-14138]. On the basis of amino acid sequence homology, the metal binding site may be related to those of rat liver PAH and other eukaryotic pterin-dependent hydroxylases, which require Fe(II) for activity. The conserved histidines at that site in CVPAH, histidines 138 and 143, were each mutated to serines. The mutant enzymes H138S and H143S were both catalytically inactive, but still able to bind Cu(II). Binding studies further demonstrated that both mutant enzymes still bind L-phenylalanine. Electron spin echo envelope modulation (ESEEM) studies on each of the mutants showed the presence of only a single copper-coordinating histidine, rather than the two histidine ligands suggested for the wild-type protein. This result supports a model in which Cu(II) is equatorially ligated to only two histidines in the Cu(II)-inhibited protein and allows us to unambiguously assign histidines 138 and 143 as these ligands. That the enzyme is inactive when these histidines are either bound with copper or when replaced with serines suggests that these histidines perform a catalytic function. Possible catalytic roles for these histidines in the hydroxylation mechanism of pterin-dependent monooxygenases are discussed along with potential future applications of the combination of ESEEM with site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1993

An examination of the copper requirement of phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) was classified as a copper metalloenzyme by virtue of a 1/1 Cu/enzyme stoichiometry and its inhibition with various chelators [Pember, S. O., Villafranca, J. J., & Benkovic, S. J. (1986) Biochemistry 25, 6611]. We have prepared "copper-free" CVPAH by extraction with DTT. These preparations retained full activity though the Cu/enzyme ratio averaged 0.015. Reconstitution by extraneous copper was disproved by measuring a Cu/enzyme ratio of 0.09 in assay mixtures after the specific activity was determined to be within 85% of a fully copper-complexed control. Several copper chelators were examined and were not inhibitory. The "copper-free" enzyme had significant activity without a thiol or other reducing agent capable of reducing the copper center whereas copper-complexed CVPAH had minimal activity under these conditions. Copper-complexed CVPAH can be activated, however, by nonreducing copper ligands such as imidazole. From these results, we conclude that copper is not a requirement of activity. Iron, cobalt, nickel, manganese, molybdenum, and chromium were not found in the "copper-free" preparation, indicating that the active hydroxylating species may not require a redox-active metal. The KdS for binding Cu2+ and Zn2+ were measured to be 0.48 and 0.85 microM, respectively. Both copper and zinc were found to be potent inhibitors of "copper-free" CVPAH in the absence of thiols. DTT reverses inhibition due to Cu2+ but not inhibition caused by Zn2+. The product stoichiometry indicates the same tightly coupled turnover found with all other pterin-dependent hydroxylases when using natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1986

Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme.

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

Chromobacterium↗