Denitrification by Chromobacterium violaceum

One host (Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO₃. Final growth yields increased in direct proportion to the quantity of added NaNO₃ over the range of 0.5 to 5 mM. Each strain reduced NO₃⁻, producing NO₂⁻, NO, and N₂O. NO₂⁻ accumulated transiently. With 2 mM NaNO₃ in the medium, N₂O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C₂H₂ (added to block N₂O reduction) was present or not. Neither N₂ production nor gas accumulation was detected during NO₃⁻ reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO₂ in lieu of NaNO₃ was delayed, and although N₂O was produced by the end of growth, NO₂⁻ -containing media did not support growth to an extent greater than did medium lacking NO₃⁻ or NO₂⁻. The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N₂O, and that NO₂⁻ reduction to N₂O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N₂ (reduced C₂H₂).     

Drug resistance in Chromobacterium violaceum

Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world. This bacterium is able to produce a large variety of products of biotechnological and pharmacological use. Although C. violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported. Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes. A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism. Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous. In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found. It is postulated that these genes contribute to the ability of C. violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium.     

DNA repair in Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.     

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.     

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.     

Chemotaxis and flagellar genes of Chromobacterium violaceum

The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism. This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C. violaceum. C. violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome. We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C. violaceum. A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins. The chemoreceptors found in C. violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C. violaceum genome. Also, the C. violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system. Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C. violaceum genome.     

Biosynthesis of a trypanocide by Chromobacterium violaceum

Radio-isotope studies indicated not only that l-tryptophan can serve as carbon source for synthesis of the trypanocide, violacein by Chromobacterium violaceum (BB-78 strain) but also that isatin and indole 3-acetic acid are both important metabolic intermediates. Using 3-indolyl [2-¹⁴C] and [1-¹⁴C] acetic acid, it was found that the carboxylic carbon was not eliminated and that indole-3-acetic acid was incorporated intact into the pigment structure. N-Ethyl(5-hydroxy-indol-3-yl)-2-indolylethylamide is also an important metabolic intermediate in the violacein biosynthesis. This is the first report of a metabolic scheme for violacein synthesis which includes an intermediate other than l-tryptophan.     

Chromobacterium violaceum infections in 13 non-human primates

Background Recently, an Indian‐origin macaque was found dead and Chromobacterium violaceum was isolated from the skin wound, and hepatic and pulmonary abscesses. Methods By searching the database, a total of thirteen cases of C. violaceum infection in pigtail macaques (n = 8), rhesus macaques (n = 4), and one baboon were identified from 2001 to 2010 at Tulane National Primate Research Center. Medical records were reviewed for breed, sex, age, clinical findings, treatment, outcome, bacteriology, and gross and histological findings. Results Seven pigtail macaques and one Indian‐origin rhesus macaque died of chromobacterial septicemia. All chromobacterial septicemic pigtail macaques were adult with higher incidence in female. Hepatic abscess and thrombosis were typical findings along with pulmonary abscess and thrombosis, renal venous thromboembolism, and necrosuppurative pleuritis, peritonitis, splenitis, myocarditis, pericarditis, and meningoencephalitis. Skin wound, uterine infection, and oral and respiratory exposure were considered the points of entry for these animals. Conclusions This represents the first report of chromobacteriosis in pigtail, rhesus macaque, and baboon. Our experience suggests that chromobacterial infections may be more common in non‐human primates than previously recognized.     

Anthranilate synthase subunit organization in Chromobacterium violaceum

Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).     

Identification of secreted virulence factors of Chromobacterium violaceum

Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identifying a large number of genes that demonstrate the vast biotechnological potential of this bacterium, genome sequencing revealed several virulence factors, including different cytolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by identifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute secreted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence similarity to previously characterized bacterial porins.     

Transport genes of Chromobacterium violaceum: an overview

The complete genome sequence of the free-living bacterium Chromobacterium violaceum has been determined by a consortium of laboratories in Brazil. Almost 500 open reading frames (ORFs) coding for transport-related membrane proteins were identified in C. violaceum, which represents 11% of all genes found. The main class of transporter proteins is the primary active transporters (212 ORFs), followed by electrochemical potential-driven transporters (154 ORFs) and channels/pores (62 ORFs). Other classes (61 ORFs) include group translocators, transport electron carriers, accessory factors, and incompletely characterized systems. Therefore, all major categories of transport-related membrane proteins currently recognized in the Transport Protein Database (http://tcdb.ucsd.edu/tcdb) are present in C. violaceum. The complex apparatus of transporters of C. violaceum is certainly an important factor that makes this bacterium a dominant microorganism in a variety of ecosystems in tropical and subtropical regions. From a biotechnological point of view, the most important finding is the transporters of heavy metals, which could lead to the exploitation of C. violaceum for bioremediation.     

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule.     

Cellulose Biosynthesis by the Beta-Proteobacterium, Chromobacterium violaceum

The Chromobacterium violaceum ATCC 12472 genome was sequenced by The Brazilian National Genome Project Consortium. Previous annotation reported the presence of cellulose biosynthesis genes in that genome. Analysis of these genes showed that, as observed in other bacteria, they are organized in two operons. In the present work, experimental evidences of the presence of cellulose in the extracellular matrix of the biofilm produced by C. violaceum in static cultures are shown. Biofilm samples were enzymatically digested by cellulase, releasing glucose units, suggesting the presence of cellulose as an extracellular matrix component. Fluorescence microscopy observations showed that C. violaceum produces a cellulase-sensitive extracellular matrix composed of fibers able to bind calcofluor. C. violaceum grows on medium containing Congo red, forming brown-red colonies. Together, these results suggest that cellulase-susceptible matrix material is cellulose. Scanning electronic microscopy analysis showed that the extracellular matrix exhibited a network of microfibrils, typical of bacterial cellulose. Although cellulose production is widely distributed between several bacterial species, including at least the groups of Gram-negative proteobacteria alpha and gamma, we give for the first time experimental evidence for cellulose production in beta-proteobacteria.     

Stable transformation of Chromobacterium violaceum with a broad-host-range plasmid

Stable transformants of Chromobacterium violaceum were obtained by high-voltage electroporation with a 7-kilobase binary plasmid. The technique was reliable, reproducible, and simple, with efficiencies of 10⁵ transformants/μg of plasmid DNA. The electrical conditions that resulted in the highest efficiencies were short pulse length (4.4–4.5 ms) and high voltage (12.5 kV/cm). The numbers of transformants were almost the same during the growth exponential phase (variation at optical density) and resulted in the highest efficiencies at DNA concentration of 250 pg/ml. Saturation appeared to begin at 4 μg/ml of DNA. This method of C. violaceum transformation should enhance the genetic and biotechnological research by providing a valuable, widely used procedure of introducing DNA into this bacterium.