Bacterium Chromobacterium Violaceum Research Articles

Two-Step Bioleaching and Spent Medium Leaching of Gold from Electronic Scrap Material Using <i>Chromobacterium violaceum</i>

Rapid technological advancement and the relatively short life time of electronic goods have resulted in an alarming growth rate of electronic waste which often contains significant quantities of toxic and precious metals. Compared to conventional chemical recovery methods, bioleaching has been shown to be an environmentally friendly process for metal extraction. In this work, gold bioleaching from electronic scrap material (ESM) was examined using batch cultures of the bacterium Chromobacterium violaceum which produces cyanide as a secondary metabolite. Gold was bioleached via gold cyanide complexation. The ESM was pretreated using nitric acid to dissolve the base metals (mainly copper) in order to reduce competition for the cyanide ion from other metals present in ESM. ESM was added to the bacterial culture after it reached maximum cyanide production during early stationary phase. Spent medium bioleaching using bacterial cell- free metabolites showed a higher gold recovery of 18%, compared to that of two-step bioleaching of 11% at 0.5% w/v pulp density of ESM. Gold bioleaching was further enhanced to 30% when the pH of the spent medium was increased to shift the equilibrium in favor of cyanide ions production. Spent medium bioleaching of pretreated ESM yield a higher gold recovery compared to two-step bioleaching at a pulp density of 0.5% w/v.

Vestigialization of arsenic resistance phenotypes/genotypes in Chromobacterium violaceum strains thriving in pristine Brazilian sites

The performance of the arsenic resistance arsRCB operon of the ubiquitous bacterium Chromobacterium violaceum has been employed as a descriptor of the genomic persistence of traits for long-ceased environmental conditions. To this end, a reliable reference for arsenic resistance/sensitivity was created in the C. violaceum ATCC 12472-type strain by deleting its genomic ars operon. This allowed tracing all of the arsenic resistance of this bacterium to the ars gene cluster. A sample of 15 environmental C. violaceum strains isolated from pristine Amazonian sites (both rain forest and savannah) were then examined for the presence of arsRCB amplicons, for arsenic resistance, and for indications of decay of ars genes into pseudogenes. While the sites of origin of such strains have no record of industrial or geological exposure to arsenic, all of them bore the standard arsRCB cluster in their genome, but they did differ in their resistance to the metalloid. This ranged from complete sensitivity to tolerance levels above the reference C. violaceum ATCC12472. Inspection of relevant DNA sequences, phenotypic analyses and monitoring expression profiles of the ars genes in each strain exposed a distinct genetic drift of the system toward vestigialization, that is, accumulation of mutations leading to the loss of a non-used character. In particular, quantitative PCR revealed inducibility by arsenic to be the first trait to be lost in the route toward decaying and eventual nonfunctionalization. The data presented fit evolutionary predictions on elimination of non-used characters through a mere genetic drift accumulating neutral mutations.

Evaluation of bioleaching factors on gold recovery from ore by cyanide-producing bacteria

The present study was conducted to investigate the gold bioleaching factors from ore by cyanide producing bacterium Chromobacterium violaceum. The optimal condition for cyanide production by C. violaceum was pH 9 and 5g/L of glycine in YP medium in 2-days of incubation. In shake flask culture, gold bioleaching from the ores by C. violaceum was determined with various experimental protocols such as particle size, pre-grown C. violaceum, pH, and biooxidative treatment. The three types of low grade ores viz., R, S, and H were used. The gold bioleaching efficiencies were recorded as 0%, 50%, and 5% for ores R, S, and H, respectively, when C. violaceum culture was used without any pretreatment (protocol 1). In the experimental protocol involving grinding and pre-grown C. violaceum, leaching efficiencies increased to 60%, 100%, and 40% for ore R, S, and H samples, respectively. Especially, the bioleaching efficiency of ore S enhanced to almost 100% with pre-grown C. violaceum (protocol 2) and that of ore R increased to 53% (i.e., 96% of cyanidable gold) with grinding (protocol 3) due to their mineralogical characteristics of the ores. For refractory gold (i.e., ore R) grinding as pretreatment was needed, and for ore S (almost all of the gold was cyanidable) cyanide production was activated by using pre-grown bacteria. Biooxidation with Acidithiobacillus ferrooxidans and pH adjustment (i.e., 9–11) did not affect the bioleaching efficiencies. The mineralogical cause of gold refractoriness was analyzed by automated SEM that showed most of gold in the ore was entrapped in pyrite and silica. The results indicated that gold bioleaching by C. violaceum from low grade ore can be enhanced by grinding and pre-grown microbe; use of appropriate experimental condition is important according to the mineralogical characteristics.

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.

Potential applications of violacein: a microbial pigment

Violacein is a versatile pigment from a bacterium Chromobacterium violaceum that exhibits several biological activities and, at present, has gained increasing importance in industrial markets, such as in medicine, cosmetics, and textiles. In this mini-review, we aimed to describe violacein production and to explore its various biological properties in a pharmacological context, including its antioxidant, immunomodulatory, antitumoral, and antiparasitic activities. In addition, its use in the fields of cosmetics, textiles, food, toys, and insecticides has emerged as unusual potential areas of application to be discussed here.

A Strategy for Antagonizing Quorum Sensing

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ∼60 Å, twice the ∼30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multidomain proteins.

Inhibition of Quorum Sensing in the Opportunistic Pathogenic Bacterium Chromobacterium violaceum by an Extract from Fruiting Bodies of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (W.Curt.:Fr.) P. Karst. (Higher Basidiomycetes)

Extracts of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, inhibited quorum sensing in Chromobacterium violaceum CV026. G. lucidum fruiting bodies were milled and extracted with ethyl acetate. The crude extract was dissolved in an appropriate concentration of methanol, sterilized by filtration through a 0.22-μm membrane filter, and added to Ch. Violaceum CV026 cultures, which were used as an indicator to monitor quorum sensing inhibition. Inhibitory activity was measured by quantifying violacein production using a microplate reader. Methanol-soluble compounds extracted from G. lucidum significantly inhibited quorum sensing-controlled behavior in Ch. Violaceum in a concentration-dependent manner. The results suggest that compounds in G. lucidum might be useful to control and handle detrimental infections caused by human, animal, and plant pathogens. Further studies are in progress in our lab to isolate the specific compounds from G. lucidum extract, evaluate them as quorum sensing inhibitors, and analyze their mechanism of action.

Effect of the cyanide-producing bacterium Chromobacterium violaceum on ultraflat Au surfaces

Solubilization, transport and re-precipitation of Au in the supergene environment can lead to the formation of secondary Au enrichment zones, revealed as geochemical anomalies and secondary gold grains. Cyanide-producing microorganisms can contribute to the solubilization of Au, and the cyanide produced may play an important role in the formation of ‘bacterioform’ structures observed on gold grains. To examine the effect of bacterial cyanide production on Au surfaces, ultra-flat Au foil was incubated in peptone meat extract (PME) medium in the presence of the cyanide-producing bacterium Chromobacterium violaceum for up to 56 days. Within seven days C. violaceum had formed biofilms on the Au surfaces, and cyanide produced by the bacteria had a measurable effect on the Au surface compared to abiotic controls. After removal of the biofilm by sonication in de-ionized water, the surfaces incubated with C. violaceum were significantly rougher compared to the negative controls. This was demonstrated by root mean square roughness (RMS) analyses and statistical t-testing following scanning tunneling microscopy (STM). Analyses of Au and cyanide in the growth medium supported the STM results: After 56 days of incubation 125 parts-per-billion (ppb) of Au was detected in solution or associated with C. violaceum cells. In contrast, Au solubilization and cyanide formation was not observed in negative controls. These experiments provide evidence that bacteria produce metabolites capable of mediating Au solubilization that may lead to the dispersion of Au in the environment, and suggest that dissolution of Au may contribute to the formation of ‘bacterioform’ structures commonly observed on secondary gold grains.

Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.

Ecosystem protection by effluent bioremediation: silver nanoparticles impregnation in a textile fabrics process

This work studied a bioremediation process of silver nanoparticles with the bacterium Chromobacterium violaceum. These nanoparticles were obtained from several washes of cotton fabrics impregnated with silver nanoparticles produced by the fungus Fusarium oxysporum. The optimized growth of C. violaceum for silver nanoparticles bioremediation was obtained. The effluents of wash process of the cotton fabric were efficiently treated with C. violaceum. This treatment was based on biosorption which was very efficient for the elimination of silver nanoparticles remaining in the wash water. The bacteria after biosorption were morphologically transformed, but the normal morphology after a new culture was completely restored. The process also allowed the recovery of silver material that was leached into the effluent for a reutilization avoiding any effect to the eco-environment.

PPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli

pPSY is a 12 kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10 kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and sub-cloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent l-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A ( recA) and an amylase ( amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividans amyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli.

Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia

Chronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Bcc (Burkholderia cepacia complex) is the major cause of morbidity and mortality among CF (cystic fibrosis) patients. PA-IIL (lecB gene), a soluble lectin from Ps. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologues have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centres, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA (B. cenocepacia lectin A), was obtained. The presence of native BclA in B. cenocepacia extracts was checked using a proteomic approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with methyl alpha-D-mannoside demonstrates a dissociation constant (K(d)) of 2.75 x 10(-6) M. The X-ray crystal structure of the complex with methyl alpha-D-mannoside was determined at 1.7 A (1 A=0.1 nm) resolution. The lectin forms homodimers with one binding site per monomer, acting co-operatively with the second dimer site. Each monomer contains two Ca2+ ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site and oligomerization mode indicate that the proteins should have different roles in the bacteria.

The Violacein Biosynthetic Enzyme VioE Shares a Fold with Lipoprotein Transporter Proteins

VioE, an unusual enzyme with no characterized homologues, plays a key role in the biosynthesis of violacein, a purple pigment with antibacterial and cytotoxic properties. Without bound cofactors or metals, VioE, from the bacterium Chromobacterium violaceum, mediates a 1,2 shift of an indole ring and oxidative chemistry to generate prodeoxyviolacein, a precursor to violacein. Our 1.21 A resolution structure of VioE shows that the enzyme shares a core fold previously described for lipoprotein transporter proteins LolA and LolB. For both LolB and VioE, a bound polyethylene glycol molecule suggests the location of the binding and/or active site of the protein. Mutations of residues near the bound polyethylene glycol molecule in VioE have identified the active site and five residues important for binding or catalysis. This structural and mutagenesis study suggests that VioE acts as a catalytic chaperone, using a fold previously associated with lipoprotein transporters to catalyze the production of its prodeoxyviolacein product.

Unusual Entropy-Driven Affinity of Chromobacterium violaceum Lectin CV-IIL toward Fucose and Mannose,

The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.

Microbe-metal-interactions for the biotechnological treatment of metal-containing solid waste

In nature, microbes are involved in weathering of rocks, in mobilization of metals from minerals, and in metal precipitation and deposition. These microbiological principles and processes can be adapted to treat particulate solid wastes. Especially the microbiological solubilization of metals from solid minerals (termed bioleaching) to obtain metal values is a well-known technique in the mining industry. We focus here on non-mining mineral wastes to demonstrate the applicability of mining-based technologies for the treatment of metal-containing solid wastes. In the case study presented, microbial metal mobilization from particulate fly ash (originating from municipal solid waste incineration) by Acidithiobacilli resulted in cadmium, copper, and zinc mobilization of >80%, whereas lead, chromium, and nickel were mobilized by 2, 11 and 32%, respectively. In addition, the potential of HCN-forming bacteria ( Chromobacterium violaceum, Pseudomonas fluorescens) was investigated to mobilize metals when grown in the presence of solid materials (e.g., copper-containing ores, electronic scrap, spent automobile catalytic converters). C. violaceum was found capable of mobilizing nickel as tetracyanonickelate from fine-grained nickel powder. Gold was microbially solubilized as dicyanoaurate from electronic waste. Additionally, cyanide-complexed copper was detected during biological treatment of shredded printed circuit-board scraps. Water-soluble copper and platinum cyanide were also detected during the treatment of spent automobile catalytic converters.

Calorimetric study of the antibacterial activity of sodium n-alkylsulfates on the metabolism of Chromobacterium violaceum

The bioactivity of a series of sodium n-alkylsulfates (C6-C10 and C12) was studied with flow calorimetry to follow in real time the calorimetric effect on the metabolic rate of the bacterium Chromobacterium violaceum. All the compounds showed a linear plot of the fraction of control metabolic heat rate against log (dose). From these plots the value of dosemax (the dose producing zero metabolic heat rate) for each compound was evaluated. The value of dosemax is correlated with the chain length of the molecule, showing that their biological activity is directly proportional to the lipophilicity of the compound.

Ingestion of bacteria in suspension Indian major carps (Catla catla, Labeo rohita) and Chinese carps (Hypophthalmichthys molitrix, Aristichthys nobilis)

Single-species groups of fry of the planktivorous, filter-feeding carps (silver carp Hypophthalmichthys molitrix, bighead carp Aristichthys nobilis, rohu Labeo rohita and catla Catla catla) were exposed to the bacterium Chromobacterium violaceum to assess if they could detect or ingest unattached bacteria. The results indicate that planktivorous carp fry are able to ingest unattached bacteria and that the numbers ingested increase with time and with bacterial concentration in the media, although probably by passive means. It is also suggested that unattached bacteria are of little nutritional significance to carps.

The ingestion of bacteria in suspension by the tilapia Oreochromis niloticus

Tilapia ( Oreochromis niloticus) fry were exposed to concentrations of the bacterium Chromobacterium violaceum in order to establish whether they can detect and ingest unattached bacteria. Groups of fish exposed to both bacteria and to cell-free bacterial extracts showed a significant increase in opercular beat rates, thus demonstrating that they are able to detect the presence of unattached bacteria in suspension. Examination of the stomach contents showed that bacteria were ingested and that there was a significant, positive correlation between ingestion and concentration in the trial media.

Serine Hydroxymethyltransferase and Glycine Cleavage Enzyme from the Cyanogenic Bacterium Chromobacterium violaceum

Chromobacterium violaceum forms cyanide as a secondary metabolite with glycine as the precursor and methionine as a stimulator. As the metabolism of glycine and methionine are interrelated by the provision of carbon-one units for the tetrahydrofolate pool by the activities of serine hydroxymethyltransferase (SHMT) and glycine cleavage enzyme (GCE), we have investigated the regulation of formation and activity of these enzymes by C. violaceum. The synthesis of SHMT was unaffected by growth under conditions of high cyanogenesis (on a medium containing glutamate, glycine and methionine) or low cyanogenesis (on glutamate alone), nor was its synthesis affected by addition of potential end-products of carbon-one metabolism to the growth medium. The activity of GCE was very low, less than 1% of the activity of SHMT, irrespective of the growth conditions. This suggests that nearly all the carbon-one units for the tetrahydrofolate pool are supplied by the activity of SHMT; this could lead to excess formation of glycine and might explain the role of cyanogenesis. The activity of SHMT was competitively inhibited by glycine and cysteine.