Violacein-producing Bacteria Research Articles

Corrigendum: 'Genome sequencing and multifaceted taxonomic analysis of novel strains of violacein-producing bacteria and non-violacein-producing close relatives'.

Corrigendum: 'Genome sequencing and multifaceted taxonomic analysis of novel strains of violacein-producing bacteria and non-violacein-producing close relatives'.

Genome sequencing and multifaceted taxonomic analysis of novel strains of violacein-producing bacteria and non-violacein-producing close relatives.

Violacein is a water-insoluble violet pigment produced by various Gram-negative bacteria. The compound and the bacteria that produce it have been gaining attention due to the antimicrobial and proposed antitumour properties of violacein and the possibility that strains producing it may have broad industrial uses. Bacteria that produce violacein have been isolated from diverse environments including fresh and ocean waters, glaciers, tropical soils, trees, fish and the skin of amphibians. We report here the isolation and characterization of six violacein-producing bacterial strains and three non-violacein-producing close relatives, each isolated from either an aquatic environment or moist food materials in northern California, USA. For each isolate, we characterized traditional phenotypes, generated and analysed draft genome sequences, and carried out multiple types of taxonomic, phylogenetic and phylogenomic analyses. Based on these analyses we assign putative identifications to the nine isolates, which include representatives of the genera Chromobacterium, Aquitalea, Iodobacter, Duganella, Massilia and Janthinobacterium. In addition, we discuss the utility of various metrics for taxonomic assignment in these groups including average nucleotide identity, whole genome phylogenetic analysis and extent of recent homologous recombination using the software program PopCOGenT.

Janthinobacterium kumbetense sp. nov., a violacein-producing bacterium isolated from spring water in Turkey, and investigation of antimicrobial activity of violacein.

Strain GKT was isolated from the Kumbet plateu of Giresun in Turkey. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GKT belonged to genus Janthinobacterium and 16S rRNA gene sequence similarities with all type strains of the genus Janthinobacterium were 98.89%-99.78%. The calculated pairwise average nucleotide identity (ANI) values between strain GKT and all type strains of Janthinobacterium species were in the range of 79.8%-93.2%. In addition, digital DNA-DNA hybridization (dDDH) values were in the range of 23.0%-51.7%. Major fatty acids are C10:03OH, C12:0, C16:1ω7c, C16:0, and C18:1ω7c, and polar lipids included phosphatidylethanolamine, phosphatidylglycerol, also one unidentified phospholipid and one unidentified aminophospholipid. The respiratory quinone of strain GKT was determinated to be Q-8. The genome sizes of strain GKT was 6197538bp with 63.16% G+C ratio. Strain GKT is Gram-stain-negative, aerobic, rod-shaped, and motile. A violet pigment was produced by strain GKT. The crude violacein pigments were separated into three diferent bands on a TLC sheet. Then violacein and deoxyviolacein were purifed by vacuum liquid column chromatography and identifed by NMR spectroscopy. The antimicrobial activities of purifed violacein and deoxyviolacein were screened for seven microorganisms. Based on the results of the morphological, biochemical, physiological, phylogenetic, and genomic characteristics, we propose classifying the strain GKT as representative of a novel species of the genus Janthinobacterium, for which the name Janthinobacterium kumbetense sp. nov. is proposed (GKT=LMG 32662T=DSM 11423T).

Investigation of potential inhibitor properties of violacein against HIV-1 RT and CoV-2 Spike RBD:ACE-2.

A violacein-producing bacterium was isolated from a mud sample collected near a hot spring on Kümbet Plateau in Giresun Province and named the GK strain. According to the phylogenetic tree constructed using 16S rRNA gene sequence analysis, the GK strain was identified and named Janthinobacterium sp. GK. The crude violacein pigments were separated into three different bands on a TLC sheet. Then violacein and deoxyviolacein were purified by vacuum liquid column chromatography and identified by NMR spectroscopy. According to the inhibition studies, the HIV-1 RT inhibition rate of 1 mM violacein from the GK strain was 94.28% and the CoV-2 spike RBD:ACE2 inhibition rate of 2 mM violacein was 53%. In silico studies were conducted to investigate the possible interactions between violacein and deoxyviolacein and three reference molecules with the target proteins: angiotensin-converting enzyme 2 (ACE2), HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain. Ligand violacein binds strongly to the receptor ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of −9.94 kcal/mol, −9.32 kcal/mol, and −8.27 kcal/mol, respectively. Deoxyviolacein strongly binds to the ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of −10.38 kcal/mol, -9.50 kcal/mol, and −8.06 kcal/mol, respectively. According to these data, violacein and deoxyviolacein bind to all the receptors quite effectively. SARS-CoV-2 spike protein and HIV-1-RT inhibition studies with violacein and deoxyviolacein were performed for the first time in the literature.Graphical abstract

Psychrotrophic violacein-producing bacteria isolated from Lake Winnipeg, Canada

Violet and purple bacterial pigmentations are uncommon when considering strict aerobes. Here we report discovery of two novel isolates, P102 and P117 from the freshwater Lake Winnipeg, each with violet to deep purple colony colouration. The relationship between pigment production and growth was investigated under different conditions, including a variety of carbon sources, pH, and temperatures. The violet compounds were purified using polar organic solvents, and then structurally characterized via mass spectrometry and nuclear magnetic resonance to be violacein and deoxyviolacein. Strain P117 produced higher concentrations of the darker pigment deoxyviolacein, which resulted in a deeper hue of violet than in cultures of P102. Strain P102, synthesized violacein maximally in liquid rich organic medium at pH 8 and 20 °C, and had 99.3% sequence similarity to the 16S rDNA from Janthinobacterium lividum. Strain P117, related to Massilia violaceinigra sharing 99.2% 16S rDNA sequence similarity, also produced violacein at similar optimal conditions, but developed higher concentrations of pigment at 15 °C. Culture-based methods found that violacein producers composed 0.001% or 0.023% CFU of environmental heterotrophic populations using BG-11 and potato-based media, respectively. Culture-independent high-throughput ribosomal 16S V4 sequencing of environmental DNA was used to detect Gram-negative species known for their production of violacein. Of all bacterial and archaeal sequences present during the fall in littoral waters and sediment of Lake Winnipeg, 5.5% and 6.3%, respectively, belonged to species that have similar pigment, demonstrating the scarcity of violacein producers in this aquatic freshwater system.

Draft Genome Sequence of Janthinobacterium sp. Strain SLB01, Isolated from the Diseased Sponge Lubomirskia baicalensis.

The draft genome sequence of Janthinobacterium sp. strain SLB01, a violacein-producing psychrotolerant bacterium isolated from the diseased sponge Lubomirskia baicalensis, was determined. We identified five genes encoding VioA, VioB, VioC, VioD, and VioE proteins related to violacein biosynthesis that were like those identified in published Janthinobacterium lividum strains MTR and RIT308.

Genomics-inspired discovery of massiliachelin, an agrochelin epimer from Massilia sp. NR 4-1.

A putative siderophore locus was detected in the genome of the violacein-producing bacterium Massilia sp. NR 4-1 and predicted to direct the biosynthesis of a molecule that is structurally related to the thiazoline-containing siderophore micacocidin. In order to track this compound, we analyzed the metabolic profiles of Massilia cultures grown under different iron concentrations. A compound which was found to be predominantly produced under iron deficiency was subsequently isolated. Its structural characterization by spectroscopic and bioinformatic analyses revealed a previously not known diastereomer of the cytotoxic alkaloid agrochelin. The structure of this natural product, which was named massiliachelin, corresponds to the assembly line encoded by the identified siderophore locus.

Draft Genome Sequences of Phenotypically Distinct Janthinobacterium sp. Isolates Cultured from the Hudson Valley Watershed

ABSTRACTInvestigation of the Hudson Valley watershed reveals many violacein-producing bacteria. These are of interest for their biotherapeutic potential in treating chytrid infections of amphibians. The draft whole-genome sequences for seven Janthinobacterium isolates with a variety of phenotypes are provided in this study.

Draft Genome Sequence of a Violacein-Producing Iodobacter sp. from the Hudson Valley Watershed.

ABSTRACTIodobacter species are among a number of freshwater Gram-negative violacein-producing bacteria. Janthinobacterium lividum and Chromobacterium violaceum have had their whole genomes sequenced and annotated. This is the first report of a draft whole-genome sequence of a violacein-producing Iodobacter strain that was isolated from the Hudson Valley watershed.

Physiological and genomic features of a novel violacein-producing bacterium isolated from surface seawater.

Strains JW1T and JW3, isolated from surface seawater of the Arabian Sea, were subjected to polyphasic taxonomic analysis. Cells of both strains were Gram-stain-negative, aerobic, and rod-shaped. They formed violet pigment and produced violacein. On the basis of 16S rRNA gene sequence analysis, strains JW1T and JW3 showed high 16S rRNA gene sequence similarity with Pseudoalteromonas byunsanensis JCM12483T (98.2%), P. shioyasakiensis SE3T (97.8%), P. arabiensis JCM 17292T (97.3%), and P. gelatinilytica NH153T (97.1%). The 16S rRNA gene sequence similarity between JW1T and JW3 was 100%. Phylogenetic analyses revealed that both strains fell within the cluster of the genus Pseudoalteromonas and represented an independent lineage. The average nucleotide identity and in silico DNA-DNA hybridization values between JW1T and type strains of the closely related Pseudoalteromonas species were 70.9–83.3% and 20.0–26.4%, respectively. The sole respiratory quinone in both strains is ubiquinone 8 (Q-8). The principal fatty acids are summed feature 3 (C16:1ω7c and/or iso-C15:0 2OH), C18:1ω7c, and C16:0. The major polar lipids are phosphatidylethanolamine, phosphatidylglycerol, one unidentified glycolipid, one unidentified aminolipid, and one unidentified phospholipid. The DNA G+C content was 43.3 mol%. Differential phylogenetic distinctiveness, chemotaxonomic differences, and phenotypic properties indicated that strains JW1T and JW3 could be differentiated from the Pseudoalteromonas species with validly published names. Therefore, it is proposed that strains JW1T and JW3 represent a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas amylolytica sp. nov. (type strain, JW1T = CGMCC 1.15681T = KCTC 52406T = MCCC 1K02162T) is proposed.

ISOLATION AND IDENTIFICATION OF CHROMOBACTERIUM SP. FROM DIFFERENT ECOSYSTEMS

ABSTRACTObjectives: The study was aimed at isolating and identifying violacein-producing bacteria from the different ecosystems.Methods: The violet color pigment (violacein) producing bacteria was isolated and identified morphologically and genomically.Results: A total of ten bacterial Chromobacterium sp. were isolated from different sources such as soil and water. The violacein productivity wasscreened by the total isolates and the selected two strains were identified as Chromobacterium violaceum CV4 and Chromobacterium vaccinii CV5 bymorphologically and genomically.Conclusion: In the present study, the two isolate was morphologically and genomically identified as Chromobacterium violaceum CV4 andChromobacterium vaccinii CV5, last one was found to be the novel strain from natural sources which possess a high violacein producing capability.Keywords: Chromobacterium sp., Violacein, Pigment production.

Violacein as a genetically-controlled, enzymatically amplified and photobleaching-resistant chromophore for optoacoustic bacterial imaging.

There is growing interest in genetically expressed reporters for in vivo studies of bacterial colonization in the context of infectious disease research, studies of the bacterial microbiome or cancer imaging and treatment. To empower non-invasive high-resolution bacterial tracking with deep tissue penetration, we herein use the genetically controlled biosynthesis of the deep-purple pigment Violacein as a photobleaching-resistant chromophore label for in vivo optoacoustic (photoacoustic) imaging in the near-infrared range. We demonstrate that Violacein-producing bacteria can be imaged with high contrast-to-noise in strongly vascularized xenografted murine tumors and further observe that Violacein shows anti-tumoral activity. Our experiments thus identify Violacein as a robust bacterial label for non-invasive optoacoustic imaging with high potential for basic research and future theranostic applications in bacterial tumor targeting.

Violacein: Properties and Production of a Versatile Bacterial Pigment.

Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein's function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. Unfortunately, the production of violacein and its related derivatives is not easy and so various groups are also seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. This review discusses the recent trends in the research and production of violacein by both natural and genetically modified bacterial strains.

Draft Genome Sequence of Violacein-Producing Marine Bacterium Pseudoalteromonas sp. 520P1.

Here, we report a draft 5.25-Mb genome sequence of Pseudoalteromonas sp. 520P1, a marine violacein-producing bacterium isolated from the Pacific coast of Japan. Genome annotation by BLAST searches revealed the presence of one acylhomoserine lactone (AHL) synthase (luxI) and five AHL receptor protein (luxR) gene homologs.

Chromobacterium vaccinii sp. nov., isolated from native and cultivated cranberry (Vaccinium macrocarpon Ait.) bogs and irrigation ponds

A large number of Gram-negative, motile, mesophilic, violacein-producing bacteria were isolated from the soils and roots of Vaccinium macrocarpon Ait. and Kalmia angustifolia L. plants and from irrigation ponds associated with wild and cultivated cranberry bogs in Massachusetts, USA. Phylogenetic analyses of 16S rRNA gene sequences placed these isolates in a clade with Chromobacterium species, but the specialized environment from which they were isolated, their low genomic DNA relatedness with Chromobacterium violaceum ATCC 12472(T) and C. subtsugae PRAA4-1(T), significant differences in fatty acid composition and colony morphology indicate that the cranberry and Kalmia isolates comprise a separate species of Chromobacterium, for which the name Chromobacterium vaccinii sp. nov. is proposed. Strain MWU205(T) ( = ATCC BAA-2314(T) = DSM 25150(T)) is proposed as the type strain for the novel species. Phenotypic analysis of 26 independent isolates of C. vaccinii sp. nov. indicates that, despite close geographical and biological proximity, there is considerable metabolic diversity among individuals within the population.

Genome Sequence of Janthinobacterium sp. Strain PAMC 25724, Isolated from Alpine Glacier Cryoconite

The draft genome of Janthinobacterium sp. strain PAMC 25724, which is a violacein-producing psychrotolerant bacterium, was determined. The strain was isolated from glacier cryoconite of the Alps mountain permafrost region. The sequence will allow identification and characterization of the genetic determination of its cold-adaptive properties.

Purple-Pigmented Violacein-Producing Duganella spp. Inhabit the Rhizosphere of Wild and Cultivated Olives in Southern Spain

Bacteria have evolved mechanisms that allow them to grow and survive in highly competitive environments like soil and the rhizosphere. Using classical microbiological, physiological, and genetic analyses, we isolated and identified for the first time Duganella spp. associated with the rhizosphere of woody plants in Mediterranean environments that are able to produce violacein, a blue-purple secondary metabolite of considerable biotechnological interest. Based on physiological and biochemical characterization and phylogenetic analysis of different genes including 16S rRNA, gyrB, and vioA (implicated in the synthesis of violacein), the seven Duganella spp. strains isolated and studied were differentiated according to their host of origin (wild versus cultivated olives) and potentially might belong to new species. All the Duganella spp. strains produced violacein in vitro, with natural production levels significantly higher than that previously reported for other violacein-producing bacteria without optimizing growing conditions. The important biological, medical, and industrial applications of violacein make these bacteria good candidates for their biotechnological exploitation because low violacein yields are considered as one of the main limitations of using wild-type strains for extensive exploitation and pigment production. Independent of violacein production, purple-pigmented strains from olives showed proteolytic and lipolytic activities and a weak siderophore production. No in vitro inhibitory activity was demonstrated for bacteria or crude violacein filtrates against plant-pathogenic Gram-negative bacteria and fungi, but they did inhibit Gram-positive bacteria.

The Bacterially Produced Metabolite Violacein Is Associated with Survival of Amphibians Infected with a Lethal Fungus

The disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis, is associated with recent declines in amphibian populations. Susceptibility to this disease varies among amphibian populations and species, and resistance appears to be attributable in part to the presence of antifungal microbial species associated with the skin of amphibians. The betaproteobacterium Janthinobacterium lividum has been isolated from the skins of several amphibian species and produces the antifungal metabolite violacein, which inhibits B. dendrobatidis. In this study, we added J. lividum to red-backed salamanders (Plethodon cinereus) to obtain an increased range of violacein concentrations on the skin. Adding J. lividum to the skin of the salamander increased the concentration of violacein on the skin, which was strongly associated with survival after experimental exposure to B. dendrobatidis. As expected from previous work, some individuals that did not receive J. lividum and were exposed to B. dendrobatidis survived. These individuals had concentrations of bacterially produced violacein on their skins that were predicted to kill B. dendrobatidis. Our study suggests that a threshold violacein concentration of about 18 microM on a salamander's skin prevents mortality and morbidity caused by B. dendrobatidis. In addition, we show that over one-half of individuals in nature support antifungal bacteria that produce violacein, which suggests that there is a mutualism between violacein-producing bacteria and P. cinereus and that adding J. lividum is effective for protecting individuals that lack violacein-producing skin bacteria.

Optimization of culture conditions for violacein production by a new strain of Duganella sp. B2

Duganella sp. B2 isolated from Xingjiang Municipality of China was found to be a novel violacein-producing bacterium. In this study, culture variables affecting violacein production were optimized statistically in shake flask experiments. The medium pH, culture volume, and medium components of potassium nitrate, l-tryptophan, and beef extract were identified by two-level Plackett–Burman design as the most significant factors for violacein production. The path of steepest ascent was undertaken to determine the optimal region of five significant factors. Box–Behnken design and response surface analysis were adopted to further investigate the interaction between the variables and determine the optimal values for violacein production. The optimal culture conditions for violacein production were: pH 6.71, potassium nitrate 1.18 g/L, ammonium ferrous sulfate 0.08 g/L, dipotassium hydrogen phosphate 0.25 g/L, magnesium sulfate 0.75 g/L, beef extract 1.53 g/L, l-tryptophan 0.74 g/L, soluble starch 13.0 g/L, 25 mL medium in a 250 mL flask, with an inoculum size of 10% (v/v). 1.62 g/L crude violacein was achieved using the optimized conditions after 32 h of cultivation. This proved to be much higher than any other violacein-producing strains so far reported.

Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates.

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.