Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination.

Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.

Theophylline as a quorum sensing and biofilm inhibitor in Pseudomonas aeruginosa and Chromobacterium violaceum.

Quorum sensing (QS) is pivotal in coordinating virulence factors and biofilm formation in various pathogenic bacteria, making it a prime target for disrupting bacterial communication. Pseudomonas aeruginosa is a member of the "ESKAPE" group of bacterial pathogens known for their association with antimicrobial resistance and biofilm formation. The current antibiotic arsenal falls short of addressing biofilm-related infections effectively, highlighting the urgent need for novel therapeutic agents. In this study, we explored the anti-QS and anti-biofilm properties of theophylline against two significant pathogens, Chromobacterium violaceum and P. aeruginosa. The production of violacein, pyocyanin, rhamnolipid, and protease was carried out, along with the evaluation of biofilm formation through methods including crystal violet staining, triphenyl tetrazolium chloride assay, and fluorescence microscopy. Furthermore, computational analyses were conducted to predict the targets of theophylline in the QS pathways of P. aeruginosa and C. violaceum. Our study demonstrated that theophylline effectively inhibits QS activity and biofilm formation in C. violaceum and P. aeruginosa. In P. aeruginosa, theophylline inhibited the production of key virulence factors, including pyocyanin, rhamnolipid, protease, and biofilm formation. The computational analyses suggest that theophylline exhibits robust binding affinity to CviR in C. violaceum and RhlR in P. aeruginosa, key participants in the QS-mediated biofilm pathways. Furthermore, theophylline also displays promising interactions with LasR and QscR in P. aeruginosa. Our study highlights theophylline as a versatile anti-QS agent and offers a promising avenue for future research to develop novel therapeutic strategies against biofilm-associated infections.

Chromobacterium indicum sp. nov., a Pigment-Producing Bacterium Isolated from Soil.

A purple colony, designated as TRC1.1.SA was isolated from a tea garden soil sample. It was a Gram-negative, rod-shaped, non-spore-forming and motile bacterium. The strain TRC1.1.SAT grew aerobically at temperatures 15-37 â„ƒ and pH levels 5.0-9.0. It showed both oxidase and catalase activity. The 16S rRNA gene sequence blast analysis revealed identity with the members of the genus Chromobacterium. The maximum identity was with the type strains of species Chromobacterium piscinae CCM 3329T (99.8%), C. vaccinii MWU205T (99.7%), and C. violaceum ATCC 12472T (98.7%). However, the average nucleotide identity (ANI) of the genome sequence showed less than 96% similarity with all species of the genus Chromobacterium. Further, digital DNA-DNA hybridization (dDDH) revealed the highest identity of 63.4% with its phylogenetic relative C. piscinae CCM 3329T. The G + C content of the strain was 63.9%. The major polar lipids identified were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphoglyceraldehyde (PG). Fatty acid analysis showed C16:0, C16:1ω7c, C17:0 cyclo, and C18:1ω7c as the major fatty acids. RAST and antiSMASH analyses of the genome revealed the presence of a biosynthetic gene cluster (BGC) involved in the production of violacein pigment, as observed for type species C. violaceum ATCC 12472T. Considering the phenotypic differences and genomic identity, strain TRC1.1.SAT is assigned as a novel species of the genus Chromobacterium, for which the name Chromobacterium indicum is proposed. The type strain of prospective species is designated as TRC1.1.SAT (= MTCC 13391T; JCM 36723T; = KCTC 8324T).

Ketoprofen, piroxicam and indomethacin-suppressed quorum sensing and virulence factors in Acinetobacter baumannii.

AIM: Quorum sensing (QS) inhibition is a promising strategy to suppress bacterial virulence and control infection caused by Gram-negative and Gram-positive bacteria. This study explores the QS inhibiting activity of the non-steroidal anti-inflammatory drugs (NSAIDs) in Acinetobacter baumannii. METHODS AND RESULTS: Ketoprofen, piroxicam and indomethacin revealed QS inhibition via elimination of violacein production of the reporter strain Chromobacterium violaceum ATCC 12472 without affecting bacterial growth. The minimal inhibitory concentration (MIC) of ketoprofen, piroxicam and indomethacin was determined against A. baumannii strains ATCC 17978, ATCC 19606, A1, A11 and A27 by the microbroth dilution method. The MICs of ketoprofen against tested isolates were 0.7-6.25 mg ml-1 , piroxicam MICs were 1.25-2.5 mg ml-1 , and indomethacin MICs were 3.12-12.5 mg ml-1 . Those compounds significantly inhibited QS-associated virulence factors such as biofilm formation, and surface motility, as well as, significantly increased bacterial tolerance to oxidative stress without affecting bacterial growth. On the molecular level, the three compounds significantly inhibited the transcription of QS regulatory genes abaI/abaR and biofilm-regulated genes cusD and pgaB. Molecular docking analysis revealed the potent binding affinity of the three compounds with AbaI via hydrogen and/or hydrophobic bonds. CONCLUSION: These results indicate that NSAIDs, ketoprofen, piroxicam and indomethacin, could be potential inhibitors of the QS and could suppress the QS-related virulence factors of A. baumannii. SIGNIFICANCE AND IMPACT: Ketoprofen, piroxicam and indomethacin could provide promising implications and strategies for combating the virulence and pathogenesis of A. baumannii.

Growth-phase specific regulation of cviI/R based quorum sensing associated virulence factors in Chromobacterium violaceum by linalool, a monoterpenoid.

Quorum sensing (QS)-dependent gene regulation in bacteria performs a vital role in synchronization of cell-density-dependent functions. In Chromobacterium violaceum QS-dependent cviI/R regulatory genes are activated during the mid- or late-exponential phase of growth. However, sufficient evidence is lacking on the role of QS inhibitors on gene regulation at different phases of growth. Hence, we report the role of linalool, a natural monoterpenoid on QS mediated gene regulation at different stages of growth in C. violaceum by performing biosensor, growth kinetic and gene expression studies. In vitro and in vivo studies were performed for establishing role of linalool in reducing the virulence and infection by using HEK-293 T cell lines and Caenorhabditis elegans models respectively. C. violaceum CV026 with C6-HSL was used as control. The results showed linalool to be a QS inhibitor with an estimated IC50 of 63 µg/mL for violacein inhibition. At this concentration the cell density difference (delta OD600) of 0.14 from the compound was observed indicating the quorum concentration. The expression of cviI/R was initiated at mid-log phase (~ 18 h) and reached the maximum at 36 h in control whereas in treatment it remained significantly downregulated at all time points. The expression of violacein biosynthetic genes vioA, vioC, vioD and vioE was also downregulated by linalool. Infection studies with linalool showed higher survival rates in HEK-293T cell lines and C. elegans compared to the infection control. Taken together, this study proves linalool to be a QS inhibitor capable of attenuation of QS by controlling the cell density through cviI/R downregulation at the early phase of growth and hence offering scope for its application for controlling infections.

The MarR family regulator OsbR controls oxidative stress response, anaerobic nitrate respiration, and biofilm formation in Chromobacterium violaceum.

BACKGROUND: Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown. RESULTS: Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor OhrR, which resulted in improved growth under organic hydroperoxide treatment, as seem by growth curve assays. We showed that the proper regulation of the nar genes by OsbR ensures optimal growth of C. violaceum under anaerobic conditions by tuning the reduction of nitrate to nitrite. Finally, the osbR overexpressing strain showed a reduction in biofilm formation, and this phenotype correlated with the OsbR-mediated repression of two gene clusters encoding putative adhesins. CONCLUSIONS: Together, our data indicated that OsbR is a MarR-type regulator that controls the expression of a large number of genes in C. violaceum, thereby contributing to oxidative stress defense (ohrA/ohrR), anaerobic respiration (narK1K2 and narGHJI), and biofilm formation (putative RTX adhesins).

Microbial exopolysaccharide production of Streptococcus thermophilus and its antiquorum sensing activity.

Interest in the production of exopolysaccharides by microorganisms has increased in the recent years. Using low-cost product is the main step of microbial production to reduce cost and compete with chemical production. In this work, EPS production of Streptococcus thermophilus isolates from yogurt (S2), kefir (S3), and S. thermophilus ATCC 19258 (S1) isolate which was used as control strains were investigated by using different fruit pulps. S. thermophilus isolates were identified by morphological and 16S sequence analysis. The amount of EPS obtained was measured spectrophotometrically using glucose as standard with phenol sulfuric acid method. All three isolates produced higher amounts of EPS on M17 medium than Nutrient medium. When the fruit pulp was added to the medium, EPS production increased in all three isolates. When different nitrogen sources were added together with fruit pulp juice, EPS production increased. The highest amount of EPS produced by ATCC 19258 strain (21.570 mg/L) and S3 isolate (29.131 mg/L) is the medium where mixed fruit pulp juice and nitrogen source is tryptophan. It has been shown that EPS production is increased by adding fruit pulps to the prepared media. It is thought that apricot pulp can be a good alternative in EPS production especially in the evaluation of wastes. Also, antiquorum sensing activity of the highest amount EPS was determined by using Chromobacterium violaceum CV026 strain and found effective on violacein pigment inhibition and C6-AHL production of biosensor strain.

Quorum sensing modulatory and biofilm inhibitory activity of Plectranthus barbatus essential oil: a novel intervention strategy.

The essential oil (EO) from the roots of Plectranthus barbatus Andr. (Syn. Coleus forskohlii Briq.) was evaluated for quorum sensing (QS) inhibitory activity. P. barbatus EO was screened for inhibition of QS regulated violacein production in Chromobacterium violaceum (ATCC 12472) wild-type strain. At inhibitory (6.25% v/v) and sub-inhibitory concentrations (3.125% v/v) of the EO, dose-dependent response in the inhibition of violacein production was observed in C. violaceum. Similarly, sub-MIC (6.25% v/v) of P. barbatus EO disrupted QS regulated biofilm formation by 27.87% and inhibited swarming and twitching motility in Pseudomonas aeruginosa PA01 implying its anti-infective and QS modulatory activity. Fluorescence microscopy studies confirmed the disruption of biofilm formation by EO in P. aeruginosa PAO1. Promising antibacterial activity was recorded at concentrations as low as 3.12% v/v for Listeria monocytogenes (ATCC 13932) and at 6.25% v/v for both Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 25241) and Escherichia coli (ATCC 11775). Furthermore, significant dose-dependent inhibition was observed for biofilm formation and motility in all the tested pathogens in different treated concentrations. GC-MS analysis revealed α-pinene, endo-borneol, bornyl acetate, 1-Hexyl-2-Nitrocyclohexane as the major phytoconstituents. P. barbatus EO or its constituent compounds with QS modulatory, antimicrobial and biofilm inhibitory property could be potential new-age dietary source based intervention and preservation technologies.

Discovery of novel ß-turn mimetic-based peptides as novel quorum sensing inhibitors of gram-negative bacteria.

To date, a very limited number of peptides reported as quorum sensing inhibitors. Herein, we report the synthesis and evaluation of a series of ß-turn mimetic-based peptides as potent quorum sensing inhibitors and antibiofilm formation. In this series, peptides P1, P4, and P5 showed very promising anti-quorum sensing activity on lasB-gfp reporter strain of Pseudomonas aeruginosa without affecting bacterial growth. Under our condition, these compounds also showed good anti-violacein production of Chromobacterium violaceum. In terms of antibiofilm formation, except P5, two ß-turn mimetic-based peptides P1 and P4 showed maximum inhibition of 80% total biomass of Pseudomonas aeruginosa. This report provides the first ß-turn mimetic-based scaffold for future drug development.

Coumarin's Anti-Quorum Sensing Activity Can Be Enhanced When Combined with Other Plant-Derived Small Molecules.

Coumarins are class of natural aromatic compounds based on benzopyrones (2H-1-benzopyran-2-ones). They are identified as secondary metabolites in about 150 different plant species. The ability of coumarins to inhibit cell-to-cell communication in bacterial communities (quorum sensing; QS) has been previously described. Coumarin and its derivatives in plant extracts are often found together with other small molecules that show anti-QS properties too. The aim of this study was to find the most effective combinations of coumarins and small plant-derived molecules identified in various plants extracts that inhibit QS in Chromobacterium violaceum ATCC 31532 violacein production bioassay. The coumarin and its derivatives: 7-hydroxycoumarin, 7.8-dihydroxy-4-methylcoumarin, were included in the study. Combinations of coumarins with gamma-octalactone, 4-hexyl-1.3-benzenediol, 3.4.5-trimethoxyphenol and vanillin, previously identified in oak bark (Quercus cortex), and eucalyptus leaves (Eucalyptus viminalis) extracts, were analyzed in a bioassay. When testing two-component compositions, it was shown that 7.8-dihydroxy-4-methylcoumarin, 4-hexyl-1.3-benzendiol, and gamma-octalactone showed a supra-additive anti-QS effect. Combinations of all three molecules resulted in a three- to five-fold reduction in the concentration of each compound needed to achieve EC50 (half maximal effective concentration) against QS in C. violaceum ATCC 31532.

Interplay between two quorum sensing-regulated pathways, violacein biosynthesis and VacJ/Yrb, dictates outer membrane vesicle biogenesis in Chromobacterium violaceum.

Outer membrane vesicles (OMVs) are lipid nanoparticles released by Gram-negative bacteria, which play multiple roles in bacterial physiology and adaptation to diverse environments. In this work, we demonstrate that OMVs released by the environmental pathogen Chromobacterium violaceum deliver the antimicrobial compound violacein to competitor bacteria, mediating its toxicity in vivo at a long distance. OMVs purified by ultracentrifugation from the wild-type strain, but not from a violacein-abrogated mutant ΔvioABCDE, contained violacein and inhibited several Gram-positive bacteria. Competition tests using co-culture and transwell assays indicated that the C. violaceum wild-type strain killed Staphylococcus aureus better than the ΔvioABCDE mutant strain. We found that C. violaceum achieves growth phase-dependent OMV release by the concerted expression of two quorum sensing (QS)-regulated pathways, namely violacein biosynthesis and VacJ/Yrb system. Although both pathways were activated at high cell density in a QS-dependent manner, the effect on vesiculation was the opposite. While the ΔvioABCDE mutant produced twofold fewer vesicles than the wild-type strain, indicating that violacein induces OMV biogenesis for its own delivery, the ΔvacJ and ΔyrbE mutants were hypervesiculating strains. Our findings uncovered QS-regulated pathways involved in OMV biogenesis used by C. violaceum to package violacein into OMVs for interbacterial competition.

Chromobacterium violaceum delivers violacein, a hydrophobic antibiotic, to other microbes in membrane vesicles.

This study describes Chromobacterium violaceum's use of extracellular membrane vesicles (MVs) to both solubilize and transport violacein to other microorganisms. Violacein is a hydrophobic bisindole with known antibiotic activities against other microorganisms. Characterization of the MVs found they carried more violacein than protein (1.37 ± 0.19-fold), suggesting they may act as a reservoir for this compound. However, MVs are not produced in response to violacein - a ΔvioA isogenic mutant, which is incapable of making violacein, actually produced significantly more MVs (3.2-fold) than the wild-type strain. Although violacein is insoluble in water (Log Poctanol:water = 3.34), 79.5% remained in the aqueous phase when it was present within the C. violaceum MVs, an increase in solubility of 1740-fold. Moreover, tests with a strain of Staphylococcus aureus showed MV-associated violacein is bactericidal, with 3.1 mg/l killing 90% of S. aureus in 6 h. Tests with the ΔvioA MVs found no loss in the S. aureus viability, even when its MVs were added at much higher concentrations, demonstrating violacein is the active component within the wild-type MVs. In conclusion, our study clearly demonstrates C. violaceum produces MVs and uses them as vehicles to solubilize violacein and transport this hydrophobic antibiotic to other microbes.

Endolichenic fungus, Aspergillus quandricinctus of Usnea longissima inhibits quorum sensing and biofilm formation of Pseudomonas aeruginosa PAO1.

Lichens are composite organisms, comprising of a fungus (mycobiont) and a blue-green alga (photobiont). Along with the mycobiont, numerous non-obligate microfungi live in lichen thalli. These microfungi are called endolichenic fungi (ELF). In recent years, the ELF are emerging as promising natural sources because of their capability to exert unique drug molecules. The current study aimed to isolate the ELF from the lichen, Usnea longissima Ach., to control of biofilm formation and quorum sensing phenomenon in Pseudomonas aeruginosa PAO1, an opportunistic multidrug resistance pathogen that uses quorum sensing network to produce an array of pathogenic agents. Therefore, inhibiting quorum sensing to manage the infection caused by PAO1 could be the paramount alternative approach to conventional antibiotics. The isolated ELF was identified by amplifying the long subunit region of the fungal genome. The extracted metabolites of ELF (MELE) using the acetone solvent was further investigated for anti-quorum sensing activity using the biomarker strain Chromobacterium violaceum 12472 which exerts violacein pigment via the AHL mediated quorum sensing signalling. Moreover, the effect of MELE was also evaluated on the production of virulence factors and biofilm formation of P. aeruginosa PAO1. The molecular identification revealed that ELF (accession number MN171299) exhibited 100% similarity with Aspergillus quandricinctus strain CBS 135.52. The MELE showed significant anti-quorum sensing activity at the concentration of 4 mg/mL without affecting the bacterial cell viability of P. aeruginosa PAO1. The MELE diminished the production of virulence factors, including pyocyanin, protease, elastase, rhamnolipids, and extracellular polysaccharides of P. aeruginosa PAO1 in a concentration-dependent manner. The MELE also disturbed biofilm formation of P. aeruginosa PAO1. The 3-D analysis of biofilm architecture showed that the thickness and surface area covered by microcolonies was decreased as the concentration of MELE was increased. The GC-MS analysis of MELE exhibited that organic acids and fatty acids are major constituents of the MELE. The present study reports first time that the ELF, A. quandricinctus possesses potential to inhibit quorum sensing and biofilm formation of P. aeruginosa and can be further exploited for hospital and healthcare facilities.

Marine bacteria associated with shallow hydrothermal systems in the Gulf of California with the capacity to produce biofilm inhibiting compounds.

Shallow hydrothermal systems are extreme environments. The sediments and fluids emitted from the vents present unusual physical and chemical conditions compared to other marine areas, which promotes unique biodiversity that has been of great interest for biotechnology for some years. In this work, a bioprospective study was carried out to evaluate the capacity of bacteria associated with shallow hydrothermal vents to produce biofilm-inhibiting compounds. Degradation assays of N-acyl homoserine lactone (AHL) autoinducers (C6HSL) involved in the quorum sensing process were carried out on 161 strains of bacteria isolated from three shallow hydrothermal systems located in Baja California Sur (BCS), Mexico. The biosensor Chromobacterium violaceum CV026 was used. Twenty-three strains showed activity, and organic extracts were obtained with ethyl acetate. The potential of the extracts to inhibit the formation of biofilms was tested against two human pathogenic strains (Pseudomonas aeruginosa PAO1 and Aeromonas caviae ScH3), a shrimp pathogen (Vibrio parahaemolyticus M8), and two marine strains identified as producing biofilms on submerged surfaces (Virgibacillus sp C29 and Vibrio alginolyticus C96). The results showed that Vibrio alginolyticus and Brevibacillus thermoruber, as well as some thermotolerant strains (mostly Bacillus), produce compounds that inhibit bacterial biofilms (B. licheniformis, B. paralicheniformis, B. firmus, B. oceanizedimenis, B. aerius and B. sonorensis).

Quorum Sensing Inhibitors against Chromobacterium violaceum CV026 Derived from an Actinomycete Metabolite Library.

Quorum sensing (QS) is a microbial signaling system that regulates the expression of many virulence genes. Herein, we studied five compounds-No. 1: (E)-2-methyl-3- (4-nitro-phenyl)-acrylaldehyde; No. 29-2: pimprinine [5-(1H-indol-3-yl)-2-methyloxazole]; No. 48: (2E,4E)-2-methyl-5-phenyl-2,4-pentadienoic acid; No. 74: (3E,5E)-5-methyl-6-(4-nitrophenyl)-hexa-3,5-dien-2-ol; and No. 130: methyphenazine-1-carboxylate-derived from an actinomycete metabolite library. These compounds were confirmed to be QS inhibitors that reduced violacein production in Chromobacterium violaceum CV026. Additionally, compounds No. 1, No. 74, and No. 130 significantly reduced fluorescent pigment production in Pseudomonas aeruginosa ATCC 27853.

Bacteria associated with marine macroorganisms as potential source of quorum-sensing antagonists.

Samples were collected from different undisturbed areas along the coast of Gujarat like Okha, Diu, Veraval, and Somnath. A total of 68 marine isolates were obtained out of which 53 were associated with various marine macroorganisms like sponges, gastropods, and algae, whereas 15 were free living. Quorum-quenching ability of all the isolates was tested against Chromobacterium violaceum MK by co-culture technique as a way to simultaneously detect signal-degrading as well as nondegrading quorum-sensing inhibitors. Nineteen macroorganism-associated bacteria and eight free-living bacteria were found to possess quorum-sensing inhibitory activity against C. violaceum MK without affecting its growth. Isolate OA22 from grape alga and OA10 from purple sponge (Haliclona sp.) were found to possess the highest C6-HSL degradation activity and extracellular non-N-acyl-homoserine lactone degrading QSI activity, respectively. OA22 was also found to degrade 3-oxo-C12 homoserine lactone. Acid recovery of both the C6- and C12-HSL after degradation by OA22 indicated the presence of lactonase enzyme in the isolate. Cell-free supernatant of OA10 was extracted with ethyl acetate to obtain the quorum-quenching compound. Pigment inhibition in C. violaceum MK treated with OA10 extract was demonstrated in various ways and was indicative of QSI activity of the extract without degradation of the quorum-sensing signaling molecule. The isolates OA22 and OA10 were identified as Desemzia incerta and Bacillus sp., respectively, by 16S ribosomal DNA sequence analysis.

Production and Uptake of Distinct Endogenous Catecholate-Type Siderophores Are Required for Iron Acquisition and Virulence in Chromobacterium violaceum.

Bacteria use siderophores to scavenge iron from environmental or host sources. The iron acquisition systems of Chromobacterium violaceum, a ubiquitous environmental bacterium that can cause infections in humans, are still unknown. In this work, we demonstrated that C. violaceum produces putative distinct endogenous siderophores, here named chromobactin and viobactin, and showed that they are each required for iron uptake and virulence. An in silico analysis in the genome of C. violaceum revealed that genes related to synthesis and uptake of chromobactin (cba) and viobactin (vba) are located within two secondary-metabolite biosynthetic gene clusters. Using a combination of gene deletions and siderophore detection assays, we revealed that chromobactin and viobactin are catecholate siderophores synthesized from the common precursor 2,3-dihydroxybenzoate (2,3-DHB) on two nonribosomal peptide synthetase (NRPS) enzymes (CbaF and VbaF) and taken up by two TonB-dependent receptors (CbuA and VbuA). Infection assays in mice revealed that both the synthesis and the uptake of chromobactin or viobactin are required for the virulence of C. violaceum, since only the mutant strains that do not produce any siderophores or are unable to take up both of them were attenuated for virulence. In addition, the mutant strain unable to take up both siderophores showed a pronounced attenuation of virulence in vivo and reduced neutrophil extracellular trap (NET) formation in in vitro assays, suggesting that extracellularly accumulated siderophores modulate the host immune response. Overall, our results revealed that C. violaceum uses distinct endogenous siderophores for iron uptake and its establishment in the host.

B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase: Evaluation of the Proline Rule as a Method for Enzyme Stabilization.

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. Here we present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60 °C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3 °C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the proline rule was deemed successful at increasing the stability of this enzyme.

Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model.

Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.

Bacterial endophytes from Artemisia nilagirica (Clarke) Pamp., with antibacterial efficacy against human pathogens.

A study was conducted to isolate and characterize endophytes from Artemisia nilagirica, a traditional medicinal plant. The plant was collected from Western Ghats, India. Endophytes isolated included Arthrobacter sp. WWAT1, Pseudomonas sp. WYAT2, Microbacterium sp. WYAT3, Psychrobacter sp. WBAT4, Enterobacter sp. WWAT5, Bacillus sp. WBAT6, Kosakonia cowanii WBAT7, Bacillus sp. WBAT8, Bacillus sp. WBAT9, Chromobacterium violaceum WVAT6, Serratia sp.WPAT8 and Burkholderia sp. WYAT7. Of these two bacteria, Chromobacterium violaceum strain WVAT6 and Burkholderia sp. strain WYAT exhibited antibacterial property against human pathogens. Similar to the environmental isolates, Burkholderia sp. WYAT7 showed pleomorphism and produced different enzymes, whereas like clinical strains they showed multidrug resistance, for their survival in different environmental conditions. Chromobacterium violaceum WVAT6 exhibited rod shape morphology and showed multiple drug resistance except to erythromycin, tetracycline and gentamicin antibiotics. Both produced biofilm and enzymes such as protease and lipase. The antimicrobial compounds from these endophytes may find application in the preparation of antimicrobial formulations.