A Quorum Sensing-Regulated Type VI Secretion System Containing Multiple Nonredundant VgrG Proteins Is Required for Interbacterial Competition in Chromobacterium violaceum.

The environmental pathogenic bacterium Chromobacterium violaceum kills Gram-positive bacteria by delivering violacein packed into outer membrane vesicles, but nothing is known about its contact-dependent competition mechanisms. In this work, we demonstrate that C. violaceum utilizes a type VI secretion system (T6SS) containing multiple VgrG proteins primarily for interbacterial competition. The single T6SS of C. violaceum contains six vgrG genes, which are located in the main T6SS cluster and four vgrG islands. Using T6SS core component-null mutant strains, Western blotting, fluorescence microscopy, and competition assays, we showed that the C. violaceum T6SS is active and required for competition against Gram-negative bacteria such as Pseudomonas aeruginosa but dispensable for C. violaceum infection in mice. Characterization of single and multiple vgrG mutants revealed that, despite having high sequence similarity, the six VgrGs show little functional redundancy, with VgrG3 showing a major role in T6SS function. Our coimmunoprecipitation data support a model of VgrG3 interacting directly with the other VgrGs. Moreover, we determined that the promoter activities of T6SS genes increased at high cell density, but the produced Hcp protein was not secreted under such condition. This T6SS growth phase-dependent regulation was dependent on CviR but not on CviI, the components of a C. violaceum quorum sensing (QS) system. Indeed, a ΔcviR but not a ΔcviI mutant was completely defective in Hcp secretion, T6SS activity, and interbacterial competition. Overall, our data reveal that C. violaceum relies on a QS-regulated T6SS to outcompete other bacteria and expand our knowledge about the redundancy of multiple VgrGs. IMPORTANCE The type VI secretion system (T6SS) is a contractile nanomachine used by many Gram-negative bacteria to inject toxic effectors into adjacent cells. The delivered effectors are bound to the components of a puncturing apparatus containing the protein VgrG. The T6SS has been implicated in pathogenesis and, more commonly, in competition among bacteria. Chromobacterium violaceum is an environmental bacterium that causes deadly infections in humans. In this work, we characterized the single T6SS of C. violaceum ATCC 12472, including its six VgrG proteins, regarding its function and regulation. This previously undescribed C. violaceum T6SS is active, regulated by QS, and required for interbacterial competition instead of acute infection in mice. Among the VgrGs, VgrG3, encoded outside the main T6SS cluster, showed a major contribution to T6SS function. These results shed light on a key contact-dependent killing mechanism used by C. violaceum to antagonize other bacteria.

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.

Calmodulin Binding Activates Chromobacterium CopC Effector to ADP-Riboxanate Host Apoptotic Caspases.

Blocking host cell death is an important virulence strategy employed by many bacterial pathogens. We recently reported that Shigella flexneri inhibits host pyroptosis by delivering a type III secretion system (T3SS) effector OspC3 that catalyzes a novel arginine ADP-riboxanation modification on caspase-4/11. Here, we investigated the OspC3 homologue CopC from Chromobacterium violaceum, an opportunistic but sometimes deadly bacterial pathogen. CopC bears the same arginine ADP-riboxanase activity as OspC3, but with a different substrate specificity. Through proteomic analysis, we first identified host calmodulin (CaM) as a binding partner of CopC. The analyses additionally revealed that CopC preferably modifies apoptotic caspases including caspase-7, -8 and -9. This results in suppression of both extrinsic and intrinsic apoptosis programs in C. violaceum-infected cells. Biochemical reconstitution showed that CopC requires binding to CaM, specifically in the calcium-free state, to achieve efficient ADP-riboxanation of the caspases. We determined crystal structure of the CaM-CopC-CASP7 ternary complex, which illustrates the caspase recognition mechanism and a unique CaM-binding mode in CopC. Structure-directed mutagenesis validated the functional significance of CaM binding for stimulating CopC modification of its caspase substrates. CopC adopts an ADP-ribosyltransferase-like fold with a unique His-Phe-Glu catalytic triad, featuring two acidic residues critical for site-specific arginine ADP-riboxanation. Our study expands and deepens our understanding of the OspC family of ADP-riboxanase effectors. IMPORTANCE Programmed cell death is a suicidal defense mechanism for eukaryotes to combat pathogen infection. In the evolutionary arms race with the host, bacteria are endowed with ingenious tactics to block host cell death to facilitate their replication. Here, we report that the C. violaceum effector CopC ADP-riboxanates caspase-7/8/9, enabled by interacting with the host factor calmodulin, to block host cell apoptosis, illustrating a unique and sophisticated strategy adopted by the pathogen to counteract host defense.

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.

Histological assessment, anti-quorum sensing, and anti-biofilm activities of Dioon spinulosum extract: in vitro and in vivo approach.

Pseudomonas aeruginosa is an opportunistic bacterium causing several health problems and having many virulence factors like biofilm formation on different surfaces. There is a significant need to develop new antimicrobials due to the spreading resistance to the commonly used antibiotics, partly attributed to biofilm formation. Consequently, this study aimed to investigate the anti-biofilm and anti-quorum sensing activities of Dioon spinulosum, Dyer Ex Eichler extract (DSE), against Pseudomonas aeruginosa clinical isolates. DSE exhibited a reduction in the biofilm formation by P. aeruginosa isolates both in vitro and in vivo rat models. It also resulted in a decrease in cell surface hydrophobicity and exopolysaccharide quantity of P. aeruginosa isolates. Both bright field and scanning electron microscopes provided evidence for the inhibiting ability of DSE on biofilm formation. Moreover, it reduced violacein production by Chromobacterium violaceum (ATCC 12,472). It decreased the relative expression of 4 quorum sensing genes (lasI, lasR, rhlI, rhlR) and the biofilm gene (ndvB) using qRT-PCR. Furthermore, DSE presented a cytotoxic activity with IC50 of 4.36 ± 0.52 µg/ml against human skin fibroblast cell lines. For the first time, this study reports that DSE is a promising resource of anti-biofilm and anti-quorum sensing agents.

Bacterial antagonism of Chromobacterium haemolyticum and characterization of its putative type VI secretion system.

This study reports the isolation of a new Chromobacterium haemolyticum strain named WI5 from a hydroponic farming facility. WI5 exhibited remarkable bacterial antagonistic properties, eliminating Salmonella, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus (initial inoculum load ∼105 CFU/ml) in dual-species co-culture biofilms. Antagonism was strictly contact-dependent and highly influenced by nutrient availability. Next, we identified a complete suite of putative Type VI secretion system (T6SS) genes in the WI5 genome, annotated the gene locus architecture, and determined the crystal structure of hallmark T6SS tube protein Hcp1, which revealed a hexameric ring structure with an outer and inner diameter of 77 and 45 Å, respectively. Structural comparison with homologs showed differences in the key loops connecting the ß-strands in which the conserved residues are located, suggesting a role of these residues in the protein function. The T6SS is well-known to facilitate interbacterial competition, and the putative T6SS characterized herein might be responsible for the remarkable antagonism by C. haemolyticum WI5. Collectively, these findings shed light on the nature of bacterial antagonism and a putative key virulence determinant of C. haemolyticum, which might aid in further understanding its potential ecological role in natural habitats.

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).

A widespread response of Gram-negative bacterial acyl-homoserine lactone receptors to Gram-positive Streptomyces γ-butyrolactone signaling molecules.

Cell-cell communication is critical for bacterial survival in natural habitats, in which miscellaneous regulatory networks are encompassed. However, elucidating the interaction networks of a microbial community has been hindered by the population complexity. This study reveals that γ-butyrolactone (GBL) molecules from Streptomyces species, the major antibiotic producers, can directly bind to the acyl-homoserine lactone (AHL) receptor of Chromobacterium violaceum and influence violacein production controlled by the quorum sensing (QS) system. Subsequently, the widespread responses of more Gram-negative bacterial AHL receptors to Gram-positive Streptomyces signaling molecules are unveiled. Based on the cross-talk between GBL and AHL signaling systems, combinatorial regulatory circuits (CRC) are designed and proved to be workable in Escherichia coli (E. coli). It is significant that the QS systems of Gram-positive and Gram-negative bacteria can be bridged via native Streptomyces signaling molecules. These findings pave a new path for unlocking the comprehensive cell-cell communications in microbial communities and facilitate the exploitation of innovative regulatory elements for synthetic biology.

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.

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.

Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

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.

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.

Structural analysis of the core and polysaccharide from the lipopolysaccharide produced by Chromobacterium violaceum strain ATCC 12472 (NCTC 9757).

The structure of the polysaccharide O-chain of the lipopolysaccharide isolated from the sequenced strain Chromobacterium violaceum ATCC 12472 (NCTC 9757) was investigated by chemical and NMR analyses, and concluded to be -4-α-Leg5Ac7Ala-4-ß-d-ManNAlaA3OAc-3-α-d-GlcNAc-where Leg5Ac7Ala indicates 5-acetamido-7-alanylamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid and ManNAlaA3OAc 3-O-acetyl-2-alanylamido-2-deoxymannopyranuronic acid. The structure of the core with one repeating unit of the polysaccharide attached was also analyzed, and it was found that the O-chain polysaccharide is linked to the core via ß-GlcpNAc, as opposite to α-GlcpNAc inside the O-chain.

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.

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.

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.

Toolkit Development for Cyanogenic and Gold Biorecovery Chassis Chromobacterium violaceum.

Chromobacterium violaceum has been of interest recently due to its cyanogenic ability and its potential role in environmental sustainability via the biorecovery of gold from electronic waste. However, as with many nonmodel bacteria, there are limited genetic tools to implement the use of this Gram-negative chassis in synthetic biology. We propose a system that involves assaying spontaneous antibiotic resistances and using broad host range vectors to develop episomal vectors for nonmodel Gram-negative bacteria. These developed vectors can subsequently be used to characterize inducible promoters for gene expressions and implementing CRISPRi to inhibit endogenous gene expression for further studies. Here, we developed the first episomal genetic toolkit for C. violaceum consisting of two origins of replication, three antibiotic resistance genes, and four inducible promoter systems. We examined the occurrences of spontaneous resistances of the bacterium to the chosen selection markers to prevent incidences of false positives. We also tested broad host range vectors from four different incompatibility groups and characterized four inducible promoter systems, which potentially can be applied in other Gram-negative nonmodel bacteria. CRISPRi was also implemented to inhibit violacein pigment production in C. violaceum. This systematic toolkit will aid future genetic circuitry building in this chassis and other nonmodel bacteria for synthetic biology and biotechnological applications.

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).