Partial genomic characterization of Chromobacterium piscinae from India reveals multi drug resistance.

Species of genus Chromobacterium have been isolated from diverse geographical settings, which exhibits significant metabolic flexibility as well as biotechnological and pathogenic properties. This study describes the isolation, characterization, draft assembly, and detailed sequence analysis of Chromobacterium piscinae strain W1B-CG-NIBSM isolated from water samples from multi use community pond. The organism was characterized by biochemical tests, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI TOF-MS) and partial genome sequencing. The partial genomic data of Chromobacterium pisciane isolate W1B NIBSM strain was submitted to GenBank with Bio project number PRJNA803347 and accession no CP092474. An integrated genome analysis of Chromobacterium piscinae has been accomplished with PATRIC which indicates good quality genome. DNA sequencing using the illumina HiSeq 4000 system generated total length of 4,155,481 bp with 63 contig with G + C content is 62.69%. This partial genome contains 4,126 protein-coding sequences (CDS), 27 repeats region and 78 transfer RNA (tRNA) genes as well as 3 ribosomal RNA (rRNA) genes. The genomic annotation of Chromobacterium W1B depicts 2,925 proteins with functional assignments and 1201 hypothetical proteins. A repertoire of specialty genes implicated in antibiotic resistance (45 genes), drug target (6 genes), Transporter (3 genes) and virulence factor (10 genes). The genomic analysis reveals the adaptability, displays metabolic varied pathways and shows specific structural complex and various virulence factors which makes this strain multi drug resistant. The isolate was found to be highly resistant to ß-lactam antibiotics whereas it showed sensitivity towards aminoglycosides and fluoroquinolone antibiotics. Hence, the recovery of Chromobacterium piscinae from community pond evidenced for uncertain hidden source of public health hazard. To the best of authors knowledge this is first report of isolation and genomic description of C. piscinae from India.

Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion.

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

Identification of 2,4-diacetylphloroglucinol production in the genus Chromobacterium.

The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.

Chromobacterium Biopesticide Exposure Does Not Select for Resistance in Aedes Mosquitoes.

Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool. IMPORTANCE Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sublethal dose of this Csp_P biopesticide over 9 to 10 generations causes resistance to arise in Aedes aegypti mosquitoes. We find no evidence of resistance at the physiological or molecular levels, confirming that the Csp_P biopesticide is a highly promising new tool for controlling mosquito populations.

Biocatalytic synthesis of chiral five-membered carbasugars intermediates utilizing CV2025 ω-transaminase from Chromobacterium violaceum.

(S)-4-(Hydroxymethyl)cyclopent-2-enone is a key intermediate in the synthesis of chiral five-membered carbasugars, which can be used to synthesize a large number of pharmacologically relevant carbocyclic nucleosides. Herein, CV2025 ω-transaminase from Chromobacterium violaceum was selected based on substrate similarity to convert ((1S,4R)-4-aminocyclopent-2-enyl)methanol to (S)-4-(hydroxymethyl)cyclopent-2-enone. The enzyme was successfully cloned, expressed in Escherichia coli, purified and characterized. We show that it has R configuration preference in contrast with the conventional S preference. The highest activity was obtained below 60 °C and at pH 7.5. Cations Ca2+ and K+ enhanced activity by 21% and 13%, respectively. The conversion rate reached 72.4% within 60 min at 50 °C, pH 7.5, using 0.5 mM pyridoxal-5'-phosphate, 0.6 µM CV2025, and 10 mM substrate. The present study provides a promising strategy for preparing five-membered carbasugars economically and efficiently.

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.

Quorum Sensing and Biofilm Disrupting Potential of Imidazole Derivatives in Chromobacterium violaceum Using Antimicrobial and Drug Discovery Approaches.

Population of drug-resistant bacteria have increased at an alarming rate in the past few decades. The major reason for increasing drug resistance is the lack of new antibiotics and limited drug targets. It has therefore been a vital task to develop new antibiotics with different drug targets. Two such targets are biofilm formation and quorum sensing. Quorum sensing is cell to cell communication used by bacteria that initiates many important survival processes and aids in establishing pathogenesis. Both biofilm and quorum sensing are inter-related processes and play a major role in physiological and pathogenesis processes. In this study, five novel imidazole derivatives (IMA-1-IMA-5) were synthesised and tested for their antibacterial and anti-quorum sensing activities against Chromobacterium violaceum using different in silico and in vitro techniques following the standard protocols. In silico results revealed that all compounds were able to effectively bind to and interact sufficiently with the target protein CviR. CviR is a protein to which autoinducers bind to initiate the quorum sensing process. In silico results also revealed that the compounds generated favourable structural dynamics implying that the compounds would be able to effectively bind to CviR and inhibit quorum sensing. Susceptibility results revealed that IMA-1 is the most active of all the derivatives against both planktonic cells and biofilms. Qualitative and quantitative evaluation of anti-quorum sensing activity at sub-inhibitory concentrations of these compounds also revealed high activity for IMA-1. Down-regulation of most of the quorum sensing genes when cells were treated with the test compounds affirmed the high anti-quorum sensing activities of these compounds. The results from this study are promising and urges on the use of anti-quorum sensing and biofilm disrupting molecules to combat multi-drug resistance problem.

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.

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.

Quorum sensing inhibitory effect of bergamot oil and aspidosperma extract against Chromobacterium violaceum and Pseudomonas aeruginosa.

Quorum sensing (QS) represents a major target for reducing bacterial pathogenicity and antibiotic resistance. This study identifies bergamot and aspidosperma as new potential sources of anti-QS agents. We investigated the anti-QS activity of plant materials on both Chromobacterium violaceum and Pseudomonas aeruginosa. Initially, we determined the minimum inhibitory concentrations (MICs) of plant materials using a broth microdilution method. Subsequently, we tested the effect of sub-MIC concentrations on QS-regulated traits and virulence factors production in test bacteria. Results revealed that bergamot and aspidosperma inhibited the ability of C. violaceum to produce violacein. Other QS-controlled phenotypes of C. violaceum, namely chitinolytic activity, motility, and biofilm formation, were also reduced by both plant materials. Moreover, QS-linked traits of P. aeruginosa were also reduced. Bergamot inhibited swarming but not swimming motility, while aspidosperma diminished both motility types in P. aeruginosa. Both plant materials also demonstrated antibiofilm activity and inhibited the production of protease and pyocyanin in P. aeruginosa. Furthermore, we tested the anti-QS effect of plant materials on the transcriptional level using RT-qPCR. Bergamot dramatically downregulated the C. violaceum autoinducer synthase gene cviI and the vioB gene involved in violacein biosynthesis, confirming the phenotypic observation on its anti-QS activity. Aspidosperma also reduced the expression of cviI and vioB but less drastically than bergamot. In P. aeruginosa, downregulation in the transcripts of the QS genes lasI, lasR, rhlI, and rhlR was also achieved by bergamot and aspidosperma. Therefore, data in the present study suggest the usefulness of bergamot and aspidosperma as sources of antivirulence agents.

Successful treatment of a patient with recurrent infection of Chromobacterium violaceum.

BACKGROUND: Chromobacterium violaceum (C. violaceum) is a Gram-negative saprophytic bacterium that is widespread in tropical and subtropical environments, and belongs to conditional pathogenic bacteria. Human infection with C. violaceum is rare, and this can be fatal when the diagnosis and treatment are delayed, especially recurrent infection patients. Since clinicians lack the knowledge for C. violaceum, rapid diagnosis and early appropriate antimicrobial treatment remains challenging. CASE PRESENTATION: A 15-year-old male student was hospitalized for dark abscess, pustules, severe pain in both legs, and fever for 11 days. There were pustules with gray-white pus and red infiltrating plaques on the back, and the subcutaneous nodules could be touched in front of both tibias, with scab, rupture and necrotic tissue of the lower limb. The patient's condition rapidly progressed. Therefore, next-generation sequencing (NGS), pustular secretion and blood culture were concurrently performed. The final diagnosis for this patient was C. violaceum infection by NGS. However, no bacterial or fungal growth was observed in the pustular secretion and blood culture. After 4 weeks of treatment, the patient was discharged from the hospital without any complications associated with C. violaceum infection. CONCLUSION: Rapid diagnosis and early appropriate antimicrobial treatment is the key to the successful treatment of C. violaceum infection, especially in patients with sepsis symptoms. This case highlights that NGS is a promising tool for the rapid diagnosis of C. violaceum infection, preventing the delayed diagnosis and misdiagnosis of C. violaceum infection in patients who tested negative for pustular secretion and blood culture.

Sepsis in cougar (Puma concolor) associated with Chromobacterium violaceum.

The genus Chromobacterium is widely distributed in the environment and is composed of Gram-negative, aerobic, or facultative anaerobic bacilli that occur in violet-colored colonies. These bacteria rarely cause infections, but when it occurs, it spreads quickly and has a high mortality. Because diseases are infrequent, the diagnosis is often delayed, and it takes time for suitable treatment to be initiated, leading to increased mortality due to the rapid progression of the disease. After the death of a cougar, serologically positive for feline leukemia virus, at the Center for Medicine and Research on Wild Animals of the Federal University of Mato Grosso, an autopsy was carried out, and fragments of its organs were sent for bacterial culture. Significant lesions were found, mainly in the liver and lungs, and upon bacterial isolation, violet-colored colonies were obtained from all of the referred organs, suggestive of C. violaceum, which was later confirmed by 16S DNA sequencing. The objective of this study was to report a case of death associated primarily with disseminated infection caused by C. violaceum in a FeLV-positive wild cougar in July 2018; no other occurrence in this species has yet been described.

Microbial synthesis of violacein pigment and its potential applications.

Violacein is a pigment synthesized by Gram-negative bacteria such as Chromobacterium violaceum. It has garnered significant interest owing to its unique physiological and biological activities along with its synergistic effects with various antibiotics. In addition to C. violaceum, several microorganisms, including: Duganella sp., Pseudoalteromonas sp., Iodobacter sp., and Massilia sp., are known to produce violacein. Along with the identification of violacein-producing strains, the genetic regulation, quorum sensing mechanism, and sequence of the vio-operon involved in the biosynthesis of violacein have been elucidated. From an engineering perspective, the heterologous production of violacein using the genetically engineered Escherichia coli or Citrobacter freundii host has also been attempted. Genetic engineering of host cells involves the heterologous expression of genes involved in the vio operon and the optimization of metabolic pathways and gene regulation. Further, the crystallography of VioD and VioE was revealed, and mass production by enzyme engineering has been accelerated. In this review, we highlight the biologically assisted end-use applications of violacein (such as functional fabric development, nanoparticles, functional polymer composites, and sunscreen ingredients) and violacein activation mechanisms, production strains, and the results of mass production with engineered methods. The prospects for violacein research and engineering applications have also been discussed.

Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli.

BACKGROUND: Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers. RESULTS: Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA-) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. CONCLUSIONS: To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Agricultural Injury-Associated Chromobacterium violaceum Infection in a Bangladeshi Farmer.

Chromobacterium violaceum is an emerging environmental pathogen that causes life-threatening infection in humans and animals. In October 2017, a Bangladeshi farmer was hospitalized with high-grade fever due to an agricultural injury-related wound infection. Bacteriological and 16S rRNA gene investigation detected C. violaceum in the wound discharge. The patient recovered successfully after a combination treatment with meropenem and ciprofloxacin, followed by prolonged medication to avoid recurrence. We strongly propose to incorporate C. violaceum in the differential diagnosis of wound and skin infections occurring in tropical and subtropical regions, especially when the injury was exposed to soil or sluggish water.

A Chemical Counterpunch: Chromobacterium violaceum ATCC 31532 Produces Violacein in Response to Translation-Inhibiting Antibiotics.

Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an antibiotic-induced response (air) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules.IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration.

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.