RESUMO
Modern crop production relies on the application of chemical pesticides and fertilizers causing environmental and economic challenges. In response, less environmentally impactful alternatives have emerged such as the use of beneficial microorganisms. These microorganisms, particularly plant growth-promoting bacteria (PGPB), have demonstrated their ability to enhance plant growth, protect against various stresses, and reduce the need for chemical inputs. Among the PGPB, Bacillus species have garnered attention due to their adaptability and commercial potential. Recent reports have highlighted Bacillus strains as biocontrol agents against phytopathogenic bacteria while concurrently promoting plant growth. We also examined Bacillus plant growth-promoting abilities in Arabidopsis thaliana seedlings. In this study, we assessed the potential of various Bacillus strains to control diverse phytopathogenic bacteria and inhibit quorum sensing using Chromobacterium violaceum as a model system. In conclusion, our results suggest that bacteria of the genus Bacillus hold significant potential for biotechnological applications. This includes developments aimed at reducing agrochemical use, promoting sustainable agriculture, and enhancing crop yield and protection.
Assuntos
Arabidopsis , Bacillus , Doenças das Plantas , Bacillus/fisiologia , Arabidopsis/microbiologia , Arabidopsis/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologia , Percepção de Quorum , Chromobacterium/fisiologia , Chromobacterium/crescimento & desenvolvimento , Agentes de Controle Biológico/farmacologia , Desenvolvimento Vegetal , Plântula/microbiologia , Plântula/crescimento & desenvolvimento , Microbiologia do SoloRESUMO
Chromobacterium violaceum is a ubiquitous environmental bacterium that causes sporadic life-threatening infections in humans. How C. violaceum acquires zinc to colonize environmental and host niches is unknown. In this work, we demonstrated that C. violaceum employs the zinc uptake system ZnuABC to overcome zinc limitation in the host, ensuring the zinc supply for several physiological demands. Our data indicated that the C. violaceum ZnuABC transporter is encoded in a zur-CV_RS15045-CV_RS15040-znuCBA operon. This operon was repressed by the zinc uptake regulator Zur and derepressed in the presence of the host protein calprotectin (CP) and the synthetic metal chelator EDTA. A ΔznuCBA mutant strain showed impaired growth under these zinc-chelated conditions. Moreover, the deletion of znuCBA provoked reductions in violacein production, swimming motility, biofilm formation, and bacterial competition. Remarkably, the ΔznuCBA mutant strain was highly attenuated for virulence in an in vivo mouse infection model and showed low capacities to colonize the liver, grow in the presence of CP, and resist neutrophil killing. Overall, our findings demonstrate that ZnuABC is essential for C. violaceum virulence, contributing to subversion of zinc-based host nutritional immunity.
Assuntos
Proteínas de Transporte/fisiologia , Chromobacterium/patogenicidade , Zinco/metabolismo , Biofilmes , Proteínas de Transporte/genética , Chromobacterium/fisiologia , Complexo Antígeno L1 Leucocitário/fisiologia , Neutrófilos/imunologia , Óperon , VirulênciaRESUMO
Aim: To determine phenotypically the anti quorum-sensing (QS) activity of 30 volatile organic products (VOPs) through the inhibition of swarming motility and pyoverdine production in Pseudomonas aeruginosa. Materials & methods: Twenty-four essential oils and six small volatile organic compounds randomly selected were screened for their anti-QS activity by violacein inhibition on Chromobacterium violaceum. The VOPs with positive results were subsequently evaluated for swarming motility and pyoverdine production on P. aeruginosa determining the colony diameter and fluorescence under UV light, respectively. Results: Fifty percent of VOPs tested showed strong violacein inhibition, 40% presented anti-swarming activity and 33% inhibited pyoverdine production. Conclusion: Our data demonstrate that VOPs have a great potential to inhibit virulence factors mediated by QS in P. aeruginosa.
Assuntos
Antibacterianos/farmacologia , Extratos Vegetais/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/biossíntese , Percepção de Quorum/efeitos dos fármacos , Compostos Orgânicos Voláteis/farmacologia , Chromobacterium/efeitos dos fármacos , Chromobacterium/fisiologia , Óleos Voláteis/farmacologia , Plantas/química , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/fisiologiaRESUMO
Iron is a highly reactive metal that participates in several processes in prokaryotic and eukaryotic cells. Hosts and pathogens compete for iron in the context of infection. Chromobacterium violaceum, an environmental Gram-negative bacterial pathogen, relies on siderophores to overcome iron limitation in the host. In this work, we studied the role of the ferric uptake regulator Fur in the physiology and virulence of C. violaceum A Δfur mutant strain showed decreased growth and fitness under regular in vitro growth conditions and presented high sensitivity to iron and oxidative stresses. Furthermore, the absence of fur caused derepression of siderophore production and reduction in swimming motility and biofilm formation. Consistent with these results, the C. violaceum Δfur mutant was highly attenuated for virulence and liver colonization in mice. In contrast, a manganese-selected spontaneous fur mutant showed only siderophore overproduction and sensitivity to oxidative stress, indicating that Fur remained partially functional in this strain. We found that mutations in genes related to siderophore biosynthesis and a putative CRISPR-Cas locus rescued the Δfur mutant growth defects, indicating that multiple Fur-regulated processes contribute to maintaining bacterial cell fitness. Overall, our data indicated that Fur is conditionally essential in C. violaceum mainly by protecting cells from iron overload and oxidative damage. The requirement of Fur for virulence highlights the importance of iron in the pathogenesis of C. violaceumIMPORTANCE Maintenance of iron homeostasis, i.e., avoiding both deficiency and toxicity of this metal, is vital to bacteria and their hosts. Iron sequestration by host proteins is a crucial strategy to combat bacterial infections. In bacteria, the ferric uptake regulator Fur coordinates the expression of several iron-related genes. Sometimes, Fur can also regulate several other processes. In this work, we performed an in-depth phenotypic characterization of fur mutants in the human opportunistic pathogen Chromobacterium violaceum We determined that fur is a conditionally essential gene necessary for proper growth under regular conditions and is fully required for survival under iron and oxidative stresses. Fur also controlled several virulence-associated traits, such as swimming motility, biofilm formation, and siderophore production. Consistent with these results, a C. violaceumfur null mutant showed attenuation of virulence. Therefore, our data established Fur as a major player required for C. violaceum to manage iron, including during infection in the host.
Assuntos
Proteínas de Bactérias/genética , Chromobacterium/fisiologia , Chromobacterium/patogenicidade , Ferro/toxicidade , Estresse Oxidativo , Proteínas Repressoras/genética , Sideróforos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Repressoras/metabolismo , VirulênciaRESUMO
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).
Assuntos
Antibacterianos/metabolismo , Antibiose/fisiologia , Biofilmes/crescimento & desenvolvimento , Chromobacterium/metabolismo , Fontes Hidrotermais/microbiologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Aeromonas caviae/efeitos dos fármacos , Bacillus/efeitos dos fármacos , Brevibacillus/efeitos dos fármacos , Chromobacterium/isolamento & purificação , Chromobacterium/fisiologia , México , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/fisiologia , Vibrio alginolyticus/efeitos dos fármacosRESUMO
BACKGROUND: Vector control is critical in reducing the disease burden caused by mosquitoes, and insecticides are an effective tool to control vector populations. Resistance to common insecticides is now widespread, and novel classes of insecticides are needed. In previous work, we described the mosquitocidal activity of Chromobacterium sp. Panama (C.sp_P), a bacterium found in association with mosquitoes in natural populations. In the current work, we further explored the effects of exposure to the bacterium on mosquito fitness and mosquito physiology. RESULTS: We found that C.sp_P has mosquitocidal activity against a broad range of mosquito taxa. When exposed to C.sp_P as adults, female An. gambiae suffered reduced longevity, but experienced no change in fecundity. The offspring of these females, however, had higher mortality as larvae and were slower to develop compared to offspring of control females. We also found that the mosquitocidal activity of C.sp_P was retained after removal of live cells from biofilm culture media, suggesting the bacteria secrete mosquitocidal compound(s) into the media during growth. Exposure to this cell-free C.sp_P-conditioned media caused female midgut transcriptional changes comprising detoxification, xenobiotic response, and stress response genes, suggesting the physiological response to C.sp_P is similar to that of insecticide exposure. Finally, we found that multiple members of the Chromobacterium genus had mosquitocidal activity, but this activity was highest in mosquitoes treated with C.sp_P. CONCLUSIONS: Our findings suggest that C.sp_P produces factor(s) with strong effects on mosquito longevity and fitness, which may be of interest for mosquitocide development. More generally, they indicate that further exploration of mosquito-associated and environmental microbes for novel insecticidal compounds or biocontrol agents is warranted.
Assuntos
Anopheles/microbiologia , Chromobacterium/fisiologia , Controle de Mosquitos/métodos , Mosquitos Vetores/microbiologia , Animais , Anopheles/efeitos dos fármacos , Anopheles/genética , Anopheles/fisiologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Inativação Metabólica , Resistência a Inseticidas , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/genética , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Mosquitos Vetores/fisiologia , Piretrinas/farmacologiaRESUMO
Chromobacterium violaceum is a free-living bacterium that inhabits low-nutrient environments such as the Amazon basin. Bacteria respond to phosphate (Pi) shortage by expressing a range of genes involved in Pi uptake and assimilation, known as the PHO regulon. Several PHO regulon genes have been annotated in the genome of C. violaceum. Here we show that C. violaceum is extremely well adapted to low-Pi conditions. Remarkably, this bacterium is able to grow in media containing only traces of Pi. The PHO regulon genes are induced upon Pi depletion, but the bacteria continued to grow under these conditions. Unlike other Proteobacteria hitherto analyzed, neither PstS nor PhoU play a role in the repression of the PHO regulon under Pi excess.
Assuntos
Chromobacterium/fisiologia , Fosfatos/metabolismo , Aclimatação , Chromobacterium/genética , Chromobacterium/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Regulon/genéticaRESUMO
To counteract bacterial resistance, we investigated the interruption of quorum sensing mediated by non-classical bioisosteres of the N-hexanoyl homoserine lactone with an azoline core. For this purpose, a set of selected 2-substituted azolines was synthesized, establishing the basis for a new protocol to synthesize 2-amino imidazolines. The synthesized compounds were evaluated as inhibitors of violacein production in Chromobacterium violaceum. Theoretical studies on bioisostere-protein interactions were performed using CviR. The results show that some azolines decreased violacein production, suggesting an antiquorum sensing profile against Gram-negative bacteria. Docking and molecular dynamic simulations together with binding free energy calculations revealed the exact binding and inhibitory profiles. These theoretical results show relationship with the in vitro activity of the azoline series.
Assuntos
4-Butirolactona/análogos & derivados , Antibacterianos/farmacologia , Chromobacterium/efeitos dos fármacos , Chromobacterium/fisiologia , Cinoxacino/farmacologia , Percepção de Quorum/efeitos dos fármacos , 4-Butirolactona/química , 4-Butirolactona/farmacologia , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Cinoxacino/química , Simulação de Dinâmica MolecularRESUMO
Many Gram-negative pathogens have the ability to produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Strategies to inhibit QS are promising for the control of infectious diseases or antibiotic resistant bacterial pathogens. The aim of the present study was to evaluate the anti-quorum sensing (anti-QS) and antibacterial potential of five essential oils isolated from Lippia alba on the Tn-5 mutant of Chromobacterium violaceum CV026, and on the growth of the gram-positive bacteria S. aureus ATCC 25923. The anti-QS activity was detected through the inhibition of the QS-controlled violacein pigment production by the sensor bacteria. Results showed that two essential oils from L. alba, one containing the greatest geranial:neral and the other the highest limonene:carvone concentrations, were the most effective QS inhibitors. Both oils also had small effects on cell growth. Moreover, the geranial/neral chemotype oil also produced the maximum zone of growth inhibition against S. aureus ATCC 25923. These data suggest essential oils from L. alba have promising properties as QS modulators, and present antibacterial activity on S. aureus.
Assuntos
Anti-Infecciosos/farmacologia , Chromobacterium/efeitos dos fármacos , Lippia/química , Óleos Voláteis/farmacologia , Percepção de Quorum/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Anti-Infecciosos/isolamento & purificação , Chromobacterium/metabolismo , Chromobacterium/fisiologia , Testes de Sensibilidade Microbiana , Óleos Voláteis/isolamento & purificação , Pigmentos Biológicos/análise , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Many Gram-negative pathogens have the ability to produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Strategies to inhibit QS are promising for the control of infectious diseases or antibiotic resistant bacterial pathogens. The aim of the present study was to evaluate the anti-quorum sensing (anti-QS) and antibacterial potential of five essential oils isolated from Lippia alba on the Tn-5 mutant of Chromobacterium violaceum CV026, and on the growth of the gram-positive bacteria S. aureus ATCC 25923. The anti-QS activity was detected through the inhibition of the QS-controlled violacein pigment production by the sensor bacteria. Results showed that two essential oils from L. alba, one containing the greatest geranial:neral and the other the highest limonene:carvone concentrations, were the most effective QS inhibitors. Both oils also had small effects on cell growth. Moreover, the geranial/neral chemotype oil also produced the maximum zone of growth inhibition against S. aureus ATCC 25923. These data suggest essential oils from L. alba have promising properties as QS modulators, and present antibacterial activity on S. aureus.
Assuntos
Anti-Infecciosos/farmacologia , Chromobacterium/efeitos dos fármacos , Lippia/química , Óleos Voláteis/farmacologia , Percepção de Quorum/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Anti-Infecciosos/isolamento & purificação , Chromobacterium/metabolismo , Chromobacterium/fisiologia , Testes de Sensibilidade Microbiana , Óleos Voláteis/isolamento & purificação , Pigmentos Biológicos/análise , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Many Gram-negative pathogens have the ability to produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Strategies to inhibit QS are promising for the control of infectious diseases or antibiotic resistant bacterial pathogens. The aim of the present study was to evaluate the anti-quorum sensing (anti-QS) and antibacterial potential of five essential oils isolated from Lippia alba on the Tn-5 mutant of Chromobacterium violaceum CV026, and on the growth of the gram-positive bacteria S. aureus ATCC 25923. The anti-QS activity was detected through the inhibition of the QS-controlled violacein pigment production by the sensor bacteria. Results showed that two essential oils from L. alba, one containing the greatest geranial:neral and the other the highest limonene:carvone concentrations, were the most effective QS inhibitors. Both oils also had small effects on cell growth. Moreover, the geranial/neral chemotype oil also produced the maximum zone of growth inhibition against S. aureus ATCC 25923. These data suggest essential oils from L. alba have promising properties as QS modulators, and present antibacterial activity on S. aureus.
Assuntos
Anti-Infecciosos/farmacologia , Chromobacterium/efeitos dos fármacos , Lippia/química , Óleos Voláteis/farmacologia , Percepção de Quorum/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Anti-Infecciosos/isolamento & purificação , Chromobacterium/metabolismo , Chromobacterium/fisiologia , Testes de Sensibilidade Microbiana , Óleos Voláteis/isolamento & purificação , Pigmentos Biológicos/análise , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Biosurfactants are produced by hydrocarbon-degrading marine bacteria in response to the presence of water-insoluble hydrocarbons. This is believed to facilitate the uptake of hydrocarbons by bacteria. However, these diffusible amphiphilic surface-active molecules are involved in several other biological functions such as microbial competition and intra- or inter-species communication. We report the isolation and characterization of a marine bacterial strain identified as Cobetia sp. MM1IDA2H-1, which can grow using the sulfur-containing heterocyclic aromatic hydrocarbon dibenzothiophene (DBT). As with DBT, when the isolated strain is grown in the presence of a microbial competitor, it produces a biosurfactant. Because the obtained biosurfactant was formed by hydroxy fatty acids and extracellular lipidic structures were observed during bacterial growth, we investigated whether the biosurfactant at its critical micelle concentration can interfere with bacterial communication systems such as quorum sensing. We focused on Aeromonas salmonicida subsp. salmonicida, a fish pathogen whose virulence relies on quorum sensing signals. Using biosensors for quorum sensing based on Chromobacterium violaceum and Vibrio anguillarum, we showed that when the purified biosurfactant was mixed with N-acyl homoserine lactones produced by A. salmonicida, quorum sensing was inhibited, although bacterial growth was not affected. In addition, the transcriptional activities of A. salmonicida virulence genes that are controlled by quorum sensing were repressed by both the purified biosurfactant and the growth in the presence of Cobetia sp. MM1IDA2H-1. We propose that the biosurfactant, or the lipid structures interact with the N-acyl homoserine lactones, inhibiting their function. This could be used as a strategy to interfere with the quorum sensing systems of bacterial fish pathogens, which represents an attractive alternative to classical antimicrobial therapies in fish aquaculture.
Assuntos
Aeromonas salmonicida/efeitos dos fármacos , Antibacterianos/metabolismo , Halomonadaceae/metabolismo , Percepção de Quorum/efeitos dos fármacos , Tensoativos/metabolismo , Aeromonas salmonicida/genética , Aeromonas salmonicida/metabolismo , Aeromonas salmonicida/fisiologia , Técnicas Biossensoriais , Biotransformação , Chromobacterium/efeitos dos fármacos , Chromobacterium/fisiologia , Perfilação da Expressão Gênica , Halomonadaceae/classificação , Halomonadaceae/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Tiofenos/metabolismo , Vibrio/efeitos dos fármacos , Vibrio/fisiologia , Fatores de Virulência/metabolismoRESUMO
Natural cembranoids have shown Quorum Sensing Inhibitory (QSI) activity, but their structure-function interactions are not well understood. Thirty-four cembranoid analogues were synthesized using six natural cembranoids (1-6) previously isolated from the Colombian Caribbean octocorals Eunicea knighti and Pseudoplexaura flagellosa as lead compounds. The analogues (7-40) obtained through the selected chemical transformations were tested in vitro against the QS systems of a Chromobacterium violaceum biosensor. Half of the cembranoid analogues assayed showed superior QSI activity to the lead compounds; three (8, 13, and 18) displayed remarkable potency up to three times higher than the natural compounds. Thereby, we have synthesized a pool of cembranoid QS inhibitors that can be used in concert with natural compounds to develop antipathogenic drugs and antifouling agents.
Assuntos
Antozoários/química , Antibacterianos/química , Antibacterianos/farmacologia , Chromobacterium/efeitos dos fármacos , Diterpenos/química , Diterpenos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Região do Caribe , Chromobacterium/fisiologia , Diterpenos/síntese químicaRESUMO
Bacteria of the genus Bradyrhizobium are able to establish a symbiotic relationship with peanut (Arachis hypogaea) root cells and to fix atmospheric nitrogen by converting it to nitrogenous compounds. Quorum sensing (QS) is a cell-cell communication mechanism employed by a variety of bacterial species to coordinate behavior at a community level through regulation of gene expression. The QS process depends on bacterial production of various signaling molecules, among which the N-acylhomoserine lactones (AHLs) are most commonly used by Gram-negative bacteria. Some previous reports have shown the production of QS signaling molecules by various rhizobia, but little is known regarding mechanisms of communication among peanut-nodulating strains. The aims of this study were to identify and characterize QS signals produced by peanut-nodulating bradyrhizobial strains and to evaluate their effects on processes related to cell interaction. Detection of AHLs in 53 rhizobial strains was performed using the biosensor strains Agrobacterium tumefaciens NTL4 (pZLR4) and Chromobacterium violaceum CV026 for AHLs with long and short acyl chains, respectively. None of the strains screened were found to produce AHLs with short acyl chains, but 14 strains produced AHLs with long acyl chains. These 14 AHL-producing strains were further studied by quantification of ß-galactosidase activity levels (AHL-like inducer activity) in NTL4 (pZLR4). Strains displaying moderate to high levels of AHL-like inducer activity were subjected to chemical identification of signaling molecules by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS). For each AHL-producing strain, we found at least four different AHLs, corresponding to N-hexanoyl-DL-homoserine lactone (C(6)), N-(3-oxodecanoyl)-L-homoserine lactone (3OC(10)), N-(3-oxododecanoyl)-L-homoserine lactone (3OC(12)), and N-(3-oxotetradecanoyl)-L-homoserine lactone (3OC(14)). Biological roles of 3OC10, 3OC12, and 3OC14 AHLs were evaluated in both AHL-producing and -non-producing peanut-nodulating strains. Bacterial processes related to survival and nodulation, including motility, biofilm formation, and cell aggregation, were affected or modified by the exogenous addition of increasing concentrations of synthetic AHLs. Our results clearly demonstrate the existence of cell communication mechanisms among bradyrhizobial strains symbiotic of peanut. AHLs with long acyl chains appear to be signaling molecules regulating important QS physiological processes in these bacteria.
Assuntos
Bradyrhizobium/fisiologia , Percepção de Quorum , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Arachis/fisiologia , Biofilmes , Bradyrhizobium/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Chromobacterium/isolamento & purificação , Chromobacterium/fisiologia , Raízes de Plantas/fisiologia , Simbiose , Espectrometria de Massas em Tandem , beta-Galactosidase/metabolismoRESUMO
AIM: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. METHODS AND RESULTS: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong ß-haemolytic activity, were nonviolacein producers and utilized i-inositol, D-mannitol and D-sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S-23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)-PCR genomic fingerprinting using the BOX-AR1 primer. tDNA- and ITS-PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to ß-lactamic antibiotics. CONCLUSION: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX-PCR analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.
Assuntos
Chromobacterium/genética , Água Doce/microbiologia , Variação Genética , Brasil , Chromobacterium/classificação , Chromobacterium/isolamento & purificação , Chromobacterium/fisiologia , Primers do DNA/genética , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Filogenia , RNA Ribossômico 16S/genética , RNA de Transferência/genéticaRESUMO
Chromobacterium violaceum is a free-living Gram-negative bacterium found in soil and aquatic habitats; abundantly present in the Brazilian Amazon, it is an important example of exploitable microbial diversity of the tropics. In this study, 24 strains from the Brazilian Amazon and ATCC 12472(T) were investigated for biocontrol potential of seven fungi pathogenic to soybean [Glycine max (L.) Merril] seed. Both cells and the supernatants of two Brazilian strains, 07-1 and 27-1, together with ATCC 12472(T) were strongly antagonistic to six out of the seven fungi. The antifungal activity of the Brazilian strains to Fusarium sp., Phomopsis sp. and Cercospora kikuchi was consistently stronger than that of ATCC 12472(T). In addition, the two Brazilian strains, but not ATCC 12472(T), were effective against Corynespora sp., and all three strains and their supernatants were equally effective against Aspergillus sp. and Colletotrichum sp. None of the strains had antifungal activity against Botroyodiplodia sp. Three potential mechanisms related to the antibiosis were investigated: violacein toxicity, cyanide production and chitinolytic activity; however, it was not possible to associate any of them with the antifungal activity. The results highlight the biotechnological potential still to be explored within the poorly characterized microbial biodiversity of the tropics.
Assuntos
Antibiose , Ascomicetos/crescimento & desenvolvimento , Chromobacterium/fisiologia , Antifúngicos/farmacologia , Ascomicetos/classificação , Ascomicetos/isolamento & purificação , Brasil , Quitinases/farmacologia , Chromobacterium/isolamento & purificação , Cianetos/farmacologia , Indóis/farmacologia , Doenças das Plantas/microbiologia , Glycine max/microbiologiaRESUMO
BACKGROUND: Chromobacterium violaceum is a free-living bacterium able to survive under diverse environmental conditions. In this study we evaluate the genetic and physiological diversity of Chromobacterium sp. isolates from three Brazilian ecosystems: Brazilian Savannah (Cerrado), Atlantic Rain Forest and Amazon Rain Forest. We have analyzed the diversity with molecular approaches (16S rRNA gene sequences and amplified ribosomal DNA restriction analysis) and phenotypic surveys of antibiotic resistance and biochemistry profiles. RESULTS: In general, the clusters based on physiological profiles included isolates from two or more geographical locations indicating that they are not restricted to a single ecosystem. The isolates from Brazilian Savannah presented greater physiologic diversity and their biochemical profile was the most variable of all groupings. The isolates recovered from Amazon and Atlantic Rain Forests presented the most similar biochemical characteristics to the Chromobacterium violaceum ATCC 12472 strain. Clusters based on biochemical profiles were congruent with clusters obtained by the 16S rRNA gene tree. According to the phylogenetic analyses, isolates from the Amazon Rain Forest and Savannah displayed a closer relationship to the Chromobacterium violaceum ATCC 12472. Furthermore, 16S rRNA gene tree revealed a good correlation between phylogenetic clustering and geographic origin. CONCLUSION: The physiological analyses clearly demonstrate the high biochemical versatility found in the C. violaceum genome and molecular methods allowed to detect the intra and inter-population diversity of isolates from three Brazilian ecosystems.
Assuntos
Chromobacterium/classificação , Chromobacterium/isolamento & purificação , Ecossistema , Variação Genética , Microbiologia da Água , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Brasil , Chromobacterium/genética , Chromobacterium/fisiologia , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Geografia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ribotipagem , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Estatística como AssuntoRESUMO
The repertoire of 4,431 open reading frames (ORFs), eight rRNA operons and 98 tRNA genes of Chromobacterium violaceum must be expressed in a regulated manner for successful adaptation to a wide variety of environmental conditions. To accomplish this feat, the organism relies on protein machineries involved in transcription, RNA processing and translation. Analysis of the C. violaceum genome showed that transcription initiation, elongation and termination are performed by the five well-known RNA polymerase subunits, five categories of sigma 70 factors, one sigma 54 factor, as well as six auxiliary elongation and termination factors. RNA processing is performed by a variety of endonucleases and exonucleases, such as ribonuclease H, ribonuclease E, ribonuclease P, and ribonuclease III, in addition to poly(A) polymerase and specific methyltransferases and pseudouridine synthases. ORFs for all ribosomal proteins, except S22, were found. Only 19 aminoacyl-tRNA synthetases were found, in addition to three aminoacyl-tRNA synthetase-related proteins. Asparaginyl-tRNA (Asn) is probably obtained by enzymatic modification of a mischarged aminoacyl-tRNA. The translation factors IF-1, IF-2, IF-3, EF-Ts, EF-Tu, EF-G, RF-1, RF-2 and RF-3 are all present in the C. violaceum genome, although the absence of selB suggests that C. violaceum does not synthesize selenoproteins. The components of trans-translation, tmRNA and associated proteins, are present in the C. violaceum genome. Finally, a large number of ORFs related to regulation of gene expression were also found, which was expected, considering the apparent adaptability of this bacterium.
Assuntos
Adaptação Fisiológica/genética , Chromobacterium/genética , Regulação Bacteriana da Expressão Gênica/genética , Chromobacterium/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Fases de Leitura Aberta/genética , RNA de Transferência/genética , Transcrição Gênica , Óperon de RNAr/genéticaRESUMO
The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism. This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C. violaceum. C. violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome. We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C. violaceum. A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins. The chemoreceptors found in C. violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C. violaceum genome. Also, the C. violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system. Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C. violaceum genome.