RESUMO
BACKGROUND: Canonical biocontrol bacteria were considered to inhibit pathogenic bacteria mainly by secreting antibiotic metabolites or enzymes. Recent studies revealed that some biocontrol bacteria can inhibit pathogenic bacteria through contact-dependent killing (CDK) mediated by contact-dependent secretion systems. The CDK was independent of antibiotic metabolites and often ignored in normal biocontrol activity assay. RESULTS: In this study, we aimed to use a pathogen enrichment strategy to isolate non-canonical bacteria with CDK ability. Rhizosphere soil samples from Chinese cabbage showing soft rot symptom were collected and Pectobacterium carotovorum subsp. carotovorum (Pcc), the pathogen of cabbage soft rot, were added into these samples to enrich bacteria which attached on Pcc cells. By co-culture with Pcc, four bacteria strains (named as PcE1, PcE8, PcE12 and PcE13) showing antibacterial activity were isolated from Chinese cabbage rhizosphere. These four bacteria strains showed CDK abilities to different pathogenic bacteria of horticultural plants. Among them, PcE1 was identified as Chryseobacterium cucumeris. Genome sequencing showed that PcE1 genome encoded a type VI secretion system (T6SS) gene cluster. By heterologous expression, four predicted T6SS effectors of PcE1 showed antibacterial activity to Escherichia coli. CONCLUSION: Overall, this study isolated four bacteria strains with CDK activity to various horticultural plant pathogens, and revealed possible involvement of T6SS of Chryseobacterium cucumeris in antibacterial activity. These results provide valuable insight for potential application of CDK activity in biocontrol bacteria. © 2024 Society of Chemical Industry.
Assuntos
Antibiose , Brassica , Pectobacterium carotovorum , Brassica/microbiologia , Pectobacterium carotovorum/genética , Microbiologia do Solo , Rizosfera , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismoRESUMO
Numerous functions in pathogenic Pectobacterium are regulated by quorum sensing (QS). Two different aiiA genes isolated from Bacillus sp. A24(aiiAA24) and Bacillus sp. DMS133(aiiADMS133) were used. Both genes encode acyl-homoserine lactonase (AiiA), which disrupts QS in Pectobacterium. To investigate the effect of different AiiAs on the inhibition of Pectobacterium carotovorum pathogenicity, two aiiA genes from different Bacillus strains were cloned and the resulting plasmids pME6863 (aiiAA24) and pME7080 (aiiADMS133) were transformed into P. carotovorum EMPCC cells. The effects of different lactonases on virulence features such as enzymatic activity, twitching and swimming motilities, and production of pellicle and biofilm formation were investigated. In EMPCC/pME6863, twitching and swimming motilities, and pellicle production were significantly reduced compared with EMPCC/pME7080. Quantitative real-time PCR (qRT-PCR) was used to measure virulence gene expression in transformed cells compared with expression levels in wild-type EMPCC. The expression of peh and hrpL genes was greatly reduced in EMPCC/pME6863 compared with EMPCC/pME7080. The sequence alignment and molecular dynamic modeling of two different AiiAA24 and AiiADMS133 proteins suggested that the replacement of proline 210 from AiiAA24 to serine in AiiADMS133 caused the reduction of enzyme activity in AiiADMS133.
Assuntos
Bacillus , Proteínas de Bactérias , Hidrolases de Éster Carboxílico , Percepção de Quorum , Bacillus/genética , Bacillus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Biofilmes/crescimento & desenvolvimento , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/patogenicidade , Virulência , Regulação Bacteriana da Expressão Gênica , Clonagem Molecular , MetaloendopeptidasesRESUMO
BACKGROUND: Pectobacterium species are necrotrophic phytopathogenic bacteria that cause soft rot disease in economically important crops. The successful infection of host plants relies on interactions among virulence factors, competition, and transmission within hosts. Pectobacteria primarily produce and secrete plant cell-wall degrading enzymes (PCWDEs) for virulence. The regulation of PCWDEs is controlled by quorum sensing (QS). Thus, the QS system is crucial for disease development in pectobacteria through PCWDEs. RESULTS: In this study, we identified a Tn-insertion mutant, M2, in the expI gene from a transposon mutant library of P. carotovorum subsp. carotovorum Pcc21 (hereafter Pcc21). The mutant exhibited reduced production and secretion of PCWDEs, impaired flagellar motility, and increased sensitivity to hydrogen peroxide, resulting in attenuated soft rot symptoms in cabbage and potato tubers. Transcriptomic analysis revealed the down-regulation of genes involved in the production and secretion in the mutant, consistent with the observed phenotype. Furthermore, the Pcc21 wild-type transiently colonized in the gut of Drosophila melanogaster within 12 h after feeding, while the mutant compromised colonization phenotype. Interestingly, Pcc21 produces a bacteriocin, carocin D, to compete with other bacteria. The mutant exhibited up-regulation of carocin D-encoding genes (caroDK) and inhibited the growth of a closely related bacterium, P. wasabiae. CONCLUSION: Our results demonstrated the significance of ExpI in the overall pathogenic lifestyle of Pcc21, including virulence, competition, and colonization in plant and insect hosts. These findings suggest that disease outcome is a result of complex interactions mediated by ExpI across multiple steps. © 2023 Society of Chemical Industry.
Assuntos
Ligases , Pectobacterium carotovorum , Pectobacterium , Animais , Virulência/genética , Pectobacterium carotovorum/genética , Drosophila melanogaster , Pectobacterium/genética , Doenças das Plantas/microbiologiaRESUMO
BACKGROUND: Quorum sensing inhibitors (QSIs) are an emerging control tool that inhibits the quorum sensing (QS) system of pathogenic bacteria. We aimed to screen for potential QSIs in the metabolites of Trichoderma and to explore their inhibitory mechanisms. RESULTS: We screened a strain of Trichoderma asperellum LN004, which demonstrated the ability to inhibit the color development of Chromobacterium subtsugae CV026, primarily attributed to the presence of emodin as its key QSI component. The quantitative polymerase chain reaction with reverse transcription results showed that after emodin treatment of Pectobacterium carotovorum subsp. carotovorum (Pcc), plant cell wall degrading enzyme-related synthetic genes were significantly downregulated, and the exogenous enzyme synthesis gene negative regulator (rsmA) was upregulated 3.5-fold. Docking simulations indicated that emodin could be a potential ligand for ExpI and ExpR proteins because it exhibited stronger competition than the natural ligands in Pcc. In addition, western blotting showed that emodin attenuated the degradation of n-acylhomoserine lactone on the ExpR protein and protected it. Different concentrations of emodin reduced the activity of pectinase, cellulase, and protease in Pcc by 20.81%-72.21%, 8.38%-52.73%, and 3.57%-47.50%. Lesion size in Chinese cabbages, carrots and cherry tomatoes following Pcc infestation was reduced by 10.02%-68.57%, 40.17%-88.56% and 11.36%-86.17%. CONCLUSION: Emodin from T. asperellum LN004 as a QSI can compete to bind both ExpI and ExpR proteins, interfering with the QS of Pcc and reducing the production of virulence factors. The first molecular mechanism reveals the ability of emodin as a QSI to competitively inhibit two QS proteins simultaneously. © 2023 Society of Chemical Industry.
Assuntos
Emodina , Pectobacterium , Trichoderma , Emodina/metabolismo , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Proteínas de Bactérias/genética , Doenças das Plantas/microbiologiaRESUMO
Pectobacterium is one of the most important genera of phytopathogenic bacteria. It can cause soft-rot diseases on a wide range of plant species across the world. In this study, three Pectobacterium strains (KC01, KC02, and KC03) were isolated from soft-rotted Chinese cabbage in Beijing, China. These three strains were identified as Pectobacterium versatile based on phylogenetic analysis of Pectobacterium 16S ribosomal RNA, pmrA, and 504 Pectobacterium core genes, as well as a genomic average nucleotide identity analysis. Their biochemical characteristics were found to be similar to the P. versatile type strain ICMP9168T but differed in response to citric acid, stachyose, D-glucuronic acid, dextrin, and N-acetyl-ß-D-mannosamine. All of the tested P. versatile strains showed different carbohydrate utilization abilities compared with P. carotovorum and P. odoriferum, particularly in their ability to utilize D-arabitol, L-rhamnose, and L-serine. Under laboratory conditions, the maceration ability of P. versatile on Chinese cabbage was the highest at 28°C, compared with those at 13, 28, 23, and 33°C. Additionally, P. versatile could infect all of the 17 known Pectobacterium host plants, except for Welsh onion (Allium fistulosum). A SYBR Green quantitative PCR (qPCR) detection system was developed to distinguish P. versatile from other soft-rot bacteria based on the combined performance of melting curve (with a single melting peak at around 85°C) and fluorescence curve (with cycle threshold <30) when the bacterial genomic DNA concentration was in the range of 10 pg/µl to 10 ng/µl. This study is the first to report the presence of P. versatile on Chinese cabbage in China, as well as a specific and sensitive qPCR assay that can be used to quickly identify P. versatile. The work contributes to a better understanding of P. versatile and will facilitate the effective diagnosis of soft-rot disease, ultimately benefitting commercial crop production.
Assuntos
Brassica , Pectobacterium , Pectobacterium carotovorum/genética , Filogenia , Pectobacterium/genética , Brassica/microbiologia , China , Plantas , Bactérias/genética , DNA Bacteriano/genética , Reação em Cadeia da PolimeraseRESUMO
Microbial-based strategy in nanotechnology offers economic, eco-friendly, and biosafety advantages over traditional chemical and physical protocols. The current study describes a novel biosynthesis protocol for chitosan nanoparticles (CNPs), employing a pioneer Streptomyces sp. strain NEAE-83, which exhibited a significant potential for CNPs biosynthesis. It was identified as Streptomyces microflavus strain NEAE-83 based on morphological, and physiological properties as well as the 16S rRNA sequence (GenBank accession number: MG384964). CNPs were characterized by SEM, TEM, EDXS, zeta potential, FTIR, XRD, TGA, and DSC. CNPs biosynthesis was maximized using a mathematical model, face-centered central composite design (CCFCD). The highest yield of CNPs (9.41 mg/mL) was obtained in run no. 27, using an initial pH of 5.5, 1% chitosan, 40 °C, and a 12 h incubation period. Innovatively, the artificial neural network (ANN), was used for validating and predicting CNPs biosynthesis based on the trials data of CCFCD. Despite the high precision degree of both models, ANN was supreme in the prediction of CNPs biosynthesis compared to CCFCD. ANN had a higher prediction efficacy and, lower error values (RMSE, MDA, and SSE). CNPs biosynthesized by Streptomyces microflavus strain NEAE-83 showed in-vitro antibacterial activity against Pectobacterium carotovorum, which causes the potato soft rot. These results suggested its potential application for controlling the destructive potato soft rot diseases. This is the first report on the biosynthesis of CNPs using a newly isolated; Streptomyces microflavus strain NEAE-83 as an eco-friendly approach and optimization of the biosynthesis process by artificial intelligence.
Assuntos
Quitosana , Nanopartículas , Solanum tuberosum , Streptomyces , Pectobacterium carotovorum/genética , RNA Ribossômico 16S/genética , Inteligência Artificial , Streptomyces/genética , Solanum tuberosum/genéticaRESUMO
Pectobacterium carotovorum is an economically important phytopathogen and has been identified as the major causative agent of bacterial soft rot in carrots. Control of this phytopathogen is vital to minimizing carrot harvest losses. As fully efficient control measures to successfully avoid the disease are unavailable, the phage-mediated biocontrol of the pathogen has recently gained scientific attention. In this study, we present a comprehensive characterization of the P. carotovorum phage vB_PcaM_P7_Pc (abbreviated as P7_Pc) that was isolated from infected carrot samples with characteristic soft rot symptoms, which were obtained from storage facilities at market places in Gampaha District, Sri Lanka. P7_Pc is a myovirus, and it exhibits growth characteristics of an exclusively lytic life cycle. It showed visible lysis against four of the tested P. carotovorum strains and one Pectobacterium aroidearum strain. This phage also showed a longer latent period (125 min) than other related phages; however, this did not affect its high phage titter (>1010 PFU/mL). The final assembled genome of P7_Pc is 147,299 bp in length with a G+C content of 50.34%. Of the 298 predicted open reading frames (ORFs) of the genome of P7_Pc, putative functions were assigned to 53 ORFs. Seven tRNA-coding genes were predicted in the genome, while the genome lacked any major genes coding for lysogeny-related products, confirming its virulent nature. The P7_Pc genome shares 96.12% and 95.74% average nucleotide identities with Cronobacter phages CR8 and PBES02, respectively. Phylogenetic and phylogenomic analyses of the genome revealed that P7_Pc clusters well within the clade with the members representing the genus Certrevirus. Currently, there are only 4 characterized Pectobacterium phages (P. atrosepticum phages phiTE and CB7 and Pectobacterium phages DU_PP_I and DU_PP_IV) that are classified under the genus, making the phage P7_Pc the first reported member of the genus isolated using the host bacterium P. carotovorum. The results of this study provide a detailed characterization of the phage P7_Pc, enabling its careful classification into the genus Certrevirus. The knowledge gathered on the phage based on the shared biology of the genus will further aid in the future selection of phage P7_Pc as a biocontrol agent. IMPORTANCE Bacterial soft rot disease, caused by Pectobacterium spp., can lead to significant losses in carrot yields. As current control measures involving the use of chemicals or antibiotics are not recommended in many countries, bacteriophage-mediated biocontrol strategies are being explored for the successful control of these phytopathogens. The successful implementation of such biocontrol strategies relies heavily upon the proper understanding of the growth characteristics and genomic properties of the phage. Further, the selection of taxonomically different phages for the formulation of phage cocktails in biocontrol applications is critical to combat potential bacterial resistance development. This study was conducted to carefully characterize and resolve the phylogenetic placement of the P. carotovorum phage vB_PcaM_P7_Pc by using its biological and genomic properties. Phage P7_Pc has a myovirus morphotype with an exclusively lytic life cycle, and the absence of genes related to lysogeny, toxin production, and antibiotic resistance in its genome confirmed its suitability to be used in environmental applications. Furthermore, P7_Pc is classified under the genus Certrevirus, making it the first reported phage of the genus of the host species, P. carotovorum.
Assuntos
Bacteriófagos , Pectobacterium carotovorum/genética , Filogenia , Myoviridae/genética , GenômicaRESUMO
Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management because different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is Pectobacterium carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of Pectobacterium versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen Pectobacterium parmentieri. This should be useful for the routine diagnosis of potato soft rot.
Assuntos
Pectobacterium carotovorum , Solanum tuberosum , Pectobacterium carotovorum/genética , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S , Solanum tuberosum/microbiologiaRESUMO
The cpxR gene, encoding a new cytoplasmic response regulator, which effects virulence, biofilm formation, chemotaxis, resistance to antimicrobials, and controls soft rot, was amplified by the polymerase chain reaction, cloned into the prokaryotic expression vector pET-15b, and expressed through the induction of isopropyl-ß-d-thiogalactoside in Escherichia coli BL21 (DE3). Then, highly purified and stable CpxR protein was produced by nickel affinity chromatography and fast protein liquid chromatography, digested by thrombin and identified by Western blotting. Furthermore, the structure of the CpxR protein was estimated by circular dichroism spectroscopy and SWISS-MODEL. The CpxR protein was a functional part in signal transduction and bacterial resistance for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The resear ch of the protein stability indicated the CpxR protein had excellent thermal stability and was suitable for crystallization. Then the small crystals of CpxR protein were found in the crystallizing tank. The latest 34 cpxR sequences from the public database were selected and analyzed by molecular clustering and multisequence alignment. These cpxR sequences were roughly divided into four categories. These results laid an important foundation for the further structural study of the CpxR protein.
Assuntos
Escherichia coli , Pectobacterium carotovorum , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Pectobacterium carotovorum/genética , Reação em Cadeia da PolimeraseRESUMO
Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Pectobacterium carotovorum/enzimologia , Poligalacturonase/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Ácidos Hexurônicos/metabolismo , Pectinas/metabolismo , Pectobacterium carotovorum/genética , Poligalacturonase/genética , Cloreto de Potássio/metabolismo , Regiões Promotoras GenéticasRESUMO
It has been shown that phages have evolved anti-CRISPR (Acr) proteins to inhibit host CRISPR-Cas systems. Most acr genes are located upstream of anti-CRISPR-associated (aca) genes, which is instrumental for identifying these acr genes. Thus far, eight Aca families (Aca1-Aca8) have been identified, all proteins of which share low sequence homology and bind to different target DNA sequences. Recently, Aca1 and Aca2 proteins were discovered to function as repressors by binding to acr-aca promoters, thus implying a potential anti-anti-CRISPR mechanism. However, the structural basis for the repression roles of Aca proteins is still unknown. Here, we elucidated apo-structures of Aca1 and Aca2 proteins and their complex structures with their cognate operator DNA in two model systems, the Pseudomonas phage JBD30 and the Pectobacterium carotovorum template phage ZF40. In combination with biochemical and cellular assays, our study unveils dimerization and DNA-recognition mechanisms of Aca1 and Aca2 family proteins, thus revealing the molecular basis for Aca1-and Aca2-mediated anti-CRISPR repression. Our results also shed light on understanding the repression roles of other Aca family proteins and autoregulation roles of acr-aca operons.
Assuntos
Bacteriófagos/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Óperon , Pectobacterium carotovorum/virologia , Pseudomonas aeruginosa/virologia , Proteínas Virais/metabolismo , Bacteriófagos/química , Bacteriófagos/genética , Modelos Moleculares , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Conformação Proteica , Multimerização Proteica , Fagos de Pseudomonas/química , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Based on Single moleculereal time(SMRT)sequencing technology, the high-quality whole genome sequence of Pectobacterium carotovorum (PC1) was obtained by the PacBio RS II sequencer. The genome is a single circular chromosome of 5.3 Mb in size, containing three kinds of m6A methylation modification by SMRT Portal analysis. Genome annotation showed that 575 virulence factor genes, 304 drug resistance genes, 774 pathogen genes, 7 secretory systems and 22 pairs of two-component regulatory system could be relevant to bacterial pathogenicity. In addition, the average nucleotide identities (ANI) analysisshowed that the PC1 exhibited the highest homology with the Pectobacteriumcarotovorumsubsp.carotovorumstrain BP201601.1 (NZ_CP034236). There are 28 unique gene families to PC1 using cluster analysis of gene families. According to the analysis of key pathogenic genes, we have obtained three kinds of highly conserved genes related to cell wall degrading enzymes, including 19 pectinase genes, 25 cellulase genes and 22 protease genes. Our studies have provided a theoretical basis for investigation of bacterial soft rot and biological specific bactercides of PC1.
Assuntos
Genes Bacterianos , Genômica , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidade , Virulência/genética , Sequenciamento Completo do Genoma , Doenças das Plantas/microbiologiaRESUMO
The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out. However, the orthologous pectate lyases Pel3 and PelI from these bacteria, which share 67% of sequence identity, are not secreted by the counterpart T2SS of each bacterium, indicating a fine-tuned control of protein recruitment. To identify the related secretion determinants, we first performed a structural characterization and comparison of Pel3 with PelI using X-ray crystallography. Then, to assess the biological relevance of the observed structural variations, we conducted a loop-substitution analysis of Pel3 combined with secretion assays. We showed that there is not one element with a definite secondary structure but several distant and structurally flexible loop regions that are essential for the secretion of Pel3 and that these loop regions act together as a composite secretion signal. Interestingly, depending on the crystal contacts, one of these key secretion determinants undergoes disorder-to-order transitions that could reflect its transient structuration upon the contact with the appropriate T2SS components. We hypothesize that such T2SS-induced structuration of some intrinsically disordered zones of secretion substrates could be part of the recruitment mechanism used by T2SS.
Assuntos
Proteínas de Bactérias/química , Dickeya/enzimologia , Pectobacterium carotovorum/enzimologia , Polissacarídeo-Liases/química , Sistemas de Secreção Tipo II/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Parede Celular/química , Parede Celular/microbiologia , Clonagem Molecular , Cristalografia por Raios X , Dickeya/classificação , Dickeya/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Pectobacterium carotovorum/classificação , Pectobacterium carotovorum/genética , Filogenia , Células Vegetais/química , Células Vegetais/microbiologia , Plantas/química , Plantas/microbiologia , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sistemas de Secreção Tipo II/genética , Sistemas de Secreção Tipo II/metabolismoRESUMO
Pectobacterium spp. are a major cause of loss in vegetable and ornamental plant production. One of these species, Pectobacterium carotovorum, can cause soft rot disease on many plants, particularly potato. These diseases lead to significant economic loss and pose food security threats by reducing crop yields in the field, in transit, and during storage. The Gram-negative enterobacterium P. carotovorum WPP14 is a particularly virulent strain for which there is no available closed genome, limiting the molecular research for this important pathogen. Here, we report a high-quality complete and annotated genome sequence of P. carotovorum WPP14. The 4,892,225-bp genome was assembled with Nanopore reads and polished with Illumina reads, yielding 394× and 164× coverage, respectively. This closed genome provides a resource for research on improved detection and biology of P. carotovorum, which could translate into improved disease management.
Assuntos
Pectobacterium , Solanum tuberosum , Bactérias , Pectobacterium/genética , Pectobacterium carotovorum/genética , Doenças das PlantasRESUMO
Multihost bacteria have to rapidly adapt to drastic environmental changes, relying on a fine integration of multiple stimuli for an optimal genetic response. Erwinia carotovora spp. are phytopathogens that cause soft-rot disease. Strain Ecc15 in particular is a model for bacterial oral-route infection in Drosophila melanogaster as it harbors a unique gene, evf, that encodes the Erwinia virulence factor (Evf), which is a major determinant for infection of the D. melanogaster gut. However, the factors involved in the regulation of evf expression are poorly understood. We investigated whether evf could be controlled by quorum sensing as, in the Erwinia genus, quorum sensing regulates pectolytic enzymes, the major virulence factors needed to infect plants. Here, we show that transcription of evf is positively regulated by quorum sensing in Ecc15 via acyl-homoserine lactone (AHL) signal synthase ExpI and AHL receptors ExpR1 and ExpR2. We also show that the load of Ecc15 in the gut depends upon the quorum sensing-mediated regulation of evf Furthermore, we demonstrate that larvae infected with Ecc15 suffer a developmental delay as a direct consequence of the regulation of evf via quorum sensing. Finally, we demonstrate that evf is coexpressed with plant cell wall-degrading enzymes (PCWDE) during plant infection in a quorum sensing-dependent manner. Overall, our results show that Ecc15 relies on quorum sensing to control production of both pectolytic enzymes and Evf. This regulation influences the interaction of Ecc15 with its two known hosts, indicating that quorum sensing signaling may impact bacterial dissemination via insect vectors that feed on rotting plants.IMPORTANCE Integration of genetic networks allows bacteria to rapidly adapt to changing environments. This is particularly important in bacteria that interact with multiple hosts. Erwinia carotovora is a plant pathogen that uses Drosophila melanogaster as a vector. To interact with these two hosts, Ecc15 uses different sets of virulence factors: plant cell wall-degrading enzymes to infect plants and the Erwinia virulence factor (evf) to infect Drosophila Our work shows that, despite the virulence factors being specific for each host, both sets are coactivated by homoserine lactone quorum sensing and by the two-component GacS/A system in infected plants. This regulation is essential for Ecc15 loads in the gut of Drosophila and minimizes the developmental delay caused by the bacteria with respect to the insect vector. Our findings provide evidence that coactivation of the host-specific factors in the plant may function as a predictive mechanism to maximize the probability of transit of the bacteria between hosts.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/genética , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Percepção de Quorum/genética , Fatores de Virulência/genética , Animais , Drosophila melanogaster/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Masculino , Fatores de Virulência/metabolismoRESUMO
Globally, there is a high economic burden caused by pre- and post-harvest losses in vegetables, fruits and ornamentals due to soft rot diseases. At present, the control methods for these diseases are limited, but there is some promise in developing biological control products for use in Integrated Pest Management. This study sought to formulate a phage cocktail which would be effective against soft rot Pectobacteriaceae species affecting potato (Solanum tuberosum L.), with potential methods of application in agricultural systems, including vacuum-infiltration and soil drench, also tested. Six bacteriophages were isolated and characterized using transmission electron microscopy, and tested against a range of Pectobacterium species that cause soft rot/blackleg of potato. Isolated bacteriophages of the family Podoviridae and Myoviridae were able to control isolates of the Pectobacterium species: Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum. Genomic analysis of three Podoviridae phages did not indicate host genes transcripts or proteins encoding toxin or antibiotic resistance genes. These bacteriophages were formulated as a phage cocktail and further experiments showed high activity in vitro and in vivo to suppress Pectobacterium growth, potentially indicating their efficacy in formulation as a microbial pest control agent to use in planta.
Assuntos
Myoviridae/metabolismo , Pectobacterium/efeitos dos fármacos , Podoviridae/metabolismo , Bacteriófagos/genética , Agentes de Controle Biológico/metabolismo , Genômica , Myoviridae/genética , Pectobacterium/crescimento & desenvolvimento , Pectobacterium/metabolismo , Pectobacterium carotovorum/genética , Controle de Pragas/métodos , Filogenia , Doenças das Plantas/microbiologia , Podoviridae/genética , Solanum tuberosum/microbiologiaRESUMO
The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella pertussis/enzimologia , Oxigênio/metabolismo , Pectobacterium carotovorum/enzimologia , Fósforo-Oxigênio Liases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Bordetella pertussis/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Cinética , Pectobacterium carotovorum/genética , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/genética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
The plant pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) regulates the expression of virulence factors by N-acylhomoserine lactone (AHL)-mediated quorum sensing. The LuxI family protein, ExpI, catalyzes AHL biosynthesis in Pcc. The structure of the predominant AHL produced by ExpI differs among Pcc strains, which may be divided into two quorum-sensing classes (QS classes) based on the AHL produced. In the present study, AHL produced by 282 Pcc strains were extracted and identified by LC-MS/MS. Seventy Pcc strains produced N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL) as the predominant AHL and were categorized into QS class I. Two hundred Pcc strains produced N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL) as the predominant AHL, and were categorized into QS class II-1. Twelve Pcc strains produced only small amounts of 3-oxo-C6-HSL, and were categorized into QS class II-2. The phylogenetic analysis revealed that the amino acid sequences of ExpI may be divided into two major clades (I and II). The Pcc strains categorized into ExpI clades I and II entirely matched QS classes I and II, respectively. A multiple alignment analysis demonstrated that only 6 amino acid substitutions were observed among ExpI from QS classes II-1 and II-2. Furthermore, many amino acid substitutions between QS classes I and II were concentrated at the C-terminal region. These amino acid substitutions are assumed to cause significant reductions in 3-oxo-C6-HSL in QS class II-2 or affect the substrate specificity of ExpI between QS classes I and II.
Assuntos
Acil-Butirolactonas/metabolismo , Variação Genética , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Doenças das Plantas/microbiologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Acil-Butirolactonas/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Homosserina/análogos & derivados , Homosserina/química , Homosserina/metabolismo , Pectobacterium carotovorum/classificação , Filogenia , Percepção de QuorumRESUMO
CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by the Pectobacterium carotovorum temperate phage ZF40, is an autoregulator that represses the anti-CRISPR-aca2 operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.
Assuntos
Sistemas CRISPR-Cas/genética , Pectobacterium carotovorum/genética , Transcrição Gênica , Proteínas Virais/genética , Bacteriófagos/genética , Escherichia coli/genética , Óperon/genética , Regiões Promotoras Genéticas/genética , Domínios Proteicos/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Pectobacterium carotovorum subsp. brasiliense is a broad host range bacterial pathogen, which causes blackleg of potatoes and bacterial soft rot of vegetables worldwide. Production of plant cell wall degrading enzymes is usually critical for Pectobacterium infection. However, other virulence factors and the mechanisms of genetic adaptation still need to be studied in detail. RESULTS: In this study, the complete genome of P. carotovorum subsp. brasiliense strain SX309 isolated from cucumber was compared with eight other pathogenic bacteria belonging to the Pectobacterium genus, which were isolated from various host plants. Genome comparison revealed that most virulence genes are highly conserved in the Pectobacterium strains, especially for the key virulence determinants involved in the biosynthesis of extracellular enzymes and others including the type II and III secretion systems, quorum sensing system, flagellar and chemotactic genes. Nevertheless, some variable regions of the T6SS and the CRISP-Cas immune system are unique for P. carotovorum subsp. brasiliense. CONCLUSIONS: The extensive comparative genomics analysis revealed highly conserved virulence genes in the Pectobacterium strains. However, several variable regions of type VI secretion system and two subtype Cas mechanism-Cas immune systems possibly contribute to the process of Pectobacterium infection and adaptive immunity.