Project description:We report the complete and annotated genome sequence of the plant-pathogenic enterobacterium Pectobacterium sp. strain SCC3193, a model strain isolated from potato in Finland. The Pectobacterium sp. SCC3193 genome consists of a 5,164,411-bp [corrected] chromosome, with no plasmids.
Project description:Quorum sensing (QS) is a cell density-dependent mechanism that regulates the expression of specific genes in microbial cells. Quorum quenching (QQ) is a promising strategy for attenuating pathogenicity by interfering with the QS system of pathogens. N-Acyl-homoserine lactones (AHLs) act as signaling molecules in many Gram-negative bacterial pathogens and have received wide attention. In this study, a novel, efficient AHL-degrading bacterium, Acinetobacter sp. strain XN-10, was isolated from agricultural contaminated soil and evaluated for its degradation efficiency and potential use against QS-mediated pathogens. Strain XN-10 could effectively degrade N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), N-hexanoyl-L-homoserine lactone (C6HSL), N-(3-oxododecanoyl)-L-homoserine lactone (3OC12HSL), and N-(3-oxooctanoyl)-L-homoserine lactone (3OC8HSL), which all belong to the AHL family. Analysis of AHL metabolic products by gas chromatography-mass spectrometry (GC-MS) led to the identification of N-cyclohexyl-propanamide, and pentanoic acid, 4-methyl, methyl ester as the main intermediate metabolites, revealing that AHL could be degraded by hydrolysis and dehydroxylation. All intermediates were transitory and faded away without any non-cleavable metabolites at the end of the experiment. Furthermore, strain XN-10 significantly attenuated the pathogenicity of Pectobacterium carotovorum subsp. carotovorum (Pcc) to suppress tissue maceration in carrots, potatoes, and Chinese cabbage. Taken together, our results shed light on the QQ mechanism of a novel AHL-degrading bacterial isolate, and they provide useful information which show potential for biocontrol of infectious diseases caused by AHL-dependent bacterial pathogens.
Project description:Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are be-lieved to play an important role in pathogenicity, and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and yields of MVs depend on medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produce MVs of a larger size (100-300 nm) apart of vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to de-grade pectates. What is more, pathogenicity test indicated that MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that Pectobacterium strains, which overexpress the green fluorescence protein (GFP), produce more MVs than wild type strains. Moreover, proteomic analysis revealed that GFP was present in MVs. Therefore, we demonstrate that protein sequestration into MVs is not limited strictly to periplasmic proteins and is a common occurrence. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an alternative secretion system.
Project description:Pectobacterium strains isolated from potato stems in Finland, Poland and the Netherlands were subjected to polyphasic analyses to characterize their genomic and phenotypic features. Phylogenetic analysis based on 382 core proteins showed that the isolates clustered closest to Pectobacterium polaris but could be divided into two clades. Average nucleotide identity (ANI) analysis revealed that the isolates in one of the clades included the P. polaris type strain, whereas the second clade was at the border of the species P. polaris with a 96 % ANI value. In silico genome-to-genome comparisons between the isolates revealed values below 70%, patristic distances based on 1294 core proteins were at the level observed between closely related Pectobacterium species, and the two groups of bacteria differed in genome size, G+C content and results of amplified fragment length polymorphism and Biolog analyses. Comparisons between the genomes revealed that the isolates of the atypical group contained SPI-1-type Type III secretion island and genes coding for proteins known for toxic effects on nematodes or insects, and lacked many genes coding for previously characterized virulence determinants affecting rotting of plant tissue by soft rot bacteria. Furthermore, the atypical isolates could be differentiated from P. polaris by their low virulence, production of antibacterial metabolites and a citrate-negative phenotype. Based on the results of a polyphasic approach including genome-to-genome comparisons, biochemical and virulence assays, presented in this report, we propose delineation of the atypical isolates as a novel species Pectobacterium parvum, for which the isolate s0421T (CFBP 8630T=LMG 30828T) is suggested as a type strain.
Project description:Bacteria of genus <i>Pectobacterium</i> are Gram-negative rods of the family <i>Pectobacteriaceae</i>. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of <i>Pectobacterium</i>. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, <i>Pectobacterium</i> produces large MVs (100-300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from <i>Pectobacterium</i> were able to induce maceration of <i>Zantedeschia</i> sp. leaves. We also show that the MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of <i>Pectobacterium</i> play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in <i>Pectobacterium</i> strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in <i>Pectobacterium</i> and an effective secretion system.
Project description:Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- ?- lactamase superfamily, has a conserved "HXHXDH" motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 ?M and the specific activity was 0.8 picomol min-1?g-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent.
Project description:Pectobacterium, which causes soft rot disease, is divided into 18 species based on the current classification. A total of 225 Pectobacterium strains were isolated from 10 main cultivation regions of potato (Solanum tuberosum), napa cabbage (Brassica rapa subsp. pekinensis), and radish (Raphanus sativus) in South Korea; 202 isolates (90%) were from potato, 18 from napa cabbage, and five from radish. Strains were identified using the Biolog test and phylogenetic analysis. The pathogenicity and swimming motility were tested at four different temperatures. Pectolytic activity and plant cell-wall degrading enzyme (PCWDE) activity were evaluated for six species (P. carotovorum subsp. carotovorum, Pcc; P. odoriferum, Pod; P. brasiliense, Pbr; P. versatile, Pve; P. polaris, Ppo; P. parmentieri, Ppa). Pod, Pcc, Pbr, and Pve were the most prevalent species. Although P. atrosepticum is a widespread pathogen in other countries, it was not found here. This is the first report of Ppo, Ppa, and Pve in South Korea. Pectobacterium species showed stronger activity at 28°C and 32°C than at 24°C, and showed weak activity at 37°C. Pectolytic activity decreased with increasing temperature. Activity of pectate lyase was not significantly affected by temperature. Activity of protease, cellulase, and polygalacturonase decreased with increasing temperature. The inability of isolated Pectobacterium to soften host tissues at 37°C may be a consequence of decreased motility and PCWDE activity. These data suggest that future increases in temperature as a result of climate change may affect the population dynamics of Pectobacterium.