Pantoea ananatis Genetic Diversity Analysis Reveals Limited Genomic Diversity as Well as Accessory Genes Correlated with Onion Pathogenicity.
ABSTRACT: Pantoea ananatis is a member of the family Enterobacteriaceae and an enigmatic plant pathogen with a broad host range. Although P. ananatis strains can be aggressive on onion causing foliar necrosis and onion center rot, previous genomic analysis has shown that P. ananatis lacks the primary virulence secretion systems associated with other plant pathogens. We assessed a collection of fifty P. ananatis strains collected from Georgia over three decades to determine genetic factors that correlated with onion pathogenic potential. Previous genetic analysis studies have compared strains isolated from different hosts with varying diseases potential and isolation sources. Strains varied greatly in their pathogenic potential and aggressiveness on different cultivated Allium species like onion, leek, shallot, and chive. Using multi-locus sequence analysis (MLSA) and repetitive extragenic palindrome repeat (rep)-PCR techniques, we did not observe any correlation between onion pathogenic potential and genetic diversity among strains. Whole genome sequencing and pan-genomic analysis of a sub-set of 10 strains aided in the identification of a novel series of genetic regions, likely plasmid borne, and correlating with onion pathogenicity observed on single contigs of the genetic assemblies. We named these loci Onion Virulence Regions (OVR) A-D. The OVR loci contain genes involved in redox regulation as well as pectate lyase and rhamnogalacturonase genes. Previous studies have not identified distinct genetic loci or plasmids correlating with onion foliar pathogenicity or pathogenicity on a single host pathosystem. The lack of focus on a single host system for this phytopathgenic disease necessitates the pan-genomic analysis performed in this study.
Project description:Pantoea ananatis is a widespread phytopathogen with a broad host range. Despite its ability to infect economically important crops, such as maize, rice and onion, relatively little is known about how this bacterium infects and colonizes host tissue or spreads within and between hosts. To study the role of motility in pathogenicity, we analysed both swimming and twitching motility in P. ananatis LMG 20103. Genetic recombineering was used to construct four mutants affected in motility. Two flagellar mutants were disrupted in the flgK and motA genes, required for flagellar assembly and flagellar rotation, respectively. Similarly, two twitching motility mutants were generated, impaired in the structure (pilA) and functioning (pilT) of the type IV pili. The role of swimming and twitching motility during the infection cycle of P. ananatis in onion seedlings was determined by comparing the mutant- and wild-type strains using several in vitro and in planta assays. From the results obtained, it was evident that flagella aid P. ananatis in locating and attaching to onion leaf surfaces, as well as in pathogenicity, whereas twitching motility is instrumental in the spread of the bacteria on the surface once attachment has occurred. Both swimming and twitching motility contribute towards the ability of P. ananatis to cause disease in onions.
Project description:BACKGROUND: The Type VI secretion system (T6SS) has been identified in several different bacteria, including the plant pathogenPantoea ananatis. Previous in silico analyses described three different T6SS loci present in the pathogenic strain of P. ananatis LMG 20103. This initial investigation has been extended to include an additional seven sequenced strains of P. ananatis together with 39 strains from different ecological niches. Comparative and phylogenetic analyses were used to investigate the distribution, evolution, intra-strain variability and operon structure of the T6SS in the sequenced strains. RESULTS: Three different T6SS loci were identified in P. ananatis strain LMG 20103 and designated PA T6SS 1-3. PA T6SS-1 was present in all sequenced strains of P. ananatis and in all 39 additional strains examined in this study. In addition, PA T6SS-1 included all 13 core T6SS genes required for synthesis of a functional T6SS. The plasmid-borne PA T6SS-2 also included all 13 core T6SS genes but was restricted to only 33% (15/46) of the strains examined. In addition, PA T6SS-2 was restricted to strains of P. ananatis isolated from symptomatic plant material. This finding raises the possibility of an association between PA T6SS-2 and either pathogenicity or host specificity. The third cluster PA T6SS-3 was present in all strains analyzed in this study but lacked 11 of the 13 core T6SS genes suggesting it may not encoded a functional T6SS. Inter-strain variability was also associated with hcp and vgrG islands, which are associated with the T6SS and encode a variable number of proteins usually of unknown function. These proteins may play a role in the fitness of different strains in a variety of ecological niches or as candidate T6SS effectors. Phylogenetic analysis indicated that PA T6SS-1 and PA T6SS-2 are evolutionarily distinct. CONCLUSION: Our analysis indicates that the three T6SSs of P. ananatis appear to have been independently acquired and may play different roles relating to pathogenicity, host range determination and/or niche adaptation. Future work will be directed toward understanding the roles that these T6SSs play in the biology of P. ananatis.
Project description:Pantoea ananatis is a Gram-negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non-pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4-formamido-4,6-dideoxy-d-glucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6-dehydratase, an aminotransferase, and an N-formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N-formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis-Menten kinetics with dTDP-4-amino-4,6-dideoxy-d-glucose as the substrate and N10 -formyltetrahydrofolate as the carbon source. In addition, the X-ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N-formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N-formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non-pathogenic apparently do not contain these genes.
Project description:Pantoea ananatis, a bacterium that is well known for its phytopathogenic characteristics, has been isolated from a myriad of ecological niches and hosts. Infection of agronomic crops, such as maize and rice, can result in substantial economic losses. In the last few years, much of the research performed on P. ananatis has been based on the sequencing and analysis of the genomes of strains isolated from different environments and with different lifestyles. In this review, we summarize the advances made in terms of pathogenicity determinants of phytopathogenic strains of P. ananatis and how this bacterium is able to adapt and survive in such a wide variety of habitats. The diversity and adaptability of P. ananatis can largely be attributed to the plasticity of its genome and the integration of mobile genetic elements on both the chromosome and plasmid. Furthermore, we discuss the recent interest in this species in various biotechnological applications. TAXONOMY:Domain Bacteria; Class Gammaproteobacteria; Family Enterobacteriaceae; genus Pantoea; species ananatis. DISEASE SYMPTOMS:Pantoea ananatis causes disease on a wide range of plants, and symptoms can range from dieback and stunted growth in Eucalyptus seedlings to chlorosis and bulb rotting in onions. DISEASE CONTROL:Currently, the only methods of control of P. ananatis on most plant hosts are the use of resistant clones and cultivars or the eradication of infected plant material. The use of lytic bacteriophages on certain host plants, such as rice, has also achieved a measure of success.
Project description:Onion thrips, Thrips tabaci Lindeman, is a primary insect pest of onions (Allium cepa) worldwide. Onion thrips cause feeding damage by destroying epidermal tissue. They are also vectors of Pantoea ananatis (Serrano) Mergaert, the bacteria that causes center rot. Onions with center rot develop white streaks with water-soaked margins along the onion leaves, which turn necrotic and lead to bulb rot during storage. The role of thrips feeding on the establishment and progression of bacterial infection in onions has not been investigated. Onions infested with thrips and inoculated with P. ananatis had more necrotic tissue and symptoms were more severe with increasing thrips density. We conducted a fluorescence microscopy study that examined how P. ananatis (expressing a fluorescence protein gene) colonized a control group of onions without thrips in comparison to a test group of onions with thrips. We found that P. ananatis colonized some onions in the control group because of naturally existing wounds in the epidermal tissue but more colonization was found in the thrips infested group because of the increased presence of entry points caused by thrips feeding. Overall, our results demonstrate that wounds caused by thrips feeding facilitate center rot development by providing entry sites for the bacteria into leaf tissue.
Project description:A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.
Project description:A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.
Project description:Pantoea ananatis is a Gram-negative plant pathogen that causes disease on a broad range of host plants, including pineapple, maize, rice, onion, melons, and Eucalyptus, and has been implicated in several cases of human disease. Here, we report the genome sequence of P. ananatis LMG20103 isolated from diseased Eucalyptus in South Africa.
Project description:The seed as a habitat for microorganisms is as yet under-explored and has quite distinct characteristics as compared to other vegetative plant tissues. In this study, we investigated three closely related P. ananatis strains (named S6, S7, and S8), which were isolated from maize seeds of healthy plants. Plant inoculation experiments revealed that each of these strains exhibited a different phenotype ranging from weak pathogenic (S7), commensal (S8), to a beneficial, growth-promoting effect (S6) in maize. We performed a comparative genomics analysis in order to find genetic determinants responsible for the differences observed. Recent studies provided exciting insight into the genetic drivers of niche adaption and functional diversification of the genus Pantoea. However, we report here for the first time on the analysis of P. ananatis strains colonizing the same ecological niche but showing distinct interaction strategies with the host plant. Our comparative analysis revealed that genomes of these three strains are highly similar. However, genomic differences in genes encoding protein secretion systems and putative effectors, and transposase/integrases/phage related genes could be observed.
Project description:Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia.