The Contributions of Motility to the Behavior of Pseudomonas syringae: Phenotypes and Genetics

Motility is considered to be a beneficial trait for microbes, as it solves many of the challenges these microorganisms face in their habitats, allowing them to find hosts, explore local environments, access protected niches and needed nutrients, and disperse to new locations. The quantitative relationship between motility and virulence, particularly of plant-associated microorganisms, remains unclear. This study has dissected the quantitative contributions of these factors in the behavior of the plant pathogenic bacterium Pseudomonas syringae within plant tissues as well as on leaf surfaces, where it commonly occurs as an epiphytic resident of leaves. P. syringae is the only known plant pathogen with pili from the widespread chaperone-usher fimbrial family as well as all subfamilies of type IV pili. These families of pili are important factors in bacterial virulence, and implicated in adhesion, invasion, and biofilm formation in mammalian systems. However, the role of pili are less well characterized in plant pathogens. As such, P. syringae is a useful model system for studying these processes in plant colonization. The availability of reproducible, quantitative variation in nearly isogenic P. syringae strains, coupled with readily generated mutants altered in expression of adhesive pili, made P. syringae an obvious choice for investigating the relative contributions of adhesive factors and motility to the behavior of plant pathogens./nNine nearly isogenic isolates of P. syringae pv. syringae strain B728a exhibited stable and reproducibly different swimming and swarming motility in culture. Swimming and swarming motility were positively correlated among the variants, suggesting that similar traits contributed to both processes. A greater rate of motility of variants was associated with their incitation of a higher incidence of lesion formation on bean leaves after topical application of bacteria. The size of lesions formed by variants after direct injection into bean pods, thus bypassing the need for invasion, was also positively correlated with both the relative rate of motility in culture and their ability to incite leaf lesions. The differences in motility among the variant strains was not associated with any difference in biosurfactant production, although mutants blocked in syringafactin production exhibited a deficiency in ability to move within the apoplast of bean pods. Re-sequencing of the variant strains using high-throughput (Illumina) sequencing followed by genomic analyses revealed five single nucleotide polymorphisms (SNPs) and two short simple repeat regions (SSRs) differing among the nine P. syringae variant strains. None of the SNPs or SSRs were associated with genes known to be directly involved in flagellar or fimbrial biogenesis and regulation. Site-directed knockout mutants in those loci altered in variant strains recapitulated the reduced swarming motility exhibited by a given strain. Phylogenetic reconstruction of the collection of strains based on variation in genome features was in agreement with historical relationships of the strains. Additional analyses of two P. syringae variant strains that were selected as having either higher or lower swarming motility than their parental strains revealed additional genetic elements involved with modulating swarming motility. A mutation leading to early truncation of ispA, encoding a protein involved in cell septation, was associated with the variant exhibiting reduced motility and thus reduced virulence to bean. While no genetic reason could be identified in the selected variant that exhibited enhanced motility, the fact that it exhibited traits specifically beneficial to motility in culture may explain why it was less virulent than expected based on its high degree of motility in vitro./nFurther investigation of factors affecting motility were explored through bioinformatic analyses of putative fimbrial biogenesis pathways. Five putative fimbrial types were identified which had structural similarities to either chaperone-usher (CU) fimbriae, type IVa fimbriae, type IVb fimbriae, or the type IVb monophyletic sub-family known as Flp fimbriae. Additional analyses of strains deficient in the production of the major structural pilin of type IVa fimbriae (PilA), CU fimbriae (FimA), and Flp fimbriae (Flp) revealed that twitching motility was not a major contributor to P. syringae motility. However, flp mutants and to a lesser extent fimA mutants exhibited higher swimming and swarming motility, suggesting that adhesive features of these fimbriae suppressed motility. Consistent with such a role, over-expression of Flp pilin resulted in a significant suppression in motility of the wild-type P. syringae strain. Co-inoculation of wild-type and fimbrial mutants onto leaf surfaces revealed that Flp fimbriae are involved in surface attachment, as the flp mutant exhibited a decreased attachment to bean leaves when compared with the wild-type strain. Attachment by Flp fimbriae appears to be an early step in the colonization process, as the reduced attachment of the flp mutant was much less pronounced after a longer incubation period. fimA and pilA mutants exhibited similar abilities to attach to leaf surfaces as the wild-type strain. Topical application of cell suspensions of pilA mutants onto bean leaves resulted in the formation of fewer lesions than conferred by the wild-type strain. When simulated rain was applied to leaves after inoculation, both the pilA and fimA mutant incited as much as 2-fold fewer lesions than the wild-type strain, suggesting that they adhered less tenaciously and thus were retained less well after the rain event. fimA and flp mutants but not a pilA mutant formed larger lesions than the wild-type strain when directly inoculated into plant tissue, suggesting that their decreased adhesiveness contributed to their ability to move within the plant after inoculation. Taken together, Flp and CU fimbriae appear to have significant roles in adhesion that affect their motility in planta, and type IVa fimbriae appear to be important in inciting disease on bean, presumably by aiding local movement to sites suitable for invasion of the leaf.

Advisor: Lindow, Steven E