No abstract

The phytopathogenic bacterium Pantoea stewartii subsp. stewartii synthesizes stewartan exo͞capsular polysaccharide (EPS) in a cell density-dependent manner governed by the EsaI͞EsaR quorumsensing (QS) system. This study analyzes biofilm development and host colonization of the WT and QS regulatory mutant strains of P. stewartii. First, we show that the cell density-dependent synthesis of stewartan EPS, governed by the EsaI͞EsaR QS system, is required for proper bacterial adhesion and development of spatially defined, 3D biofilms. Second, a nonvirulent mutant lacking the esaI gene adheres strongly to surfaces and develops densely packed, less structurally defined biofilms in vitro. This strain appears to be arrested in a low cell density developmental mode. Exposure of this strain to exogenous N-acyl-homoserine lactone counteracts this adhesion phenotype. Third, QS mutants lacking the EsaR repressor attach poorly to surfaces and form amorphous biofilms heavily enmeshed in excess EPS. Fourth, the WT strain disseminates efficiently within the xylem, primarily in a basipetal direction. In contrast, the two QS mutant strains remain largely localized at the site of infection. Fifth, and most significantly, epifluorescence microscopic imaging of infected leaf tissue and excised xylem vessels reveals that the bacteria colonize the xylem with unexpected specificity, particularly toward the annular rings and spiral secondary wall thickenings of protoxylem, as opposed to indiscriminate growth to fill the xylem lumen. These observations are significant to bacterial plant pathogenesis in general and may reveal targets for disease control.maize ͉ xylem ͉ pathogenesis ͉ dissemination

SummaryThe LuxR-type quorum-sensing transcription factor EsaR functions as a repressor of exopolysaccharide (EPS) synthesis in the phytopathogenic bacterium Pantoea stewartii ssp. stewartii . The cell densitydependent expression of EPS is critical for Stewart's wilt disease development. Strains deficient in the synthesis of a diffusible acyl-homoserine lactone inducer remain repressed for EPS synthesis and are consequently avirulent. In contrast, disruption of the esaR gene leads to hypermucoidy and attenuated disease development. Ligand-free EsaR functions as a negative autoregulator of the esaR gene and responds to exogenous acyl-homoserine lactone for derepression. The focus of this study was to define the mechanism by which EsaR governs the expression of the cps locus, which encodes functions required for stewartan EPS synthesis and membrane translocation. Genetic and biochemical studies show that EsaR directly represses the transcription of the rcsA gene. RcsA encodes an essential coactivator for RcsA/ RcsB-mediated transcriptional activation of cps genes. In vitro assays identify an EsaR DNA binding site within the rcsA promoter that is reasonably well conserved with the previously described esaR box. We also describe that RcsA positively controls its own expression. Interestingly, promoter proximal genes within the cps cluster are significantly more acyl-homoserine lactone responsive than genes located towards the middle or 3 ¢ ¢ ¢ ¢ end of the gene cluster. We will discuss a possible role of EsaR-mediated quorum sensing in the differential expression of the cps operon.

Pantoea stewartii subsp. stewartii is a plant-pathogenic bacterium that causes Stewart's vascular wilt in maize. The organism is taxonomically described as aflagellated and nonmotile. We recently showed that P. stewartii colonizes the xylem of maize as sessile, cell-wall-adherent biofilms. Biofilm formation is a developmental process that generally involves some form of surface motility. For that reason, we reexamined the motility properties of P. stewartii DC283 based on the assumption that the organism requires some form of surface motility for biofilm development. Here, we show that the organism is highly motile on agar surfaces. This motility is flagella dependent, shown by the fact that a fliC mutant, impaired in flagellin subunit synthesis, is nonmotile. Motility also requires the production of stewartan exopolysaccharide. Moreover, surface motility plays a significant role in the colonization of the plant host.