Pseudomonas syringae Lipopolysaccharide Synthesis Gene wbpL Displays Heterogeneous Expression Within In Vitro and In Planta Populations

Abstract

Phenotypic heterogeneity usually refers to phenotypic variation not associated with genetic variation, nor induced by environmental stimuli. The phenotypic heterogeneity processes described for some complex bacterial traits are causing a shift in how bacterial phenotypes are studied, from traditional assessments by averaging populations to single-cell analysis focused on bacterial individual phenotypes and how these distribute within the population. The structure of the lipopolysaccharide (LPS) layer on the outer membrane in Gram-negative bacteria is often subjected to phenotypic variation, a process that is critical for virulence in animal pathogens. Here, we apply single-cell expression analyses to wbpL, a conserved Pseudomonas syringae glycosyltransferase-encoding gene essential for the synthesis of the O-antigen component of LPS. We show that expression of wbpL displays phenotypic heterogeneity in P. syringae pv. phaseolicola growing in rich medium and reaches bistable expression in minimal medium, where the population splits into WbpL^ON and WpbL^OFF subpopulations. In planta, wbpL expression is also heterogeneous, displaying intermingled ON/OFF with comparable viability. Finally, we followed the expression of wbpL within the spatial context of apoplastic microcolonies, and not only detected heterogeneity within each microcolony, but also found that microcolonies displayed overall differences in fluorescence intensity that correlated with size, with smaller microcolonies displaying higher levels of wbpL expression.