The Gram-negative enterobacterium Erwinia amylovora causes fire blight disease in apple and pear trees. Lipopolysaccharides and the exopolysaccharide amylovoran are essential E. amylovora virulence factors. We found that mutations in rfbX disrupted amylovoran production and virulence in apple fruits and tree shoots and that the deletion of yibD suppressed the rfbX mutant phenotype. The level of expression of yibD was about 10-fold higher in the ΔrfbX mutant than the wild type. A forward genetic suppressor screen in the ΔrfbX mutant uncovered multiple mutations in yibD and supported the conclusion that the virulence defect of rfbX mutants is due to reduced amylovoran production. The yibD and rfbX genes are expressed as a two-gene operon, yibD rfbX The rfbX gene encodes a previously uncharacterized putative polysaccharide subunit transporter, while yibD encodes a predicted glycosyltransferase. Mutation of rfbX did not have a detectable effect on lipopolysaccharide patterns; however, the overexpression of yibD in both the wild-type and ΔyibD ΔrfbX genetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, the overexpression of yibD in the ΔyibD ΔrfbX mutant inhibited bacterial growth in amylovoran-inducing medium. This growth inhibition phenotype was used in a forward genetic suppressor screen and reverse-genetics tests to identify several genes involved in lipopolysaccharide production, which, when mutated, restored the ability of the ΔyibD ΔrfbX mutant overexpressing yibD to grow in amylovoran-inducing medium. Remarkably, all the lipopolysaccharide gene mutants tested were defective in lipopolysaccharide and amylovoran production. These results reveal a genetic connection between amylovoran and lipopolysaccharide production in E. amylovoraIMPORTANCE This study discovered previously unknown genetic connections between exopolysaccharide and lipopolysaccharide production in the fire blight pathogen Erwinia amylovora This represents a step forward in our understanding of the biology underlying the production of these two macromolecules. Fire blight is an economically important disease that impacts the production of apples and pears worldwide. Few fire blight control measures are available, and growers rely heavily on antibiotic applications at bloom time. Both exopolysaccharide and lipopolysaccharide are E. amylovora virulence factors. Our results indicate that the overexpression of the yibD gene in E. amylovora disrupts both lipopolysaccharide production and exopolysaccharide production. This effect could potentially be used as the basis for the development of an antivirulence treatment for the prevention of fire blight disease.
All Science Journal Classification (ASJC) codes
- Molecular Biology