Virulence Genetics of anErwinia amylovoraPutative Polysaccharide Transporter Family Member

Abstract

ABSTRACTThe Gram-negative enterobacteriumErwinia amylovoracauses fire blight disease in apple and pear trees. Lipopolysaccharides and the exopolysaccharide amylovoran are essentialE. amylovoravirulence factors. We found that mutations inrfbXdisrupted amylovoran production and virulence in apple fruits and tree shoots and that the deletion ofyibDsuppressed therfbXmutant phenotype. The level of expression ofyibDwas about 10-fold higher in the ΔrfbXmutant than the wild type. A forward genetic suppressor screen in the ΔrfbXmutant uncovered multiple mutations inyibDand supported the conclusion that the virulence defect ofrfbXmutants is due to reduced amylovoran production. TheyibDandrfbXgenes are expressed as a two-gene operon,yibD rfbX. TherfbXgene encodes a previously uncharacterized putative polysaccharide subunit transporter, whileyibDencodes a predicted glycosyltransferase. Mutation ofrfbXdid not have a detectable effect on lipopolysaccharide patterns; however, the overexpression ofyibDin both the wild-type and ΔyibDΔrfbXgenetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, the overexpression ofyibDin the ΔyibDΔrfbXmutant 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 ΔyibDΔrfbXmutant overexpressingyibDto 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 inE. amylovora.IMPORTANCEThis study discovered previously unknown genetic connections between exopolysaccharide and lipopolysaccharide production in the fire blight pathogenErwinia 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 areE. amylovoravirulence factors. Our results indicate that the overexpression of theyibDgene inE. amylovoradisrupts 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.