Putative transcription antiterminator RfaH contributes to Erwinia amylovora virulence.

The gram-negative bacterium Erwinia amylovora causes fire blight disease of apple and pear trees. The exopolysaccharide amylovoran and lipopolysaccharides are essential E. amylovora virulence factors. Production of amylovoran and lipopolysaccharide is specified in part by genes that are members of long operons. Here, we show that full virulence of E. amylovora in apple fruitlets and tree shoots depends on the predicted transcription antiterminator RfaH. RfaH reduces pausing in the production of long transcripts having an operon polarity suppressor regulatory element within their promoter region. In E. amylovora, only the amylovoran operon and a lipopolysaccharide operon have such regulatory elements within their promoter regions and in the correct orientation. These operons showed dramatically increased polarity in the ΔrfaH mutant compared to the wild type as determined by RNA sequencing. Amylovoran and lipopolysaccharide production in vitro was reduced in rfaH mutants compared to the wild type, which probably contributes to the rfaH mutant virulence phenotype. Furthermore, type VI secretion cluster 1, which contributes to E. amylovora virulence, showed reduced expression in ΔrfaH compared to the wild type, although without an increase in polarity. The data suggest that E. amylovora RfaH directly, specifically, and exclusively suppresses operon polarity in the amylovoran operon and a lipopolysaccharide operon.

Virulence Genetics of an Erwinia amylovora Putative Polysaccharide Transporter Family Member.

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.

Extragenic Suppression of Elongation Factor P Gene Mutant Phenotypes in Erwinia amylovora.

Elongation factor P (EF-P) facilitates the translation of certain peptide motifs, including those with multiple proline residues. EF-P must be posttranslationally modified for full functionality; in enterobacteria, this is accomplished by two enzymes, namely, EpmA and EpmB, which catalyze the ß-lysylation of EF-P at a conserved lysine position. Mutations to efp or its modifying enzymes produce pleiotropic phenotypes, including decreases in virulence, swimming motility, and extracellular polysaccharide production, as well as proteomic perturbations. Here, we generated targeted deletion mutants of the efp, epmA, and epmB genes in the Gram-negative bacterium Erwinia amylovora, which causes fire blight, an economically important disease of apples and pears. As expected, the Δefp, ΔepmA, and ΔepmB mutants were all defective in virulence on apples, and all three mutants were complemented in trans with plasmids bearing wild-type copies of the corresponding genes. By analyzing spontaneous suppressor mutants, we found that mutations in the hrpA3 gene partially or completely suppressed the colony size, extracellular polysaccharide production, and virulence phenotypes in apple fruits and apple tree shoots but not the swimming motility phenotypes of the Δefp, ΔepmA, and ΔepmB mutants. The deletion of hrpA3 alone did not produce any alterations in any characteristics measured, indicating that the HrpA3 protein is not essential for any of the processes examined. The hrpA3 gene encodes a putative DEAH-box ATP-dependent RNA helicase. These results suggest that the loss of the HrpA3 protein at least partially compensates for the lack of the EF-P protein or ß-lysylated EF-P.IMPORTANCE Fire blight disease has relatively few management options, with antibiotic application at bloom time being chief among them. As modification to elongation factor P (EF-P) is vital to virulence in several species, both EF-P and its modifying enzymes make attractive targets for novel antibiotics. However, it will be useful to understand how bacteria might overcome the hindrance of EF-P function so that we may be better prepared to anticipate bacterial adaptation to such antibiotics. The present study indicates that the mutation of hrpA3 could provide a partial offset for the loss of EF-P activity. In addition, little is known about EF-P functional interactions or the HrpA3 predicted RNA helicase, and our genetic approach allowed us to discern a novel gene associated with EF-P function.