Virulence Genetics of anErwinia amylovoraPutative Polysaccharide Transporter Family Member

Abstract

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 to rfbX disrupted amylovoran production and virulence in apple fruits and tree shoots, and that deletion of yibD suppressed the rfbX mutant phenotype. Expression of yibD was about ten-fold higher in ΔrfbX compared to 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, yibDrfbX. 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, overexpression of yibD in both the wild-type and ΔyibDrfbX genetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, overexpression of yibD in ΔyibDrfbX 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 ΔyibDrfbX 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. amylovora. IMPORTANCE 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 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 development of an antivirulence treatment for the prevention of fire blight disease.

Natural Products-Based Botanical Bactericides Discovery: Novel Abietic Acid Derivatives as Anti-Virulence Agents for Plant Disease Management

“…EPS levels decreased by 4.8, 10.3, 26.0, 45.0, 94.9, and 96.8%, respectively (Figure 3C,D). These results were similar to those of other studies in which EPS biosynthesis was regulated by gum gene clusters 29,30 and suggested that synthetic compound C 2 was able to effectively prevent bacterial EPS production. The outcomes indicated that compound C 2 reduced bacterial biofilm formation (Figure 3E).…”

A Mutation of a Putative NDP-Sugar Epimerase Gene in Ralstonia pseudosolanacearum Attenuates Exopolysaccharide Production and Bacterial Virulence in Tomato Plant

“…The putative NDP-sugar epimerase Rsp1004 in GMI1000 is known to be involved in EPS biosynthesis and virulence ( Capela et al, 2017 ; Wang et al, 2022 ). Additionally, the homologues of putative NDP-sugar epimerase in SL341 and other phytopathogen contain “Polysacc_synt_2” motif domain, which is found in diverse bacterial polysaccharide biosynthesis proteins, including the ExoT protein from Rhizobium meliloti , the AmsL protein from Erwinia amylovora , the CapD protein from Staphylococcus aureus , the WalL protein from Vibrio cholerae , and several putative epimerases ( Becker et al, 1993 ; Fallarino et al, 1997 ; Klee et al, 2020 ; Lin et al, 1994 ). Thus, our results suggested that the putative NDP-sugar epimerase is a novel virulence gene regulating EPS biosynthesis in SL341.…”

Putative transcription antiterminator RfaH contributes to Erwinia amylovora virulence

“…A copy of rfaH (Figure 1a) cloned into cosmid vector pCE (Figure S2, and Tables S1 and S2) restored the ability of the three rfaH mutants to cause oozing and necrosis in apple fruitlets (Figures 1c and S3). Fire blight symptoms caused by rfaHTn5-474 in apple tree branches inoculated by shoot tip wounding (Klee et al, 2020) were significantly reduced and delayed compared to the wild type (Figure 1d). promoter-proximal genes (Bailey et al, 1996).…”

“…Genes in the ams operon, which is involved in amylovoran production (Bugert & Geider, 1995), had reduced transcript abundance in ΔrfaH compared to the wild type, with LFCs ranging from −1 to −4 (Figure 2a and Table S3). Genes in the rfb operon, which is involved in LPS and EPS production (Klee et al, 2020), also had reduced transcript abundance in ΔrfaH relative to the wild type, with LFCs from −1.8 to −5.3 (Figure 2b and Table S3). In both the rfb and ams clusters, ops-distal genes tended to be more down-regulated than opsproximal genes (Figure 2a,b).…”

“…To examine effects of deletion of rfaH on gene expression, transcriptomes of wild‐type and Δ rfaH bacteria were compared using RNA‐sequencing (RNA‐Seq; Methods S1). Three independent cultures of each strain were grown to mid‐log phase in amylovoran minimal medium and RNA was isolated as previously described (Klee et al, 2020 ). Following rRNA depletion, single‐end, 75 bp RNA‐Seq was performed at the Penn State Genomics Core Facility on an Illumina NextSeq 550.…”