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HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family.

ABSTRACT: HrpI, a 78-kDa protein, functions in the secretion of harpin, a proteinaceous elicitor of the hypersensitive response from Erwinia amylovora. The predicted amino acid sequence of HrpI is remarkably similar to that of LcrD of Yersinia species, the first member of a recently described protein family. Other proteins of the family are MixA from Shigella flexneri, InvA from Salmonella typhimurium, FlhA from Caulobacter crescentus, HrpI from Pseudomonas syringae pv. syringae, HrpO from Pseudomonas solanacearum, and HrpC2 from Xanthomonas campestris pv. vesicatoria. Cells of E. amylovora containing mutated hrpI genes or cells of Escherichia coli containing the cloned hrp gene cluster with mutated hrpI produce but do not export harpin. When similar cells with functional hrpI genes were grown at 25 degrees C, but not at 37 degrees C, harpin was exported to the culture supernatant. Direct evidence that HrpI is involved in the secretion of a virulence protein has been offered. Two other loci of the hrp gene cluster are involved in the regulation of harpin, and four other loci also are involved in the secretion of harpin. Since harpin and other proteins likely to be secreted by the LcrD family of proteins lack typical signal peptides, their secretion mechanism is distinct from the general protein export pathway.

SUBMITTER: Wei ZM

PROVIDER: S-EPMC206975 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family.

Wei Z M ZM Beer S V SV

Journal of bacteriology 19931201 24

HrpI, a 78-kDa protein, functions in the secretion of harpin, a proteinaceous elicitor of the hypersensitive response from Erwinia amylovora. The predicted amino acid sequence of HrpI is remarkably similar to that of LcrD of Yersinia species, the first member of a recently described protein family. Other proteins of the family are MixA from Shigella flexneri, InvA from Salmonella typhimurium, FlhA from Caulobacter crescentus, HrpI from Pseudomonas syringae pv. syringae, HrpO from Pseudomonas sol ...[more]

PMID: 8253684

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Project description: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. 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 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.

Project description:A 6.2-kb region of DNA corresponding to complementation groups II and III of the Erwinia amylovora hrp gene cluster was analyzed. Transposon mutagenesis indicated that the two complementation groups are required for secretion of harpin, an elicitor of the hypersensitive reaction. The sequence of the region revealed 10 open reading frames in two putative transcription units: hrpA, hrpB, hrcJ, hrpD, and hrpE in the hrpA operon (group III) and hrpF, hrpG, hrcC, hrpT, and hrpV in the hrpC operon (group II). From promoter regions of the hrpA, hrpC, and hrpN operons, sequences similar to those of the HrpL-dependent promoters of Pseudomonas syringae pathovars were identified with a consensus sequence of 5'-GGAAC-N17-18-CACTNAA-3'. The protein products of seven genes, hrpA, hrcJ, hrpE, hrpF, hrpG, hrcC, and hrpV, were visualized with a T7 polymerase/promoter expression system. HrcC, HrcJ, and HrpT sequences contained potential signal peptides, and HrcC appeared to be envelope associated based on a TnphoA translational fusion. Comparison of deduced amino acid sequences indicated that many of the proteins are homologous to proteins that function in the type III protein secretion pathway. HrcC is a member of the YscC-containing subgroup in the PulD/pIV superfamily of outer membrane proteins. HrcJ is a member of a lipoprotein family that includes YscJ of Yersinia spp., MxiJ of Shigella flexneri, and NolT of Rhizobim fredii. Additional similarities were detected between HrpB and YscI and between HrpE and YscL. HrcJ and HrpE were similar to flagellar biogenesis proteins FliF and FliH, respectively. In addition, HrpA, HrpB, HrcJ, HrpD, HrpE, HrpF, and HrcC showed various degrees of similarity to corresponding proteins of P. syringae. Comparison of hrp clusters with respect to gene organization and similarity of individual proteins confirms that the hrp systems of E. amylovora and P. syringae are closely related to each other and distinct from those of Ralstonia (Pseudomonas) solanacearum and Xanthomonas campestris. Possible implications of extensive similarities between the E. amylovora and P. syringae hrp systems in pathogenesis mechanisms are discussed.

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Dataset Information

HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family.

Publications

HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family.

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