Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production

Identification, characterization and regulation of two secreted polygalacturonases of the emerging rice pathogen Burkholderia glumae

“…Polygalacturonases have been studied in various bacterial plant pathogens including Erwinia chrysanthemi, Erwinia carotovora (Ried & Collmer, 1986), Ralstonia solanacearum (Gonzalez & Allen, 2003), B. cepacia (Gonzalez et al, 1997;Aguilar et al, 2003;Massa et al, 2007) and Agrobacterium vitis (Herlache et al, 1997); in most cases, they contribute to the fitness of these plant pathogens. In B. cepacia, pehA is involved in the maceration of onion bulbs (Gonzalez et al, 1997;Aguilar et al, 2003); in E. carotovora, PehA and regulators of pehA gene expression are required for virulence (Saarilahti et al, 1992;Flego et al, 2000); in A. vitis, pehA mutants produce smaller tumors and fail to form necrotic lesions (Rodriguez-Palenzuela et al, 1991;Herlache et al, 1997); in E. chrysanthemi, the four polygalacturonases pehV, pehW, pehX and pehN do not play a major role in maceration of plant tissue and pathogenicity (Nasser et al, 1999;Hugouvieux-Cotte-Pattat et al, 2002); and in R. solanacearum, pehA and pehB mutants are severely reduced in virulence, as these enzymes may aid the bacteria to penetrate plant roots and colonize vessels, whereas pehC mutants are not affected in terms of virulence potential Huang & Allen, 1997;Gonzalez & Allen, 2003). Interestingly, bacterial polygalacturonases have an unexpectedly low primary structural identity and belong to family 28 of glycosyl hydrolases (Davies & Henrissat, 1995).…”

The extreme C‐terminus is required for secretion of both the native polygalacturonase (PehA) and PehA‐Bla hybrid proteins in Erwinia carotovora subsp. carotovora

“…All the peh variants contained an intact promoter region, and thus, the expression of the chimeric genes occurred under the regulatory control of pehA. When expressed in E. coil and in the Peh-strain of Ecc, HSE2201 (see the Experimental procedures), the resulting PehA-Bla hybrid proteins exhibited a functionally active C-terminal !3lactamase which rendered the cells ampicillin resistant (ApR), implicating that the hybrid proteins were exported out of the cytoplasm (Saarilahti and Palva, 1990;Saarilahti et aL, 1992). Cells expressing the gene fusions were Fig.…”

“…The approximately 2.4 kb DNA fragments containing the 3' terminally truncated pehA gene were ligated to the Klenow filled-in BamHI site 5' of the 13-1actamase gene ('bla) devoid of its own promoter and signal sequence. In-frame fusions were scored by Ap resistant phenotype in E. coil In addition, a fusion containing the entire pehA gene was constructed by first amplifying the pehA gene in PCR, using the oligonucieotides 935 and 934 (see the Experimental procedures), followed by ligation of the 3' terminal BstEII-BamHI segment of the PCR product to the corresponding sites of a previously constructed pehA-bla fusion in pHSK75-5 (Saarilahti et al, 1992). The pehA ~, pehA d2, pehA d4 and pehA d7 denote the wild-type pehA sequence and deletions of last 2, 4 and 7 codons of the coding sequence fused to bla via the linker region, respectively.…”

“…Erwinia carotovora subsp, carotovora (Ecc) is a phytopathogenic enterobacterium causing soft rot in a variety of crops through a massive production of extracellular, plant-cell-wall-degrading enzymes, including pectate lyase, pectin lyase, polygalacturonase, cellulase and protease (see Perombelon and Kelman, 1980;Collmer and Keen, 1986). The ability to secrete enzymes is essential for virulence, as mutations affecting either their production or secretion cause reduced virulent or avirulent phenotypes (Pirhonen et aL, 1991;Saarilahti et aL, 1992). The pectinases and the cellulases appear to employ a common secretion pathway because in secretion-deficient (Out-) mutants these proteins accumulate in the periplasmic space (Chatterjee et aL, 1985;Murata et aL, 1990;Pirhonen et aL, 1991).…”

“…A single gene, pehA, is responsible for all detectable Peh activity produced by the bacterium (Saarilahti et aL, 1990a;: a feature that makes this protein useful for protein-secretion studies. Previously, a study of PehA-Bla hybrid proteins showed that up to 87% of the mature PehA present in the fusions could not direct secretion of the hybrid protein (Saarilahti et aL, 1992). The aim of this work was to investigate whether the C-terminal sequences might contain information relevant for secretion of PehA and a PehA-Bla hybrid protein.…”