Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi.

Soft-rotting Erwinia spp. export degradative enzymes to the cell exterior (Out+), a process contributing to their ability to macerate plant tissues. Transposon (Tn5, Tn10, Tn10-lacZ) insertion Out- mutants were obtained in Erwinia carotovora subsp. carotovora 71 by using plasmid and bacteriophage lambda delivery systems. In these mutants, pectate lyases, polygalacturonase, and cellulase, which are normally excreted into the growth medium, accumulated in the periplasm. However, localization of the extracellular protease was not affected. The Out- mutants were impaired in their ability to macerate potato tuber tissue. Out+ clones were identified in a cosmid library of E. carotovora subsp. carotovora 71 by their ability to complement mutants. Localization of cyclic phosphodiesterase in the periplasm indicated that the Out+ plasmids did not cause lysis or a nonspecific protein release. The Out+ derivatives of the E. carotovora subsp. carotovora 71 mutants regained the ability to macerate potato tuber tissue. Our data indicate that a cluster of several genes is required for the Out+ phenotype. While one plasmid, pAKC260, restored the Out+ phenotype in each of the 31 mutants of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi, it failed to render Escherichia coli export proficient. Homologs of E. carotovora subsp. carotovora 71 out DNA were detected by Southern hybridizations in subspecies of E. carotovora under high-stringency conditions. In contrast, E. chrysanthemi sequences bearing homology to the E. carotovora subsp. carotovora 71 out DNA were detectable only under low-stringency hybridization. Thus, although the out genes are functional in these two soft-rotting bacterial groups, the genes appear to have diverged.     

Molecular cloning of ompRS, a regulatory locus controlling production of outer membrane proteins in Erwinia carotovora subsp. carotovora

Summary A locus, ompRS, controlling synthesis of outer membrane proteins was cloned from Erwinia carotovora subsp. carotovora (Ecc) by complementation of an Escherichia coli ompR—envZ mutant. The Ecc ompRS locus was both structurally and functionally similar to the ompR—envZ operon controlling porin gene expression in E. coli as shown by DNA hybridization and complementation of E. coli ompR and envZ mutants. Furthermore, introduction of ompRS into E. coli (ompR—envZ) strains restored the osmoregulation of the major outer membrane protein genes ompC and ompF Maxicell analysis of ompRS-carrying plasmids suggested that proteins similar in size to the E. coli ompR and envZ gene products were encoded by the Ecc ompR and ompS genes, respectively. Introduction of an ompRS transposon mutant onto the Ecc chromosome by marker exchange mutagenesis showed that ompRS is essential for production of a major outer membrane porin in Ecc. This mutational defect could be complemented by clones carrying Ecc ompRS or E. coli ompR envZ. The lack of the porin did not, however, compromise the virulence of these Ecc mutants.     

Production of cell wall dissolving enzymes by Erwinia carotovora subsp. atroseptica in vitro at 27°C and 30°5C

Cultures of Erwinia carotovora subsp. atroseptica , grown at 27°C and 30·°C in different liquid media were assayed for activities of pectate lyase, polygalacturonase and cellulase. Total production of both pectate lyase and of polygalacturonase was 3–6 times less at 30·5°C than at 27°C; secretion of pectate lyase was similarly affected. Cellulase was cell bound and its production was not affected by the temperatures investigated. Growth, protein synthesis and protease activity were similar at the two temperatures and production of enzyme activity at 27°C and 30·5°C was independent of the growth medium.     

The Rcs phosphorelay modulates the expression of plant cell wall degrading enzymes and virulence in Pectobacterium carotovorum ssp. carotovorum

Production of plant cell wall degrading enzymes, the major virulence factors of soft-rot Pectobacterium species, is controlled by many regulatory factors. Pectobacterium carotovorum ssp. carotovorum SCC3193 encodes an Rcs phosphorelay system that involves two sensor kinases, RcsC(Pcc) and RcsD(Pcc), and a response regulator RcsB(Pcc) as key components of this system, and an additional small lipoprotein RcsF(Pcc). This study indicates that inactivation of rcsC(Pcc), rcsD(Pcc) and rcsB(Pcc) enhances production of virulence factors with the highest effect detected for rcsB(Pcc). Interestingly, mutation of rcsF(Pcc) has no effect on virulence factors synthesis. These results suggest that in SCC3193 a parallel phosphorylation mechanism may activate the RcsB(Pcc) response regulator, which acts as a repressor suppressing the plant cell wall degrading enzyme production. Enhanced production of virulence factors in Rcs mutants is more pronounced when bacteria are growing in the absence of plant signal components.     

AepA of Pectobacterium is not involved in the regulation of extracellular plant cell wall degrading enzymes production

Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239–246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.