Tol-Pal proteins are critical cell envelope components of Erwinia chrysanthemi affecting cell morphology and virulence

Abstract

The tol-pal genes are necessary for maintaining the outer-membrane integrity of Gram-negative bacteria. These genes were first described in Escherichia coli, and more recently in several other species. They are involved in the pathogenesis of E. coli, Haemophilus ducreyi, Vibrio cholerae and Salmonella enterica. The role of the tol-pal genes in bacterial pathogenesis was investigated in the phytopathogenic enterobacterium Erwinia chrysanthemi, assuming that this organism might be a good model for such a study. The whole Er. chrysanthemi tol-pal region was characterized. Tol-Pal proteins, except TolA, showed high identity scores with their E. coli homologues. Er. chrysanthemi mutants were constructed by introducing a uidA-kan cassette in the ybgC, tolQ, tolA, tolB, pal and ybgF genes. All the mutants were hypersensitive to bile salts. Mutations in tolQ, tolA, tolB and pal were deleterious for the bacteria, which required high concentrations of sugars or osmoprotectants for their viability. Consistent with this observation, they were greatly impaired in their cell morphology and division, which was evidenced by observations of cell filaments, spherical forms, membrane blebbing and mislocalized bacterial septa. Moreover, tol-pal mutants showed a reduced virulence in a potato tuber model and on chicory leaves. This could be explained by a combination of impaired phenotypes in the tol-pal mutants, such as reduced growth and motility and a decreased production of pectate lyases, the major virulence factor of Er. chrysanthemi. INTRODUCTIONThe Tol-Pal system of Gram-negative bacteria is required for outer-membrane stability (Lazzaroni et al., 1999). It comprises five envelope proteins, TolQ, TolR, TolA, TolB and Pal, which form two complexes. The TolQ, TolR and TolA inner-membrane proteins interact via their transmembrane domains (Derouiche et al., 1995;Lazzaroni et al., 1995). The b-propeller domain of the periplasmic protein TolB is responsible for its interaction with Pal (Ray et al., 2000). TolB also interacts with the outer-membrane peptidoglycan-associated proteins Lpp and OmpA (Cascales et al., 2002;Clavel et al., 1998). TolA undergoes a conformational change in response to changes in the protonmotive force (Germon et al., 2001), and interacts with Pal in an energy-dependent manner (Cascales et al., 2001). The Cterminal periplasmic domain of TolA also interacts with the N-terminal domain of TolB (Dubuisson et al., 2002;Walburger et al., 2002).The transcriptional organization of the E. coli tol-pal genes has been characterized. The genes ybgC (orf1), tolQ, tolR, tolA and tolB, and pal and ybgF (orf2) form two operons (Muller & Webster, 1997); a large ybgC-ybgF transcript has also been postulated (Vianney et al., 1996). ybgC and ybgF encode proteins of unknown function located in the cytoplasm and the periplasm, respectively (Clavel et al., 1996;Sun & Webster, 1987). Inactivation of these two ORFs induces no obvious phenotype in E. coli. In contrast, mutations in the tol-pal genes cause the disruption of outermembr...

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