Erwinia chrysanthemi tolCIs Involved in Resistance to Antimicrobial Plant Chemicals and Is Essential for Phytopathogenesis†

Abstract

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.The bacterium Erwinia chrysanthemi is a gram-negative broad-host-range phytopathogen causing soft-rot disease. Production of plant cell wall-degrading enzymes and avirulence proteins by the phytopathogen contributes to the destruction of plant structural barriers and evasion of certain host defense responses, respectively (4, 9, 10, 21, 37). Nutrient acquisition in E. chrysanthemi from degraded plant cell-wall pectin is facilitated by transport systems for pectin-derived oligomers and monomers (18,20,22,46). Secretion of pathogenesis-related proteins across the bacterial cytoplasmic and outer membranes requires several export systems that have been extensively studied. Pectin-degrading enzymes are secreted by a type IIdependent mechanism (encoded by the out genes), and secretion of avirulence proteins into the host is mediated by a type III-dependent process (encoded by the hrp genes) (9,33,35). Finally, secretion of metalloproteases by a sec-independent type I mechanism (prt genes) in E. chrysanthemi has also been demonstrated (11,25,26).TolC is an important though low-abundance protein in the outer membrane of gram-negative bacteria (17). The crystal structure of this protein has recently been determined (24). The protein exists functionally as a trimer forming a ␤-barrel with a large internal diameter, facilitating movement of both large and small molecules through the outer membrane (2). TolC functions as a component of multidrug resistance (MDR) efflux systems in the removal of a broad range of toxic chemicals from the cell (16, 41). In Escherichia coli, type I-dependent secretion of certain virulence-associated proteins also requires TolC (48). The role of TolC in virulence and survival strategies of the pathogenic enteric bacteria E. coli (49), Vibrio cholerae (5), Salmonella enterica serovar Enteritidis (40), and Serratia marcescens (6) has been established. While the existence and ...

Identification of Genes in Xanthomonas campestris pv. vesicatoria Induced during Its Interaction with Tomato

“…(5) In vitro induced strains were diluted out of the population by sequential growth in NB supplemented with Km prior to plant inoculation. (6) Tomato plants were inoculated with pools of unresolved clones. (7) In vivo induced/resolved strains were selected on NA containing 10% sucrose, and clones that were able to grow on sucrose were verified for their inability to grow in NA with Km.…”

The Role of Secretion Systems and Small Molecules in Soft-Rot Enterobacteriaceae Pathogenicity

“…Numerous MDR systems are encoded in SRE genomes, and several of these contribute to virulence or allow bacteria to successfully compete with secondary microbial invaders (7,91,148). As with many other SRE virulence genes, genes encoding efflux pumps are induced by plant-produced phenolic acids, such as salicylic acid (132).…”

TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…Berberine resembles ethidium bromide, accumulates inside cells driven by the membrane potential, (Mikes and Dadak, 1983) and is extruded by multidrug resistance pumps (MDRs) found in bacteria (Stermitz et al, 2000;Barabote et al, 2003). Figure 3B shows that while the alkaloid is incorporated by the wildtype and tdeA mutant strains, the tdeA mutant accumulates it at a higher rate (left panel).…”

“…The machinery required for drug efflux is similar to that involved in the secretion of protein toxins. TolC is also an important virulence factor as exemplified by the fact that TolC has been shown to be required for several bacteria to cause disease including Salmonella enterica serovar Enteritidis in mice (Stone and Miller, 1995;Nishino et al, 2006), S. enterica serovar Typhimurium in chicks (Baucheron et al, 2005), Vibrio cholerae in mice (Bina and Mekalanos, 2001), and Erwinia chrysanthemi in plants (Barabote et al, 2003) To date, there is no published report or communication regarding the existence of a functional TolC-like protein in A. actinomycetemcomitans. Because LtxA is secreted from A. actinomycetemcomitans (Kachlany et al, 2000) and LtxB and LtxD are both required for this process (Guthmiller et al, 1995); M. P. Palacio, M. S. Duncan, and S. C. Kachlany, unpublished), we asked whether a TolC-like protein exists in A. actinomycetemcomitans.…”