Two Host-Induced Ralstonia solanacearum Genes, acrA and dinF , Encode Multidrug Efflux Pumps and Contribute to Bacterial Wilt Virulence

Abstract

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10 7 CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.Many bacteria can survive and even grow in the presence of toxic compounds (48). One of the means by which bacteria survive in toxic environments is by extruding toxins through membrane-bound efflux pumps (7, 69). These efflux proteins, called multidrug resistance efflux pumps (MDRs), transport a broad range of structurally unrelated compounds out of the cell and can confer resistance to a wide variety of toxins, including antibiotics (7, 36).The following five MDR families have been characterized: (i) the ATP binding cassette (ABC) superfamily (13), (ii) the major facilitator superfamily (54,55,66), (iii) the resistance nodulation-cell division (RND) superfamily (66), (iv) the small multidrug resistance (SMR) superfamily (47), and (v) the multidrug and toxic compound extrusion (MATE) superfamily (10). These superfamilies vary in the mechanism of transport, the number of transmembrane domains, and substrate specificity. The SMR superfamily has been found only in prokaryotes, while members of the RND, major facilitator, ABC, and MATE superfamilies are present in all domains of life (48).The role of MDRs in human and animal pathogens in association with the emergence of antibiotic-resistant strains has been well studied (36). However, comparative genomic analyses have revealed that MDRs are widely distributed in both pathogenic and nonpathogenic bacteria (55). This ubiquity underscores the importance of MDRs in bacterial life cycles. Despite the genomic abundan...

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

“…Correct deletion construct insertion was confirmed by sequencing. The deletion construct was introduced into wildtype (WT) R. solanacearum chromosome by double homologous recombination following electroporation, as previously described (22). Correct allelic replacement was confirmed with PCR.…”

Characterization of the MSMEG_2631 Gene ( mmp ) Encoding a Multidrug and Toxic Compound Extrusion (MATE) Family Protein in Mycobacterium smegmatis and Exploration of Its Polyspecific Nature Using Biolog Phenotype MicroArray

“…In Staphylococcus aureus, the mepRAB operon encodes a MarR-like transcriptional regulator (mepR), a MATE family efflux pump (mepA), and a hypothetical protein (mepB) (24). In Ralstonia solanacearum, dinF is flanked by genes encoding a hypothetical amino acid permease and a putative transmembrane protein (58). These studies demonstrate that bacterial dinF-flanking regions lack conserved synteny.…”

“…The genomic organization of MATE efflux pump gene flanking regions has been studied in DinFs in several bacteria (24,(56)(57)(58). In E. coli and Streptococcus pneumoniae, dinF is in operons with lexA and recA, respectively (56,57).…”

“…The norM promoter is induced at 18°C in Erwinia amylovora (61), DNA-damaging compounds induce dinF in Clostridium difficile (62) and R. solanacearum (58), and mepR negatively regulates S. aureus mepB MATE transcription (24). Our data indicate that MSMEG_2626-2628-2629-2630-2631 operon transcription is initiated from a single TSS and suggest that the promoter is constitutive, since no tested drugs induce promoter activity.…”

The Virulence Factors of the Bacterial Wilt Pathogen Ralstonia solanacearum

“…Inactivation of oxyR, which encodes the only identified regulator of oxidative stress gene in R. solanacearum significantly reduced virulence [37]. Two genes, acrA and dinF encoding multidrug efflux pumps, were also found to contribute to bacterial wilt virulence [38]. Some metabolic pathways also appear to be required during pathogenesis of R. solanacearum, and deletion of metER methionine biosynthesis genes produced significantly reduced disease symptoms without causing auxotrophy or affecting growth inside the plant [39,40].…”