Antimicrobial Peptide Resistance Genes in the Plant Pathogen Dickeya dadantii

Abstract

Modification of teichoic acid through the incorporation of d-alanine confers resistance in Gram-positive bacteria to antimicrobial peptides (AMPs). This process involves the products of the dltXABCD genes. These genes are widespread in Gram-positive bacteria, and they are also found in a few Gram-negative bacteria. Notably, these genes are present in all soft-rot enterobacteria (Pectobacterium and Dickeya) whose dltDXBAC operons have been sequenced. We studied the function and regulation of these genes in Dickeya dadantii dltB expression was induced in the presence of the AMP polymyxin. It was not regulated by PhoP, which controls the expression of some genes involved in AMP resistance, but was regulated by ArcA, which has been identified as an activator of genes involved in AMP resistance. However, arcA was not the regulator responsible for polymyxin induction of these genes in this bacterium, which underlines the complexity of the mechanisms controlling AMP resistance in D. dadantii Two other genes involved in resistance to AMPs have also been characterized, phoS and phoH dltB, phoS, phoH, and arcA but not dltD mutants were more sensitive to polymyxin than the wild-type strain. Decreased fitness of the dltB, phoS, and phoH mutants in chicory leaves indicates that their products are important for resistance to plant AMPs.

Importance: Gram-negative bacteria can modify their lipopolysaccharides (LPSs) to resist antimicrobial peptides (AMPs). Soft-rot enterobacteria (Dickeya and Pectobacterium spp.) possess homologues of the dlt genes in their genomes which, in Gram-positive bacteria, are involved in resistance to AMPs. In this study, we show that these genes confer resistance to AMPs, probably by modifying LPSs, and that they are required for the fitness of the bacteria during plant infection. Two other new genes involved in resistance were also analyzed. These results show that bacterial resistance to AMPs can occur in bacteria through many different mechanisms that need to be characterized.

FIG 1

Organization of dlt operons in Bacillus subtilis 168 and Dickeya dadantii 3937. Homology between equivalent proteins is indicated.

FIG 2

Regulation of dltB and phoS. The dltB-uidA (A) and phoS-lacZ (B) fusions of strains A5394 and A5749, respectively, were assayed in the presence of protamine or polymyxin in a phoP background. Activities shown are the mean values ± standard deviations (SDs) from at least four separate experiments and are expressed in micromoles of o-nitrophenol or p-nitrophenol produced per minute and per milligram of bacterial dry weight.

FIG 3

Activation of genes involved in AMP resistance by arcA. Plasmid pGEM-T or pGEM-T-arcA was introduced into strains containing a dltB-uidA, dltD-uidA, phoS-lacZ, or arnB-uidA transcriptional fusion. The ratios of expression in strains with pGEM-T-arcA versus those in strains with pGEM-T are shown. Values represent the means ± SDs from at least four separate experiments.

FIG 4

arcA is not involved in induction by polymyxin. Fusions in the indicated genes in the WT or arcA background were assayed in the absence (dark bars) or presence (gray bars) of 5 μg/ml polymyxin. Values represent the means ± SDs from at least four separate experiments. All results comparing gene expression in the presence or absence of polymyxin for each strain were statistically different by the Wilcoxon test (P < 0.05).

FIG 5

Analysis of the LPS of various mutants. Whole cells from different strains (lane 1, A350; lane 2, A5256; lane 3, A5394; lane 4, A5823; lane 5, A5749; lane 6, A5632; lane 7, A5537; lane 8, A3092) or purified LPS from strain A5256 was separated by SDS-PAGE and blotted onto a PVDF membrane. LPS was revealed with anti-KdgM antibodies.

FIG 6

Survival of various mutants against polymyxin. Wild-type and various mutants in genes involved in resistance to AMPs (arnB, dltB, dltD, phoS, phoH, arcA, and gmd) were incubated in the presence of 1 μg/ml (dark bars) or 10 μg/ml (gray bars) polymyxin for 1 h. Samples were diluted and plated onto LB agar plates to assess bacterial viability. Survival values are relative to the original inocula. This experiment was repeated three times. The trends found in the three independent experiments were the same, but variations prevented statistical analysis. Results of a representative experiment are shown.

FIG 7

Competitivity of various mutants in plant infection. (A) Induction of fusions in genes involved in resistance to AMP in the presence of plant chips. Bacteria were grown in M63 medium with glycerol in the absence or presence of chicory chips. Values are the ratios of the activity of the fusion under induced conditions versus those under noninduced conditions. Values are the means ± SDs from at least four separate experiments. (B) Competitivity indexes of various mutants in chicory leaf infections. The wild-type and mutant strains were inoculated in a 1/1 ratio in a chicory leaf. After 24 h, rotten tissue was collected, homogenized, and diluted, and the numbers of bacteria were counted. The competitivity index is the ratio (number of mutant bacteria/number of WT bacteria) in the rotten tissue/(number of mutant bacteria/number of WT bacteria) in the inoculum. Values are the averages ± standard deviations of data from at least five infected leaves.