SlyA, a MarR family transcriptional regulator, is essential for virulence in Dickeya dadantii 3937

Abstract

SlyA, a MarR family transcriptional regulator, controls an assortment of biological functions in several animal-pathogenic bacteria. In order to elucidate the functions of SlyA in the phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) 3937, a slyA gene deletion mutant (denoted DeltaslyA) was constructed. The mutant exhibited increased sensitivity to sodium hypochlorite, the cationic antimicrobial peptide polymyxin B, and oxidative stress. The mutant showed reduced production of pectate lyase and exopolysaccharide and an inability to form a pellicle. The mutant lacking a functional slyA gene showed a significantly reduced ability to cause maceration of potato tubers. Accordingly, the mutant exhibited significantly reduced bacterial growth and failed to hyperinduce pectate lyase production in planta. Introduction of a plasmid containing slyA into the DeltaslyA mutant caused all of these phenotypes to recover to wild-type levels. These results suggest that SlyA plays an important role in virulence to plants by positively regulating the expression of multiple pathogenicity-related traits of D. dadantii 3937.

FIG. 1.

Inactivation of slyA attenuates the virulence of D. dadantii 3937. The bacterial inoculum of 20 μl contained 10⁴ CFU/ml of the wild type (D. dadantii 3937), the ΔslyA mutant, and the complementation strain [ΔslyA(pSlyA)]. The disease symptoms (A), mass of the rotted tissues (B), and bacterial populations in macerated tissue (C) were documented after 36 h of incubation at 27°C with high relative humidity. The error bars indicate standard deviations.

FIG. 2.

Pectate lyase specific activity. Strains were grown in M63 glycerol minimal medium (noninduced), M63 glycerol minimal medium plus 0.4% PGA (induced), and M63 glycerol minimal medium plus 0.4% PGA plus 1% potato tuber extract (PTE) (hyperinduced) to an OD₆₆₀ of 1.0 before the total pectate lyase specific activity was determined with a spectrophotometer. The mean of pectate lyase activities from five independent experiments was expressed as the specific activity (U/OD₆₆₀). The error bars indicate the standard deviations.

FIG. 3.

Sensitivity tests. (A) Sensitivities of the different bacterial strains to H₂O₂. The data represent one of three separate experiments, which gave similar results. (B) Sensitivity to polymyxin B, showing the mean and standard deviation of five separate experiments. (C) Colony morphologies of the wild type and the ΔslyA mutant after exposure to hydrogen peroxide. The photographs represent one of three separate experiments, which gave similar results.

FIG. 4.

(A) Pellicle formation following growth in SOBG medium without shaking at 27°C for 48 h. (B) Strains were grown in M63 glycerol minimal medium agar plates with calcofluor at 27°C for 48 h and then exposed to UV light. The photographs represent one of three separate experiments, which gave similar results.

FIG. 5.

Effect of slyA on phoP-phoQ expression. Bacterial strains were grown in M63 glycerol minimal medium containing a low concentration (10 μM) of magnesium plus 0.4% PGA plus 1% potato tuber extract to an OD₆₆₀ of 1.0 before total RNA was harvested. The expression levels of phoP and phoQ in a ΔslyA background were compared to those in the wild type by quantitative reverse transcription-PCR and normalized to the level of expression of the 16S rRNA gene. The results show the means of three replicates. The error bars indicate the standard deviations.

FIG. 6.

SDS-PAGE of recombinant His₆-SlyA. E. coli BL21(DE3) carrying pETSlyA was induced by IPTG and purified as described in Materials and Methods. The purified protein (lane 2) was subjected to 12% SDS-PAGE. Molecular mass standards were loaded in lane 1.

FIG. 7.

EMSA using the promoter region of phoP and purified His-SlyA protein. Labeled probe (25 ng) was incubated in the absence (lane 1) or presence of increasing amounts of protein (50, 100, 200, and 400 nM) (lanes 2 to 5, respectively) or in the presence of labeled probe (25 ng), protein (400 nM), and a 50-fold excess of unlabeled probe as a specific competitor (lane 6).