Contribution of a lectin, LecM, to the quorum sensing signalling pathway of Ralstonia solanacearum strain OE1-1

Abstract

The soil-borne bacterium Ralstonia solanacearum invades the roots and colonizes the intercellular spaces and then the xylem. The expression of lecM, encoding a lectin LecM, is induced by an OmpR family response regulator HrpG in R. solanacearum strain OE1-1. LecM contributes to the attachment of strain OE1-1 to the host cells of intercellular spaces. OE1-1 produces methyl 3-hydroxymyristate (3-OH MAME) through a methyltransferase (PhcB) and extracellularly secretes the chemical as a quorum sensing (QS) signal, which activates QS. The expression of lecM is also induced by the PhcA virulence regulator functioning through QS, and the resulting LecM is implicated in the QS-dependent production of major exopolysaccharide EPS I and the aggregation of OE1-1 cells. To investigate the function of LecM in QS, we analysed the transcriptome of R. solanacearum strains generated by RNA sequencing technology. In the lecM mutant, the expression of positively QS-regulated genes and negatively QS-regulated genes was down-regulated (by >90%) and up-regulated (by ~60%), respectively. However, phcB and phcA in the lecM mutant were expressed at levels similar to those in strain OE1-1. The lecM mutant produced significantly less ralfuranone and exhibited a significantly greater swimming motility, which were positively and negatively regulated by QS, respectively. In addition, the extracellular 3-OH MAME content of the lecM mutant was significantly lower than that of OE1-1. The application of 3-OH MAME more strongly increased EPS I production in the phcB-deleted mutant and strain OE1-1 than in the lecM mutant. Thus, the QS-dependent production of LecM contributes to the QS signalling pathway.

Keywords: Ralstonia solanacearum; LecM; quorum sensing.

Figure 1

Influence of the lecM mutation on the expression of phc quorum sensing (QS)‐related genes phcB and phcA, and the positively phc QS‐mediated virulence genes epsB and ralA, of Ralstonia solanacearum. Ralstonia solanacearum OE1‐1, lecM mutant (OE1‐1‐lecM::EZ Tn5) and native lecM‐expressing complemented lecM mutant (lecM‐comp) strains were grown in ¼ × M63 medium [to an optical density at 600 nm (OD₆₀₀) = 0.3]. Total RNA was then extracted from the bacterial cells. The rpoD gene was used as an internal control for quantitative reverse transcription‐polymerase chain reaction. The gene expression levels are presented relative to the rpoD expression level. The experiment was conducted at least twice using independent samples, with similar results. Results for a single representative sample are provided. Values are presented as the means ± standard deviations of three replicates. Asterisks indicate values significantly different from those of lecM mutant cells (P < 0.05, t‐test).

Figure 2

Cell aggregation by Ralstonia solanacearum OE1‐1, the phcB‐deleted mutant (ΔphcB) and ΔphcB supplemented with methyl 3‐hydroxymyristate (3‐OH MAME). The OE1‐1 and ΔphcB cells were incubated in ¼ × M63 medium in the wells of polyvinylchloride microtitre plates. ΔphcB cells were also incubated in ¼ × M63 medium supplemented with 3‐OH MAME at concentrations of 0.001–1.0 µm. The wells were stained with crystal violet. The experiment was repeated three times, with seven technical replicates in each experiment. Asterisks indicate values significantly different from those of strain OE1‐1 (P < 0.05, t‐test).

Figure 3

Number of genes for which expression was regulated by methyl 3‐hydroxymyristate (3‐OH MAME), PhcA and LecM encoded in lecM in Ralstonia solanacearum strain OE1‐1. The transcriptome analyses using RNA sequencing (RNA‐seq) were performed on RNA from the R. solanacearum phcB‐deleted mutant (ΔphcB), ΔphcB supplemented with 1.0 µm methyl 3‐hydroxymyristate (3‐OH MAME), phcA‐deleted mutant (ΔphcA) and lecM mutant (OE1‐1‐lecM::EZ Tn5). Numbers of genes that exhibited expression level log₂(fold changes) of ≤−1 (a) or ≥1 (b) in ΔphcB supplemented with 1.0 µm 3‐OH MAME relative to the expression levels in ΔphcB, and expression level log₂(fold changes) of ≤−1 (a) or ≥1 (b) in ΔphcA and lecM mutant relative to the expression levels of strain OE1‐1. The fragments per kilobase of open reading frame per million fragments mapped (FPKM) values of OE1‐1, ΔphcB, ΔphcA and lecM mutant strains were normalized prior to the analysis of differentially expressed genes.

Figure 4

Influence of the lecM mutation on the production of ralfuranones by Ralstonia solanacearum strains. High‐performance liquid chromatography (HPLC) analysis of culture extracts from R. solanacearum strain OE1‐1 (a) and lecM mutant (OE1‐1‐lecM::EZ Tn5, b). The peaks of ralfuranones are marked as A, B, J and K.

Figure 5

Influence of the lecM mutation on swimming motility (a) and fliC expression (b) of Ralstonia solanacearum strains. (a) Ralstonia solanacearum OE1‐1, lecM mutant (OE1‐1‐lecM::EZ Tn5) and native lecM‐expressing complemented lecM mutant (lecM‐comp) strains were grown on ¼ × M63 medium solidified with 0.25% agar. Values are presented as the means ± standard deviations of three replicates. The experiment was repeated three times, with five technical replicates in each experiment. Asterisks indicate values significantly different from those of WT strain OE1‐1 (P < 0.05, t‐test). (b) The R. solanacearum strains were grown in ¼ × M63 medium [to an optical density at 600 nm (OD₆₀₀) = 0.3]. Total RNA was then extracted from the bacterial cells. The rpoD gene was used as an internal control for quantitative reverse transcription‐polymerase chain reaction. The gene expression levels are presented relative to the rpoD expression level. The experiment was conducted at least twice using independent samples, with similar results. Results for a single representative sample are provided. Values are presented as the means ± standard deviations of three replicates. Asterisks indicate values significantly different from those of OE1‐1 cells (P < 0.05, t‐test).

Figure 6

Influence of the lecM mutation on methyl 3‐hydroxymyristate (3‐OH MAME) purified from the Ralstonia solanacearum strains OE1‐1, lecM mutant (OE1‐1‐lecM::EZ Tn5) and native lecM‐expressing complemented lecM mutant (lecM‐comp). The experiment was conducted three times using independent samples. Asterisks indicate values significantly different from those of OE1‐1 (P < 0.05, t‐test).

Figure 7

Production of exopolysaccharide I (EPS I) in Ralstonia solanacearum strains. Immunological quantification of EPS I in supernatants of R. solanacearum strains was performed using an enzyme‐linked immunosorbent assay with anti‐R. solanacearum EPS I antibodies. The R. solanacearum cells were also incubated in ¼ × M63 medium supplemented with methyl 3‐hydroxymyristate (3‐OH MAME) at concentrations of 1.0 µm. EPS I productivity was quantified by absorbance at 650 nm (A₆₅₀). Bars indicate the standard errors. The experiment was repeated three times, with five technical replicates in each experiment. Asterisks indicate significant differences (P < 0.05, t‐test).