Contribution of the murI Gene Encoding Glutamate Racemase in the Motility and Virulence of Ralstonia solanacearum

Abstract

Bacterial traits for virulence of Ralstonia solanacearum causing lethal wilt in plants were extensively studied but are not yet fully understood. Other than the known virulence factors of Ralstonia solanacearum, this study aimed to identify the novel gene(s) contributing to bacterial virulence of R. solanacearum. Among the transposon-inserted mutants that were previously generated, we selected mutant SL341F12 strain produced exopolysaccharide equivalent to wild type strain but showed reduced virulence compared to wild type. In this mutant, a transposon was found to disrupt the murI gene encoding glutamate racemase which converts L-glutamate to D-glutamate. SL341F12 lost its motility, and its virulence in the tomato plant was markedly diminished compared to that of the wild type. The altered phenotypes of SL341F12 were restored by introducing a full-length murI gene. The expression of genes required for flagella assembly was significantly reduced in SL341F12 compared to that of the wild type or complemented strain, indicating that the loss of bacterial motility in the mutant was due to reduced flagella assembly. A dramatic reduction of the mutant population compared to its wild type was apparent in planta (i.e., root) than its wild type but not in soil and rhizosphere. This may contribute to the impaired virulence in the mutant strain. Accordingly, we concluded that murI in R. solanacearum may be involved in controlling flagella assembly and consequently, the mutation affects bacterial motility and virulence.

Keywords: bacterial wilt disease; glutamate racemase; tomato plant.

Fig. 1

(A) Genomic position and surrounding regions of the insertion site of transposon insertion mutant, SL341F12. (B) Phylogenetic tree of glutamate racemase homologues from Ralstonia solanacearum and others bacteria inferred from MurI comparisons. The tree was constructed by using the maximum likelihood method. Bootstrap values are shown for each node that had >50% support in a bootstrap analysis of 1,000 replicates. The scale bar indicates the genetic distance of 0.20 substitution per site.

Fig. 2

Growth curves of Ralstonia solanacearum SL341, SL341F12, and SL341F12C. Strains grown in casamino acid-peptone-glucose (A) and M9 (B) broth at 30°C. Colony forming unit (cfu) was observed every 12 h, until 72 h using plate dilution method and cfu counting. Error bars represent the standard deviation of the mean from three replicates.

Fig. 3

Motility assay of Ralstonia solanacearum strains. SL341F12C is a complemented strain of SL341F12 with murI gene. Growth of each strain on casamino acid-peptone-glucose soft agar at 30°C through 48-72 h (+, motile; -, non-motile).

Fig. 4

Bar chart of the relative normalized expression of 3 genes involved in flagella assembly of Ralstonia solanacearum SL341, SL341F12, and SL341F12C assessed by reverse transcription quantitative PCR. Error bars represent the standard deviation of the mean from three replicates.

Fig. 5

Evaluation of bacterial wilt occurrence on a susceptible (A) and a resistant (B) tomato cultivars inoculated by Ralstonia solanacearum strain SL341, SL341F12, and SL341F12C using soil-soaking method. Disease severity was scored through 2 weeks after inoculation of 3 weeks old tomato cultivars grown at 28°C, in a day/night regime (14 h light/10 h dark). Control was treated with distilled water. All experiments were performed in triplicate (each replicate include 10 plants). Vertical bars represent standard deviations (n = 30). Significant difference was noticed by repeated measures ANOVA (***P < 0.001).

Fig. 6

Comparison of Ralstonia solanacearum SL341 and a mutant SL341F12 population in Moneymaker bulk soil (A), rhizosphere (B), and root (C) by colony forming unit (cfu). Tomato plants were treated with each R. solanacearum SL341 and a mutant suspension. Bacterial population in Moneymaker in 5 days after inoculation (***P < 0.001).