A LuxR-type regulator, AcrR, regulates flagellar assembly and contributes to virulence, motility, biofilm formation, and growth ability of Acidovorax citrulli

Abstract

LuxR-type regulators regulate many bacterial processes and play important roles in bacterial motility and virulence. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. We identified a LuxR-type, nonquorum sensing-related regulator, AcrR, in the group II strain Aac-5 of A. citrulli. We found that the acrR mutant lost twitching and swimming motilities, and flagellar formation. It also showed reduced virulence, but increased biofilm formation and growth ability. Transcriptomic analysis revealed that 394 genes were differentially expressed in the acrR mutant of A. citrulli, including 33 genes involved in flagellar assembly. Our results suggest that AcrR may act as a global regulator affecting multiple important biological functions of A. citrulli.

Keywords: Acidovorax citrulli; AcrR; LuxR-type regulator; flagellar assembly.

Figure 1

Effect of acrR on virulence of Acidovorax citrulli on watermelon leaves. (a) Appearance of watermelon seedlings 10 days after inoculation with sterile water, the wild‐type strain Aac‐5, the acrR mutant strain ∆acrR, and the mutant complementation strain ∆acrRcomp of A. citrulli. (b) Virulence of A. citrulli strains 10 days after inoculation, calculated based on a disease index of 0 to 100 (Wang et al., 2016). The bars represent standard errors of the means from three experiments, each containing six inoculated watermelon seedlings in three pots per tested strain. **p < .01 by Student's t test

Figure 2

Effect of acrR on growth ability of Acidovorax citrulli in watermelon cotyledons. (a) Appearance of watermelon cotyledons inoculated with sterile water (CK), the wild‐type strain Aac‐5 (WT), the acrR mutant strain ∆acrR (∆acrR), and the mutant complementation strain ∆acrRcomp (∆acrRcomp) of A. citrulli at 1, 24, 48, 72 and 96 hr after inoculation (hai). (b) Count of colonies isolated from leaf disks of watermelon cotyledons inoculated with the WT, ∆acrR, and ∆acrRcomp strains 1, 24, 48, 72 and 96 hai. The bars represent standard errors of the means from three experiments, each containing six cotyledon discs per stain per time point. The asterisks indicate a significant difference compared to the WT strain, calculated by Student's t test (p < .05)

Figure 3

Effect of acrR on biofilm formation in Acidovorax citrulli, as visualized by the violet ring formed on the inner surface of the glass flasks (a) and measured under OD₅₈₀ using stained biofilm solubilized with ethanol (b). The error bar represents standard errors of the means from three experiments, each containing three replicates per treatment. **p < .01 by Student's t test. Aac‐5, wild‐type strain of A. citrulli; ∆acrR, acrR deletion mutant of Aac‐5; ∆acrRcomp, acrR complemented strain of ∆acrR

Figure 4

Effect of acrR on growth ability of Acidovorax citrulli in liquid King's B medium. Optical density (OD₆₀₀) of the cultured cell suspensions of the tested A. citrulli strains measured at time points of 0–72 hr. The error bar represents standard errors of the means from three experiments, each containing five replicates per strain. Aac‐5, wild‐type strain of A. citrulli; ∆acrR, acrR deletion mutant of Aac‐5; ∆acrRcomp, acrR complemented strain of ∆acrR

Figure 5

Effect of acrR on twitching and swimming motilities of Acidovorax citrulli. (a) The wild‐type strain Aac‐5 and the complementation strain ∆acrRcomp produced typical haloes around bacterial colonies, indicating bacterial motility via twitching. The mutant strain ∆acrR produced few or no haloes. Thirty colonies were observed for each strain. (b) Comparison of swimming motility of A. citrulli strains 36 hr after inoculation of 10 µl each bacterial suspension to the centre of a soft basal medium plate at 28 °C. (c) Swimming motility measured by colony diameters of each strain on basal medium plate. The bars represent standard errors of the means from three experiments, and each experiment contained three replicates for each strain. **p < .01 by Student's t test

Figure 6

Effect of acrR on formation of polar flagellum of Acidovorax citrulli. The formation of polar flagellum observed under transmission electron microscopy. Clear filament polar flagella (indicated by arrows) were observed in the wild‐type strain Aac‐5 and complementation strain ∆acrRcomp, but not in the mutant strain ∆acrR

Figure 7

Gene ontology of highly (a) and lowly (b) differentially expressed genes between acrR mutant and wild‐type strains of Acidovorax citrulli. The y axis represents terms of gene ontology, which are divided into three categories: biological process indicated in blue, molecular function in green, and cellular component in orange. The x axis represents the number of genes

Figure 8

Schematic diagram of the differentially expressed genes involved in flagellar assembly. Layers of inner and outer membrane, peptidoglycan layer, C‐, MS‐, P‐, and L‐rings, motor proteins (MotAB), rotor (FlgBCF), hollow rod, hook, and distal part of flagellum are shown. Components of flagellum transport apparatus, ATPase complex, chaperones of flagellar transport, and their substrates are presented schematically. Highly expressed genes are shaded grey and lowly expressed genes are highlighted in green