Role and regulation of the Flp/Tad pilus in the virulence of Pectobacterium atrosepticum SCRI1043 and Pectobacterium wasabiae SCC3193

Abstract

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers.

Figure 1. Predicted Flp/Tad pilus-encoding gene cluster in Pectobacterium.

Comparative genomics analysis revealed that the synteny of the gene clusters encoding the putative novel virulence determinant Flp/Tad pilus in Pectobacterium and in one Dickeya species is similar to that in the well-studied Flp/Tad model species Aggregatibacter actinomycetemcomitans. Flp = fimbrial low-molecular-weight protein. Tad = tight adherence protein. Rcp = rough colony protein. The gene cluster comparison was based on genomic and protein sequence comparisons utilizing blastn and blastp.

Figure 2. VasH regulates hcp genes but not flp/tad genes in Pectobacterium atrosepticum.

A) Microarray data showing that 15 genes were downregulated in the ΔvasH mutant (FDR<0.05) compared with the wild-type strain P. atrosepticum SCRI1043. The two T6SS-related Hcp-encoding genes are marked with blue, and the three Flp/Tad pilus-related genes are marked with red. The microarray results represent the average of three independent experiments. B) Relative qPCR validation of the microarray results and complementation of the mutant vasH (ECA3435) in trans indicate that VasH is likely to regulate hcp genes (p<0.03) but is not likely to regulate the predicted Flp/Fap pilin component-encoding gene (2^−ΔΔCt: SCRI1043; 0.7, ΔvasH; 1, ΔvasH(pMW119); 0.5, ΔvasH (pMW119::vasH); 0.7, p value for all relevant comparisons >0.08) in P. atrosepticum SCRI1043 C) or other flp/tad genes observed in the microarrays (ECA0789, ECA0790 and ECA0792) or any other genes that were downregulated in the microarrays. ECA4044 is a negative control and was not differentially expressed in the microarrays. The qPCR experiments were repeated a minimum of three times, and the graphs show the averages and standard deviations of three independent experiments. D) Growth of P. atrosepticum SCRI1043 and ΔvasH in hrp-inducing minimal medium salts supplemented with 10% v/v potato tuber extract at 15°C. The sampling point for the microarray and qPCR experiments is marked with an arrow. The growth curves show the averages and standard deviations of three replicates in a single experiment, which was repeated a minimum of three times with similar results.

Figure 3. A novel two-component system in Pectobacterium regulates predicted Flp/Tad pilus-encoding genes.

A) Genes encoding the Flp/Fap pilin component, RcpA (ECA0789), RcpB (ECA0790) and TadA (ECA0792) were upregulated under in planta-mimicking conditions (hrp-inducing minimal medium salts supplemented with 0.4% polygalacturonic acid = PGA) compared with their levels in rich medium (Luria broth) when measured by relative qPCR. However, despite relatively large fold changes, only the Flp/Fap pilin component result was statistically significant (p<0.02). Due to the analysis method used for the relative qPCR data (2^−ΔΔCt), the values obtained from the Luria broth samples were normalized to be 1 and are thus very close to the x-axis on the left side of the column, which represents the relative fold change of PGA samples. B) A novel response regulator (ECA0785) affected the expression levels of genes encoding Flp/Fap pilin component, RcpA (ECA0789), RcpB (ECA0790) and TadA (ECA0792) (p<0.05) under conditions that induce flp/tad gene expression (hrp-inducing minimal medium salts supplemented with 0.4% polygalacturonic acid = PGA). The reduction of gene expression in the ΔECA0785 mutant was restored by in trans complementation (p<0.05). C) In vitro growth of P. atrosepticum SCRI1043 and its derivative ΔECA0785 in hrp-inducing minimal medium salts supplemented with 0.4% PGA at 28°C. The sampling point for the qPCR experiments is marked with an arrow. The experiments were repeated independently a minimum of three times, and the figures represent the averages and standard deviations of three independent experiments (A and B) or the averages of three replicates in a single experiment (C).

Figure 4. The predicted Flp/Tad pilus is necessary for full virulence of Pectobacterium in potato tubers.

A) A mutant strain of P. atrosepticum SCRI1043 deficient in the expression of the full flp/tad gene cluster (Δflp/tad) exhibited impaired maceration capacity in potato tubers compared with the wild-type strain (p = 0.004). The maceration capacity was complemented in trans (p = 0.047). B) A mutant strain of P. atrosepticum SCRI1043 deficient in the expression of a putative response regulator of the flp/tad gene cluster (ΔECA0785) also displayed impaired virulence in potato tubers (p = 0.0002), and the phenotype was restored by complementation in trans (p = 0.027). C) P. wasabiae SCC3193 lacking the predicted Flp/Fap pilin component-encoding gene (Δflp) also displayed impaired maceration of potato tubers (p = 0.015), and the phenotype was complemented in trans (p = 0.007). As a negative control, 10 mM MgSO₄ buffer was used, confirming that the symptoms are a consequence of the inoculated bacterial strains as opposed to the natural population of soft-rot bacteria. The virulence assays were repeated independently a minimum of three times, and the figures represent a single biological replicate (n = 10–15 tubers per strain).

Figure 5. The predicted Flp/Tad pilus has no effect on growth, PCWDEs, motility or biofilm formation in vitro.

A) In vitro growth of P. atrosepticum SCRI1043, P. wasabiae SCC3193 and their derivatives (Δflp/tad, ΔECA0785 and Δflp) in hrp-inducing minimal media supplemented with 10% v/v potato tuber extract. P. wasabiae and P. atrosepticum reached different cell densities, although there was no significant difference between the wild-type strains and the corresponding mutant strains. B) Production of plant cell wall-degrading enzymes (PCWDEs) in P. atrosepticum SCRI1043, P. wasabiae SCC3193 and their derivatives (Δflp/tad, ΔECA0785 and Δflp) growing on indicator plates containing 0.7% polygalacturonic acid (PGA) or 0.5% carboxymethylcellulose (CMC). In the figure, “average Ø” indicates the diameter of the halo around the bacteria in centimeters. The averages and standard deviations (SD) of four replicates (n = 4) are provided, and the experiment was repeated a minimum of three times with similar results. C) Flagella-based motility of P. atrosepticum SCRI1043, P. wasabiae SCC3193 and their derivatives (Δflp/tad, ΔECA0785 and Δflp) on 0.25% agar plates. All strains were motile, and no significant differences in spreading were observed. D) The in vitro biofilm formation ability of P. atrosepticum SCRI1043 and P. wasabiae SCC3193 differed in 0.4% glycerol after 18 h of incubation, but there was no significant difference between the wild-type strains and the corresponding mutant strains (Δflp/tad, ΔECA0785, Δflp). E) The in vitro biofilm formation of P. atrosepticum SCRI1043 and P. wasabiae SCC3193 differed in Luria broth after 6 h of incubation, but there was no difference between the wild-type strains and the corresponding mutant strains (Δflp/tad, ΔECA0785 and Δflp). All experiments were repeated a minimum of three times with a minimum of three replicates. The figures represent the averages and standard deviations of one experiment.