Systems level analysis of two-component signal transduction systems in Erwinia amylovora: role in virulence, regulation of amylovoran biosynthesis and swarming motility

Abstract

Background: Two-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in Erwinia amylovora have either not been identified or have not yet been studied.

Results: We used a systems approach to identify TCST and related signal transduction genes in the genome of E. amylovora. Comparative genomic analysis of TCSTs indicated that E. amylovora TCSTs were closely related to those of Erwinia tasmaniensis, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in E. amylovora including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in E. amylovora control amylovoran biosynthesis, one of two major virulence factors in E. amylovora. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified.

Conclusion: Our results demonstrated that TCSTs in E. amylovora played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora.

Figure 1

Comparison of motility on swarming plates for WT and TCST mutants. Bacterial strains were spotted on the swarming plate (0.3% agar) and incubated at 28°C. Photos were taken at one or two days post inoculation. DPI: days post inoculation. A1 to D1: WT strain; A2: grrS mutant; A3: grrA mutant; B2: envZ mutant; B3: ompR mutant; B4: envZ/ompR double mutant; C2 to C4: representative mutants with irregular movements at two days post inoculation; D2 to D4: flhD, flhC and fliA mutant, respectively. Flagella mutants were used as negative controls.

Figure 2

Comparison of the swarming distance of WT and TCST mutants. The diameters of the swarming circle were measured 24, 48 and 72 hrs after incubation. The experiments were repeated at least three times.

Figure 3

TCSTs regulate amylovoran biosynthesis and gene expression. (A) Amylovoran production of E. amylovora WT and TCST mutants in vitro. Bacterial strains were grown in MBMA media with 1% sorbitol for 48 hrs at 28°C with shaking. The amount of amylovoran was measured with the CPC assay and normalized to a cell density of 1. Amylovoran operon (amsA-L) deletion mutant (Δams) was used as a negative control [47]. (B) Gene expression of the amsG gene in WT and TCST mutants in vitro. GFP intensity in WT and TCST mutants containing amsG promoter-gfp fusion plasmid was measured by flow cytometry. GFP-A: Green fluorescence protein absorbance; Count: Number of cells.