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

Zhao Y, Wang D, Nakka S, Sundin GW, Korban SS - BMC Genomics (2009)

Bottom Line: Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. zhao888@illinois.edu

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.

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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.
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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.

Mentions: All generated mutants did not exhibit any distinctive phenotypes on rich LB medium, except for a single mutant which was mucoid (ybjN mutant). Therefore, phenotypes of the 59 deletion mutant strains were evaluated using a swarming plate assay [50,51]. WT cells can swarm via the combined effects of flagellar motility, chemotaxis, and growth, thus creating a circular colony. Defects in cell motility, chemotaxis, or growth can produce alterations in swarming size or density [51]. For E. amylovora WT and mutant strains, bacterial suspensions were plated onto swarming agar plates containing 0.3% agar, as previously described [50,51]. Swarming diameter and density were determined following incubation at 28°C for up to 72 hr, and three different swarming phenotypes were identified. Two mutants, grrA and grrS, exhibited substantially larger and lower density swarms than the WT (Figure 1A; Table 3, significance level a). In addition, three mutants, envZ, ompR, and envZ/ompR double-mutant, showed dramatic reduction in swarm size, but with increased density (Figure 1B; Table 3, significance level v). All these five mutants exhibited circular swarming (Figure 1A and 1B). A third group of mutants showed smaller swarms, colonies exhibited irregular circular patterns, and these included arcAB, baeRS, cpxA1R1, kdpE, luxPQ, rstAB, and yciR mutants (Figure 1C; Table 3, significance levels o to u). Although the distance of the cpxA1R1 mutants had similar significant level as that of envZ/ompR mutants at 48 hr, these mutants were included in the third group due to its irregular movements and with increased distance at 72 hr in contrast to envZ/ompR mutants which remained the same at 72 hr (Figure 2B). The rest of mutants showed even less change in swarm distance. As negative controls, all flagella-deficient mutants, flhDC and fliA, were non-motile on swarming plates (Figure 1D).


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

Zhao Y, Wang D, Nakka S, Sundin GW, Korban SS - BMC Genomics (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698875&req=5

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.
Mentions: All generated mutants did not exhibit any distinctive phenotypes on rich LB medium, except for a single mutant which was mucoid (ybjN mutant). Therefore, phenotypes of the 59 deletion mutant strains were evaluated using a swarming plate assay [50,51]. WT cells can swarm via the combined effects of flagellar motility, chemotaxis, and growth, thus creating a circular colony. Defects in cell motility, chemotaxis, or growth can produce alterations in swarming size or density [51]. For E. amylovora WT and mutant strains, bacterial suspensions were plated onto swarming agar plates containing 0.3% agar, as previously described [50,51]. Swarming diameter and density were determined following incubation at 28°C for up to 72 hr, and three different swarming phenotypes were identified. Two mutants, grrA and grrS, exhibited substantially larger and lower density swarms than the WT (Figure 1A; Table 3, significance level a). In addition, three mutants, envZ, ompR, and envZ/ompR double-mutant, showed dramatic reduction in swarm size, but with increased density (Figure 1B; Table 3, significance level v). All these five mutants exhibited circular swarming (Figure 1A and 1B). A third group of mutants showed smaller swarms, colonies exhibited irregular circular patterns, and these included arcAB, baeRS, cpxA1R1, kdpE, luxPQ, rstAB, and yciR mutants (Figure 1C; Table 3, significance levels o to u). Although the distance of the cpxA1R1 mutants had similar significant level as that of envZ/ompR mutants at 48 hr, these mutants were included in the third group due to its irregular movements and with increased distance at 72 hr in contrast to envZ/ompR mutants which remained the same at 72 hr (Figure 2B). The rest of mutants showed even less change in swarm distance. As negative controls, all flagella-deficient mutants, flhDC and fliA, were non-motile on swarming plates (Figure 1D).

Bottom Line: Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. zhao888@illinois.edu

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.

Show MeSH
Related in: MedlinePlus