Figure 1.
Schematic representation of the B728a genome showing the different sigma factors.
The 6.09 Mb genome of B728a encodes 15 distinct sigma factors. rpoD (Psyr_4641) is the essential housekeeping sigma factor for this bacterium belonging to the σ70 family, while rpoN (Psyr_4147) belongs to the σ54 type of sigma factors. The other sigma factors belong to the alternative sigma factor family and include fliA (Psyr_3437), which controls the flagellar biosynthesis genes; rpoS (Psyr_1374), the starvation phase σ factor; rpoH (Psyr_4748), the heat shock sigma factor; and 10 ECF sigma factors, which are summarized in Table 2. Five of these belong to the FecI-type of ECF sigma factors, including ecf5 (Psyr_1040), ecf7 (Psyr_1107), pvdS (Psyr_1943), acsS (Psyr_2580), and ecf6 (Psyr_4731). The five ECF sigma factors characterized in this study are identified by rectangular boxes.
Table 1.
Strains and plasmids used in this study.
Figure 2.
Genomic neighborhood of ecf5, ecf7, ecf6, ecf18, and ecf11 sigma factor genes.
The genes for ECF sigma factor genes are shown in red, the anti-sigma factor genes in brown, the FecA-like genes in yellow, a transmembrane (TM) helix protein in light green, and the toxin/antitoxin (Tx/Atx) genes in olive green; other nearby genes are shown in gray. Additional genes are labeled as T-Reg, transcriptional regulator; gapA, glyceraldehyde-3-phosphate dehydrogenase; betA, choline dehydrogenase; nhaA, Na+/H+antiporter NhaA; cysE, serine O-acetyltransferase; and aroA, 5-enolpyruvylshikimate-3-phosphate synthase.
Table 2.
ECF sigma factors in the genome of Pseudomonas syringae pv. syringae strain B728a.
Figure 3.
Assay for swarming activity by P. syringae pv. syringae ECF sigma factor mutants of strain B728a.
Parental strain B728a and mutant derivatives were spotted on sterile filter discs placed in the center of semisolid NBY, and incubated in a humid chamber for 24 h at 25°C. Swarming phenotypes of B728a and the ECF sigma factor mutants, B728aΔecf5, B728aΔecf7, B728aΔecf18, B728aΔecf11, and B728aΔecf6 are shown; strain B728aΔecf7 carrying pPKT::ecf7 shows restoration of the swarming phenotype. B728aΔgacS is unable to swarm and was used as a negative control.
Figure 4.
Pathogenicity assays to evaluate the contribution of the ecf5 and ecf7 genes to disease development in a susceptible bean (Phaseolus vulgaris cv. Blue Lake 274) host. (A) Disease symptoms.
Bean leaves were inoculated using vacuum infiltration with cell suspensions containing 106 CFU/ml of strain B728a, ECF sigma factor mutants B728aΔecf5 and B728aΔecf7, and the avirulent B728aΔgacS as a negative control. Plants were maintained at 25°C in a growth chamber for 6 days. The experiment was performed twice; representative results are shown 4 days after inoculation. (B) Bacterial populations. Bean leaf populations of the four bacterial strains identified in panel A were determined at days 0, 2, 4 and 6 days after inoculation with 106 CFU/ml for each strain. Bacterial populations are shown in terms of the logarithm of CFU/cm2 of leaf surface. Values are the average counts from four individual plants sampled at each time point; the experiment was repeated on two occasions. Error bars represent the standard errors (SE) of the respective means.
Table 3.
Expression analysis in strain B728a of three FecI-like ECF σ factor genes (ecf5, ecf7, and ecf6) and associated putative iron-responsive genes in low and high iron media.a
Table 4.
Expression analysis in low iron media of putative iron-responsive genes in B728aΔecf5, B728aΔecf6, and B728aΔecf7 mutant strains as compared to B728a.a
Figure 5.
Quantitative real-time PCR analysis of Type I fimbrial gene expression in Δecf5 and Δecf7 mutants of B728a.
The pilus assembly/fimbrial biogenesis gene cluster (Psyr_1131-Psyr_1134) analyzed is located in close proximity to the ecf7 sigma factor gene. Values represent the average fold differences in gene expression from parental strain B728a; results are averages of three technical replicates (to measure reproducibility from a single source) from each of three biological samples grown in NBY liquid medium at 25°C (∼5×108 CFU/ml final concentrations). Gene expression levels were normalized to the 16S-rRNA and recA internal control genes, and standard deviations from the mean are denoted by the error bars; asterisks denote greater than a 2-fold change in gene expression. A Student’s t-test was performed using 95% confidence interval to calculate p-values between biological replicates. Negative values indicate a decrease in expression levels as computed by taking the negative inverse of a fold change value less than 1.
Table 5.
Putative pilus assembly/fimbrial genes downstream of ecf7 in B728a.a