Table 1.
Strains and plasmids used in the study.
Table 2.
List of genes differentially expressed 4-fold or more in the ΔrcsA RNA-Seq data.
Table 3.
List of genes differentially expressed 4-fold or more in the ΔlrhA RNA-Seq data.
Fig 1.
Impact of RcsA and LrhA on phenotype of P. stewartii.
Panel A shows an analysis of capsule production in P. stewartti DC283 wild-type, ΔrcsA mutant and ΔrcsA/rcsA+ complementation strains (left to right). Differences in capsule production are apparent in the regions between the arms of the X-cross streak. Panel B shows an analysis of swarming motility in wild-type, ΔlrhA mutant and ΔlrhA/lrhA+ complementation strains (left to right). All pictures for panel A or B, respectively, were taken at the same magnification after 48 hours of incubation.
Fig 2.
Differential mRNA expression during QS.
Whole transcriptome data of the P. stewartii DC283 wild-type strain compared to the ΔrcsA strain (panel A) or the ΔlrhA strain (panel B). An open circle is used to represent each gene. Those filled with green are activated (levels of expression are four-fold or lower in the deletion strain) and those filled with red are repressed (levels of expression are four-fold or higher in the deletion strain), by either RcsA (panel A) or LrhA (panel B). The two extreme outlier points, with RPM expression >100 in the wild-type and ~1 in the deletion strains, represent the deleted genes rcsA or lrhA, respectively. All of the other colored points with RPM expression >1 in both samples are tabulated in Tables 2 and 3. The RPM change of normalized expression for most genes fall tightly around a line of slope of 1, indicating that they are approximately equally expressed in both strains.
Fig 3.
Validating transcriptional control of select genes by RcsA.
Changes in gene expression were compared between the RNA-Seq analysis (light grey) and qRT-PCR assays (dark grey) for five genes regulated by RcsA. Y-axis represents the fold activation (panel A) or repression (panel B) on a logarithmic scale in the presence of RcsA. RNA-Seq results are averages of two experimental samples and qRT-PCR data represent two experimental samples analyzed in triplicate. Error bars were estimated using the sample standard error of the fold-change across the two independent biological replicates for both RNA-Seq and qRT-PCR.
Fig 4.
Validating transcriptional control of select genes by LrhA.
Changes in gene expression were compared between the RNA-Seq analysis (light grey) and qRT-PCR assays (dark grey) for five genes regulated by LrhA. Y-axis represents the fold activation (panel A) or repression (panel B) on a logarithmic scale in the presence of LrhA. RNA-Seq results are averages of two experimental samples and qRT-PCR data represent two experimental samples analyzed in triplicate. Error bars were estimated using the sample standard error of the fold-change across the two independent biological replicates for both RNA-Seq and qRT-PCR.
Fig 5.
Expression from the rcsA promoter.
A GFP reporter was used to measure levels of transcription from the rcsA promoter. Strains were grown to an OD600 of 0.5 and the average fluorescence/OD600 was measured. As indicated by an asterisk, expression from the ΔlrhA strain is significantly higher (p< 0.05) than either the wild-type strain or the ΔlrhA/lrhA+ strain using a two-tailed homoscedastic Student’s t-test. This indicates that LrhA normally represses expression of rcsA in the wild-type strain. Data represents three experimental samples analyzed in triplicate. Error bars denote standard error.
Fig 6.
Plant assays testing the role of RcsA or LrhA in virulence.
Data shown is the average score of disease for Day 12 of an infection assay performed with 15 plants inoculated with P. stewartii DC283 strains: wild type (WT), ΔrcsA, ΔrcsA/rcsA+, ΔlrhA, ΔlrhA/lrhA+, or PBS as a negative control. The asterisks (*) represent strains that are statistically significantly different (p< 0.05) from the wild-type strain using a two-tailed homoscedastic Student’s t-test. Error bars denote standard error.
Fig 7.
Model of the quorum-sensing regulatory network in P. stewartii.
See the text for details. Solid lines indicate known direct regulatory control. Dashed lines indicate either direct or indirect control found in the present study. Arrows represent activation and T lines represent repression.