Figure 1.
(A) MA plot showing the fold change in transcript expression of all B. cenocepacia H111 genes versus the mean of identified reads in an rpfFBc mutant and wild type. The 112 down-regulated transcripts/proteins in the rpfFBc mutant are indicated in red, the 38 up-regulated genes in green. (B) Box plot demonstrating that supplementing the medium with 10 µM BDSF rescues the gene expression defects in the rpfFBc mutant.
Table 1.
List of genes and proteins differentially expressed using RNA-Seq and proteomics in the rpfFBc mutant compared to the wild type.
Table 2.
List of genes differentially expressed in the rpfFBc and cepR mutant, using the wild type as baseline.
Table 3.
Validation of RNA-Seq results using quantitative PCR analysis.
Figure 2.
The role of the two QS systems in the regulation of selected genes.
The bapA (A), bclA (B), and aidA (C) promoter activities were assessed by means of transcriptional lacZ fusions in the H111 wild type strain and in the mutant defective in AHL and BDSF synthesis (ΔcepI rpfFBc). The strains were grown to late exponential growth phase in LB Lennox broth in the absence or presence of signal molecules (200 nM C8-HSL; 10 µM BDSF) as indicated by+and - below each bar. Error bars indicate SEM, n = 3. * P<0.05, ** P<0.01, *** P<0.001 (t-test, two-tailed) compared to ΔcepI rpfFBc without signalling molecule (ns, not significant) (D) Expression of BclB and AidA in the H111 wild type and the double mutant ΔcepI rpfFBc as assessed by Western Blot analysis. The strains were grown on plates in the presence or absence of signal molecules as indicated by+and - below each band.
Figure 3.
AHL-levels are reduced in an rpfFBc mutant.
The amount of AHLs produced by the cepI and the rpfFBc mutant with cepI constitutively expressed from plasmid pBBRcepI or with the empty plasmid control pBBR1MCS5 was quantified by the aid of the biosensor P. putida/pAS-C8. Error bars indicate SEM, n≥3.
Figure 4.
Biofilm formation and protease activity are co-regulated by AHL and BDSF.
(A) Biofilm formation in ABC minimal. (B) Protease activity in NYG medium. The strains tested are the wild type H111 and the cepI rpfFBc double mutant. Strains were grown in the presence of absence of signal molecules (200 nM C8-HSL; 10 µM BDSF) as indicated by+and - below each bar. Error bars indicate SEM, n≥3. ** P<0.01, *** P<0.001 (t-test, two-tailed) compared to ΔcepI rpfFBc without signalling molecule (ns, not significant).
Figure 5.
Schematic presentation of the two B. cenocepacia H111 QS circuitries.
The CepI/CepR system consists of the AHL synthase CepI directing the synthesis of C8-HSL, and of the transcriptional regulator CepR. The RpfF/RpfR system consists of RpfF which directs the synthesis of BDSF, and of its cognate receptor RpfR. Upon binding of BDSF to RpfR the c-di-GMP phosphodiesterase activity of the protein is stimulated and as a consequence the intracellular c-di-GMP level is lowered. The two QS systems operate in parallel to control specific as well as overlapping sets of genes. Our working model assumes an unknown c-di-GMP receptor protein × that stimulates transcription of target genes. Alternatively, the two QS cascades converge and control the expression or the activity status of an unknown common regulator Y, which in turn regulates expression of target genes. C-di-GMP has a negative regulatory effect on AHL levels via an unknown mechanism (depicted by the dashed line).