Figure 4
From: An iron detection system determines bacterial swarming initiation and biofilm formation
pvc cluster regulated by RssB is involved in regulating swarming and biofilm formation.
(a) Schematic map of the pvc cluster, RssB-P binding site, and pvc cluster deletion mutant. Red dash lines represent RssB-P binding sites (−349 to +38) of the pvcA promoter region. For construction of pvc cluster deletion mutants (Δpvc), genomic region between two asterisks (*) was replaced with Smr cassette. (b) EMSA was employed to confirm the interaction between phosphorylated RssB (RssB-P) and promoter region of pvcA (PpvcA). Digoxigenin (DIG)-labeled DNA fragments were incubated with purified GST, GST-RssBD51E or GST-RssB-P, followed by analysis by non-denaturing PAGE. Negative control (NC) was performed by incubating GST-RssB-P with the DNA sequence between M13F/M13R in the plasmid pBIISK. (c) During swarming progression (2–6 hr) with different iron conditions, relative expression of RssB downstream genes (flhDC and pvcA), normalized to 16S rRNA, in WT and ΔrssBA was respectively determined by qRT-PCR. (d,e) Swarming migration radius (d) and biofilm formation (e) of each strain of S. marcescens. Strain harboring pPvc encoding pvc cluster under pBAD promoter with 0.01% arabinose. The results shown represent means ± SEM from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA. For Fig. 4c, * and ** corresponding to P-value < 0.05 and <0.01 in comparison with LB group of each strain, respectively. For Fig. 4e, * corresponding to P-value < 0.05 compared to WT.
