The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus
Fig 1
vtrB gene expression exhibits an autoactivating feature.
(A) Schematic representation of the Vp-PAI region (VPA1370–VPA1321: top) and the vtrB upstream region with adjacent genes (bottom). The arrows indicate open reading frames (ORFs) and their orientation. The ORFs are colored according to their function: T3SS2 apparatus genes, blue; genes encoding T3SS2-secreted proteins, orange; other T3SS2-associated proteins, yellow; regulator genes, leaf green; hypothetical genes, gray. A long operon upstream of the vtrB gene predicted by Operon-mapper (https://biocomputo.ibt.unam.mx/operon_mapper/) [20] is indicated above the arrows. The region targeted by the qRT–PCR primers used in B and C is indicated by a red line as vtrB 5’-UTR. (B) Relative expression of the vtrB 5’-UTR region in V. parahaemolyticus WT, ΔvtrA, and ΔvtrB strains grown in LB medium containing 0.3 M NaCl at 37°C with or without TDC. Total RNA was extracted from each culture at an OD600 of 1 and was analyzed by qRT–PCR. The mean fold-change and standard deviation (SD) values are indicated relative to the WT strains (n = 3). *, p < 0.05, as revealed by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Effect of vtrB complementation under the control of the PBAD promoter on vtrB 5’-UTR expression. V. parahaemolyticus WT, ΔvtrB and ΔvtrAΔvtrB strains with pBAD18-Cm empty vector (indicated as p) or pBAD18-Cm-vtrB (indicated as pvtrB) were grown under TDC and arabinose induction. The mean and SD values are indicated relative to the WT strain harboring the empty vector (n = 3). *, p < 0.05, as indicated by one-way ANOVA followed by Dunnett’s multiple comparison test. (D) β-galactosidase activity from the lacZ fusion reporter of the vtrB promoter region (PvtrB) in V. parahaemolyticus WT, ΔvtrA, and ΔvtrB strains grown under TDC induction. The values show the means, and the error bars represent S.D. (n = 3). *, p < 0.05; ns, not significant, as revealed by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) β-galactosidase activity from the PvtrB-lacZ reporter of the V. parahaemolyticus ΔvtrAΔvtrB strain with pBAD18-Cm empty vector (indicated as p), pBAD18-Cm-vtrA (indicated as pvtrA), or pBAD18-Cm-vtrB (indicated as pvtrB) was grown under TDC and arabinose induction. The values show the means, and the error bars represent S.D. (n = 3). *, p < 0.05, as revealed by Student’s t test.