csrB Gene Duplication Drives the Evolution of Redundant Regulatory Pathways Controlling Expression of the Major Toxic Secreted Metalloproteases in Vibrio tasmaniensis LGP32

VibtasCsrB4 can complement the absence of csrB genes in V. cholerae. Luminescence was measured during growth (see Materials and Methods) in strains carrying a pLux plasmid as follows: V. cholerae wild-type strain (blue diamonds), V. cholerae strain ΔcsrBCD (red squares), and V. cholerae strain ΔcsrBCD/pCsrB4 (green triangles). Relative light unit (RLU) represent counts per minute per milliliter per OD₆₀₀ value.

Regulation of csrB genes in V. tasmaniensis. (A) The level of each CsrB product was determined by dot blot experiments performed with the V. tasmaniensis wild-type strain and ΔvarA and ΔvarS single mutant and ΔvarA ΔvarS double mutant strains as described in Materials and Methods. The values are expressed as fold differences from the tmRNA level. The error bars correspond to standard deviations of the means (SEM). (B) The level of CsrB1 was determined in duplicate by dot blot experiments performed at an OD₆₀₀ of 0.4 (exponential phase [Exp]) and at an OD₆₀₀ of 1.4 (stationary phase [St]). Results are expressed as fold changes from the average value corresponding to the WT level in exponential phase, after normalization to the tmRNA level. *, P > 0.05; **, P > 0.01; ***, P > 0.001.

Production of Vsm and PrtV is HapR dependent whereas only Vsm production is σS dependent. (A) (Left panel) The protein profiles of vesicle-free supernatants of WT LGP32 culture supernatants at different time points during growth were analyzed by SDS-PAGE (12% acrylamide) after TCA precipitation. The sizes of the molecular weight markers are indicated at the left of the gel. Bands corresponding to proteins analyzed by mass spectroscopy are indicated by an asterisk (*). (Right panel) Supernatants of an O/N culture of WT LGP32 and a ΔhapR mutant were subjected to TCA precipitation and analyzed by SDS-PAGE (4% to 12% gradient gel). Identities of the bands are indicated between the two panels. (B) (Left panel) Vesicle-free supernatants of an LGP32 ΔrpoS mutant collected at different time points during growth were subjected to TCA precipitation and analyzed by SDS-PAGE (12% acrylamide). Sizes of the molecular weight markers are indicated at the left of the gels. (Right panel) Supernatants of an O/N culture of WT LGP32 and the ΔrpoS mutant were subjected to TCA precipitation and analyzed by SDS-PAGE (4% to 12% gradient gel). Identities of the bands are indicated between the two panels. (C) Secreted proteolytic activity was assayed for different strains on milk agar nutrient plates as described in Materials and Methods and detected as a clearing zone around the colonies.

A redundant VarS/A-CsrB pathway controls protease production through LuxO. (A) O/N culture supernatants of the WT strain and different mutants were subjected to trichloroacetic acid (TCA) precipitation as described above and analyzed by SDS-PAGE (4% to 12% acrylamide gradient gels). MW, molecular weight. (B and C) Secreted proteolytic activity was assayed on milk marine agar plates without (B) or with (C) supplementation with 2 µg/ml of Cm for the different strains, and the size of the clearing zones was measured after 48 h of incubation at 20°C. Values represent averages of data from a minimum of three independent determinations, and error bars correspond to the SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by pairwise t tests. Values considered to be significantly different (P < 0.05) are indicated by different letters.

CsrBs are redundant in V. tasmaniensis. (A) Secreted proteolytic activity was assayed in triplicate for different strains on milk agar nutrient plates as described in Materials and Methods and were detected as a clearing zone around the colonies. The size of the clearing zones was measured after 48 h. Means of results from a minimum of three replicates are presented, and error bars correspond to the SEM. (B) The level of each CsrB was determined by dot blot experiments in V. tasmaniensis strains in which the three other csrB genes had been deleted. Relative density values are expressed as fold change from the tmRNA level and correspond to averages of results from three independent measurements. The error bars correspond to standard deviations of the means (SEM).

The VarS/VarA/CsrB system controls production of σS via quorum sensing in a redundant manner. Identical amounts of whole-cell extract from various strains as indicated were analyzed by Western blotting using polyclonal antibodies directed against S. enterica serovar Typhimurium σS. (Top panel) A representative Western blot is shown. (Lower panel) σS levels were quantified using ImageJ. Results (normalized to the WT signal) are expressed as fold change from the WT level and represent averages of four independent determinations (four different samples from four independent cultures). Error bars correspond to SEM. Data were analyzed by one-way ANOVA as described for Fig. 5.