The two faces of ToxR: activator of ompU, co-regulator of toxT in Vibrio cholerae

Abstract

ToxR of Vibrio cholerae directly activates the ompU promoter, but requires a second activator, TcpP to activate the toxT promoter. ompU encodes a porin, while toxT encodes the transcription factor, ToxT, which activates V. cholerae virulence genes including cholera toxin and the toxin co-regulated pilus. Using an ompU-sacB transcriptional fusion, toxR mutant alleles were identified that encode ToxR molecules defective for ompU promoter activation. Many toxR mutants defective for ompU activation affected residues involved in DNA binding. Mutants defective for ompU activation were also tested for activation of the toxT promoter. ToxR-F69A and ToxR-V71A, both in the α-loop of ToxR, were preferentially defective for ompU activation, with ToxR-V71A nearly completely defective. Six mutants from the ompU-sacB selection showed more dramatic defects in toxT activation than ompU activation. All but one of the affected residues map to the wing domain of the winged helix-turn-helix of ToxR. Some ToxR mutants preferentially affecting toxT activation had partial DNA-binding defects, and one mutant, ToxR-P101L, had altered interactions with TcpP. These data suggest that while certain residues in the α-loop of ToxR are utilized to activate the ompU promoter, the wing domain of ToxR contributes to both promoter binding and ToxR/TcpP interaction facilitating toxT activation.

Figure 1

Activation of ompU-lacZ or toxT-lacZ chromosomal reporter constructs in V. cholerae by various ToxR mutant derivatives. ΔtoxR V. cholerae strains were complemented with HA-tagged wild-type ToxR, the empty vector pSK Bluescript, or various ToxR mutant proteins. β-galactosidase activity was measured after a 3-4 hour induction with 100μM IPTG at 30°C. Miller units were expressed as percent, relative to activation seen with wild-type ToxR-HA. Data shown are from at least two experiments performed in triplicate. ToxR-HA from the same samples used in the μ-galactosidase assay was detected with an anti-HA antibody to assess protein stability.

Figure 2

Modeling of the N-terminal DNA-binding/transactivation domain of ToxR (residues 1-114) using threading of the ToxR sequence onto structurally related winged-HTH family members. A) labeling of putative domains of interest within the ToxR transcription activation and DNA-binding domain, model assembled using I-TASSER (Zhang, 2008, Zhang, 2009, Roy et al., 2010) B) homology modeling of residues within ToxR that affect ompU promoter activation more dramatically than toxT promoter activation. C) homology modeling of residues within ToxR that affect toxT promoter activation more dramatically than ompU promoter activation.

Figure 3

Gel-shift assays to assess promoter recognition by various ToxR mutant proteins identified in a random mutagenesis ompU-sacB selection strategy. V. cholerae membranes prepared from a ΔtoxRΔtcpP V. cholerae strain (EK459) expressing each mutant ToxR derivative (or wild-type ToxR-HA) were mixed at 0.05 and 0.25 mg/ml (two lanes for each sample) with radiolabeled ompU promoter DNA (A) or 0.1 and 0.5 mg/ml with toxT promoter DNA (B) prior to running samples in a non-denaturing PAGE.

Figure 4

Gel-shift assays to assess promoter recognition by putative α-loop site-directed ToxR mutant proteins. V. cholerae membranes prepared from a ΔtoxRΔtcpP V. cholerae strain (EK459) expressing each mutant ToxR derivative (or wild-type ToxR-HA) were mixed at 0.05 and 0.25 mg/ml with radiolabeled ompU promoter DNA (A) or 0.2 and 1.0 mg/ml with toxT promoter DNA (B) prior to running samples in a non-denaturing PAGE.

Figure 5

Assessment of TcpP protein interaction by various ToxR mutant proteins that preferentially affect toxT transcription. V. cholerae membranes from cells expressing each ToxR mutant derivative were assessed for their ability to interact with wild-type TcpP-HSV or TcpP-K101E. ToxR mutant proteins were expressed from the chromosomal toxR locus while TcpP-HSV was expressed from the IPTG-inducible plasmid pEK41 in a ΔtcpP V. cholerae background. TcpP-HSV was captured to 96-well microtiter plates using an anti-HSV monoclonal antibody. TcpP capture was assessed by probing the wells with an anti-TcpP polyclonal antibody (Krukonis & DiRita, 2003, Matson & DiRita, 2005), while ToxR co-capture was assessed by probing parallel wells with an anti-ToxR polyclonal antibody. Levels of each ToxR mutant protein and TcpP-HSV were assessed by Western blot on the same protein extracts used for crosslinking. Statistical comparisons were made using the students’ t-test and compare samples relative to a strain expressing wild-type ToxR and wild-type TcpP-HSV. * p=0.05, ** p<0.02

Figure 6

Assessment of ToxR/TcpP interaction by various ToxR and TcpP mutants using an adenylate cyclase-based membrane-anchored bacterial two-hybrid system. A) Interaction between wild-type TcpPH fused to the cya25 fragment of B. pertussis CyaA and ToxRS fused to the cya18 fragment of CyaA were assessed in strain BTH101. Interaction was determined by measuring β-galactosidase activity produced as a result of reconstitution of a functional adenylate cyclase enzyme. Known non-interacting mutants of TcpP were fused to cya25 as controls. As additional controls cya18-ToxR and cya25-TcpP alone were tested B) Western blot analysis of ToxR mutant derivatives fused to cya18. C) Western blot analysis of wild-type ToxR and EpsM fused to cya18. D) Western blot analysis of TcpP fused to cya25. All western blots were probed with rabbit anti-CyaA antibodies (Santa Cruz Biotechnology).

Figure 7

Model for differential activation of the ompU and toxT promoters of V. cholerae by ToxR. A) ToxR directly activates the ompU promoter in the absence of TcpP using residues F69 and V71 of the α-loop, and possibly E39 of α3, to stimulate RNA polymerase transcription. B) ToxR activates the toxT promoter in conjunction with TcpP by facilitating the ability of TcpP to interact with RNA polymerase. At the toxT promoter, ToxR and TcpP may interact via wing-wing contacts between the proteins. Thus, in this model, ToxR is inverted in its binding orientation on the DNA relative to the ompU promoter to present the wing face of ToxR to TcpP. Both promoters are shown from a backside view to allow easier visualization of the side chains that contribute preferentially to ompU (magenta) or toxT (orange) activation. The DNA template used is from the structure of PhoB bound to DNA (Yamane et al., 2008).