Identification of the target DNA sequence and characterization of DNA binding features of HlyU, and suggestion of a redox switch for hlyA expression in the human pathogen Vibrio cholerae from in silico studies

Abstract

HlyU, a transcriptional regulator common in many Vibrio species, activates the hemolysin gene hlyA in Vibrio cholerae, the rtxA1 operon in Vibrio vulnificus and the genes of plp-vah1 and rtxACHBDE gene clusters in Vibrio anguillarum. The protein is also proposed to be a potential global virulence regulator for V. cholerae and V. vulnificus. Mechanisms of gene control by HlyU in V. vulnificus and V. anguillarum are reported. However, detailed elucidation of the interaction of HlyU in V. cholerae with its target DNA at the molecular level is not available. Here we report a 17-bp imperfect palindrome sequence, 5′-TAATTCAGACTAAATTA-3′, 173 bp upstream of hlyA promoter, as the binding site of HlyU. This winged helix-turn-helix protein binds necessarily as a dimer with the recognition helices contacting the major grooves and the β-sheet wings, the minor grooves. Such interactions enhance hlyA promoter activity in vivo. Mutations affecting dimerization as well as those in the DNA–protein interface hamper DNA binding and transcription regulation. Molecular dynamic simulations show hydrogen bonding patterns involving residues at the mutation sites and confirmed their importance in DNA binding. On binding to HlyU, DNA deviates by ∼68º from linearity. Dynamics also suggest a possible redox control in HlyU.

The Transcriptional Regulator HlyU Positively Regulates Expression of exsA , Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus

“…6A). A similar observation was previously reported for V. cholerae HlyU (27). The current data cannot differentiate if the DNA shifts were due to (i) increased HlyU binding at multiple sites, creating larger DNA-protein complexes, or (ii) different physical conformations of DNA-protein complexes.…”

“…3) and therefore strongly implied a role in exsA regulation. In silico analysis of Vp0529 using NCBI BLAST indicated sequence similarity to HlyU, a DNA binding protein of many Vibrio species that acts as a positive regulator of virulence genes (27)(28)(29)(30)(31). Vp1133 encodes a histone-like nucleoid-structuring protein (H-NS) and has previously been implicated in exsA repression (32).…”

“…Purified HlyU binds upstream of exsA. HlyU is a member of the SmtB/ArsR family of regulator proteins that bind to DNA using a winged helix-turn-helix (wHTH) domain structure (27). Based on the hlyU1 deficiency for T3SS-1-associated phenotypes, we set out to investigate whether HlyU binds to V. parahaemolyticus DNA.…”

“…HlyU protein sequences are highly conserved among Vibrio species (ranging from 81 to 100% identity) (27). Numerous structure-function studies have established that HlyU proteins form stable homodimers and possess a winged helix-turn-helix (wHTH) domain structure which is modeled to bind DNA within two major grooves (27,44). Furthermore, HlyU dimerization is necessary for DNA binding activity in V. cholerae, and specific amino acids within HlyU contribute to DNA binding (27).…”

“…Numerous structure-function studies have established that HlyU proteins form stable homodimers and possess a winged helix-turn-helix (wHTH) domain structure which is modeled to bind DNA within two major grooves (27,44). Furthermore, HlyU dimerization is necessary for DNA binding activity in V. cholerae, and specific amino acids within HlyU contribute to DNA binding (27). These critical amino acids are conserved in all V. parahaemolyticus HlyU homologues within databases.…”