Molecular characterization of a Salmonella specific locus coding for a heavy metal-responsive transcriptional regulador

SUSANA KARINA CHECA; BOTTA, P.; SPINELLI, S. V.; ESPARIZ, M.; SONCINI, F. C.

Washington, D.C.

Congreso; 104 th General Meeting of the American Society for Microbiology; 2004

American Society for Microbiology

Molecular characterization of a Salmonella specific locus coding for a heavy metal-responsive transcriptional regulator Checa, Susana K., Botta, Pablo E., Spinelli, Silvana V., Espariz, Mart¨ªn, and Soncini, Fernando C. IBR-CONICET, School of Biochemistry, Rosario National University, Rosario, ARGENTINA. Instituto de Biolog¨ªa Molecular y Celular de Rosario (IBR-CONICET), Fac.  Cs. Bioqu¨ªmicas y Farmace¨²ticas,  Rosario, Argentina. E-mail: pat-bact@citynet.net.ar The MerR family is a group of transcriptional regulators with similar N-terminal helix-turn-helix DNA binding domains and C-terminal effector recognition regions that respond specifically to certain environmental stimuli, such as heavy metals, oxidative stress or antibiotics.  We have performed an in silico screen, searching for new metal-responsive transcriptional regulators in the Salmonella enterica serovar Typhimurium genome, and identified a gene located within a Salmonella specific region that we named hmrR.  HmrR displays a 40 to 50% sequence homology to members of the MerR family of metalloregulatory proteins responsive to copper.  The hmrR gene forms a two-gene operon together with hmrT, a gene coding for a putative metal-transporting P-type ATPase harboring the conserved CXXC metal-binding motif in its N-terminal sequence.  The hmrT-hmrR operon is located upstream to hmrB, a gene showing a 20 to 40% amino acid sequence similarity to members of the MerP/CopZ family of periplasmic metal-binding proteins.  HmrR regulates the expression of the two neighboring genes, and its own synthesis.  By measuring ¦Â-galactosidase activity, we found that HmrR functions both as a repressor and an activator of the expression of hmrT and hmrB depending on the environmental condition.  We mapped hmrT and hmrB promoters by primer extension analysis and detected that their promoter/operator regions have signature elements that distinguish promoters controlled by transcriptional regulators of the MerR family, such as a suboptimal spacing between -10 and -35 elements and sequences of dyad symmetry.  Surprisingly, by measuring cuiD expression in high and low extracellular copper concentration, we determined that HmrR does not affect the SctR-controlled copper homeostasis, indicating that these two proteins belong to independent regulatory pathways.