OH 44106-4965, USA Salmonella typhimurium has three distinct Mg2+ transport systems, the constitutive high-capacity CorA transporter and two P-type ATPases, MgtA and MgtB, whose transcription is repressed by normal concentrations of Mg2+ in the growth medium. The latter Mg2+-transporting ATPase is part of a two-gene operon, mgtCB, with mgtC encoding a 23 kDa protein of unknown function. Transcriptional regulation using fusions of the promoter regions of mgtA and mgtCB to luxAB showed a biphasic time and Mg2+ concentration dependence. Between 1 and 6 h after transfer to nitrogen minimal medium containing defined concentrations of Mg2+, transcription increased about 200-fold for mgtCB and up to 400-fold for mgtA, each with a half-maximal dependence on Mg2+ of 0 5 mM. Continued incubation revealed a second phase of increased transcription, up to 2000-fold for mgtCB and up to 10000-fold for mgtA. This secondary increase occurred between 6 and 9 h after transfer to defined medium for mgtCB but between 12 and 24 h for mgtA and had a distinct halfmaximal dependence for Mg2+ of 0.01 mM. A concomitant increase of at least 1000-fold in uptake of cation was seen between 8 and 24 h incubation with either system, showing that the transcriptional increase was followed by functional incorporation of large amounts of the newly synthesized transporter into the membrane. Regulation of transcription by Mg2+ was not dependent on a functional stationary-phase sigma factor encoded by rpoS, but it was dependent on the presence of a functional phoPQ two-component regulatory system. Whereas mgtCB was completely dependent on regulation via phoPQ, the secondary late Mg2+-dependent phase of mgtA transcription was still evident in strains carrying a mutation in either phoP or phoQ, albeit substantially diminished. Several divalent cations blocked the early phase of the increase in transcription elicited by the decrease in Mg2+ concentration, including cations that inhibit Mg2+ uptake (Co2+, NiZ+ and Mn2+) and those which do not (Ca2+ and Zn2+). In contrast, the second later phase of the transcriptional increase was not well blocked by any cation except those which inhibit uptake. Overall, the data suggest that at least two distinct mechanisms for transcriptional regulation of the mgtA and mgtCB loci exist.

CorA is a primary Mg2؉ transporter for Bacteria and Archaea. The C-terminal domain of ϳ80 amino acids forms three transmembrane (TM) segments, which suggests that CorA is a homo-oligomer. A Cys residue was added to the cytoplasmic C terminus (C317) of Salmonella enterica serovar Typhimurium CorA with or without mutation of the single periplasmic Cys191 to Ser; each mutant retained function. Oxidation of the Cys191Ser Cys317 CorA gave a dimer. Oxidation of Cys317 CorA showed a dimer plus an additional band, apparently cross-linked via both Cys317 and C191. To determine oligomer order, intact cells or purified membranes were treated with formaldehyde or carbon disulfide. Higher-molecular-mass bands formed, consistent with the presence of a tetramer. Cross-linking of the Bacillus subtilis CorA expressed in Salmonella serovar Typhimurium similarly indicated a tetramer. CorA periplasmic soluble domains from both Salmonella serovar Typhimurium and the archaeon Methanococcus jannaschii were purified and shown to retain structure. Formaldehyde treatment showed formation of a tetramer. Finally, previous mutagenesis of the CorA membrane domain identified six intramembrane residues forming an apparent pore that interacts with Mg 2؉ during transport. Each was mutated to Cys. In mutants carrying a single intramembrane Cys residue, spontaneous disulfide bond formation that was enhanced by oxidation with Cu(II)-1,10-phenanthroline was observed between monomers, indicating that these Mg 2؉ -interacting residues within the membrane are very close to their cognate residue on another monomer. Thus, CorA appears to be a homotetramer with a TM segment of one monomer physically close to the same TM segment of another monomer.