Salmonella typhimurium contains two inducible Mgz+ transport systems, MgtA and MgtB, the latter encoded by a two-gene operon, mgtC6. Mg2+ deprivation of S. typhimurium increases transcription of both mgtA and mgtCB over a thousandfold and a similar increase occurs upon S. ephimurium invasion of epithelial cells. These increases are mediated by the phoPQ two-component signal transduction system, an essential system for 5. typhimurium virulence. It was therefore hypothesized that expression of MgtA and MgtCB is increased upon invasion of eukaryotic cells because of a lack of intravacuolar Mg? However, when S. typhimurium was grown a t pH 5.2. the capacity of the constitutive CorA transporter in mediating Mgz+ was greater than that a t pH 74. Furthermore, induction of mgtA and mgtCB transcription was greater in the presence of a wild-type corA allele than in its absence. This implies that intravacuolar 5. typhimurium could obtain sufficient Mg2* via the CorA system. The effect of acid pH on mgtA and mgtCf? transcription was also measured. Compared to induction at pH 74, exposure to pH 5.2 almost completely abolished induction of mgtA at low Mg2+ concentrations but diminished induction of m g K B only twofold. Adaptation of cells to acid pH by overnight growth resulted in normal levels of induction of mgtA and mgtCB at low Mg2+ concentrations. These results imply an additional level of regulation for mgtA that is not present for mgtCB. Conversely, repression of mgtA and mgtCB expression by increased extracellular Mgz* was relatively insensitive to acid.Transcription of both loci was strongly induced upon invasion of the Hep-2 or CMT-93 epithelial-like or J774 macrophage-like cell lines. However, the presence or absence of functional alleles of either or both mgtA or -5 had no effect on invasion efficiency or short-term survival of S. typhimurium within the eukaryotic cells. It was concluded that the strong Mgz*-dependent induction of mgtA and mgtCB upon invasion of eukaryotic cells is not required because 5. typhimurium lacks sufficient Mgz+ during eukaryotic cell invasion and initial intravacuolar growth.

The CorA transport system is the major Mg 2؉ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg 2؉ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. , albeit only at extracellular concentrations that are toxic to the cell. The amino acid sequence of CorA lacks homology to other known families of proteins (3). Studies of its phylogenetic distribution (4) and the recent plethora of microbial genome sequences have demonstrated that CorA is virtually ubiquitous in bacteria and the Archaea and likely forms the major Mg 2ϩ influx system for these kingdoms (5). The membrane topology of CorA has been previously studied using C-terminal protein fusions to ␤-lactamase and ␤-galactosidase (3). Like its amino acid sequence, its membrane topology is also unlike that of other known transport proteins. The N-terminal 235 residues reside in the periplasm while the remaining 80 amino acids form three transmembrane segments, including a short 6 residue C-terminal sequence in the cytosol. Three transmembrane segments are unlikely to be sufficient to form a transport channel or pore; thus, CorA probably functions as a homoligomer.The mechanism of ion transport through CorA may also be unique. Unlike many ion transporters and channels, CorA contains only two charged residues, both within the first membrane domain; neither is conserved in other homologs (5). This suggests that the most charge dense of the common biological cations passes through the membrane without involvement of electrostatic bonds. Mg 2ϩ coordinates virtually exclusively with oxygen rather than nitrogen or sulfur (6) which suggests backbone carbonyl groups and hydroxyl bearing residues within the membrane environment would be important. Sequence alignment of the CorA homologs currently available suggest a high degree of conservation of such groups in the second and third transmembrane segments. In this study, conserved residues in the third transmembrane segment (TM3) 1 of CorA were mutated. All but one mutation resulted in stable expression of protein and protein insertion into the membrane. Conserved residues Phe 290 , Tyr 309 , and Phe 310 at the termini of TM3 could be substituted by other aromatic residues without significant change in transport properties suggesting a structural rather than a transport role. In contrast, mutations at Tyr 292 , Met 299 , and Tyr 307 showed large decreases i...