L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14CJproline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 ,M; maximum rate of transport [VmaxJ = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 ,uM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na+-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14CJproline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.

Plasmid pWG115 isolated from a methicillin-resistant Staphylococcus aureus encodes resistance to cationic surface-active agents and trimethoprim. It has a molecular weight of ca 14.6 megadaltons and can be transferred to other strains of staphylococci in mixed-culture transfer with propamidine isethionate as a selective agent. Gentamicin resistance in Australian methicillin-resistant Staph. aureus isolates can be either chromosomal or plasmid-borne. The most common gentamicin resistance plasmid is 18.0 megadaltons and also encodes resistance to trimethoprim and cationic surface-active agents. This suggested that pWG115 was related to gentamicin resistance plasmids and that it may provide a target for the postulated gentamicin resistance transposon. This paper demonstrates that the chromosomal gentamicin resistance determinant from WG523 can transpose into pWG115 to generate an 18.0 megadalton plasmid, phenotypically indistinguishable from the naturally occurring gentamicin resistance plasmids such as pWG53. EcoR1 restriction enzyme analysis demonstrated that gentamicin resistance can transpose into at least two sites on pWG115. One of these sites generates EcoR1 restriction fragments identical to pWG53. The 5.2 kilobase pair (3.4 megadalton) element involved confers low-level resistance to gentamicin, cross resistance to tobramycin and kanamycin, and has been designated Tn3851.

The MM281 strain of Salmonella typhimurium possesses mutations in each of its three Mg 2؉ transport systems, requires 100 mM Mg 2؉ for growth, and was used to screen a genomic library from the gram-negative bacterium Providencia stuartii for clones that could restore the ability to grow without Mg 2؉ supplementation. The clones obtained also conferred sensitivity to Co 2؉ , a phenotype similar to that seen with the S. typhimurium corA Mg 2؉ transport gene. The sequence of the cloned P. stuartii DNA revealed the presence of a single open reading frame, which was shown to express a protein with a gel molecular mass of 37 kDa in agreement with the deduced size of 34 kDa. Despite a phenotype similar to that of corA and the close phylogenetic relationship between P. stuartii and S. typhimurium, this new putative Mg 2؉ transporter lacks similarity to the CorA Mg 2؉transporter and is instead homologous to MgtE, a newly discovered Mg 2؉ transport protein from the grampositive bacterium Bacillus firmus OF4. The distribution of mgtE in bacteria was studied by Southern blot hybridization to PCR amplification products. In contrast to the ubiquity of the corA gene, which encodes the dominant constitutive Mg 2؉ influx system of bacteria, mgtE has a much more limited phylogenetic distribution.Gram-negative bacteria possess at least two distinct classes of Mg 2ϩ transport systems: the inducible P-type ATPases exemplified by the MgtA and MgtB systems of Salmonella typhimurium and Escherichia coli (4, 17) and the constitutive CorA Mg 2ϩ transporter, which is ubiquitous in gram-negative bacteria (4,5,(12)(13)(14)17 -independent growth and restoration of Co 2ϩ sensitivity (4, 5). Of five similar overlapping clones, pRB6 containing a 3.0-kb insert was selected for further study; the complementing insert DNA was localized to a 2.2-kb SphI restriction fragment and subcloned into the SphI site of pBS KSϩ to create pDT3.The nucleotide sequence of this DNA insert (Fig. 1) contains a single open reading frame between nucleotides 343 and 1419, predicting a protein with a size of about 38 kDa. An additional ATG codon is present at bp 469, predicting a protein with a size of 34 kDa and 314 amino acids (Fig. 1). There is no Shine-Dalgarno site near the ATG at bp 343, and there is only a relatively poor one at bp 469. Comparison of the predicted amino acid sequence against sequences contained in GenBank by using the BLAST program (1) identified no proteins that shared significant similarity, including the CorA Mg 2ϩ transporter. This laboratory recently reported the cloning of a CorA-like Mg 2ϩ transporter with a length of 312 amino acids from the gram-positive alkaliphile B. firmus OF4 by similar functional complementation of the Mg 2ϩ transport defect of MM281 (16). Comparison of the deduced protein sequences of MgtE from B. firmus OF4 with the newly cloned locus from P. stuartii shows 55% overall similarity (Fig. 1) to the smaller of the two open reading frames. The larger open reading frame in the P. stuartii insert potentially encodes an ...

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