CLONING AND CHARACTERIZATION OF MGTE, A PUTATIVE NEW CLASS OF MG2+ TRANSPORTER FROM BACILLUS-FIRMUS OF4

The MM281 strain of Salmonella typhimurium which possesses mutations in each its three known Mg2+ transport systems and requires 100 mM Mg2+ for growth aas used to screen a genomic library from the gram-positive alkaliphilic bacterium Bacillus firmus OF4 for clones that could restore the ability to grow without Mg2+ supplementation. Of the clones obtained, five also conferred sensitivity to Co2+, similar to the phenotype of mutants,vith mutations in the S. typhimurium corA Mg2+ transport locus, All five contained identical inserts by restriction analysis. Using Ni-63(2+) as a surrogate for the unavailable Mg-28(2+), the plasmid insert was shown to restore cation uptake with properties similar but not identical to those of the S. typhimurium CorA Mg2+ transporter. Sequence analysis of one clone identified a single open reading frame with multiple possible initiation sites, Deletion and mutation analysis identified a minimum open reading frame of 939 bp encoding a polypeptide with a predicted molecular mass of 34 kDa. Disruption of the open reading frame eliminated cation influx activity and restored resistance to Co2+. This putative transporter, designated MgtE, has no sequence similarity to any known protein including CorA and appears to represent a new class of Mg2+ transport system.