TOPOLOGICAL ANALYSIS OF THE LYSINE-SPECIFIC PERMEASE OF ESCHERICHIA-COLI

Escherichia coli accumulates lysine via two systems, one specific for lysine (LysP) and a second inhibited by arginine or ornithine (LAO). The lysP gene encodes a polypeptide of 489 residues. A topological analysis of the LysP protein was performed using gene fusions. Random in-frame fusions of the lysP gene with the lacZ or blaM genes were generated. Site-directed mutagenesis was also used to generate additional blaM fusions at specific locations in the lysP gene. Two methods were used to alleviate the problem of lethal expression of some lysP::blaM fusions. First, ternary fusions were constructed in which the arsD gene was fused at the 5' end of the lysP gene and the blaM gene fused at specific sites within the lysP gene. In these plasmids lysP expression was controlled by the ars promoter. Secondly, an E. coli strain with a pcnB mutation was used with some fusions to maintain the plasmids at a reduced copy number. From analysis of 30 gene fusions, a topological model of the LysP protein is proposed in which the protein has 12 membrane-spanning regions, with the N- and C-termini in the cytosol.