Leucine is transported into E coli by two osmotic shock-sensitive, high-affinity systems (LIV-I and leucine-specific systems) and one membrane bound, low-affinity system (LIV-II). Expression of the high-affinity transport systems is altered by mutations in livR and 1stR, genes for negatively acting regulatory elements, and by mutations in rho, the gene for transcription termination. All four genes for high-affinity leucine transport (livJ, livK, livH, and livG) are closely linked and have been cloned on a plasmid vector, pOX1. Several subcloned fragments of this plasmid have been prepared and used in complementation and regulation studies. The results of these studies suggest that livJ and livK are separated by approximately one kilobase and give a gene order of livJ-livK-livH. livJ and livK appear to be regulated in an interdependent fashion; livK is expressed maximally when the livJ gene is activated by mutation or deletion. The results support the existence of separate promotors for the livJ and livK genes. The effects of mutations in the rho and livR genes are additive on one another and therefore appear to be involved in independent regulatory mechanisms. Mutations in the rho gene affect both the LIV-I and leucine-specific transport systems by increasing the expression of livJ and livK, genes for the LIV-specific and leucine-specific binding proteins, respectively.

Leucine is transported into E. coli cells by high‐affinity transport systems (LIV‐I and leucine‐specific systems) which are sensitive to osmotic shock and require periplasmic binding proteins. In addition leucine is transported by a low‐affinity system (LIV‐II) which is membrane bound and retained in membrane vesicle preparations. The LIV‐I system serves for threonine and alanine in addition to the 3 branched‐chain amino acids. The LIV‐II system is more specific for leucine, isoleucine, and valine while the high‐affinity leucine‐specific system has the greatest specificity. A regulatory locus, livR at minute 22 on the E. coli chromosome produces negatively regulated leucine transport and synthesis of the binding proteins. Valine‐resistant strains have been selected to screen for transport mutants. High‐affinity leucine transport mutants that have been identified include a LIV‐binding protein mutant, livJ, a leucine‐specific binding protein mutant livK and a nonbinding protein component of the LIV‐I system, livH. A fourth mutant, livP, appears to be required only for the low‐affinity LIV‐II system. The existence of this latter mutant indicates that LIV‐I and LIV‐II are parallel transport systems. The 4 mutations concerned with high‐affinity leucine transport form a closely linked cluster of genes on the E. coli chromosome at minute 74. The results of recent studies on the regulation of the high‐affinity transport systems suggests that an attenuator site may be operative in its regulation. This complex regulation appears to require a modified leucyl‐tRNA along with the transcription termination factor rho. Regulation of leucine transport is also defective in relaxed strains. Among the branched‐chain amino acids only leucine produces regulatory changes in LIV‐I activity suggesting a special role of this amino acid in the physiology of E. coli. It was shown that the rapid exchange of external leucine for intracellular isoleucine via the LIV‐I system could create an isolucine pseudoauxotrophy and account for the leucine sensitivity of E. coli.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOXl plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.