Cloning and Characterization of the Genes Coding for the Membrane Components of the Leucine Transport in Escherichia Coli.

The high-affinity transport of the branched chain amino acids in E. coli is mediated by two binding protein dependent transport systems, LIV-I and LS, which are composed of multiple components. Genetic analyses have identified four complementation groups, livJ, livK, livH and livG which form a gene cluster at minute 74 on the E. coli chromosome. The livJ and livK genes code for LIV-BP and for LS-BP, respectively. Mutations in livH and livG have similar phenotypes which lack both LIV-I and LS transport activities. The liv genetic locus has recently been cloned into the pACYC184 plasmid vector to give the pOX1 plasmid. This thesis is concerned with the characterization of the livH and livG transport components. The genetic evidence indicating the presence of two genes has been confirmed by physically and functionally identifying the genes on the pOX1 plasmid using transposon mediated mapping, subcloning and DNA sequencing analysis. These experiments indicated that the livK, livH and livG genes form a polycistronic operon which appears to also contain an additional, previously unidentified gene, livM, located between the livH and livG genes. The livH, livM and livG genes have been subcloned into multicopy plasmid vectors and their products have been tentatively identified in vitro. The molecular weights of these components are 30,000, 27,000 and 22,000 respectively. All three proteins appear to be membrane-bound and are poorly expressed. The DNA sequence of the livH gene and its intergenic regions have been determined. The livH gene product is a rather basic protein containing several long hydrophobic segments which might be expected to transverse the cell membrane. An intergenic region of 47 bp exists between the livK and livH genes that contains a dyad symmetry of 7 GC base pairs which has the potential to form a stable stem-loop structure when transcribed. Finally livH-lacZ gene fusions containing the first ten amino acids of the livH protein have been constructed in vitro. The regulation of livH-lacZ gene expression has been studied and preliminary results indicate that the livH is under leucine control.PhDBiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/160005/1/8412216.pd