Date of Award
1996
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Isolation, characterization and definition of the function of the gmhA locus in Escherichia coli could lead to a better understanding of the contribution of lipopolysaccharide (LPS) to bacterial disease. Such a description of the gmhA locus was the objective of the present studies. LPS mutants of Escherichia coli with defects in the biosynthesis and assembly of the heptose component of the core oligosaccharide display a shorter core that compromises the structural integrity of the outer membrane, resulting in a marked inability of these cells to grow in the presence of detergents, bile salts, and hydrophobic antibiotics. A heptoseless lipopolysaccharide E. coli strain, {dollar}\chi{dollar}711, demonstrating sensitivity to novobiocin has been shown to possess an alteration in a genetic locus, gmhA. In this study, gmhA has been cloned and characterized as containing a single gene which maps around 5.3 min (246.4 kb) on the E. coli K-12 chromosome and encodes a 22.6 kDa cytosolic protein. A plasmid carrying gmhA restored the structural defect in the core LPS of E. coli strain {dollar}\chi{dollar}711. Experiments in this work suggest that this strain contains a chromosomal deletion mediated by the IS5 transposon that removes 35-kb of chromosomal DNA including the gmhA locus and the proAB genes. Colony hybridizations indicated that gmhA was conserved among different members of enteric bacteria suggesting this gene has an important function in LPS biosynthesis. Reverse-phase high pressure liquid chromatography (RP-HPLC) analysis indicated that the GmhA protein is a phosphoheptose isomerase used for the conversion of sedoheptulose 7-phosphate to glyceromannoheptose 7-phosphate, the first reaction in the biosynthesis of L-glycero-D-mannoheptose, a component of inner core lipopolysaccharide. In this study the gmhA homologue in Haemophilus influenzae was also cloned and characterized. This gmhA homologue was able to complement the score LPS defect of E. coli {dollar}\chi{dollar}711. Evidence is presented in this work that these two GmhA proteins and other proteins in the database belong to a new family of phosphosugar isomerases.
Recommended Citation
Brooke, Joanna Susan, "Identification And Characterization Of Thegmha Gene Used For Inner Core Lipopolysaccharide Synthesis In Escherichia Coli" (1996). Digitized Theses. 2682.
https://ir.lib.uwo.ca/digitizedtheses/2682