Cloning and characterization of the galE locus of Pasteurella haemolytica A1.
The enzyme UDP-galactose 4-epimerase (GalE) is involved in one of the major steps of galactose metabolism in bacteria. In many cases, GalE is required for the biosynthesis of extracellular polysaccharide materials such as lipopolysaccharide (LPS) and capsule. Mutants defective in galE have been shown to exhibit reduced virulence. Here we describe the cloning and characterization of the galE gene from the bovine pathogen Pasteurella haemolytica A1. This was achieved by the complementation of a Salmonella typhimurium galE mutant with a P. haemolytica A1 plasmid bank. Analysis of six clones recovered on minimal media with galactose as the carbon source showed that they all contained the same recombinant plasmid with a 5-kbp DNA insert. The galE-complementing activity was localized to a 2.2-kbp DNA region by subcloning. Biochemical, immunological, and phage sensitivity analyses of the recombinant LPS in S. typhimurium showed that it is essentially identical to that of the wild type. In vivo expression studies showed that a 37-kDa protein is expressed from the complementing plasmids, and the presence of GalE activity was confirmed by an assay for epimerase activity. Nucleotide sequence analysis of the cloned DNA identified the galE gene. Comparison of the deduced amino acid sequence analysis of P. haemolytica A1 GalE with published data showed high-level homology, 81.6%, with the GalE of Haemophilus influenzae type b. However, the sequences flanking galE do not show similarity with any other gal gene, suggesting that P. haemolytica A1 galE is not linked to the other genes of the gal operon, as is the case for Neisseria meningitidis, Neisseria gonorrhoeae, and H. influenzae. The separation of galE from the classical gal operon genes was confirmed by Southern blot hybridization studies, and a physical map showing the relative positions of galE, galT, and galK was constructed.
Potter
M D
MD
Department of Microbiology, University of Guelph, Ontario, Canada.
Lo
R Y
RY
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D005798:Genes, Bacterial; D008070:Lipopolysaccharides / Q000032:analysis; D016978:Mannheimia haemolytica / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D014534:UDPglucose 4-Epimerase / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D008070:Lipopolysaccharides; D014534:UDPglucose 4-Epimerase