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 i s required for the biosynthesis of extracellular polysaccharide materi als such as lipopolysaccharide (LPS) and capsule. Mutants defective in galE have been shown to exhibit reduced virulence, Here we describe t he cloning and characterization of the galE gene from the bovine patho gen Pasteurella haemolytica A1. This was achieved by the complementati on of a Salmonella typhimurium galE mutant with a P. haemolytica A1 pl asmid bank, Analysis of six clones recovered on minimal media with gal actose as the carbon source showed that they all contained the same re combinant plasmid with a 5-kbp DNA insert, The galE-complementing acti vity 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 t he 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, Compari son of the deduced amino acid sequence of P. haemolytica A1 GalE with published data showed high-level homology, 81.6%, with the GalE of Hae mophilus influenzae type b. However, the sequences flanking galE do no t show similarity with any other gal gene, suggesting that P. haemolyt ica A1 galE is not linked to the other genes of the gal operon, as is the case for Neisseria meningitidis, Neisseria gonorrhoeae, and H. inf luenzae. The separation of galE from the classical gal operon genes wa s confirmed by Southern blot hybridization studies, and a physical map showing the relative positions of galE, galT, and galK was constructe d.