Cloning and characterization of the galE locus of Pasteurella haemolytica A1

Abstract

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 galEcomplementing 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 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.

The Genome of Melanoplus sanguinipes Entomopoxvirus

“…5C). These enzymes, which transfer sugar residues to lipid moieties or other sugar residues, have not been described previously in poxviruses (83,145,178). The presence of a transmembrane domain at the carboxyl terminus of MSV206 (amino acids 252 to 276) suggests that the protein is membrane associated.…”

Importance of the galE gene on the virulence of Pasteurella multocida

“…Downstream from the P. multocida galE , a sequence similar to the putative HI125 protein of H. influenzae was identified. Moreover, the sequence located upstream of the P. haemolytica galE gene has significant similarity with the orfE of E. coli , whereas no apparent homology with any known gene was detected downstream from the P. haemolytica galE gene [14]. It has been described that flanking the P. haemolytica galE gene there are two direct repeats which could explain the separation of this gene from the rest of the gal operon by a transpositional event [14].…”

Immunoproteomic to Analysis the Pathogenicity Factors in Leukopenia Caused by Klebsiella Pneumonia Bacteremia

“…The role of glutathione synthetase in bacteria is very important, it can protect bacteria from damage of osmotic stress, low pH, toxicity of ethylglyoxal and oxidative stress [19] – [22] .UDP-galactose-4-epimerase is an essential enzyme of the Leloir pathway of galactose metabolism and catalyzes the interconversion of UDP-galactose and UDP-glucose [23] . UDP-galactose thus formed serves as galactose donor for the biosynthesis of galactosyl residues in glycoproteins and complex polysaccharides, including both the core and O-antigen polysaccharide of the LPS [24] . Acetate kinase, a conserved enzyme that is widespread in bacteria, is responsible for the phosphorylation of acetate [25] .…”