Iron acquisition and characterization of bacteriophage-like genes of haemophilus somnus

Abstract

Potential virulence mechanisms of the gram-negative bovine pathogen Haemophilus somnus include the abilities to acquire iron from bovine transferrin, to resist killing by complement, and to survive in leukocytes. The original objective of this project was to investigate the iron-uptake mechanisms of H. somnus. When grown in the presence of iron-specific chelators, H. somnus expressed seven novel iron-regulated outer membrane proteins with relative molecular weights of 125-, 115-, 105-, 80-, 75-, 60-, and 50-kDa. Transferrin-bound iron, free heme, or hemopexin-bound heme, could act a sole source of iron for H. somnus. Therefore, a cosmid library of H. somnus genomic DNA was screened for clones able to bind hemin (Hmb$\sp+)$ on agar containing hemin. Cosmid pRAP117 contained a 7.8 kb HindIII fragment that endowed E. coli with Hmb$\sp+$ and hemolytic phenotypes. The. 7.8 kb fragment, and a 2.9 kb XbaI fragment from within it, were subcloned into plasmid pTZ19R, creating plasmids pRAP401 and pRAP501, respectively. E. coli cells containing pRAP501 were Hmb$\sp+,$ but were not hemolytic. DNA sequencing and transposon mutagenesis of pRAP501 identified a gene (hmb) which encoded a polypeptide (Hmb) of 178 amino acids whose expression correlated with the Hmb$\sp+$ phenotype. Hmb had significant homology to bacteriophage-encoded lysozymes and had lytic activity in a crude lysozyme assay. The hmb gene acted in trans to complement mutations in the lysozyme genes of bacteriophages P2 and $\lambda.$ The gene upstream from hmb encodes a polypeptide similar to holins, another phage-encoded lysis protein. The DNA sequence of plasmid pRAP401 had 13 open reading frames (orfs), including the holin gene (orf6) and hmb (orf7). orfs1-5, and orfsr11-12, encode polypeptides that are similar to the DNA packaging, capsid, and tail synthesis proteins of phage P2. All attempts to characterize the hemolytic phenotype were unsuccessful. Antiserum raised against the Orf2 protein detected recombinant Orf2, but native Orf2 was not detected in H. somnus. A Southern blot detected the phage-like DNA sequence in two serum-sensitive strains, but not in two serum-resistant strains of H. somnus. The phage-like DNA had no observable influence on the ability of H. somnus to survive in bovine leukocytes.