Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1

• Published in: Journal of Bacteriology Vol. 179; no. 6; pp. 1872 - 1879
• Main Authors: Pontarollo, R.A. (Defence Research Establishment Suffield, Medicine Hat, Alberta, Canada.), Rioux, C.R, Potter, A.A
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.03.1997
• Subjects: ADN; Amino Acid Sequence; Bacteria; BACTERIOFAGOS; Bacteriology; BACTERIOPHAGE; Bacteriophage P2 - genetics; Bacteriophages - genetics; Base Sequence; Blood Bactericidal Activity; Capsid - chemistry; Capsid - genetics; CLONACION; CLONAGE; Clones; Cloning, Molecular; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Deoxyribonucleic acid; DNA; DNA, Viral - analysis; DNA, Viral - genetics; ESCHERICHIA COLI; GENE; GENES; Genetic Complementation Test; GENOMAS; GENOME; Haemophilus - genetics; Haemophilus - metabolism; Haemophilus - pathogenicity; Haemophilus - virology; HAEMOPHILUS SOMNUS; Hemin - metabolism; HEMOGLOBINA; HEMOGLOBINE; Molecular Sequence Data; Muramidase - chemistry; Muramidase - genetics; Open Reading Frames; PATOGENICIDAD; phage P2; Phenotype; POUVOIR PATHOGENE; Protein Precursors - chemistry; Protein Precursors - genetics; PROTEINAS; PROTEINE; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; Viral Proteins - chemistry; Viral Proteins - genetics; Virulence; VIRUS DE LOS ANIMALES; VIRUS DES ANIMAUX
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/jb.179.6.1872-1879.1997

In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-hp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-cML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage A. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21, when expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-Mr protein expressed from pRAP401 but did-not detect Orf2 in H. somnus lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-r