CLONING AND CHARACTERIZATION OF BACTERIOPHAGE-LIKE DNA FROM HAEMOPHILUS-SOMNUS HOMOLOGOUS TO PHAGES P2 AND HP1

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-bp HindIII fragme nt of H. somnus DNA that was subcloned and sequenced, Thirteen open re ading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated wit h the Hmb(+) phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi- vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay u sing supernatants from whole-cell lysates off. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product ) exhibited significant levels of lysozyme activity, The orf6 gene ups tream 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 lac promote r, the orf6 gene product caused immediate cell death without lysis, wh ile cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform, Based on this dat a, we concluded that the Hmb(+) phenotype was an artifact resulting fr om the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs th at are similar to structural and DNA packaging genes of phage P2, Poly clonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein e xpressed from pRAP401 but did not detect Orf2 in H. somnus lysates. Th e phage-like DNA was detected in the serum-susceptible preputial strai ns HS-124P and HS-127P but was absent from the serum-resistant preputi al strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the N. somnus life cycle requires more study.