Colitis phage lytic enzyme and its gene.

Abstract

Colitis bacteriophage is a virulent phage of Escherichia coli and has a growth cycle of 35 minutes with a burst size of 100. It contains 65% protein and 35% DNA. The DNA is doublestranded, 33 kilobases (kb) in size, and is highly transfective. The phage induces a lysozyme which is involved in the lysis of infected cells. The lysozyme acts on Micrococcus lysodeikticus and E. coli cells. A lysozyme mutant of the colitis phage was unable to produce the lysozyme protein, as demonstrated by its inability to act on M. lysodeikticus cells and by the absence of immunoreactivity with antibodies raised against lysozyme. However, the lysozymemutant phage was able to lyse the host cell by inducing a lytic enzyme that acts on E. coli but not on M. lysodeikticus cells. Expression of the cloned colitis phage DNA showed that a 1.2 kb BamHI DNA fragment codes for the lysozyme, while a 2.3 kb BamHI fragment codes for the lytic enzyme. The lytic enzyme was partially purified from lysozymemutant phageinfected cells, and to near homogeneity from E. coli cells harbouring the cloned lyticenzyme gene, by a singlestep purification on a CMcellulose column. The lytic enzyme has:

Molecular weight: 19,000 Optimum pH: 7.5 Requires 0.5 mM MgCl for optimum activity

It was found to be different from T4, colitisphage, and heneggwhite lysozymes in immunoreactivity and substrate specificity. DNA from the lysozymemutant colitis phage was digested with BamHI and cloned into pBR322 at the BamHI site. Recombinant clones were screened for lyticenzyme activity. A clone producing the lytic enzyme was identified and contained a 2.3 kb insert. This insert also encoded a 20 kDa structural protein. The 2.3 kb insert contained a single ClaI site, yielding 1.2 kb and 1.1 kb ClaI-BamHI fragments, both cloned into pBR322. The clone containing the 1.1 kb fragment produced the lytic enzyme. The structural gene for the lytic enzyme and its promoter were mapped to a 0.8 kb BglI-BamHI segment of the 1.1 kb fragment using an invitro transcription-translation system derived from E. coli MRE600. The translated products were immunoprecipitated with antibodies raised against the colitisphage lytic enzyme, resolved by SDS-PAGE, and radioactive protein bands were detected by fluorography. The 1.1 kb ClaI-BamHI DNA fragment was sequenced by the dideoxy chaintermination method, yielding 1071 nucleotides. A sequence of 163 amino acids was derived, corresponding to a protein of molecular weight 18,050, containing 13 Arg and 8 Lys residues, making the protein basic. The molecular weight, basicity, and Nterminal aminoacid sequence (Met-Arg-Phe) matched those of the lytic enzyme. The derived Nterminal aminoacid sequence showed 18-34% homology with those of T4, P22, and 29 phage lysozymes. A Shine-Dalgarno-like sequence (GGGGT) is located 5 nucleotides upstream of the initiator codon (ATG). Primerextension studies showed that transcription initiates at G, 166 nucleotides upstream of ATG. The sequences TAACGTG and TTATCA, at the 10 and 35 positions from the transcription start site, may represent the Pribnow box and the RNApolymerase recognition site, respectively. The leader sequence of the lytic enzyme mRNA is 166 nucleotides long and can form stem-loop structures that may protect the mRNA from nuclease digestion. The transcriptiontermination signal (invert repeat) and the termination site (oligoT stretch) occur at 7 and 28 nucleotides, respectively, downstream of the translationtermination codon TAA. Another inverted repeat occurs 85 nucleotides downstream, which may act as a rhodependent termination signal. RNAs isolated from colitisphageinfected cells hybridized with the cloned lyticenzyme gene and were resolved into four species of sizes 1100, 800, 700, and 560 nucleotides. This indicates that initiation and/or termination of transcription of the lyticenzyme mRNA occurs at multiple sites.