Molecular characterization of genes and promoters of mycobacteriophage I3

Abstract

Although nearly 250 mycobacteriophages have been isolated, they have not been subjected to detailed biochemical studies. Our laboratory has been investigating various aspects of mycobacteriophage 13 and its interaction with the host Mycobacterium smegmatis. The present study deals with the molecular analysis of the structural proteins, genes, and promoters of mycobacteriophage 13. Phage 13 genome is linear doublestranded DNA of 135-145 kbp. The DNA harbors 6-7 random singlestranded gaps of 6-10 nucleotides on each strand. Restriction analysis revealed the presence of multiple sites for many enzymes (e.g., PstI, SalI, BglII, KpnI, etc.) and the absence of sites for enzymes such as EcoRI and DraI (whose recognition sequences are rich in AT base pairs). The sites for XbaI, XhoI, HindIII and BamHI were few (1, 3, 4 and 9 sites respectively), and hence digestion with these enzymes resulted in large fragments. These large fragments were resolved by pulsedfield gel electrophoresis. The XbaI and XhoI fragments, as well as native 13 DNA, did not band sharply under any experimental conditions tested because of the random singlestranded gaps on both strands. However, the BamHI and HindIII fragments were resolved and their molecular sizes determined. A restriction map for these two enzymes has been constructed. The structural proteins of phage 13 were characterized by SDSpolyacrylamide gel electrophoresis, immunoprecipitation of iodinated phage proteins, and Western blotting (immunoblotting). The number of structural proteins associated with phage 13 was found to be 36, though this number may be overestimated. The molecular mass of the proteins ranged from 13 kDa to 340 kDa. A genomic library of phage 13 was constructed in vector pUC18. One clone carrying a 1 kbp DNA insert reacted positively with phage 13 antibodies. Immunoblotting of the protein extract from this clone showed expression of the 17 kDa structural protein of phage 13. The insert was completely sequenced and analyzed. The structural protein gene was oriented opposite to the lacZ reading frame and possessed its own transcriptional and translational regulatory sequences. The deduced aminoacid sequence showed an unusually high abundance of proline residues. Codonusage analysis indicated a bias, with preference for ‘G’ or ‘C’ in the third position. Neither the DNA nor the protein sequence showed homology to known sequences in existing databases. Downstream of the 17 kDa gene another incomplete ORF was detected, with its own upstream regulatory elements. Phage 13 promoters were analyzed by cloning restriction fragments into a promoterselection vector. The growth pattern of recombinants on chloramphenicol gradient plates indicated relative promoter strengths. Enzyme assays were performed for quantitative comparison. Two cloned 13 promoters exhibited strengths comparable to endogenous E. coli promoters. DNA sequence analysis revealed potential 10 and 35 regions. The 35 sequences showed similarity to E. coli consensus sequences, while the 10 regions differed. A likely promoter consensus for phage 13 has been proposed. A 70 kDa structural protein gene of phage 13 was cloned and expressed in an E. coli expression system. Expression was confirmed by immunodot blot and Western blot analyses. The gene was completely sequenced using unidirectional nested deletions generated by exonuclease III. Sequence analysis suggested the presence of at least a bicistronic transcript in this genomic fragment. The deduced protein sequence showed high abundance of proline, threonine, glycine and asparagine residues. Codonusage analysis again showed a characteristic bias. The DNA and protein sequences exhibited similarity to collagen and to Epstein-Barr virus nuclear protein antigens. This similarity appears to arise from the proline and glycinerich composition, and therefore no functional significance can be attributed to the phage protein.