Journal of Biochemistry and Molecular Biology, Vol. 37, No. 2, March 2004, pp. 254-259 © KSBMB & Springer-Verlag 2004 Cloning and Sequencing Analysis of the Repressor Gene of Temperate Mycobacteriophage L1 Subrata Sau*, Partho Chattoraj, Tridib Ganguly, Chia Yen Lee † and Nitai Chandra Mandal* Department of Biochemistry, Bose Institute, P1/12-CIT Scheme VII M, Calcutta 700 054, India † Department of Microbiology, Molecular Genetics and Immunology, KU Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66103, USA Received 24 June 2003, Accepted 13 August 2003 The wild-type and temperature-sensitive (ts) repressor genes were cloned from the temperate mycobacteriophage L1 and its mutant L1cIts391, respectively. A sequencing analysis revealed that the 131 st proline residue of the wild- type repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative N- terminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor. Keywords: Helix-turn-helix (HTH) motif, M. smegmatis, Mycobacteriophage L1, Repressor gene, Temperature- sensitive (ts) Introduction The phages of mycobacteria are extremely diverse in nature and carry highly mosaic genomes (Pedulla et al., 2003). Several molecular tools have been developed from mycobacteriophages during the last ten years. They are very useful for mycobacterial research and the diagnosis of mycobacterial infections (Hatfull, 2000). Among the mycobacteriophages, L5, Bxb1, I3 (Hatfull, 2000), Ms6 (Garcia et al., 2002), and L1 (Chaudhuri et al., 1993) were studied to some extent at the molecular level. Both the cis- and trans-acting regulatory elements that are involved in the integration of L5 into its host genome were identified and characterized at length (Hatfull, 2000). Several promoters of I3 (Ramesh and Gopinathan, 1995), L5 (Nesbit et al., 1995), Bxb1 (Jain and Hatfull, 2000), and Ms6 (Garcia et al., 2002) were reported. The repressors of both L5 and Bxb1 negatively regulate the expression of their respective early promoters by binding at the cognate operators (Nesbit et al., 1995; Jain and Hatfull, 2000). Currently, the molecular mechanism of the interaction between the repressor of any mycobacteriophage and its operator DNA is poorly understood, though it has immense potential in deciphering the gene regulation in both mycobacteriophage and mycobacterial systems. Also, the information could lead to the construction of the tightly regulated expression vector (for the mycobacterial system) by assembling the early promoter and the repressor gene of temperate mycobacteriophage. Mycobacteriophage L1, a sister homoimmune phage of L5, has a 50-kb double-stranded DNA genome. The genes that regulate both the lysogenic and lytic development of L1 were mapped and some were to some extent characterized (Chaudhuri et al., 1993). The G27 gene of L1was shown to be an early positive regulator as it controls the expression of both the delayed early and late genes at the transcriptional level (Datta and Mandal, 1998). A few promoters of L1 have been cloned in a promoter-cloning vector having β-galactosidase as *To whom correspondence should be addressed. Fax: 91-33-2337-9416 E-mail: sau@bic.boseinst.ernet.in or ncmandal@boseinst.ernet.in