Bacterial and phage strains, plasmids and growth conditions
M. smegmatis mc2155 and E. coli were routinely grown in Middlebrook 7H9  and Luria-Bertani  media (supplemented with appropriate antibiotics), respectively. The vectors pSD5S30 and pMPMK4 were obtained from Drs. A. Tyagi (University of Delhi, India) and S. Yasuda (Japan), respectively. Mycobacteriophage L1 and its growth conditions were described previously .
Purification of L1 repressor
To purify CI, cells harvested from one liter induced E. coli (pSAU1049) culture  were resuspended in 1/20 volume of lysis buffer A [20 mM Na-phosphate buffer (pH 6.0), 50 mM NaCl, 1 mM EDTA, 5% glycerol, and 100 μg/ml PMSF] followed by preparation of crude extract by appropriate sonication. Crude extract without cell debris was subjected sequentially to ultracentrifugation, 40 – 65% ammonium sulfate precipitation, SP-Sepharose column chromatography and hydroxyapatite column chromatography and fractions collected from each step were analyzed by SDS-12%PAGE (Fig. S1A). The elute from final step mainly shows a protein of ~22 kDa protein. It might be L1 CI as its molecular weight closely matched to that estimated from amino acid sequence of CI and binds to L1 operator DNA (Fig. S1C). The putative repressor was estimated to be around 97% pure.
To overexpress CI as an N-terminal histidine-tagged variant (His-CI), a vector pSAU1180 was constructed by cloning an L1 DNA [12, 17] (amplified with primers, LCP2: 5'AAGCTTCCTTTCGTTGCGCGGC and LCP3: 5'GAATTCATGAGCGGCAAAATC) to pET28a (Novagen, USA). This cloning has added extra 36 amino acid residues (including six histidine residues) to N-terminal end of CI.
Histidine-tagged CI (His-CI) overexpressed in E. coli BL21 (DE3) (pSAU1180) cells was purified by Ni-NTA resin (QIAGEN, Germany) according to manufacturer's protocol. Analysis of elution fraction showed only one protein of nearly 25 kDa (Fig. S1B). This seems to be the His-CI as its molecular mass matched to that estimated from its primary structure and it binds to L1 operator DNA (Fig. S1C).
Limited proteolysis of His-CI
It was carried out at 25°C in 20 μl phosphate buffer [50 mM phosphate buffer (pH 6.0), 50 mM NaCl]. Nearly 4 μg His-CI was mixed with 16 ng enzyme and reactions were performed for different times ranging from 0 – 30 mins followed by analysis of samples by Tris-Tricine SDS-16.5% PAGE .
Protein fragments generated from limited proteolysis of His-CI were transferred to nitrocellulose membrane followed by treatment of membrane sequentially with 3% BSA, mouse anti-his antibody (QIAGEN, Germany), goat anti-mouse antibody IgG1-AP (Santa Cruz Biotechnology, Germany), and NBT – BCIP (Bangalore Genei, India) solution for 1–2 h at room temperature. Each incubation step follows adequate washing step.
N-terminal protein sequencing
Stable His-CI fragments obtained from limited proteolysis were transferred to PVDF membrane. A PVDF paper strip carrying the fragment of interest was utilized for its N-terminal sequencing according to a standard protocol (Applied Biosystems, USA).
Purification of CTD
To purify CTD, nearly 200 μg of His-CI was digested with 800 ng of trypsin in 400 μl for 30 minutes at 25°C. After dialysis against buffer B, digested protein was loaded onto Ni-NTA column followed by the collection of flow-through. Analysis shows that flow-through contains mainly CTD (data not shown).
Cloning of O
64 and O
The 40426 – 40812 bp co-ordinate of L5 genome carries gp64 gene and an operator (5'GGTGGATGTCAAG) . A 386 bp DNA was amplified from L1 genomic DNA using primers designed on the basis of L5 gp64 and neighboring sequences and analysis revealed that it is 100% identical to the above mentioned L5 region at nucleotide level (data not shown). Next, polymerase chain reaction was carried out using 386 bp L1 DNA as a template and a suitable primer pair and the resulting ~120 bp DNA fragment harboring 5'GGTGGATGTCAAG sequence was designated O
Cloning of a 97 bp L1 DNA fragment that harbors a promoter and an operator (5'GGTGGCTGTCAAG) was reported previously  and designated O
Gel shift assay
To study the equilibrium binding of CI to O
64 and O
L operators, several gel shift assay were performed according to a modified method described earlier . Briefly, a 20 μl reaction mixture in Buffer A containing repressor, [32P-γ] ATP (BARC, India) end labeled operator DNA and 10 μg/ml bovine serum albumin was incubated at 25°C for 20 mins. As a reaction between L1 repressor and cognate operator is very fast , we assumed that 20 mins are sufficient for reaching equilibrium between the two species. Analysis of reaction mixtures was performed by a standard method as described earlier [14, 18].
Using different temperature-controlled water baths, equilibrium dissociation constant (Keq) of operator – CI interaction at each of 32°, 37°, and 42°C was determined from respective gel shift assay picture data.
To study the rate of dissociation of CI – operator complexes, a 200 μl reaction mixture in Buffer A containing ~0.1 nM operator and saturating amount of repressor was incubated for 20 mins at 25°C. Then a 300-fold excess of cold operator was added to the reaction mixture and 20 μl aliquot taken out at 0, 10, 20, 30, 60, 90, 120, 150 and 180 mins. Analysis of reaction mixture was done by same procedure as described above.
CD spectra of CI and CTD
Nearly 20 μM protein was taken in a cuvette (1 mm path length) and incubated at 32° or 42°C for 10 min. Next, Circular Dichroism (CD) spectrum (200 – 260 nm) of the protein was recorded by JASCO J600 spectrophotometer.
Analytical gel filtration chromatography
Analytical gel filtration chromatography was performed in an HPLC system using a gel filtration column Protein Pak (Waters, USA) after equilibration with 1× Buffer A (minus PMSF).
Cross-linking reactions of His-CI and CTD [56–183] were performed in Buffer A in 20 μl total volume at 25°C. Repressor containing solution was incubated at 25°C for 20 mins. Next, glutaraldehyde solution (0.1%) was added to repressor solution and incubated for 2 mins. The reaction was stopped by adding 5 μl of 4× SDS gel loading dye. After boiling the sample for 2 mins, it was analyzed by 10% SDS PAGE.