Among these, Pham7 was shown to contain genes encoding lysin A proteins, one of two lysins from mycobacteriophages (Garcia et al., 2002). Phage TM4 is one of the best-documented mycobacteriophages. It is a dsDNA-tailed phage that infects both fast-growing and slow-growing strains of mycobacteria (Ford et al., 1998) and has been shown to be active against a number of Mycobacterium species including M. tuberculosis and Mycobacterium ulcerans (Rybniker et al., 2006). Its genome structure has been analysed by Ford et al. (1998). Given the lytic spectrum of this phage, it selleck products was of interest to clone, express and purify the putative

lysin and assess its mureinolytic activity. A standard plaque assay was performed and the plates were used to harvest phage. High-titre phage suspension (up to 1014 PFU mL−1) was obtained by adding 5 mL of mycobacteriophage buffer (50 mM Tris, 150 mM NaCl, 10 mM MgCl2,

2 mM CaCl2, pH 8) to a plate from a plaque assay for 2 h with shaking. The buffer was then removed, centrifuged (1000 g for 10 min) and the supernatant was filtered through a 0.2-μm filter (Filtropur, Sarstedt). The approximate phage titre of the suspension was subsequently evaluated using a spot plaque assay method (20 μL of diluted phage suspension spotted on Middlebrook 7H9 agar; Becton Dickinson) seeded with 5%M. smegmatis. Mycobacterium smegmatis that was grown overnight in Middlebrook broth with 5% OADC supplement (Becton Dickinson) was inoculated (10%) into 100 μL of fresh broth in individual wells of a 96-well plate. 109 PFU mL−1 TM4 in mycobacteriophage PS-341 concentration buffer was added to certain wells. The final volume in all wells was 300 μL. Cell growth was measured spectrophotometrically over 72 h at 37 °C by determining OD600 nm in a temperature-controlled automatic plate reader (Multiskan FC, ThermoScientific). The Mycobacterium phage TM4 complete genome sequence (NC_003387) was accessed via SPTLC1 the National Center for Biotechnology Information (NCBI) Genome database (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome).

Gp29 amino acid sequence (NP_569764) was analysed using a variety of web-based programs including UniProt (http://www.uniprot.org/), the NCBI Conserved Domains Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and ProtParam (http://www.expasy.ch/tools/protparam.html). Homology searches were performed using the blast database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). DNase (1 μL) (New England Biolabs) and 2 μL of 25 mg mL−1 RNase A (Roche) were added to 750 μL of high-titre phage suspension and incubated for 10 min at 37 °C. Lysis buffer (150 μL) [400 mM EDTA, 0.01% sodium dodecyl sulphate (SDS), 50 mM Tris pH 8] and 10 μL of 10 mg mL−1 Proteinase K were then added and the sample was incubated for 30 min at 65 °C. DNA was extracted using a standard procedure of phenol : chloroform : isoamyl alcohol (25 : 24 : 1) and chloroform : isoamyl alcohol (24 : 1) extraction.