Primer sequences are given in the 5′-3′ direction; restriction MLN8237 ic50 sites included in the primer sequences are underlined. DNA manipulation and cloning of constructs All molecular biology techniques were carried out according to standard procedures [26]. Restriction or DNA modifying enzymes and other molecular biology reagents were obtained from Roche Diagnostics or New England Biolabs.

Genomic DNA of M. smegmatis was isolated as described previously [13]. All primer sequences are listed in Table 1. To create a transcriptional fusion of the pitA promoter to lacZ, a fragment containing 750 bp of upstream sequence to pitA (MSMEG_1064) was amplified with primers PitA6 and PitA5 and cloned into the BamHI and SphI sites of the low copy-number vector (3-10 copies per cell) pJEM15 [27], resulting in plasmid pAH1. Assays for β-galactosidase activity were carried out as described previously

[13]. Cells of M. smegmatis harbouring the empty vector pJEM15 displayed β-galactosidase activities of less than 2 MU. Statistical analysis of reporter-strain experiments after LY2874455 price starvation or stress-exposure was performed

using one-way ANOVA followed by a Dunnett’s post-test comparison of each sample to the control condition. Data from experiments of the phnD-lacZ and pstS-lacZ constructs in various YH25448 genetic backgrounds were analyzed by one-way ANOVA followed by Bonferroni post-test comparison of all pairs of data-sets. All statistical analyses were performed using GraphPad Prism 4 software. To create a construct for markerless deletion of pitA, an 833 bp fragment Non-specific serine/threonine protein kinase flanking pitA on the left, including 62 bp coding sequence, was amplified with primers PitA1 and PitA2, and a 1022 bp fragment flanking pitA on the right, including 4 bp coding sequence, was amplified with primers PitA3 and PitA4. The two products were fused by PCR-overlap extension [28], cloned into the SpeI site of the pPR23-derived [29] vector pX33 [13], creating pPitAKO, and transformed into M. smegmatis mc2155. Deletion of pitA was carried out using the two-step method for integration and excision of the plasmid as described previously [20]. Correct integration and excision were confirmed by Southern hybridization analysis as described previously [13].