coli RNA polymerase (Abcam), which also recognizes SigA of M. smegmatis [38]. Western signals were quantified by using the Quantity One software (Bio-Rad). To test the localization

of wild-type Wag31 in the presence or absence YH25448 manufacturer of pknA Mtb – or pknB Mtb -overexpression, pCK314 was transformed into a M. smegmatis strain KMS2 or KMS4. Transformants were grown in 7H9 liquid medium until early-log phase (approximate OD600 = 0.2), split into two flasks, and 0.1% acetamide was added to express pknA Mtb or pknB Mtb for 2 hr. Both cultures were further incubated with tetracycline (20 ng ml-1) for 2 hr to express gfp-wag31 Mtb . For Van-Alexa568 staining, 5 μg ml-1 of Van-Alexa568 was added to both cultures, and incubated for 20 min at 37°C before microscopic examination. To examine the phosphorylation of wild-type Wag31Mtb under pknB Mtb -overexpression, total protein was purified and cleaned up with the ReadyPrep 2 D Cleanup Kit (Bio-Rad). 200 μg of total Selleck Eltanexor protein from each sample was rehydrated into isoelectric focusing strips with a pH range of 4 to 7 (Bio-Rad). Isoelectric focusing was performed for 35,000 V-h in a PROTEAN IEF Cell (Bio-Rad). 2-D SDS-PAGE was performed

using 10% Tris-HCl gels (Bio-Rad), and immunoblot blot analysis was performed using a phospho-(S/T)Q polyclonal antibody (Cell Signaling Technology), stripped, and then re-probed with anti-GFP antibody (Sigma). Yeast two-hybrid analysis Constructs of PD0332991 pJZ4-G-wag31 (pCK145), pJZ4-G-wag31T73A (pCK143) and pJZ4-G-wag31T73E (pCK142) were individually transformed into the yeast strain RFY231 by plating on agar minimal media lacking tryptophan [16]. Each of pHZ5-NRT-wag31 (pCK146), pHZ5-NRT-wag31T73A (pCK147) and pHZ5-NRT-wag31T73E (pCK148) was also transformed into another yeast strain Y309 by plating on agar minimal media lacking

histidine and uracil. Oxymatrine Four independent colonies from each transformation were mated on YPD plates, re-streaked onto minimal media lacking uracil, histidine, and tryptophan. As negative controls, mated cells containing empty vectors alone or cells containing pHZ5-NRT-wag31 Mtb (pCK146) and pJZ4-G vector were included. Mated cells that we recently showed the interaction between Rv1102c and Rv1103c (with pCK227 and pCK228) [39] were included as a positive control. Quantitative measurements (β-galactosidase activity in Miller unit) of interactions were conducted by using the Yeast β-Galactosidase Assay Kit (Pierce). Enzymatic assay for peptidoglycan synthetic enzymes The wag31 Msm deletion mutants containing each wag31 Mtb allele behind the Ptet promoter (KMS41, KMS42, and KMS43) were cultured to mid-log phase (approximate OD600 = 0.4), and a cell-wall enriched envelope fraction (P60) was prepared as previously described [22]. Briefly, 8 g of harvested cells were resuspended in 30 ml of buffer A (50 mM MOPS (pH 8.