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Figure 3 | BMC Microbiology

Figure 3

From: Regulation of Polar Peptidoglycan Biosynthesis by Wag31 Phosphorylation in Mycobacteria

Figure 3

Effect of Wag31 phosphorylation on polar localization. A. Each gfp-wag31 Mtb allele behind the Pacet promoter in the replicating plasmid pMV261 (pCK174, pCK175 and pKC176) was introduced into the wag31 Msm deletion mutant expressing wag31 Mtb (KMS41), wag31T73A Mtb (KMS42) or wag31T73E Mtb (KMS43) under a Ptet promoter, respectively. Transformants (KMS69, KMS70, and KMS71) were cultured in the presence of tetracycline (20 ng ml-1) until early-log phase where the expression of each gfp-wag31 allele was induced with acetamide (0.1%) for 3 hr before cells were observed under a fluorescence microscope, and the polar GFP-Wag31 signal was measured by using ImageJ software. Top, GFP signal from fluorescence microscopy; Middle, DIC image of the cells shown at the top panel; Bottom, enlarged overlap image of GFP signal and DIC. Average GFP intensity from cells expressing gfp-wag31T73A Mtb or gfp- wag31T73E Mtb relative to those of cells expressing wild-type gfp-wag31 is shown at the bottom. p-values for the difference in GFP signals (one-tailed, unpaired t-tests): wild-type Wag31Mtb vs. Wag31T73EMtb = 1.2 × 10-14, significant, and wild-type Wag31Mtb vs. Wag31T73AMtb = 1.2 × 10-36, significant (significant to p < 0.05). bar, 5 μm. B. Western blot analysis to examine the total Wag31 levels (GFP-Wag31 from Pacet and non-tagged Wag31 from Ptet) relative to those of SigAMsm. Total protein was purified from each strain at the same time cells were examine for fluorescence, and 20 μg of total protein was used for Western blot analysis with the anti-Wag31 mAb, stripped of the antibody, and subsequently for another Western blot with a monoclonal antibody against the Sig70 of E. coli RNA polymerase (Abcam). The ratio of total Wag31/SigA signal intensity from cells expressing wild-type gfp-wag31 was set as 1. Data shown are from a representative experiment done in duplicate.

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