subtilis, the PrkC kinase,
a homolog of PknBMtb with three ARN-509 manufacturer PASTA domains, induces germination in response to muropeptide fragments released by surrounding growing bacteria . In stationary phase, however, Wag31 remains non- or lowly-phosphorylated but can still be recruited to the cell poles and lead to polar peptidoglycan synthesis. This idea is consistent with our observation that the phosphoablative Wag31T73A does localize at the cell poles (Figure 3A), and that wild-type GFP-Wag31 shows clear localization and peptidoglycan biosynthesis at cell poles at late stationary phase, albeit lower than in exponential phase (data not shown). This model is also consistent with previous reports that a fairly high capacity for peptidoglycan biosynthesis is maintained in slow-growing and stationary phase bacterial cells . Either way, Wag31 itself is essential for mycobacterial survival as we observed in our previous report  because Wag31 must be present and localized to the cell poles for polar peptidoglycan synthesis. Conclusions This study demonstrated that Wag31Mtb phosphorylation, which is unique among DivIVA homologues, regulates polar peptidoglycan biosynthesis
and optimal growth of mycobacterial cells through modulating the localization of Wag31 and the activity of peptidoglycan biosynthetic enzymes. Methods Bacterial growth condition, media and strains LGK-974 M. smegmatis mc2155 cultures were grown
at 37°C in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% ADC (5% (W/V) BSA fraction V, 2% (W/V) glucose and 0.85% (W/V) NaCl) and 0.05% (W/V) Tween-80, or on Middlebrook 7H9-ADC agar plates. Kanamycin (50 μg ml-1), hygromycin (50 μg ml-1) or apramycin (50 μg ml-1) was added to culture media as indicated. E. coli TOP10 strain (Life Technologies) was used as host strain for cloning experiments, and was grown in LB broth or solid medium with kanamycin (20 μg ml-1). Plasmid construction All plasmid constructs and primers are shown in Additional file 1 and 4 (Table A1 and A2). For localization of different forms of Wag31Mtb, wild-type gfp-wag31 Mtb , gfp-wag31T73A Mtb or gfp-wag31T73E Mtb was cloned under the acetamide-inducible promoter (Pacet) in a replicating plasmid Adenosine pMV261 (Kmr) to make pCK174, pCK175, and pCK176, respectively. The gfp gene was amplified from pTracerCMV plasmid (Invitrogen) using Ngfp-wag-1 and Ngfp-wag-2 primers. Genes for Wag31Mtb, Wag31T73AMtb and Wag31T73EMtb were amplified from plasmids pCK89, pCK90, and pCK91 using Ngfp-TBwag-3 and Ngfp-TBwag-4 primers. Second overlap PCR to fuse gfp and each wag31 Mtb gene was conducted by using Ngfp-wag-1 and Ngfp-TBwag-4 primers. To test localization of Wag31 in the presence of pknA Mtb – or pknB Mtb -overexpression in M.