Background: Mycobacterium tuberculosis PknB plays a critical role in modulating cell division and cell wall synthesis. Results: We present a comprehensive evaluation of the importance of various domains of PknB in modulating cell survival. Conclusion:The intracellular kinase domain and extracytoplasmic PASTA domains of PknB are essential for cell survival. Significance: PknB is essential for both in vitro growth and survival of the pathogen in vivo.

InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.The characteristic nature of the cell envelope of Mycobacterium tuberculosis is linked to its pathogenicity. The thick layer of lipids on the outer surface of mycobacteria is protective in nature, and mycolic acids comprise the bulk of this layer. Mycolic acids also constitute structural components of the cell wall and envelope (1, 2). Mycobacteria are unique in having two fatty-acid synthase (FAS) 5 systems, FAS-I and FAS-II, and both of these pathways are involved in the synthesis of mycolic acids. Eukaryotic-like FAS-I is a single multidomain enzyme, whereas FAS-II includes discrete monofunctional enzymes that carry out the various sequential steps involved in the process of synthesis (3). The FAS-I system is responsible for the de novo fatty acid biosynthesis (4), producing C20-and C26-S-enzyme derivatives that are converted to the respective coenzyme A (CoA) forms and then released. The C20 fatty acid is the probable starting point for the FAS-II system (2), and the C26 fatty acid is incorporated as the ␣-branch of mycolic acid (5).FabH (␤-ketoacyl-ACP synthase III) forms the link between the two FAS systems by condensing the long chain acyl-CoA products (produced by FAS-I) and malonyl-ACP (produced from malonyl-CoA by the action of FabD (malonyl-CoA:ACP transacylase) (6)). The ␤-ketoacyl-S-ACP product (7) thus formed is subjected to keto-reduction, dehydration, and en...

Background: Protein kinase A has been shown to be involved in modulating critical functions. Results: Although the activity of PknA is crucial for cell growth, the extracellular domain is expendable. Conclusion: Although the activation of PknA is necessary for its function, this is independent of PknB. Significance: PknA plays an indispensable role and is required for both in vitro and in vivo growth.

Segregation of replicated chromosomes in bacteria is poorly understood outside some prominent model strains and even less is known about how it is coordinated with other cellular processes. Here we report that RocS is crucial for chromosome segregation in the opportunistic human pathogen Streptococcus pneumoniae. RocS is membrane-bound and interacts both with DNA and the chromosome partitioning protein ParB to properly segregate the origin of replication region to new daughter cells. In addition, we show that RocS interacts with the tyrosineautokinase CpsD required for polysaccharide capsule biogenesis, which is crucial for S. pneumoniae's ability to prevent host immune detection. Altering the RocS-CpsD interaction drastically hinders chromosome partitioning and cell division. Altogether, this work reveals that RocS is the cornerstone of an atypical nucleoid occlusion system ensuring proper cell division in coordination with the biogenesis of a protective capsular layer..

No abstract