The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes including two, pknA and pknB, that are part of an operon encoding genes involved in cell shape control and cell wall synthesis. Here we demonstrate that pknA and pknB are predominantly expressed during exponential growth, and that overexpression of these kinases slows growth and alters cell morphology. We determined the preferred substrate motifs of PknA and PknB, and identified three in vivo substrates of these kinases: PknB; Wag31, an ortholog of the cell division protein DivIVA; and Rv1422, a conserved protein of unknown function. Expression of different alleles of wag31 in vivo alters cell shape, in a manner dependent on the phosphoacceptor residue in the protein produced. Partial depletion of pknA or pknB results in narrow, elongated cells. These data indicate that signal transduction mediated by these kinases is a novel mechanism for the regulation of cell shape in mycobacteria, one that may be conserved among gram-positive bacteria.[Keywords: Kinase; DivIVA; peptide library screen; mycobacteria] Supplemental material is available at http://www.genesdev.org.

The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.signal transduction | phosphorylation motif | phosphoproteomics A key feature of all living cells is the ability to sense environmental signals and implement adaptive changes. These inputs propagate through complex signal transduction networks whose activity is often regulated by reversible protein phosphorylation. Although Ser/Thr/Tyr protein phosphorylation-based signaling in eukaryotes has been intensively studied, the extent to which this mechanism is used in prokaryotes has only recently begun to be appreciated (1). The number of protein kinases in prokaryotes varies widely. Although many bacteria have only a few or none of these enzymes, some cyanobacteria and streptomycetes have dozens of them (2). Bacteria that do possess Ser/Thr or Tyr kinases often have complex lifestyles and depend on these kinases to regulate critical processes, such as stress adaptation, development, and virulence (2).Mycobacterium tuberculosis is an extraordinarily versatile pathogen that can exist in distinct states in the host, leading to asymptomatic latent tuberculosis (TB) infection in which bacteria are thought to be dormant, or active TB disease in which the organisms are actively replicating. To achieve these different physiologic states M. tuberculosis requires mechanisms to sense a wide range of signals from the host and to coordinately regulate multiple cellular processes. In most bacterial pathogens, the predominant phosphorylation-based signal transduction mechanism is the two-component system. The M. tuberculosis genome, however, encodes 11 Ser/Thr protein kinases (STPKs) and an equal number of two-component system sensor kinases, suggesting that these two phospho-based signaling systems are of comparable importance in this organism (3).Knowledge of the substrates of each of the M. tuberculosis STPKs is essential for understanding their function; however, only a small number of kinase-sub...

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in B:acillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.

The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes, and the products of two of these, PknA and PknB, are key components of a signal transduction pathway that regulates cell division and/or morphology. Previously, we have shown that one substrate of these kinases is Wag31, a homologue of the cell division protein DivIVA that is present, but not known to be phosphorylated, in other Gram-positive bacteria. Here, we investigate the localization and function of Wag31 and its phosphorylation. We demonstrate that Wag31 is localized to the cell poles. We further show that wag31 is an essential gene and that depletion of its product causes a dramatic morphological change in which one end of the cell becomes round rather than rod-shaped. This abnormal morphology appears to be caused by a defect in polar peptidoglycan synthesis. Finally, expression of M. tuberculosis wag31 in the wag31 conditional mutant of Mycobacterium smegmatis altered the growth rate in a manner that depended on the phospho-acceptor residue encoded by the allele being expressed. Taken together, these results indicate that Wag31 regulates cell shape and cell wall synthesis in M. tuberculosis through a molecular mechanism by which the activity of Wag31 can be modulated in response to environmental signals.

An incorrect version of Fig. 4 was published. The correct version is shown below.

BackgroundSensing and responding to environmental changes is a central aspect of cell division regulation. Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis.ResultsWe demonstrate that the differential growth among cells expressing different wag31 alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway.ConclusionsThe Wag31Mtb phosphorylation is a novel molecular mechanism by which Wag31Mtb regulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.

S-Adenosyl-L-methionine synthetase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. Despite extensive research with many organisms, its role in Streptomyces sp. remains unclear. In the present study, the putative SAM-s gene was isolated from a spectinomycin producer, Streptomyces spectabilis. The purified protein from the transformed Escherichia coli with the isolated gene synthesized SAM from L-methionine and ATP in vitro, strongly indicating that the isolated gene indeed encoded the SAM-s protein.The overexpression of the SAM-s gene in Streptomyces lividans TK23 inhibited sporulation and aerial mycelium formation but enhanced the production of actinorhodin in both agar plates and liquid media. Surprisingly, the overexpressed SAM was proven by Northern analysis to increase the production of actinorhodin through the induction of actII-ORF4, a transcription activator of actinorhodin biosynthetic gene clusters. In addition, we found that a certain level of intracellular SAM is critical for the induction of antibiotic biosynthetic genes, since the control strain harboring only the plasmid DNA did not show any induction of actII-ORF4 until it reached a certain level of SAM in the cell. From these results, we concluded that the SAM plays important roles as an intracellular factor in both cellular differentiation and antibiotic production in Streptomyces sp.

SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in Mycobacterium tuberculosis, and this sigma factor is required for virulence in animal models of infection. SigH is negatively regulated by RshA, its cognate anti-sigma factor, which functions as a stress sensor and redox switch. While RshA provides a direct mechanism for sensing stress and activating transcription, bacteria use several types of signal transduction systems to sense the external environment. M. tuberculosis encodes several serine-threonine protein kinase signaling molecules, 2 of which, PknA and PknB, are essential and have been shown to regulate cell morphology and cell wall synthesis. In this work, we demonstrate that SigH and RshA are phosphorylated in vitro and in vivo by PknB. We show that phosphorylation of RshA, but not SigH, interferes with the interaction of these 2 proteins in vitro. Consistent with this finding, negative regulation of SigH activity by RshA in vivo is partially relieved in strains in which pknB is over-expressed, resulting in increased resistance to oxidative stress. These findings demonstrate an interaction between the signaling pathways mediated by PknB and the stress response regulon controlled by SigH. The intersection of these apparently discrete regulatory systems provides a mechanism by which limited activation of the SigH-dependent stress response in M. tuberculosis can be achieved. Coordination of the PknB and SigH regulatory pathways through phosphorylation of RshA may lead to adaptive responses that are important in the pathogenesis of M. tuberculosis infection.anti-sigma factor ͉ sigma factor ͉ transcription regulation ͉ phosphorylation S igH, an alternative sigma factor of Mycobacterium tuberculosis and other mycobacterial species, is a central regulator of the response to oxidative, nitrosative, and heat stresses. SigH directly regulates both effectors of the response to these stresses and additional transcription regulators that control expression of a broad range of stress response genes (1-4). The SigHdependent activation of this extensive stress response regulon is critical for M. tuberculosis virulence, as a sigH mutant is highly attenuated in the mouse model of infection (5).SigH activity is regulated at the transcriptional level via autoregulation of the sigH promoter, and posttranslationally via interaction with its cognate anti-sigma factor, RshA. This protein is a member of the Zinc-associated anti-sigma (ZAS) family, several members of which, including RshA, have been shown to function as redox switch proteins (3, 6-8). The interaction of RshA with SigH is disrupted under oxidizing conditions, allowing SigH to associate with core RNA polymerase and activate transcription of stress response genes and additional transcription regulators. The autoregulation of the sigH promoter results in rapid, strong induction of the SigH regulon following oxidative stress, which is maintained until redox homeostasis is reestablished and th...