1. Top of page
  2. Summary
  3. Role of the STPK-dependent phosphorylation in mycobacterial cell wall metabolism
  4. Role of the Ser/Thr-mediated phosphorylation in mycobacterial cell division
  5. A common theme to actinomycetes?
  6. Concluding remarks and perspectives
  7. Acknowledgements
  8. References

Mycobacterium tuberculosis (M. tb) has a complex lifestyle in different environments and involving several developmental stages. The success of M. tb results from its remarkable capacity to survive within the infected host, where it can persist in a non-replicating state for several decades. The survival strategies developed by M. tb are linked to the presence of an unusual cell envelope. However, little is known regarding its capacity to modulate and adapt production of cell wall components in response to environmental conditions or to changes in cell shape and cell division. Signal sensing leading to cellular responses must be tightly regulated to allow survival under variable conditions. Although prokaryotes generally control their signal transduction processes through two-component systems, signalling through Ser/Thr phosphorylation has recently emerged as a critical regulatory mechanism in bacteria. The genome of M. tb possesses a large family of eukaryotic-like Ser/Thr protein kinases (STPKs). The physiological roles of several mycobacterial STPK substrates are connected to cell shape/division and cell envelope biosynthesis. Although these regulatory mechanisms have mostly been studied in Mycobacterium, Ser/Thr phosphorylation appears also to regulate cell division and peptidoglycan synthesis in Corynebacterium and Streptomyces. This review focuses on the proteins which have been identified as STPK substrates and involved in the synthesis of major cell envelope components and cell shape/division in actinomycetes. It is also intended to describe how phosphorylation affects the activity of peptidoglycan biosynthetic enzymes or cell division proteins.

In response to its environment, Mycobacterium tuberculosis (M. tb) activates or represses the expression of a number of genes in order to adjust promptly to new conditions (Schnappinger et al., 2003). Protein phosphorylation/dephosphorylation represents a central mechanism for transduction of specific signals to various cellular processes, such as regulation of growth, differentiation, mobility and survival (Stock et al., 1989). Many of these stimuli are transduced via sensor kinases present on the mycobacterial membrane, enabling the pathogen to adapt its cellular response to survive in hostile environments.

Protein kinases are classified into two families based on their similarities and enzymatic specifications: (i) the histidine kinase superfamily belonging to the two-component systems, which autophosphorylates a conserved histidine residue (Stock et al., 1989), and (ii) the serine, threonine and tyrosine kinase superfamily that phosphorylates serine, threonine and tyrosine residues, respectively (Hanks et al., 1988). Although the two-component systems represent the classical prokaryotic mechanism for detection and response to environmental changes, the serine/threonine and tyrosine protein kinases associated with their phosphatases have recently emerged as important regulatory systems (Av-Gay and Everett, 2000; Greenstein et al., 2005; Wehenkel et al., 2008).

Mycobacterium tuberculosis contains 11 Ser/Thr protein kinases (STPKs) (Cole et al., 1998; Av-Gay and Everett, 2000) and their physiological roles are being investigated because they are potential targets for antitubercular drug development (Wehenkel et al., 2008). Through phosphorylation, these STPKs influence a wide range of biological functions, such as adaptation to various environmental conditions, stress, cell wall synthesis, cell division and pathogenicity (Narayan et al., 2007). The cell wall of M. tb plays a critical role in combating host defences, and changes in cell wall composition in response to various environmental stimuli are critical to M. tb adaptation during infection (Daffe and Draper, 1998). Although little is known about the cell wall regulatory mechanisms in M. tb, there is now an increasing body of evidence indicating that control of cell wall synthesis and cell division relies largely on STPK-dependent mechanisms.

The mycobacterial cell wall core consists of three interconnected macromolecules: peptidoglycan, arabinogalactan (AG) and mycolic acids. Mycolic acids are the outermost components of the cell wall. They are long chain α-alkyl-β-hydroxy fatty acids that are unique to mycobacteria and related taxa and which are believed to play a crucial role in the architecture of the mycobacterial envelope and in mycobacterial virulence (Brennan and Nikaido, 1995; Daffe and Draper, 1998). The mycolic acids can be linked via esterification to the heteropolysaccharide, AG, which is composed primarily of d-arabinofuranosyl and d-galactofuranosyl residues. This entire polymer (mAG) is connected via a unique linker disaccharide phosphate to a muramic acid residue of peptidoglycan. AG plays a crucial role in the assembly of the mycobacterial cell wall.

This review article is not dedicated to the description of mycobacterial kinases/phosphatases, which has been the subject of several excellent reviews (Av-Gay and Everett, 2000; Greenstein et al., 2005; Wehenkel et al., 2008). It rather addresses the recent advances made in the field of Ser/Thr phosphorylation through the description and role of STPK substrates in relation to cell division, as well as biosynthesis and metabolism of major cell wall-associated components, including arabinan, mycolic acids and peptidoglycan. Moreover, recent data shedding light on the phosphorylation mechanisms involved in the activity and/or regulation of the target proteins are also discussed.

Role of the STPK-dependent phosphorylation in mycobacterial cell wall metabolism

  1. Top of page
  2. Summary
  3. Role of the STPK-dependent phosphorylation in mycobacterial cell wall metabolism
  4. Role of the Ser/Thr-mediated phosphorylation in mycobacterial cell division
  5. A common theme to actinomycetes?
  6. Concluding remarks and perspectives
  7. Acknowledgements
  8. References

Phosphorylation and arabinan synthesis

EmbR belongs to the Streptomyces coelicolor antibiotic regulatory protein (SARP) family (Wietzorrek and Bibb, 1997) that is known to regulate genes involved in secondary metabolite synthesis. Early studies proposed that EmbR influences the expression of the Mycobacterium avium embAB operon (Belanger et al., 1996). Membranes from Mycobacterium smegmatis carrying the M. avium embAB and embR genes and treated with ethambutol, a front-line drug known to target EmbAB, retain significantly more arabinosyltransferase activity than membranes originating from similarly treated M. smegmatis carrying only the embAB cluster (Belanger et al., 1996). The M. avium embR gene is located immediately upstream of embAB, while the embR gene of M. tb is separated from the embCAB gene cluster (Telenti et al., 1997; Cole et al., 1998). Most of the work characterizing the role and the expression of Emb proteins has been carried out in M. smegmatis, where the role of each protein has been determined. Biochemical analyses of the deletion mutants demonstrated that EmbABC proteins are all arabinosyltransferases, but with segregated biological functions. Whereas EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif in the cell wall polysaccharide arabinogalactan (Escuyer et al., 2001), EmbC is involved in the biosynthesis of the arabinan portion of lipoarabinomannan (LAM) (Zhang et al., 2003). That Ser/Thr phosphorylation participates in arabinan synthesis is supported by the original demonstration that EmbR (Rv1267c) is phosphorylated by the STPK PknH (Rv1268c) in vitro, and that interaction between the two partners is mediated by the EmbR C-terminal FHA (ForkHead Associated) domain (Molle et al., 2003a). FHA domains are small protein modules capable of mediating protein–protein interactions through phosphothreonine (pThr) recognition, and they often participate in protein–protein interactions in STPK-dependent signal transduction pathways (Durocher et al., 2000a,b). Truncation of the EmbR FHA domain or introduction of single amino acid substitutions of conserved residues in the phosphothreonine-binding pocket (Arg312, Ser326 and Asn348) dramatically diminished PknH-mediated threonine phosphorylation of EmbR (Molle et al., 2003a). The three-dimensional structure revealed that, in addition to the FHA domain, EmbR possesses two major domains, an N-terminal winged helix–turn–helix DNA-binding domain followed by a central bacterial transcription activation domain, suggesting that it might act as a transcriptional regulator in M. tb (Alderwick et al., 2006). This is supported by the fact that phosphorylation of EmbR enhances its ability to bind to the promoter regions of the embCAB genes (Sharma et al., 2006a), and that deletion of PknH in M. tb results in decreased transcription of embB and embC in cultures treated with sublethal concentrations of ethambutol (Papavinasasundaram et al., 2005).

Since activation of EmbR upon phosphorylation by PknH induces transcription of the embCAB operon, and because the embCAB gene products participate in the biosynthesis of arabinan (Belanger et al., 1996; Telenti et al., 1997; Escuyer et al., 2001; Zhang et al., 2003), this signalling cascade may control the LAM/lipomannan (LM) ratio (Fig. 1). LAM is an important determinant of virulence because it suppresses the host immune system, whereas LM represents a potent proinflammatory-inducing factor (Briken et al., 2004). Therefore, by increasing the LAM/LM balance, the PknH/EmbR couple is likely to play an important role in immunopathogenesis. EmbR is also phosphorylated by other STPKs, including PknA and PknB and is dephosphorylated by the Ser/Thr phosphatase PstP, indicating that several signalling pathways might be involved in generating a global response by integrating diverse signals (Sharma et al., 2006b). Moreover, the PknH-mediated increase in embAB transcription also alters resistance to ethambutol (Sharma et al., 2004; Sharma et al., 2006a), and PknH is upregulated in vivo inside host macrophages (Sharma et al., 2004). Therefore, these observations suggest a functionally relevant signalling mechanism via phosphorylation involving PknH/EmbR/EmbCAB in the modulation of arabinan synthesis.


Figure 1. Role and implication of Ser/Thr protein kinases in mycobacterial cell wall metabolism. STPKs are autophosphorylated in response to environmental signals, and subsequently phosphorylate their target proteins that, in turn, modulate their biological activities/functions. Among the targets identified, only those participating in the cell wall metabolism are depicted. Ser/Thr phosphorylation can directly affect the targets, such as the condensing enzymes of the FAS-II system (mtFabH, KasA, KasB and Pks13) or indirectly via phosphorylation of transcriptional factors (VirS and EmbR). In the case of virS/mymA, phosphorylation of VirS affects transcriptional regulation of the mymA operon, whose gene products can also be directly phosphorylated by PknK. It is noteworthy that phosphorylation of kinases and substrates is reversible and the Ser/Thr phosphatase (PstP) can dephosphorylate both kinases and the targets substrates in response to environmental cues, thus adding a further level of regulation.

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Phosphorylation of the mycolic acid biosynthetic machinery

Mycolic acids are a homologous series of C60–C90α-alkyl-β-hydroxy fatty acids produced by all mycobacteria (Kremer et al., 2000). Similar but shorter-length mycolic acids are found in related genera like Corynebacterium and Nocardia. Mycolic acids are primarily found as esters of the non-reducing arabinan terminus of arabinogalactan (Brennan and Nikaido, 1995) but they are also present as extractable free lipids within the cell wall, mainly associated with trehalose to form trehalose dimycolate (TDM) or ‘cord factor’. TDM is the most abundant granulomagenic and toxic lipid extractable from the cell surface of virulent M. tb (Hunter et al., 2006). Mycolic acids confer important properties, including resistance to chemical injury and dehydration, low permeability to hydrophobic antibiotics, virulence (Dubnau et al., 2000; Glickman et al., 2000; Glickman and Jacobs, 2001), acid-fast staining (Bhatt et al., 2007a), biofilm formation (Ojha et al., 2008), and persistence within the host (Daffe and Draper, 1998; Yuan et al., 1998; Glickman et al., 2000; Bhatt et al., 2007a). In addition, the enzymes involved in mycolate biosynthesis are essential for mycobacterial survival and, thus, represent excellent drug targets.

Mycolic acids biosynthesis involves two types of fatty acid-synthesizing systems, the type I and type II fatty acid synthases FAS-I and FAS-II, respectively (Fig. 1). The eukaryotic-like FAS-I system catalyses the de novo synthesis of fatty acids from acetyl-CoA. In contrast, FAS-II is similar to systems found in bacteria, apicomplexa parasites and plants, and is composed of several enzymes that act successively and repetitively to elongate the growing acyl chain. Acyl-primers are continually activated via a thioester linkage to the prosthetic group of coenzyme A (CoA) for FAS-I, or of an acyl carrier protein (ACP) for FAS-II. The biosynthesis involves five distinct stages (Takayama et al., 2005): (i) the synthesis of the C26 saturated straight chain fatty acids by FAS-I to provide the α-alkyl branch of the mycolic acids, (ii) the synthesis of the meromycolic acid by FAS-II, (iii) the introduction of functional groups to the meromycolate chain by numerous cyclopropane synthases, (iv) the condensation reaction catalysed by the polyketide synthase Pks13 between the α-branch and the meromycolate chain before a final reduction to generate the mycolic acid, and (v) the transfer of mycolic acids to arabinogalactan and other acceptors such as trehalose.

Phosphorylation of the FAS-II condensing enzymes (mtFabH, KasA, KasB, Pks13).  The condensation reaction that leads to carbon–carbon bond formation during acyl-group elongation is an extremely important step that is catalysed by condensing enzymes (Heath and Rock, 2002). These enzymes can be of three different types, depending on the step in the elongating process in which they are involved. The initiation condensing enzyme mtFabH, which might be the pivotal link between FAS-I and FAS-II, uses acyl-CoA primers (Choi et al., 2000), whereas the elongating condensing enzymes KasA and KasB exclusively use acyl-ACP thioesters (Schaeffer et al., 2001; Kremer et al., 2002). The termination condensing enzyme Pks13 uses both a meromycolyl-AMP and an acyl-CoA as a substrate (Portevin et al., 2004). Although they all catalyse carbon–carbon bond formation, they are defined by differences in their active sites, chain-length specificity and sensitivity to inhibitors.

Molle et al. (2006) proposed a model for mycolic acid synthesis regulation based on the phosphorylation of the β-ketoacyl-ACP synthases KasA and KasB. Both enzymes are phosphorylated at high rates in vivo in Mycobacterium bovis BCG, mainly at two positions, and can be dephosphorylated by the mycobacterial Ser/Thr phosphatase PstP. Interestingly, phosphorylation of KasA and KasB differentially modulates their condensing activities (Molle et al., 2006). While phosphorylation reduces the condensation activity of KasA, it increases the activity of KasB in the presence of either malonyl-ACP or C16-ACP. The differential effect of phosphorylation of two highly similar enzymes sharing the same enzymatic activity, but with different substrate specificities, represents an unusual mechanism of regulation in which dampening KasA might allow time to produce immature mycolates. Conversely, boosting KasB activity might ensure that mycobacteria produce only full-length mycolates required for virulence and intracellular survival and virulence (Bhatt et al., 2007a,b). Thus, STPK-dependent phosphorylation might induce either positive or negative signalling to the different interconnected FAS-II complexes. Different expression profiles of the various mycobacterial STPKs in response to stress conditions might directly affect the phosphorylation profile of the condensing enzymes and, consequently, modulate mycolic acid biosynthesis in order to promote adaptation to environmental changes.

Future work is now required to address whether phosphorylation of KasA/B regulates the mycolic acid profile in vivo, and affects M. tb virulence in infected mice. This could be achieved by generating isogenic M. tb strains in which the phosphorylation sites in KasA/B are mutated either into Ala (to prevent phosphorylation) or into Asp (which mimics constitutive phosphorylation due to the introduction of a negative charge). This approach could greatly benefit from a newly developed genetic system that allows to introduce single point mutations in both fast- and slow-growing mycobacterial species (Vilcheze et al., 2006).

This view is supported by recent observations that, in addition to KasA and KasB, the two other condensases mtFabH and Pks13 are also substrates for mycobacterial STPKs (Fig. 1) (Veyron-churlet et al., 2009). The β-ketoacyl-ACP synthase III (mtFabH) links FAS-I and FAS-II, catalysing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP, thus representing a potential regulatory key point of the mycolic acid pathway. MtFabH is efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo (Veyron-churlet et al., 2009). Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues, and mass spectrometry analyses identified Thr45 as the unique phosphoacceptor (Veyron-churlet et al., 2009). Furthermore, PknF- or PknA-dependent phosphorylation was completely defective in a mtFabH T45A variant. Interestingly, Thr45 is located at the entrance of the substrate channel on the crystal structure of mtFabH, suggesting that the phosphate group alters substrate accessibility, thereby affecting mtFabH enzymatic activity. Importantly, a T45D variant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-ACP decarboxylation and condensing activities compared with the wild-type protein or the T45A variant. These findings represent the first demonstration of phosphorylation of a KASIII enzyme, and indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which might have important consequences in regulating mycolic acid biosynthesis.

The fact that most, if not all, FAS-II enzymes are phosphorylated is intriguing. Why would M. tb phosphorylate multiple enzymes that all belong to the same pathway? One explanation of the biological relevance of this specific post-translational regulation could be that altering the activity of most of these enzymes leads to a tightly regulated system that allows survival and/or adaptation under variable growth conditions. While phosphorylation of each individual FAS-II enzyme is accompanied by only a partial reduction of activity, the cumulative effects of partial reduced enzymatic activities could result in a complete cessation of mycolic acid production. Then, rather than acting on a single and defined target enzyme in the mycolic biosynthesis pathway, M. tb prefers to tackle multiple biosynthetically linked targets. This attractive hypothesis should be tested by addressing whether the remaining FAS-II components, notably the β-ketoacyl-ACP reductase MabA, as well as the enoyl-ACP reductase InhA, known as essential enzymes and potential regulatory checkpoints, are STPK substrates.

Phosphorylation of VirS by PknK controls the mymA operon.  The mymA operon (Rv3083–Rv3089) of M. tb might participate in the biosynthesis of mycolic acids, presumably via a FAS-II-independent pathway, and is important for maintaining cell wall integrity (Singh et al., 2005). This operon is located in reverse orientation to the virS gene, encoding a member of the AraC/XylS transcriptional regulator family (Singh et al., 2003). Transcription of the mymA operon depends on VirS (Singh et al., 2003). Disruption of the virS or mym genes alters cell wall structure and persistence in the spleen of infected guinea pigs (Singh et al., 2005). mymA operon transcription increases at acidic pH, indicating a role for the encoded proteins in survival of the pathogen in severely acidic conditions of activated macrophages (Singh et al., 2005). A recent study identified VirS as a substrate of PknK (Fig. 1) and, importantly, demonstrated that PknK-mediated phosphorylation of VirS increases its affinity for the mym promoter DNA (Kumar et al., 2009). In addition to VirS, the authors also demonstrated that several mymA operon-encoded proteins (MymA, LipR, Rv3085 and Rv3088) are direct targets of PknK, suggesting their activities are modulated by phosphorylation. This study illustrates the multi-layered complexity of Ser/Thr phosphorylation: kinases can phosphorylate both the regulator and the regulator targets in the same pathway.

In two-component systems, the phosphorylated response regulator interacts with promoter regions, which in turn up- or downregulates the target genes. Although the two-component systems and the STPKs families do not share sequence similarity, STPKs might somehow fulfil the role of typical bacterial two-component systems. As detailed above, PknK phosphorylates the transcriptional regulator VirS, which in turns regulates expression of the mycobacterial monooxygnease (mymA) operon and probably also other genes. Moreover, the PknH/EmbR pair also presents functional analogy with two-component systems in that PknH can be compared with the sensor kinase, and EmbR to the transcriptional factor that is regulated by phosphorylation. In conclusion, these examples highlight the functional analogy between M. tb STPKs that regulate the activity of DNA-binding proteins and classical two-component couples, and might explain why this organism possesses a larger number of STPKs and fewer two-component systems than other bacteria (Cole et al., 1998; Av-Gay and Everett, 2000).

Phosphorylation of cell wall glycolipids transporters

Through the use of a M. tb mutant lacking the PknD kinase domain, Perez et al. (2006) identified MmpL7 as an endogenous phosphorylated substrate. MmpL7 and the other members of the MmpL family (Domenech et al., 2005) belong to the RND (resistance, nodulation and cell division) family of transporters. MmpL7 was shown to be essential for virulence (Camacho et al., 2001), presumably because its transports phthiocerol dimycocerosate (PDIM) (Cox et al., 1999) and a related but distinct phenolic glycolipid (Reed et al., 2004). These findings suggest that phosphorylation of MmpL7 could regulate the deposition of cell wall-associated glycolipids, or at least the transport of components to the cell membrane (Fig. 1). In another study, mutants in any of the six members of the family had compromised virulence (MmpL7, MmpL4, MmpL11, MmpL8, MmpL5 and MmpL10) (Lamichhane et al., 2005) and MmpL8 transports sulpholipids, which are other important cell wall-associated virulence factors (Converse et al., 2003). It is therefore tempting to speculate that phosphorylation plays a critical role in the cell wall composition by affecting the transport of several of the MmpL proteins, a hypothesis that awaits to be addressed experimentally.

Role of the Ser/Thr-mediated phosphorylation in mycobacterial cell division

  1. Top of page
  2. Summary
  3. Role of the STPK-dependent phosphorylation in mycobacterial cell wall metabolism
  4. Role of the Ser/Thr-mediated phosphorylation in mycobacterial cell division
  5. A common theme to actinomycetes?
  6. Concluding remarks and perspectives
  7. Acknowledgements
  8. References

Ser/Thr kinase substrates involved in peptidoglycan synthesis and cell division

PknA and PknB are encoded by an operon involved in cell shape and cell wall synthesis. The markedly higher expression of the operon during exponential growth relative to stationary phase suggests that the regulatory function of these essential kinases is required during active cell replication. This interpretation is in agreement with microarray data indicating that both pknA and pknB are downregulated in response to nutrient starvation (Betts et al., 2002). Overexpression of PknA and PknB alters cell growth and morphology (Kang et al., 2005). In addition, partial depletion of PknA or PknB resulted in elongated cells, suggesting a critical role of these two kinases in regulating cell shape in mycobacteria (Kang et al., 2005). These authors also found that PknA and PknB phosphorylate several proteins including Wag31 (Rv2145c) (Kang et al., 2005), a homologue of the cell shape/cell division protein DivIVA that is essential for mycobacterial growth (Nguyen et al., 2007) (Fig. 2). DivIVA mediates polar cell wall synthesis in Streptomyces, Corynebacterium and Mycobacterium (Nguyen et al., 2007; Kang et al., 2008), and is essential for elongation of Corynebacterium glutamicum where it is recruited to the septum only after cell division-related peptidoglycan synthesis has started (Letek et al., 2008). Interestingly, changing the PknA target phosphorylation site on Wag31 alters cell morphology, implicating Wag31 phosphorylation in maintaining cell shape (Kang et al., 2005). Alteration of Wag31 activity or localization resulting from phosphorylation by PknA could account, at least partly, for the phenotypes observed in the mycobacterial kinase overexpression and depletion strains. The role of PknA in controlling cell division was also confirmed in studies demonstrating direct interaction between PknA and M. tb FstZ, a protein central to bacterial septum formation. In addition, phosphorylation of FtsZ by PknA impairs its GTP-dependent polymerization ability and, thereby, reduces septum formation (Thakur and Chakraborti, 2006).


Figure 2. Role and implication of Ser/Thr protein kinases in mycobacterial peptidoglycan synthesis, cell shape and cell division. STPKs are autophosphorylated in response to external stimuli and subsequently phosphorylate their target proteins. For instance, PknL phosphorylates Rv2175c, a transcriptional regulator possibly involved in modulating expression of regulons including the dcw cluster that participate in peptidoglycan synthesis, cell shape and cell division. Genes belonging to the dcw cluster are likely to be regulated by the PknL/Rv2175c pair at a transcriptional level and, in addition, their translational products are PknA kinase substrates. Therefore, by analogy with the virS/mymA system, it appears that cell wall/cell division-associated regulons are regulated by Ser/Thr phosphorylation at both transcriptional and post-translational levels. +/− indicates that the effect of phosphorylation (inhibition or activation) remains to be established experimentally.

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Alteration in cell shape is often correlated with changes in peptidoglycan biosynthesis (Nanninga, 1991). This prompted investigators to consider peptidoglycan biosynthetic enzymes as potential cellular substrates of STPKs in mycobacteria. A recent study demonstrated that PknA interacts in vitro with the mycobacterial UDP-N-acetylmuramoyl-l-alanine:d-glutamate-ligase (MurD), a cytoplasmic enzyme that catalyses the addition of d-glutamate to the UDP-N-acetylmuramoyl-l-alanine precursor during peptidoglycan biosynthesis (Thakur and Chakraborti, 2008), thus establishing a link between STPK-dependent phosphorylation and peptidoglycan biosynthesis (Fig. 2). However, the mechanisms by which phosphorylation modulates the activity of MurD have not yet been investigated.

Interestingly, pknA, pknB and pstpP (encoding the ser/thr protein phosphatase) are in an operon that also includes rodA and pbpA, two genes encoding proteins involved in peptidoglycan biosynthesis, as well as Rv0019c and Rv0020c of unknown function (Fig. 4) (Cole et al., 1998; Boitel et al., 2003). Penicillin-binding proteins (PBPs) synthesize cross-linked peptidoglycan during cell elongation and cell division and participate in cell wall expansion, cell shape maintenance, as well as in septum formation and cell division. The mycobacterial PBP, PbpA (Rv0016c), is phosphorylated by PknB and dephosphorylated by PstP (Dasgupta et al., 2006). Therefore, PknA, PknB and PstP might control the regulation of cell elongation of cell division proteins. Furthermore, the FHA-containing protein Rv0020c is phosphorylated by several STPKs (Grundner et al., 2005), although its function and role in peptidoglycan synthesis and/or cell division is unknown. PknB also phosphorylates Rv0019c in a reaction that is dependent on conserved residues in the Rv0019c FHA domain as well as the activation loop of PknB (Gupta et al., 2009). Interestingly, the M. tb PapA5 protein, involved in PDIM biosynthesis, interacts with Rv0019c and is itself phosphorylated on threonine residue(s) by PknB and completely dephosphorylated by Ser/Thr phosphatase PstP. These data provide the first indication that PknB might regulate PapA5 activity in PDIM biosynthesis, either by the direct phosphorylation of PapA5 or indirectly through Rv0019c.


Figure 4. Conservation of the ‘pknB’ gene cluster in actinomycetes. This putative operon comprises important signal transduction components such as the STPKs pknA and pknB, the Ser/Thr phosphatase pstP and proteins bearing a FHA domain (Rv0019c and Rv0020c) and is close to several genes participating in peptidoglycan biosynthesis (rodA or pbpA). One notable exception is the absence of a pknA orthologue in Streptomyces.

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Moreover, the N-acetylglucosamine-1-phosphate uridyltransferase GlmU of M. tb has recently been added to the list of the PknB substrates participating in peptidoglycan biosynthesis (Fig. 2) (Parikh et al., 2009). GlmU, which catalyses an important step in peptidoglycan synthesis, carries out two distinct activities. The C-terminal domain transfers an acetyl group from acetyl-CoA to glucosamine-1-phosphate, which is converted into UDP-N-acetylglucosamine by transfer of UMP, a reaction catalysed by the N-terminal domain. GlmU is phosphorylated on threonine residues located in the C-terminal domain and, importantly, the phosphorylated isoform exhibits decreased acyltransferase activity (Parikh et al., 2009). Although glmU is not clustered with other peptidoglycan-biosynthetic genes, these results imply that STPK-dependent phosphorylation of GlmU regulates peptidoglycan synthesis by modulating its acetyltransferase activity.

The link between STPK-dependent phosphorylation and peptidoglycan biosynthesis in mycobacteria is further supported by the fact that DacB1, a probable PBP, is also phosphorylated by PknH in M. tb (Zheng et al., 2007). Bacillus subtilis DacB1 is a sporulation-specific protein, leading to speculation that phosphorylation of DacB1 by PknH regulates a specific step in the synthesis of peptidoglycan and cell envelope (Fig. 2).

Potential role of the DNA-binding protein Rv2175c in controlling cell division

The gene encoding PknL has recently received close attention because of its close proximity to the ∼30 kb dcw (division cell wall) gene cluster, which includes several genes involved in cell wall synthesis and cell division (Narayan et al., 2007; Canova et al., 2008) (Fig. 2), leading to the hypothesis that this kinase may participate in cell division regulation. These studies also identified a novel substrate/kinase pair, PknL/Rv2175c. Indeed, pknL (Rv2176) is adjacent to Rv2175c, a gene encoding a putative DNA-binding transcriptional regulator. PknL recruits and phosphorylates Rv2175c in a reaction dependent on a specific phosphorylated residue in the PknL activation loop. Together, these observations suggest that Rv2175c/PknL are part of the signal transduction cascade controlling cell wall synthesis and cell division (Canova et al., 2008).

However, although Rv2175c has a DNA-binding domain, it shares only weak similarity to transcriptional regulatory proteins. Further indications of the function of Rv2175c were recently provided by its soluble NMR structure (Cohen-gonsaud et al., 2009). The three-dimensional structure unambiguously confirmed that Rv2175c is a DNA-binding protein possessing a winged helix–turn–helix domain. Gel shift mobility assays also confirmed that Rv2175c binds DNA (Cohen-gonsaud et al., 2009), and mass spectrometry analyses identified Thr9 as the unique phosphoacceptor, a feature supported by the complete loss of PknL-dependent phosphorylation of an Rv2175c_T9A mutant. The DNA-binding activity was completely abrogated in a Rv2175c_T9D derivative, which behaves as constitutively phosphorylated, but not in a variant lacking the first 13 residues. Importantly, Rv2175c phosphorylated in vitro by PknL also failed to bind to DNA, thus confirming the critical role of Thr9 phosphorylation to prevent DNA binding (Cohen-gonsaud et al., 2009). Interestingly, the M. smegmatis homologue of Rv2175c, which lacks the first 12 amino acids, is not phosphorylated by PknL. Therefore, the 12-amino-acid extension of M. tb Rv2175c probably represents a flexible region that carries/presents the unique phosphorylation site (Thr9) to the kinase. Since this N-terminal region is restricted to members of the M. tb complex, we propose that Rv2175c regulation by phosphorylation is unique to the highly pathogenic species.

Interestingly, wag31 (Rv2145c), a phosphorylated cell division protein, is in the 30 kb dcw gene cluster mentioned above, thus providing a possible link between cell division proteins and the PknL/Rv2175c pair (Fig. 2). Future work dedicated to examine the role of the phosphorylation on Rv2175c and identifying the regulons controlled by Rv2175c in response to environmental changes should unravel original and unexpected mechanisms of cell division in M. tb. Such studies could greatly benefit from the recent development and use of the Chip-to-chip technology (Molle et al., 2003b; Sala et al., 2009) that produces a direct in situ snapshot of the specific DNA regions that transcriptional regulators (such as Rv2175c, VirS or EmbR) bind in vivo under various environments. This should help to establish whether the sets of bound target genes uncovered under these different conditions vary, and how phosphorylation of the regulators influences the composition of these regulons. Use of these innovative and powerful technologies should further our understanding of the specific pathways that allow M. tb to adapt to environmental changes and the control of pathogenesis.

A common theme to actinomycetes?

  1. Top of page
  2. Summary
  3. Role of the STPK-dependent phosphorylation in mycobacterial cell wall metabolism
  4. Role of the Ser/Thr-mediated phosphorylation in mycobacterial cell division
  5. A common theme to actinomycetes?
  6. Concluding remarks and perspectives
  7. Acknowledgements
  8. References

Recent advances in understanding the role of Ser/Thr phosphorylation in the biosynthesis and metabolism of major cell wall-associated components, including arabinogalactan, peptidoglycan and mycolic acids and cell division in M. tb, have recently paved the way to investigate other actinomycetes such as C. glutamicum or S. coelicolor.

In contrast to closely related pathogenic bacteria like Corynebacterium diphtheriae, Mycobacterium leprae or M. tb, C. glutamicum is generally considered as a non-hazardous organism. Therefore, based on its well-studied central metabolism and well-established genetic tools, C. glutamicum is recognized as a model organism for high GC-rich Gram-positive organisms in general and mycolic acid-containing actinomycetes in particular (Portevin et al., 2004; Alderwick et al., 2005; Tropis et al., 2005). Ser/Thr/Tyr phosphorylation was revealed by analysis of the C. glutamicum phosphoproteome (Bendt et al., 2003). Most of the phosphorylated proteins identified are enzymes acting on central metabolic pathways such as glycolysis (enolase, fructose-1,6 bisphosphate aldolase, Gap dehydrogenase, pyruvate decarboxylase, pyruvate kinase), the tricarboxylic cycle and fatty-acid metabolism (acyl-CoA synthetase, acyl-CoA carboxylase, succinyl-CoA:CoA-transferase). Furthermore, chaperones (DnaK, GroEL2, DnaJ2), as well as components of the protein synthesis machinery (translation elongation factors and ribosomal proteins) were found to be phosphorylated.

Corynebacterium glutamicum has four STPKs that are named CgPknA, CgPknB, CgPknG and CgPknL by analogy with their mycobacterial counterparts (Fig. 3). Fiuza et al. recently demonstrated that all STPKs, except CgPknG, are autokinases (Fiuza et al., 2008a), although a recent work provided evidence that CgPknG is capable of autophosphorylation (Schultz et al., 2009). Disruption of either CgPknL or CgPknG in C. glutamicum ATCC 13869 resulted in viable mutants presenting a typical cell morphology and growth rate. However, CgPknA or CgPknB turned out to be essential genes in this strain (Fiuza et al., 2008a), similar to the situation reported for mycobacteria (Kang et al., 2005; Fernandez et al., 2006). However, in an independent study, mutants lacking CgpknA or CgpknB were obtained in C. glutamicum ATCC 13032 (Schultz et al., 2009). These discrepancies may be due to the use of different C glutamicum strains and/or different gene inactivation methods. The use of conditional of CgPknA and CgPknB mutants in the ATCC 13869 strain to reduce levels of CgPknA or CgPknB resulted in elongated cells (Fiuza et al., 2008a). Overall, these data suggest that low production of CgPknA or CgPknB profoundly alters cell division. Interestingly, CgPknA and CgPknB are in a gene cluster that also includes rodA and pbp2b, genes encoding proteins involved in cell division and cell wall biosynthesis in corynebacteria (Kalinowski et al., 2003), and that are conserved in M. tb and S. coelicolor (Fig. 4). Furthermore, overproduction of CgPknA or CgPknB in C. glutamicum resulted in a lack of apical growth and a coccoid morphology. This coccoid phenotype differs from that seen in mycobacterial strains overexpressing the M. tb pknA or pknB genes (Kang et al., 2005). The cell division protein FtsZ was also identified as a substrate of CgPknA, CgPknB and CgPknL and the phosphatase Ppp (Fig. 3), suggesting that these proteins also play a role in cell division (Schultz et al., 2009). Taken collectively, the effects on cell morphology indicate that PknA and PknB kinases play a role in the regulation of the cell shape and morphology in both corynebacteria and mycobacteria.


Figure 3. The C. glutamicum Ser/Thr protein kinases and their role in cell division and peptidoglycan synthesis. In contrast to CgPknA, CgPknB and CgPknL, CgPknG is a soluble kinase. The kinase domain and putative transmembrane domains are shown. The extracellular PASTA (penicillin-binding protein and serine/threonine kinase-associated) motifs are represented by different blocks. CgPknA phosphorylates MurC and FtsZ, two proteins involved in peptidoglycan biosynthesis and cell division respectively.

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Moreover, CgPknA interacts in vitro with the corynebacterial UDP-N-acetylmuramoyl l-alanine ligase (MurC), thus connecting STPK-dependent phosphorylation and peptidoglycan biosynthesis (Fiuza et al., 2008b) (Fig. 3). The Mur ligases play an essential role in bacterial cell-wall peptidoglycan biosynthesis as catalysts of the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC adds the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. The possibility that C. glutamicum MurC activity is regulated by phosphorylation was recently confirmed by showing that CgPknA is the kinase responsible (Fiuza et al., 2008b). Importantly, in vitro assays indicated that the activity of phosphorylated MurC isoform is much lower than that of the non-phosphorylated protein. Therefore, this study confirmed the participation of the corynebacterial STPKs in the regulation of peptidoglycan biosynthesis.

Streptomyces coelicolor is a filamentous actinomycete that has been extensively studied as a model of bacterial differentiation (Bentley et al., 2002; Flardh and Buttner, 2009). The S. coelicolor A3(2) genome includes 34 putative STPKs encoding genes (Bentley et al., 2002), but their roles remain unclear. In S. coelicolor, the best-characterized kinase, AfsK (PK17), is necessary for antibiotic production, whereas in Streptomyces griseus (AfsK-g), it needed for morphological differentiation (Umeyama et al., 1999). The surrounding genes might provide clues to a predicted possible role of a STPK: the genes for PK14 and its close homologue PknB of M. tb (Av-gay et al., 1999) are clustered with genes implicated in cell wall biosynthesis and with other regulatory genes involved in protein Ser/Thr phosphorylation/dephosphorylation (Fig. 4). A highly similar architecture of the chromosomal regions together with the lack of a comparable clustering in the genomes of distantly related prokaryotes (Escherichia coli, Helicobacter pylori and B. subtilis) indicates the existence of a specific, cell division-associated regulatory pathway common to the high-GC branch of the Gram-positive bacteria.

Concluding remarks and perspectives

  1. Top of page
  2. Summary
  3. Role of the STPK-dependent phosphorylation in mycobacterial cell wall metabolism
  4. Role of the Ser/Thr-mediated phosphorylation in mycobacterial cell division
  5. A common theme to actinomycetes?
  6. Concluding remarks and perspectives
  7. Acknowledgements
  8. References

The morphology, replication and growth of mycobacteria are linked to the synthesis and extension of their complex cell wall. Cell elongation, septation and cell division must be co-ordinated both temporally and spatially. This co-ordinated activity must be tightly regulated in order to adapt appropriately to the prevailing conditions. Among the multiple regulatory strategies employed by M. tb in order to achieve this, Ser/Thr phosphorylation appears to occupy a previously poorly appreciated central role. Nevertheless, many important questions remain.

How does phosphorylation affect the structure of the target proteins and modulate their biological activities? Both biophysical and structural approaches may be very helpful to investigate, at an atomic level, how phosphorylation induces conformational changes influencing enzymatic activity. This can be illustrated by recent studies conducted on the M. tb GarA protein. GarA is a FHA-containing protein and represents a major substrate of PknB in M. tb cell extracts (Villarino et al., 2005). The functional importance of this observation was illuminated by the discovery that phosphorylation of the C. glutamicum orthologue, termed OdhI, abrogates its inhibition of a tricarboxylic cycle (TCA) enzyme (Niebisch et al., 2006). Biochemical and biophysical experimentation revealed that phosphorylation of GarA resulted in a conformational compaction of the phosphorylated form in which the FHA domain interacts strongly with the phosphorylated N-terminal extension of the protein (England et al., 2009). Subsequent structural studies confirmed that phosphorylation of GarA triggers this intra-molecular association, and allows to understand how this phospho-switch blocks the access of several target enzymes of the TCA cycle to a common FHA interaction surface (Nott et al., 2009). In an independent study Barthe et al. (2009) reported the three-dimensional structures of the phosphorylated and unphosphorylated isoforms of OdhI, regulating the TCA cycle in C. glutamicum. Comparison of the NMR structures of the unphosphorylated and phosphorylated OdhI isoforms revealed a major conformational change, characterized by intramolecular binding of the phosphorylated N-terminus of OdhI to the FHA domain of OdhI, in a manner similar to GarA (Barthe et al., 2009). This intramolecular switch reported in GarA and OdhI represents an unexpected and original mechanism of auto-inhibition that might be extended to other FHA-containing proteins. These observations revealed unsuspected versatility in the FHA domain and represent new mechanisms of autoregulation that might also occur in eukaryotic cells.

Another important question that remains to be answered concerns the possible signals sensed by the different kinases. Many bacteria, including mycobacteria, exist in a state of metabolic dormancy (Keep et al., 2006) that increases their resistance to antibiotics or to other stresses encountered in nutrient-limited conditions. In an elegant study, Shah et al. (2008) demonstrated that the B. subtilis Ser/Thr kinase PrkC contains an extracellular domain capable to bind to peptidoglycan fragments and this signals the bacteria to exit dormancy by stimulating germination. Like PrkC, M. tb PknB possesses the PASTA (Penicillin And Ser/Thr kinase Associated) domains found in the extracellular portion of membrane-associated STPK and proposed to bind to peptidoglycan. Therefore, although this has not been demonstrated yet, PknB may also recognize peptidoglycan fragments as a signal when growth-promoting conditions are present.

Clearly, the recent developments involving STPK regulatory mechanisms in Mycobacterium, and subsequently in other bacteria, have opened up a new discipline in bacteriology. The sophisticated phospho-signalling networks in M. tb has recently pushed further with the identification of a tyrosine kinase named PtkA (Bach et al., 2009), although its link to cell wall biosynthesis and/or cell division is not known. Therefore, we anticipate that Ser/Thr/Tyr phosphorylation networks will soon be shown to play central roles in many different bacteria.


  1. Top of page
  2. Summary
  3. Role of the STPK-dependent phosphorylation in mycobacterial cell wall metabolism
  4. Role of the Ser/Thr-mediated phosphorylation in mycobacterial cell division
  5. A common theme to actinomycetes?
  6. Concluding remarks and perspectives
  7. Acknowledgements
  8. References
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