Agents of change – concepts in Mycobacterium tuberculosis Ser/Thr/Tyr phosphosignalling

The flow of information from the outside to the inside of bacterial cells is largely directed by protein kinases. In addition to histidine/aspartate phosphorelays of two‐component response regulators, recent work in Mycobacterium tuberculosis (Mtb) reinforces the idea that phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) is central to bacterial physiology and pathogenesis, and that the corresponding phosphosystems are highly similar to those in eukaryotes. In this way, eukaryotes are a useful guide to understanding Ser/Thr/Tyr phosphorylation (O‐phosphorylation) in prokaryotes such as Mtb. However, as novel functions and components of bacterial O‐phosphorylation are identified, distinct differences between pro‐ and eukaryotic phosphosignalling systems become apparent. The emerging picture of O‐phosphorylation in Mtb is complicated, goes beyond the eukaryotic paradigms, and shows the limitations of viewing bacterial phosphosignalling within the confines of the ‘eukaryotic‐like’ model. Here, we summarize recent findings about Ser/Thr and the recently discovered Tyr phosphorylation pathways in Mtb, highlight the similarities and differences between eukaryotic and prokaryotic O‐phosphorylation, and pose additional questions about signalling components, pathway organization, and ultimately, the cellular roles of O‐phosphorylation in Mtb physiology and pathogenesis.     

Interaction of Penicillin‐Binding Protein 2x and Ser/Thr protein kinase StkP,two key players in Streptococcus pneumoniae R6 morphogenesis

Bacterial cell growth and division require the co‐ordinated action of peptidoglycan biosynthetic enzymes and cell morphogenesis proteins. However, the regulatory mechanisms that allow generating proper bacterial shape and thus preserving cell integrity remain largely uncharacterized, especially in ovococci. Recently, the conserved eukaryotic‐like Ser/Thr protein kinase of Streptococcus pneumoniae (StkP) was demonstrated to play a major role in cell shape and division. Here, we investigate the molecular mechanisms underlying the regulatory function(s) of StkP and show that it involves one of the essential actors of septal peptidoglycan synthesis, Penicillin‐Binding Protein 2x (PBP2x). We demonstrate that StkP and PBP2x interact directly and are present in the same membrane‐associated complex in S. pneumoniae. We further show that they both display a late‐division localization pattern at the division site and that the positioning of PBP2x depends on the presence of the extracellular PASTA domains of StkP. We demonstrate that StkP and PBP2x interaction is mediated by their extracellular regions and that the complex formation is inhibited in vitro in the presence of cell wall fragments. These data suggest that the role of StkP in cell division is modulated by an interaction with PBP2x.     

Biochemical and Spatial Coincidence in the Provisional Ser/Thr Protein Kinase Interaction Network of Mycobacterium tuberculosis

Many Gram-positive bacteria coordinate cellular processes by signaling through Ser/Thr protein kinases (STPKs), but the architecture of these phosphosignaling cascades is unknown. To investigate the network structure of a prokaryotic STPK system, we comprehensively explored the pattern of signal transduction in the Mycobacterium tuberculosis Ser/Thr kinome. Autophosphorylation is the dominant mode of STPK activation, but the 11 M. tuberculosis STPKs also show a specific pattern of efficient cross-phosphorylation in vitro. The biochemical specificity intrinsic to each kinase domain was used to map the provisional signaling network, revealing a three-layer architecture that includes master regulators, signal transducers, and terminal substrates. Fluorescence microscopy revealed that the STPKs are specifically localized in the cell. Master STPKs are concentrated at the same subcellular sites as their substrates, providing additional support for the biochemically defined network. Together, these studies imply a branched functional architecture of the M. tuberculosis Ser/Thr kinome that could enable horizontal signal spreading. This systems-level approach provides a biochemical and spatial framework for understanding Ser/Thr phospho-signaling in M. tuberculosis, which differs fundamentally from previously defined linear histidine kinase cascades.     

Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis

Although many membrane Ser/Thr‐kinases with PASTA motifs have been shown to control bacterial cell division and morphogenesis, inactivation of the Ser/Thr‐kinase PrkC does not impact Bacillus subtilis cell division. In this study, we show that PrkC localizes at the division septum. In addition, three proteins involved in cell division/elongation, GpsB, DivIVA and EzrA are required for stimulating PrkC activity in vivo. We show that GpsB interacts with the catalytic subunit of PrkC that, in turn, phosphorylates GpsB. These observations are not made with DivIVA and EzrA. Consistent with the phosphorylated residue previously detected for GpsB in a high‐throughput phosphoproteomic analysis of B. subtilis, we show that threonine 75 is the single PrkC‐mediated phosphorylation site in GpsB. Importantly, the substitution of this threonine by a phospho‐mimetic residue induces a loss of PrkC kinase activity in vivo and a reduced growth under high salt conditions as observed for gpsB and prkC null mutants. Conversely, substitution of threonine 75 by a phospho‐ablative residue does not induce such growth and PrkC kinase activity defects. Altogether, these data show that proteins of the divisome control PrkC activity and thereby phosphorylation of PrkC substrates through a negative feedback loop in B. subtilis.     

The Streptococcus pyogenes orphan protein tyrosine phosphatase,SP‐PTP,possesses dual specificity and essential virulence regulatory functions

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP‐PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP‐PTP is, therefore, questionable. Here, we demonstrate that SP‐PTP possesses dual phosphatase specificity for Tyr‐ and Ser/Thr‐phosphorylated GAS proteins, such as Ser/Thr kinase (SP‐STK) and the SP‐STK‐phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP‐PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP‐PTP displayed increased Ser‐/Thr‐/Tyr‐phosphorylation. SP‐PTP also differentially regulates the expression of ～50% of the total GAS genes, including several virulence genes potentially through the two‐component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP‐PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr‐phosphorylation in GAS and the relevance of SP‐PTP as an important therapeutic target.     

Analysis of the phosphoproteome of the multicellular bacterium Streptomyces coelicolor A3(2) by protein/peptide fractionation,phosphopeptide enrichment and high‐accuracy mass spectrometry

The serine (Ser)/threonine (Thr)/tyrosine (Tyr) phosphoproteome of exponentially growing Streptomyces coelicolor A3(2) was analysed using the gel‐free approaches of preparative IEF for protein fractionation, followed by strong cation exchange peptide fractionation and phosphopeptide enrichment by TiO₂ metal oxide affinity chromatography. Phosphopeptides were identified using LC‐ESI‐LTQ‐Orbitrap^? MS. Forty‐six novel phosphorylation sites were identified on 40 proteins involved in gene regulation or signalling, central metabolism, protein biosynthesis, membrane transport and cell division, as well as several of unknown function. In contrast to other studies, Thr phosphorylation appeared to be preferred, with relative levels of Ser, Thr and Tyr phosphorylation of 34, 52 and 14%, respectively. Genes for most of the 40 phosphorylated proteins reside in the central “housekeeping” region of the linear S. coelicolor chromosome, suggesting that in general Ser, Thr and Tyr phosphorylation play a role in regulating essential aspects of metabolism in streptomycetes. A greater number of regulators and putative regulators were also identified compared with other bacterial phosphoproteome studies, potentially reflecting the complex heterotrophic and developmental life style of S. coelicolor. This study is the first analysis of the phosphoproteome of a member of this morphologically complex and industrially important group of microorganisms.     

An FHA phosphoprotein recognition domain mediates protein EmbR phosphorylation by PknH, a Ser/Thr protein kinase from Mycobacterium tuberculosis

In bacteria, regulatory phosphorylation of proteins at serine and/or threonine residues by Ser/Thr protein kinase (STPK) is an emerging theme in prokaryotic signaling, particularly since the prediction of the occurrence of several STPKs from genome sequencing and sequence surveys. Here we show that protein PknH possesses an autokinase activity and belongs to the large STPK family found in Mycobacterium tuberculosis. Evidence is presented that PknH can also phosphorylate EmbR, a protein suspected to modulate the level of arabinosyltransferase activity involved in arabinan biosynthesis of arabinogalactan, a key molecule of the mycobacterial cell wall. Interestingly, EmbR possesses an FHA (forkhead-associated) domain, a newly described phosphoprotein recognition domain, which plays an essential role in PknH-EmbR interaction and phosphorylation of EmbR by PknH. It is demonstrated that mutation of each of three particular residues of this FHA domain, Arg312, Ser326, and Asn348, totally abolishes the PknH-mediated phosphorylation of EmbR, thus highlighting the critical role of this domain in the direct interaction between EmbR and PknH.     

Phosphorylation statuses at different residues of lamin B2, B1, and A/C dynamically and independently change throughout the cell cycle

Lamins, major components of the nuclear lamina, undergo phosphorylation at multiple residues during cell cycle progression, but their detailed phosphorylation kinetics remain largely undetermined. Here, we examined changes in the phosphorylation of major phosphorylation residues (Thr14, Ser17, Ser385, Ser387, and Ser401) of lamin B2 and the homologous residues of lamin B1, A/C during the cell cycle using novel antibodies to the site-specific phosphorylation. The phosphorylation levels of these residues independently changed during the cell cycle. Thr14 and Ser17 were phosphorylated during G₂/M phase to anaphase/telophase. Ser385 was persistently phosphorylated during mitosis to G₁ phase, whereas Ser387 was phosphorylated discontinuously in prophase and G₁ phase. Ser401 phosphorylation was enhanced in the G₁/S boundary. Immunoprecipitation using the phospho-antibodies suggested that metaphase-phosphorylation at Thr14, Ser17, and Ser385 of lamins occurred simultaneously, whereas G₁-phase phosphorylation at Ser385 and Ser387 occurred in distinct pools or with different timings. Additionally, we showed that lamin B2 phosphorylated at Ser17, but not Ser385, Ser387 and Ser401, was exclusively non-ionic detergent soluble, depolymerized forms in growing cells, implicating specific involvement of Ser17 phosphorylation in lamin depolymerization and nuclear envelope breakdown. These results suggest that the phosphorylations at different residues of lamins might play specific roles throughout the cell cycle.     

A new factor stimulating peptidoglycan hydrolysis to separate daughter cells in Caulobacter crescentus

Cell division in Gram‐negative bacteria involves the co‐ordinated invagination of the three cell envelope layers to form two new daughter cell poles. This complex process starts with the polymerization of the tubulin‐like protein FtsZ into a Z‐ring at mid‐cell, which drives cytokinesis and recruits numerous other proteins to the division site. These proteins are involved in Z‐ring constriction, inner‐ and outer‐membrane invagination, peptidoglycan remodelling and daughter cell separation. Three papers in this issue of Molecular Microbiology, from the teams of Lucy Shapiro, Martin Thanbichler and Christine Jacobs‐Wagner, describe a novel protein, called DipM for Division Involved Protein with LysM domains, that is required for cell division in Caulobacter crescentus. DipM localizes to the mid‐cell during cell division, where it is necessary for the hydrolysis of the septal peptidoglycan to remodel the cell wall. Loss of DipM results in severe defects in cell envelope constriction, which is deleterious under fast‐growth conditions. State‐of‐the‐art microscopy experiments reveal that the peptidoglycan is thicker and that the cell wall is incorrectly organized in DipM‐depleted cells compared with wild‐type cells, demonstrating that DipM is essential for reorganizing the cell wall at the division site, for envelope invagination and cell separation in Caulobacter.     

The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation

Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages.     

Evidence for a peptidoglycan‐like structure in Orientia tsutsugamushi

Bacterial cell walls are composed of the large cross‐linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan‐like structure in Orientia, as well as an outer membrane containing a network of cross‐linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.     

DipM links peptidoglycan remodelling to outer membrane organization in Caulobacter

Cell division in Gram‐negative organisms requires coordinated invagination of the multilayered cell envelope such that each daughter receives an intact inner membrane, peptidoglycan (PG) layer and outer membrane (OM). Here, we identify DipM, a putative LytM endopeptidase in Caulobacter crescentus, and show that it plays a critical role in maintaining cell envelope architecture during growth and division. DipM localized to the division site in an FtsZ‐dependent manner via its PG‐binding LysM domains. Although not essential for viability, ΔdipM cells exhibited gross morphological defects, including cell widening and filamentation, indicating a role in cell shape maintenance and division that we show requires its LytM domain. Strikingly, cells lacking DipM also showed OM blebbing at the division site, at cell poles and along the cell body. Cryo electron tomography of sacculi isolated from cells depleted of DipM revealed marked thickening of the PG as compared to wild type, which we hypothesize leads to loss of trans‐envelope contacts between components of the Tol–Pal complex. We conclude that DipM is required for normal envelope invagination during division and to maintain a sacculus of constant thickness that allows for maintenance of OM connections throughout the cell envelope.     

EspC forms a filamentous structure in the cell envelope of Mycobacterium tuberculosis and impacts ESX‐1 secretion

Pathogenicity of Mycobacterium tuberculosis (M. tb) is mediated by the ESX‐1 secretion system, which exports EsxA and EsxB, the major virulence factors that are co‐secreted with EspA and EspC. Functional information about ESX‐1 components is scarce. Here, it was shown that EspC associates with EspA in the cytoplasm and membrane, then polymerizes during secretion from M. tb. EspC was localized by immuno‐gold electron microscopy in whole cells or cryosections as a surface‐exposed filamentous structure that seems to span the cell envelope. Consistent with these findings, purified EspC homodimerizes via disulphide bond formation, multimerizes and self‐assembles into long filaments in vitro. The C‐terminal domain is required for multimerization as truncation and selected point mutations therein impact EspC filament formation, thus reducing secretion of EsxA and causing attenuation of M. tb. The data are consistent with EspC serving either as a modulator of ESX‐1 function or as a component of the secretion apparatus.     

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor‐like kinases

The Arabidopsis thaliana somatic embryogenesis receptor‐like kinase (SERK) family consists of five leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)‐mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC‐MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C‐terminally located residue Ser‐562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr‐462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP‐tagged SERK1 from plant extracts followed by MS/MS identified Ser‐303, Thr‐337, Thr‐459, Thr‐462, Thr‐463, Thr‐468, and Ser‐612 or Thr‐613 or Tyr‐614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser‐299 and Thr‐462. This suggests both intra‐ and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser‐887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.     

Cell Wall Stress Stimulates the Activity of the Protein Kinase StkP of Streptococcus pneumoniae,Leading to Multiple Phosphorylation

Streptococcus pneumoniae is an opportunistic human pathogen that encodes a single eukaryotic-type Ser/Thr protein kinase StkP and its functional counterpart, the protein phosphatase PhpP. These signaling enzymes play critical roles in coordinating cell division and growth in pneumococci. In this study, we determined the proteome and phosphoproteome profiles of relevant mutants. Comparison of those with the wild-type provided a representative dataset of novel phosphoacceptor sites and StkP-dependent substrates. StkP phosphorylates key proteins involved in cell division and cell wall biosynthesis in both the unencapsulated laboratory strain Rx1 and the encapsulated virulent strain D39. Furthermore, we show that StkP plays an important role in triggering an adaptive response induced by a cell wall-directed antibiotic. Phosphorylation of the sensor histidine kinase WalK and downregulation of proteins of the WalRK core regulon suggest crosstalk between StkP and the WalRK two-component system. Analysis of proteomic profiles led to the identification of gene clusters regulated by catabolite control mechanisms, indicating a tight coupling of carbon metabolism and cell wall homeostasis. The imbalance of steady-state protein phosphorylation in the mutants as well as after antibiotic treatment is accompanied by an accumulation of the global Spx regulator, indicating a Spx-mediated envelope stress response. In summary, StkP relays the perceived signal of cell wall status to key cell division and regulatory proteins, controlling the cell cycle and cell wall homeostasis.     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.