Suppression and synthetic‐lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin‐binding protein interactions in Streptococcus pneumoniae D39

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side‐wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division‐protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co‐IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP‐PBP2x.     

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.     

Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis

The characteristic shape of bacterial cells is mainly determined by the cell wall, the synthesis of which is orchestrated by penicillin-binding proteins (PBPs). Rod-shaped bacteria have two distinct modes of cell wall synthesis, involved in cell elongation and cell division, which are believed to employ different sets of PBPs. A long-held question has been how these different modes of growth are co-ordinated in space and time. We have now identified the cell division protein, EzrA, and a newly discovered protein, GpsB, as key players in the elongation-division cycle of Bacillus subtilis. Mutations in these genes have a synthetic phenotype with defects in both cell division and cell elongation. They also have an unusual bulging phenotype apparently due to a failure in properly completing cell pole maturation. We show that these phenotypes are tightly associated with disturbed localization of the major transglycosylase/transpeptidase of the cell, PBP1. EzrA and GpsB have partially differentiated roles in the localization cycle of PBP1, with EzrA mainly promoting the recruitment of PBP1 to division sites, and GpsB facilitating its removal from the cell pole, after the completion of pole maturation.     

The GpsB files: the truth is out there

Peptidoglycan (PG), an essential stress‐bearing component of the bacterial cell wall, is synthesised by penicillin binding proteins (PBPs). PG synthesis at the cell division septum is necessary for constructing new poles of progeny cells, and cells cannot elongate without inserting new PG in the side‐wall. The cell division regulator GpsB appears to co‐ordinate PG synthesis at the septum during division and at the side‐wall during elongation in rod‐shaped and ovococcoid Gram‐positive bacteria. How the control over PG synthesis is exerted is unknown. In this issue of Molecular Microbiology, Rued et al. show that in pneumococci GpsB forms complexes with PBP2a and PBP2b, and that deletion or depletion of GpsB prevents closure of the septal ring that in itself is PBP2x‐dependent. Loss of GpsB can be suppressed by spontaneous mutations, including within the gene encoding the only PP2C Ser/Thr phosphatase in Streptococcus pneumoniae, indicating that GpsB plays a key – but unknown – role in protein phosphorylation in pneumococci. Rued et al. combine phenotypic and genotypic analyses of mutant strains that suggest discrepancies in the literature concerning GpsB might have arisen from accumulation of unidentified suppressors, highlighting the importance and power of strain validation and whole genome sequencing in this context.     

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.     

Structural basis for interaction of DivIVA/GpsB proteins with their ligands

DivIVA proteins and their GpsB homologues are late cell division proteins found in Gram‐positive bacteria. DivIVA/GpsB proteins associate with the inner leaflet of the cytosolic membrane and act as scaffolds for other proteins required for cell growth and division. DivIVA/GpsB proteins comprise an N‐terminal lipid‐binding domain for membrane association fused to C‐terminal domains supporting oligomerization. Despite sharing the same domain organization, DivIVA and GpsB serve different cellular functions: DivIVA plays diverse roles in division site selection, chromosome segregation and controlling peptidoglycan homeostasis, whereas GpsB contributes to the spatiotemporal control of penicillin‐binding protein activity. The crystal structures of the lipid‐binding domains of DivIVA from Bacillus subtilis and GpsB from several species share a fold unique to this group of proteins, whereas the C‐terminal domains of DivIVA and GpsB are radically different. A number of pivotal features identified from the crystal structures explain the functional differences between the proteins. Herein we discuss these structural and functional relationships and recent advances in our understanding of how DivIVA/GpsB proteins bind and recruit their interaction partners, knowledge that might be useful for future structure‐based DivIVA/GpsB inhibitor design.     

Mechanism of regulation of prokaryotic tubulin-like GTPase FtsZ by membrane protein EzrA

At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed.     

Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching

Escherichia coli cells lacking low-molecular-weight penicillin-binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape. We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches. Branches always originated from the remnants of abnormal septation events, cementing the relationship between aberrant cell division and branch formation. In the absence of PBP5, PBP6 and DacD localized to nascent septa, suggesting that these PBPs can partially substitute for the loss of PBP5. We propose that branching begins when mislocalized FtsZ triggers the insertion of inert peptidoglycan at unusual positions during cell division. Only later, after normal cell wall elongation separates the patches, do branches become visible. Thus, a relationship between the LMW PBPs and cytoplasmic FtsZ ultimately affects cell division and overall shape.     

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi‐functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N‐deacetylase PgdA and the peptidoglycan O‐acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA‐dependent and OatA‐independent. Protein‐protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB‐like cell division protein and factors important for escape from the host immune system.     

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

Polar targeting of DivIVA in Bacillus subtilis is not directly dependent on FtsZ or PBP 2B

DivIVA is involved in Bacillus subtilis cell division and is located at the cell poles. Previous experiments suggested that the cell division proteins FtsZ and PBP 2B are required for polar targeting of DivIVA. By using outgrowing spores, we show that DivIVA accumulates at the cell poles independent of the presence of FtsZ or PBP 2B.     

ZipA Is Required for FtsZ-Dependent Preseptal Peptidoglycan Synthesis prior to Invagination during Cell Division

Rod-shaped bacteria grow by a repetitive cycle of elongation followed by division, and the mechanisms responsible for these two processes have been studied for decades. However, little is known about what happens during the transition between the two activities. At least one event occurs after elongation ends and before division commences, that being the insertion of new cell wall peptidoglycan into a narrowly circumscribed ribbon around midcell where septation is destined to take place. This insertion does not depend on the presence of the septation-specific protein PBP3 and is therefore known as PBP3-independent peptidoglycan synthesis (PIPS). Here we report that only FtsZ and ZipA are required to generate PIPS in wild-type Escherichia coli. PIPS does not require the participation of other members of the divisome, the MreB-directed cell wall elongation complex, alternate peptidoglycan synthases, the major peptidoglycan amidases, or any of the low-molecular-weight penicillin binding proteins. ZipA-directed PIPS may represent an intermediate stage that connects cell wall elongation to septal invagination and may be the reason ZipA is essential in the gammaproteobacteria.     

A novel component of the division‐site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD

Cell division in bacteria is governed by a complex cytokinetic machinery in which the key player is a tubulin homologue, FtsZ. Most rod‐shaped bacteria divide precisely at mid‐cell between segregated sister chromosomes. Selection of the correct site for cell division is thought to be determined by two negative regulatory systems: the nucleoid occlusion system, which prevents division in the vicinity of the chromosomes, and the Min system, which prevents inappropriate division at the cell poles. In Bacillus subtilis recruitment of the division inhibitor MinCD to cell poles depends on DivIVA, and these proteins were thought to be sufficient for Min function. We have now identified a novel component of the division‐site selection system, MinJ, which bridges DivIVA and MinD. minJ mutants are impaired in division because MinCD activity is no longer restricted to cell poles. Although MinCD was thought to act specifically on FtsZ assembly, analysis of minJ and divIVA mutants showed that their block in division occurs downstream of FtsZ. The results support a model in which the main function of the Min system lies in allowing only a single round of division per cell cycle, and that MinCD acts at multiple levels to prevent inappropriate division.     

Maturation of the Escherichia coli divisome occurs in two steps

Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno- and GFP-fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14-21 min, depending on the growth rate, between Z-ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two-step model for bacterial division in which the Z-ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.     

Cell wall growth during elongation and division: one ring to bind them?

The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. In this issue of Molecular Microbiology, the groups of Christine Jacobs-Wagner and Waldemar Vollmer provide compelling evidence that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis in non-dividing cells. During elongation (cell growth) FtsZ is responsible for the incorporation of CW material in a zone at the midcell by recruiting MurG, a protein involved in peptidoglycan (PG) precursor synthesis. This resembles earlier findings of FtsZ mediated PG synthesis activity in Escherichia coli. A role of FtsZ in PG synthesis during elongation forces a rethink of the current model of CW synthesis in rod-shaped bacteria.     

The division inhibitor EzrA contains a seven-residue patch required for maintaining the dynamic nature of the medial FtsZ ring

The essential cytoskeletal protein FtsZ assembles into a ring-like structure at the nascent division site and serves as a scaffold for the assembly of the prokaryotic division machinery. We previously characterized EzrA as an inhibitor of FtsZ assembly in Bacillus subtilis. EzrA interacts directly with FtsZ to prevent aberrant FtsZ assembly and cytokinesis at cell poles. EzrA also concentrates at the cytokinetic ring in an FtsZ-dependent manner, although its precise role at this position is not known. Here, we identified a conserved patch of amino acids in the EzrA C terminus that is essential for localization to the FtsZ ring. Mutations in this patch (designated the “QNR patch”) abolish EzrA localization to midcell but do not significantly affect EzrA's ability to inhibit FtsZ assembly at cell poles. ezrA QNR patch mutant cells exhibit stabilized FtsZ assembly at midcell and are significantly longer than wild-type cells, despite lacking extra FtsZ rings. These results indicate that EzrA has two distinct activities in vivo: (i) preventing aberrant FtsZ ring formation at cell poles through inhibition of de novo FtsZ assembly and (ii) maintaining proper FtsZ assembly dynamics within the medial FtsZ ring, thereby rendering it sensitive to the factors responsible for coordinating cell growth and cell division.     

A protein critical for cell constriction in the Gram‐negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan‐binding LysM domains

During division of Gram‐negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast‐growth conditions. Under slow‐growth conditions, the ΔdipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non‐canonical. DipM also carries two tandems of the PG‐binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non‐septal, monolayered PG.     

FtsZ dynamics in bacterial division: What,how, and why?

Bacterial cell division is orchestrated by the divisome, a protein complex centered on the tubulin homolog FtsZ. FtsZ polymerizes into a dynamic ring that defines the division site, recruits downstream proteins, and directs peptidoglycan synthesis to drive constriction. Recent studies have documented treadmilling of FtsZ polymer clusters both in cells and in vitro. Emerging evidence suggests that FtsZ dynamics are regulated largely by intrinsic properties of FtsZ itself and by the membrane anchoring protein FtsA. Although FtsZ dynamics are broadly required for Z-ring assembly, their role(s) during constriction may vary among bacterial species. These recent advances set the stage for future studies to investigate how FtsZ dynamics are physically and/or functionally coupled to peptidoglycan metabolic enzymes to direct efficient division.     

The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu⁷⁵–Gln⁹³, known as ^EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that ^EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with ^EFtsN, which demonstrates that ^EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the ^EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponA^ts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that ^EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.