PASTA Domains Research Articles

Cytosolic Factors Controlling PASTA Kinase-Dependent ReoM Phosphorylation.

Bacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram-positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram-positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of invivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth phase-dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must be able to bind its substrates and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases were not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation.

Penicillin-Binding Protein 1 (PBP1) of Staphylococcus aureus Has Multiple Essential Functions in Cell Division.

ABSTRACTBacterial cell division is a complex process requiring the coordination of multiple components to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multistage PG architecture that develops during septation. Penicillin-binding proteins (PBPs) are essential for the final steps of PG biosynthesis; their transpeptidase activity links the peptide side chains of nascent glycan strands. PBP1 is required for cell division in S. aureus, and here, we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thereby potentially coordinate the cell division process. The transpeptidase activity of PBP1 is also essential, but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific β-lactam, meropenem. Together, these results lead to a model for septal PG synthesis where PBP1 enzyme activity is required for the characteristic architecture of the septum and PBP1 protein molecules enable the formation of the septal plate.

A cortex-specific penicillin-binding protein contributes to heat resistance in Clostridioides difficile spores

BackgroundSporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridioides difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile. MethodsBioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity. ResultsDeletion of CDR20291_2544 (SpoVDCd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVDCd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVDCd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVDCd was also seen to localise to the developing spore by super-resolution confocal microscopy. ConclusionsWe have identified and characterised a cortex specific PBP in C. difficile. This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.

Integrative structural biology of the penicillin-binding protein-1 from Staphylococcus aureus, an essential component of the divisome machinery

The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis ofPBP1 ofStaphylococcus aureus(saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the “penicillin-binding protein and serine/threonine kinase associated” (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domainswas determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains ofsaPBP1 as conformationally mobile and separated from the transpeptidasedomain.This conclusion was confirmed by SAXS experiments on the full-length protein in solution.A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain insaPBP1 could play a crucial role forin vivointeraction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.

The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance in Clostridium difficile.

Clostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkC mutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkC mutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the ΔprkC mutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkC mutant had a colonization delay that did not significantly affect overall virulence.

PASTA sequence composition is a predictive tool for protein class identification.

PASTA domains are small modules expressed in bacteria and found in one or multiple copies at the C-terminal end of several penicillin binding proteins (PBPs) and Ser/Thr protein kinases (STPKs) and represent potential targets for a new class of antibiotics. PASTA domains are currently annotated as sensor domains, as they are thought to activate their cognate proteins in response to binding to opportune ligands. However, recent studies have shown that PASTA domains linked to proteins of different classes, STPKs or PBPs,do not share the same binding abilities. Despite this, there is currently no way to distinguish between PASTA domains from the two classes, since all of them share the same fold, independent of the class they belong to. To identify a predictive tool of class identification, we here analyse a pool of parameters, including amino acid compositions and total charges of PASTA domains either linked to PBPs or to STPKs. We screened sequences from Actinobacteria, Firmicutes and Bacteroidetes. The first two phyla include some of the most dangerous micro-organisms for human health such as Mycobacterium tuberculosis and Staphylococcus aureus. Based on this analysis, our study proposes a predictive method to assign PASTA domains with unknown origin to their corresponding enzyme class, based solely on sequence information.

Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth

The activity of the PrkC protein kinase is regulated in a sophisticated manner in Bacillus subtilis cells. In spores, in the presence of muropeptides, PrkC stimulates dormancy exit. The extracellular region containing PASTA domains binds peptidoglycan fragments to probably enhance the intracellular kinase activity. During exponential growth, the cell division protein GpsB interacts with the intracellular domain of PrkC to stimulate its activity. In this paper, we have reinvestigated the regulation of PrkC during exponential and stationary phases. We observed that, during exponential growth, neither its septal localization nor its activity are influenced by the addition of peptidoglycan fragments or by the deletion of one or all PASTA domains. However, Dynamic Light Scattering experiments suggest that peptidoglycan fragments bind specifically to PrkC and induce its oligomerization. In addition, during stationary phase, PrkC appeared evenly distributed in the cell wall and the deletion of one or all PASTA domains led to a non-activated kinase. We conclude that PrkC activation is not as straightforward as previously suggested and that regulation of its kinase activity via the PASTA domains and peptidoglycan fragments binding occurs when PrkC is not concentrated to the bacterial septum, but all over the cell wall in non-dividing bacillus cells.

Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae.

Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by β-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.

PASTA in Penicillin Binding Proteins and Serine/Threonine Kinases: A Recipe of Structural, Dynamic and Binding Properties.

Penicillin binding proteins (PBPs) and Serine Threonine kinases (STPKs) are two classes of bacterial enzymes whose involvement in a series of vital processes in bacterial growth and division is well assessed. Many PBPs and STPKs show linked an ancillary domain named PASTA, whose functional role is not completely deciphered so far. It has been proposed that PASTAs are sensor modules that by binding opportune ligands (i.e. muropeptides) activate the cognate proteins to their functions. However, based on recent data, the sensor annotation sounds true for PASTA from STPKs, and false for PASTA from PBPs. Different PASTA domains, belonging or not to different protein classes, sharing or not appreciable sequence identities, always show identical folds. This survey of the structural, binding and dynamic properties of PASTA domains pursues the reasons why identical topologies may turn in different roles. Amino acid compositions, total charges and distribution of the hydrophobic/hydrophilic patches on the surface, significantly vary among PASTAs from STPKs and PBPs and appear to correlate with different functions. A possible criterion to discriminate between PASTA modules of STPKs or PBPs solely based on their sequences is proposed. Possibly reflecting different species as well as functional roles and evolutionary profile, our routine represents a fast even though approximate method to distinguish between PASTA belonging to different classes.

Structural model of the full-length Ser/Thr protein kinase StkP from S. pneumoniae and its recognition of peptidoglycan fragments

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivoEnterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivoIMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The prevailing model for signaling by PASTA kinases proposes that the extracellular PASTA domains bind ligands to drive kinase dimerization, enhanced autophosphorylation, and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo We show that the PASTA kinase IreK of Enterococcus faecalis responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires the PASTA domains and phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling.

The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus

The assembly of the bacterial cell wall requires synchronization of a multitude of biosynthetic machineries and regulatory networks. The eukaryotic-like serine/threonine kinase PknB has been implicated in coordinating cross-wall formation, autolysis and cell division in Staphylococcus aureus. However, the signal molecule sensed by this kinase remained elusive so far. Here, we provide compelling biochemical evidence that PknB interacts with the ultimate cell wall precursor lipid II, triggering kinase activity. Moreover, we observed crosstalk of PknB with the two component system WalKR and identified the early cell division protein FtsZ as another PknB phosphorylation substrate in S. aureus. In agreement with the implied role in regulation of cell envelope metabolism, we found PknB to preferentially localize to the septum of S. aureus and the PASTA domains to be crucial for recruitment to this site. The data provide a model for the contribution of PknB to control cell wall metabolism and cell division.

Self-resistance in Streptomyces, with Special Reference to β-Lactam Antibiotics.

Antibiotic resistance is one of the most serious public health problems. Among bacterial resistance, β-lactam antibiotic resistance is the most prevailing and threatening area. Antibiotic resistance is thought to originate in antibiotic-producing bacteria such as Streptomyces. In this review, β-lactamases and penicillin-binding proteins (PBPs) in Streptomyces are explored mainly by phylogenetic analyses from the viewpoint of self-resistance. Although PBPs are more important than β-lactamases in self-resistance, phylogenetically diverse β-lactamases exist in Streptomyces. While class A β-lactamases are mostly detected in their enzyme activity, over two to five times more classes B and C β-lactamase genes are identified at the whole genomic level. These genes can subsequently be transferred to pathogenic bacteria. As for PBPs, two pairs of low affinity PBPs protect Streptomyces from the attack of self-producing and other environmental β-lactam antibiotics. PBPs with PASTA domains are detectable only in class A PBPs in Actinobacteria with the exception of Streptomyces. None of the Streptomyces has PBPs with PASTA domains. However, one of class B PBPs without PASTA domain and a serine/threonine protein kinase with four PASTA domains are located in adjacent positions in most Streptomyces. These class B type PBPs are involved in the spore wall synthesizing complex and probably in self-resistance. Lastly, this paper emphasizes that the resistance mechanisms in Streptomyces are very hard to deal with, despite great efforts in finding new antibiotics.

Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

The PASTA domain of penicillin-binding protein SpoVD is dispensable for endospore cortex peptidoglycan assembly in Bacillus subtilis.

Peptidoglycan is the major structural component of the bacterial cell wall. Penicillin-binding proteins (PBPs), located at the exterior of the cytoplasmic membrane, play a major role in peptidoglycan synthesis and remodelling. A PASTA domain (penicillin-binding protein and serine/threonine kinase associated domain) of about 65 residues is found at the C-terminal end of some PBPs and eukaryotic-like protein serine/threonine kinases in a variety of bacteria. The function of PASTA domains is not understood, but some of them are thought to bind uncross linked peptidoglycan. Bacillus subtilis has 16 different PBPs, but only 2 of them, Pbp2b and SpoVD, contain a PASTA domain. SpoVD is specific for sporulation and essential for endospore cortex peptidoglycan synthesis. We have studied the role of the PASTA domain in SpoVD by deleting this domain and analysing the effects on endospore formation and subcellular localization of SpoVD. Our results demonstrate that the PASTA domain in SpoVD is not essential for cortex synthesis and not important for targeting SpoVD to the forespore outer membrane during sporulation.

Protein Kinase B (PknB) of Mycobacterium tuberculosis Is Essential for Growth of the Pathogen in Vitro as well as for Survival within the Host

The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host.

Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis

PonA2 is one of the two class A penicillin binding proteins of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. It plays a complex role in mycobacterial physiology and is spotted as a promising target for inhibitors. PonA2 is involved in adaptation of M. tuberculosis to dormancy, an ability which has been attributed to the presence in its sequence of a C-terminal PASTA domain. Since PASTA modules are typically considered as β-lactam antibiotic binding domains, we determined the solution structure of the PASTA domain from PonA2 and analyzed its binding properties versus a plethora of potential binders, including the β-lactam antibiotics, two typical muropeptide mimics, and polymeric peptidoglycan. We show that, despite a high structural similarity with other PASTA domains, the PASTA domain of PonA2 displays different binding properties, as it is not able to bind muropeptides, or β-lactams, or polymeric peptidoglycan. These results indicate that the role of PASTA domains cannot be generalized, as their specific binding properties strongly depend on surface residues, which are widely variable.

Streptococcus pneumoniae PBP2x mid‐cell localization requires the C‐terminal PASTA domains and is essential for cell shape maintenance

The transpeptidase activity of the essential penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N-terminal GFP-PBP2x fusions under the control of a zinc-inducible promoter. The ectopic fusion protein localized at mid-cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP-PBP2x is functional. Depletion of GFP-PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP-PBP2x still localized at septal sites. Remarkably, the same was true for a GFP-PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C-terminal PASTA domains, identifying HtrA as targeting GFP-PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex.

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.

Mutations in Streptococcus pneumoniae Penicillin-Binding Protein 2x: Importance of the C-Terminal Penicillin-Binding Protein and Serine/Threonine Kinase-Associated Domains for Beta-Lactam Binding

Penicillin-binding protein 2x (PBP2x) mutations that occur during the selection with beta-lactams are located within the central penicillin-binding/transpeptidase (TP) domain, and are believed to mediate resistance by interfering with the formation of a covalent complex of the active site serine with the antibiotic. We now investigated the effect of two point mutations found in two independently obtained laboratory mutants that are located at the surface of the TP domain with their side chains facing outside (G422D respectively R426C). They have no significant effect on resistance to cefotaxime in vivo or on binding to Bocillin™FL to the active site in vitro using purified PBP2x derivatives, thus apparently do not affect the active site directly. In contrast, in silico modeling revealed that they affect van der Waal's interactions with the PASTA1 (PBP and serine/threonine kinase associated) domain of the C-terminal extension and a noncovalent cefuroxime molecule found in the X-ray structure of an acylated PBP2x, suggesting some effect of the mutations on the interaction of the TP domain with PASTA1 and/or with the antibiotic associated with PASTA1. The effect of the PASTA domains on covalent binding of PBP2x to Bocillin FL was then investigated using a series of soluble truncated PBP2x derivatives. Deletion of 127 C-terminal residues, that is, of both PASTA domains, decreased binding dramatically by ∼90%. Surprisingly, deletion of only 40 amino acids resulted in the same phenotype, whereas the absence of 30 amino acids affected binding marginally by 10%, documenting a crucial role of the C-terminal domain for beta-lactam binding.