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.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Evolution of transmembrane protein kinases implicated in coordinating remodeling of gram-positive peptidoglycan: inside versus outside

Members of a family of serine/threonine protein kinases (STPKs), unique to gram-positive bacteria, comprise an intracellular kinase domain and reiterated extracellular PASTA (for "penicillin-binding protein and serine/threonine kinase associated") domains. PASTA domains exhibit low affinity for beta-lactam antibiotics that are structurally similar to their likely normal ligands: stem peptides of unlinked peptidoglycan. The PASTA-domain STPKs are found in the actinobacteria and firmicutes and, as exemplified by PknB of Mycobacterium tuberculosis, they are functionally implicated in aspects of growth, cell division, and development. Whereas the kinase domains are well conserved, there is a wide divergence in the sequences of the multiple PASTA domains. Closer inspection reveals position-dependent evolution of individual PASTA domains: a domain at one position within a gene has a close phylogenetic relationship with a domain at a similar position in an orthologous gene, whereas neighboring domains have clearly diverged one from one another. A similar position-dependent relationship is demonstrated in the second family of proteins with multiple PASTA domains: the high-molecular-weight type II penicillin-binding protein (PBP2x) family. These transpeptidases are recruited to the division site by a localized pool of unlinked peptidoglycan. We infer that protein localization is guided by low-affinity interactions between structurally different unlinked peptidoglycan stem peptides and individual PASTA domains. The STPKs possess a greater multiplicity and diversity of PASTA domains, allowing interactions with a wider range of stem-peptide ligands. These interactions are believed to activate the intracellular kinase domain, allowing an STPK to coordinate peptidoglycan remodeling and reproduction of a complex cell wall structure.     

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.     

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.     

Colocalization and interaction between elongasome and divisome during a preparative cell division phase in Escherichia coli

The rod‐shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome, respectively, which catalyse peptidoglycan extension and maturation. Endogenous immunolabelled PBP2 localized in the cylindrical part of the cell as well as transiently at midcell. Using the novel image analysis tool Coli‐Inspector to analyse protein localization as function of the bacterial cell age, we compared PBP2 localization with that of other E. coli cell elongation and division proteins including PBP3. Interestingly, the midcell localization of the two transpeptidases overlaps in time during the early period of divisome maturation. Försters Resonance Energy Transfer (FRET) experiments revealed an interaction between PBP2 and PBP3 when both are present at midcell. A decrease in the midcell diameter is visible after 40% of the division cycle indicating that the onset of new cell pole synthesis starts much earlier than previously identified by visual inspection. The data support a new model of the division cycle in which the elongasome and divisome interact to prepare for cell division.     

Functional redundancy of division specific penicillin‐binding proteins in Bacillus subtilis

Bacterial cell division involves the dynamic assembly of a diverse set of proteins that coordinate the invagination of the cell membrane and synthesis of cell wall material to create the new cell poles of the separated daughter cells. Penicillin‐binding protein PBP 2B is a key cell division protein in Bacillus subtilis proposed to have a specific catalytic role in septal wall synthesis. Unexpectedly, we find that a catalytically inactive mutant of PBP 2B supports cell division, but in this background the normally dispensable PBP 3 becomes essential. Phenotypic analysis of pbpC mutants (encoding PBP 3) shows that PBP 2B has a crucial structural role in assembly of the division complex, independent of catalysis, and that its biochemical activity in septum formation can be provided by PBP 3. Bioinformatic analysis revealed a close sequence relationship between PBP 3 and Staphylococcus aureus PBP 2A, which is responsible for methicillin resistance. These findings suggest that mechanisms for rescuing cell division when the biochemical activity of PBP 2B is perturbed evolved prior to the clinical use of β‐lactams.     

Penicillin‐binding protein folding is dependent on the PrsA peptidyl‐prolyl cis‐trans isomerase in Bacillus subtilis

The PrsA protein is a membrane‐anchored peptidyl‐prolyl cis‐trans isomerase in Bacillus subtilis and most other Gram‐positive bacteria. It catalyses the post‐translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin‐binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross‐linkage index. Misfolded PBP2a was detected in PrsA‐depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.     

Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli

In Escherichia coli, penicillin-binding protein 3 (PBP3), also known as FtsI, is a central component of the divisome, catalyzing cross-linking of the cell wall peptidoglycan during cell division. PBP3 is mainly periplasmic, with a 23 residues cytoplasmic tail and a single transmembrane helix. We have solved the crystal structure of a soluble form of PBP3 (PBP3_57–577) at 2.5 Å revealing the two modules of high molecular weight class B PBPs, a carboxy terminal module exhibiting transpeptidase activity and an amino terminal module of unknown function. To gain additional insight, the PBP3 Val88-Ser165 subdomain (PBP3_88–165), for which the electron density is poorly defined in the PBP3 crystal, was produced and its structure solved by SAD phasing at 2.1 Å. The structure shows a three dimensional domain swapping with a β-strand of one molecule inserted between two strands of the paired molecule, suggesting a possible role in PBP3_57–577 dimerization.     

Functional domain analysis of the Mycoplasma pneumoniae co‐chaperone TopJ

Colonization of conducting airways of humans by the prokaryote Mycoplasma pneumoniae is mediated by a differentiated terminal organelle important in cytadherence, gliding motility and cell division. TopJ is a predicted J‐domain co‐chaperone also having domains unique to mycoplasma terminal organelle proteins and is essential for terminal organelle function, as well as stabilization of protein P24, which is required for normal initiation of terminal organelle formation. J‐domains activate the ATPase of DnaK chaperones, facilitating peptide binding and proper protein folding. We performed mutational analysis of the predicted J‐domain, central acidic and proline‐rich (APR) domain, and C‐terminal domain of TopJ and assessed the phenotypic consequences when introduced into an M. pneumoniae topJ mutant. A TopJ derivative with amino acid substitutions in the canonical J‐domain histidine–proline–aspartic acid motif restored P24 levels but not normal motility, morphology or cytadherence, consistent with a J‐domain co‐chaperone function. In contrast, TopJ derivatives having APR or C‐terminal domain deletions were less stable and failed to restore P24, but resulted in normal morphology, intermediate gliding motility and cytadherence levels exceeding that of wild‐type cells. Results from immunofluorescence microscopy suggest that both the APR and C‐terminal domains, but not the histidine–proline–aspartic acid motif, are critical for TopJ localization to the terminal organelle.     

Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole

The localization of penicillin-binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)-PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot-like pattern and also at mid-cell during cell division. PBP2 disappeared from mid-cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid-cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid-cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.     

Overexpression of a soybean nuclear localized type–III DnaJ domain‐containing HSP40 reveals its roles in cell death and disease resistance

Heat‐shock proteins such as HSP70 and HSP90 are important molecular chaperones that play critical roles in biotic and abiotic stress responses; however, the involvement of their co‐chaperones in stress biology remains largely uninvestigated. In a screen for candidate genes stimulating cell death in Glycine max (soybean), we transiently overexpressed full‐length cDNAs of soybean genes that are highly induced during soybean rust infection in Nicotiana benthamiana leaves. Overexpression of a type‐III DnaJ domain‐containing HSP40 (GmHSP40.1), a co‐chaperone of HSP70, caused hypersensitive response (HR)‐like cell death. The HR‐like cell death was dependent on MAPKKKα and WIPK, because silencing each of these genes suppressed the HR. Consistent with the presence of a nuclear localization signal (NLS) motif within the GmHSP40.1 coding sequence, GFP‐GmHSP40.1 was exclusively present in nuclear bodies or speckles. Nuclear localization of GmHSP40.1 was necessary for its function, because deletion of the NLS or addition of a nuclear export signal abolished its HR‐inducing ability. GmHSP40.1 co‐localized with HcRed‐SE, a protein involved in pri‐miRNA processing, which has been shown to be co‐localized with SR33‐YFP, a protein involved in pre‐mRNA splicing, suggesting a possible role for GmHSP40.1 in mRNA splicing or miRNA processing, and a link between these processes and cell death. Silencing GmHSP40.1 enhanced the susceptibility of soybean plants to Soybean mosaic virus, confirming its positive role in pathogen defense. Together, the results demonstrate a critical role of a nuclear‐localized DnaJ domain‐containing GmHSP40.1 in cell death and disease resistance in soybean.     

Dual localized kinesin‐12 POK2 plays multiple roles during cell division and interacts with MAP65‐3

Kinesins are versatile nano‐machines that utilize variable non‐motor domains to tune specific motor microtubule encounters. During plant cytokinesis, the kinesin‐12 orthologs, PHRAGMOPLAST ORIENTING KINESIN (POK)1 and POK2, are essential for rapid centrifugal expansion of the cytokinetic apparatus, the phragmoplast, toward a pre‐selected cell plate fusion site at the cell cortex. Here, we report on the spatio‐temporal localization pattern of POK2, mediated by distinct protein domains. Functional dissection of POK2 domains revealed the association of POK2 with the site of the future cell division plane and with the phragmoplast during cytokinesis. Accumulation of POK2 at the phragmoplast midzone depends on its functional POK2 motor domain and is fine‐tuned by its carboxy‐terminal region that also directs POK2 to the division site. Furthermore, POK2 likely stabilizes the phragmoplast midzone via interaction with the conserved microtubule‐associated protein MAP65‐3/PLEIADE, a well‐established microtubule cross‐linker. Collectively, our results suggest that dual localized POK2 plays multiple roles during plant cell division.     

Conditional KIAA1549:BRAF mice reveal brain region‐ and cell type‐specific effects

Low‐grade brain tumors (pilocytic astrocytomas) that result from a genomic rearrangement in which the BRAF kinase domain is fused to the amino terminal of the KIAA1549 gene (KIAA1549:BRAF fusion; f‐BRAF) commonly arise in the cerebellum of young children. To model this temporal and spatial specificity in mice, we generated conditional KIAA1549:BRAF strains that coexpresses green fluorescent protein (GFP). Although both primary astrocytes and neural stem cells (NSCs) from these mice express f‐BRAF and GFP as well as exhibit increased MEK activity, only f‐BRAF‐expressing NSCs exhibit increased proliferation in vitro. Using Cre driver lines in which KIAA1549:BRAF expression was directed to NSCs (f‐BRAF; BLBP‐Cre mice), astrocytes (f‐BRAF; GFAP‐Cre mice), and NG2 progenitor cells (f‐BRAF; NG2‐Cre mice), increased glial cell numbers were observed only in the cerebellum of f‐BRAF; BLBP‐Cre mice in vivo. The availability of this unique KIAA1549:BRAF conditional transgenic mouse strain will enable future mechanistic studies aimed at defining the developmentally–regulated temporal and spatial determinants that underlie low‐grade astrocytoma formation in children. genesis 51:708–716. © 2013 Wiley Periodicals, Inc.     

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N‐terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear‐membrane constituents. Results. Binding mutants of LBR—GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild‐type constructs was employed to examine the retention of LBR—GFP in the plant NE. wtLBR—GFP (wild‐type LBR—GFP) was shown to have significantly lower mobility in the NE than the lamin‐binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin‐binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. Conclusions. As expression of truncated LBR—GFP in plant cells results in altered targeting and retention compared with wtLBR—GFP, we conclude that plant cells can recognize the INE (inner NE)‐targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

A role for FtsA in SPOR‐independent localization of the essential Escherichia coli cell division protein FtsN

FtsN is a bitopic membrane protein and the last essential component to localize to the Escherichia coli cell division machinery, or divisome. The periplasmic SPOR domain of FtsN was previously shown to localize to the divisome in a self‐enhancing manner, relying on the essential activity of FtsN and the peptidoglycan synthesis and degradation activities of FtsI and amidases respectively. Because FtsN has a known role in recruiting amidases and is predicted to stimulate the activity of FtsI, it follows that FtsN initially localizes to division sites in a SPOR‐independent manner. Here, we show that the cytoplasmic and transmembrane domains of FtsN (FtsN_Cyto‐TM) facilitated localization of FtsN independently of its SPOR domain but dependent on the early cell division protein FtsA. In addition, SPOR‐independent localization preceded SPOR‐dependent localization, providing a mechanism for the initial localization of FtsN. In support of the role of FtsN_Cyto‐TM in FtsN function, a variant of FtsN lacking the cytoplasmic domain localized to the divisome but failed to complement an ftsN deletion unless it was overproduced. Simultaneous removal of the cytoplasmic and SPOR domains abolished localization and complementation. These data support a model in which FtsA–FtsN interaction recruits FtsN to the divisome, where it can then stimulate the peptidoglycan remodelling activities required for SPOR‐dependent localization.     

AtTRB1, a telomeric DNA‐binding protein from Arabidopsis,is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double‐stranded DNA‐binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N‐terminus, a central H1/H5‐like domain and a C‐terminally located coiled‐coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double‐stranded DNA in vitro, while the central H1/H5‐like domain interacts non‐specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5‐like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle‐regulated, as the level of nuclear‐associated GFP diminishes during mitotic entry and GFP progressively re‐associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell‐cycle progression, with the other involving rapid exchange.     

A functional NR4A nuclear receptor DNA‐binding domain is required for organ development in Caenorhabditis elegans

NR4A nuclear receptors are a diverse group of orphan nuclear receptors with critical roles in regulating cell proliferation and cell differentiation. The ortholog of the NR4A nuclear receptor in Caenorhabditis elegans, NHR‐6, also has a role in cell proliferation and cell differentiation during organogenesis of the spermatheca. Here we show that NHR‐6 is able to bind the canonical NR4A monomer response element and can transactivate from this site in mammalian HEK293 cells. Using a functional GFP‐tagged NHR‐6 fusion, we also demonstrate that NHR‐6 is nuclear localized during development of the spermatheca. Mutation of the DNA‐binding domain of NHR‐6 abolishes its activity in genetic rescue assays, demonstrating a requirement for the DNA‐binding domain. This study represents the first genetic demonstration of an in vivo requirement for an NR4A nuclear receptor DNA‐binding domain in a whole organism. genesis 48:485–491, 2010. © 2010 Wiley‐Liss, Inc.