How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae , StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

SUMMARY 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.

SummaryStreptococcus pneumoniae is an oval-shaped Grampositive coccus that lives in intimate association with its human host, both as a commensal and pathogen. The seriousness of pneumococcal infections and the spread of multi-drug resistant strains call for new lines of intervention. Bacterial cell division is an attractive target to develop antimicrobial drugs. This review discusses the recent advances in understanding S. pneumoniae growth and division, in comparison with the best studied rod-shaped models, Escherichia coli and Bacillus subtilis. To maintain their shape, these bacteria propagate by peripheral and septal peptidoglycan synthesis, involving proteins that assemble into distinct complexes called the elongasome and the divisome, respectively. Many of these proteins are conserved in S. pneumoniae, supporting the notion that the ovococcal shape is also achieved by rounds of elongation and division. Importantly, S. pneumoniae and close relatives with similar morphology differ in several aspects from the model rods. Overall, the data support a model in which a single large machinery, containing both the peripheral and septal peptidoglycan synthesis complexes, assembles at midcell and governs growth and division. The mechanisms generating the ovococcal or coccal shape in lactic-acid bacteria have likely evolved by gene reduction from a rod-shaped ancestor of the same group.

SUMMARY Suppressor mutations were isolated that obviate the requirement for essential PBP2b in peripheral elongation of peptidoglycan from the midcells of dividing Streptococcus pneumoniae D39 background cells. One suppressor was in a gene encoding a single KH-domain protein (KhpA). ΔkhpA suppresses deletions in most, but not all (mltG), genes involved in peripheral PG synthesis and in the gpsB regulatory gene. ΔkhpA mutations reduce growth rate, decrease cell size, minimally affect shape, and induce expression of the WalRK cell-wall stress regulon. Reciprocal co-immunoprecipitations show that KhpA forms a complex in cells with another KH-domain protein (KhpB/JAG/EloR). ΔkhpA and ΔkhpB mutants phenocopy each other exactly, consistent with a direct interaction. RNA-immunoprecipitation showed that KhpA/KhpB bind an overlapping set of RNAs in cells. Phosphorylation of KhpB reported previously does not affect KhpB function in the D39 progenitor background. A chromosome duplication implicated FtsA overproduction in Δpbp2b suppression. We show that cellular FtsA concentration is negatively regulated by KhpA/B at the post-transcriptional level and that FtsA overproduction is necessary and sufficient for suppression of Δpbp2b. However, increased FtsA only partially accounts for the phenotypes of ΔkhpA mutants. Together, these results suggest that multimeric KhpA/B may function as a pleiotropic RNA chaperone controlling pneumococcal cell division.

An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-,I-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing I-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the I-lactamase II of Bacillus cereus and the CfiA I-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-,I-lactamases.In recent classification schemes for ,B-lactamases, a distinct grouping has been reserved for those enzymes requiring a metal cofactor for activity (metallo-1-lactamases) (1, 4, 5). The prototype for this group of enzymes is the 1-lactamase II of Bacillus cereus, which has long been studied as a model of metallo-13-lactamases and for which molecular sequence data, along with enzymologic and crystallographic data, are available (3,12,17,27). In the molecular classification of 1-lactamases, a class B was devised for this enzyme (1).Interest in this group of enzymes has grown recently since an increasing number of metallo-1-lactamases have been described in several gram-negative species which share the important characteristic of being able to hydrolyze carbapenem compounds (2,7,9,22,25,26,31). Carbapenems are new P-lactam antibiotics of great therapeutic potential, since they are not hydrolyzed by most bacterial 3-lactamases and show a broad spectrum of activity.Information currently available on carbapenem-hydrolyzing (CH) metallo-1-lactamases is limited, in most cases, to a few biochemical features and suggests the existence of different molecular species within this group of enzymes (5). Molecular sequence data are presently available only for the CfiA P-lactamase of Bacteroides fragilis TAL2480 (28) and for the ,B-lactamase II of B. cereus 569/H (12) and 5/B/6 (17). A strong sequence similarity exists between these two enzymes (28), suggesting that the CfiA enzyme also belongs to molecular class B...

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner.

SummaryWe studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae . FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis , FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae , we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro , in a nucleotide-dependent manner, forming long corkscrew-like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.

How bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement in Streptococcus pneumoniae. We show that locZ is not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division.