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.

To clarify the function of DivIVA in Streptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found that S. pneumoniae DivIVA (DivIVA SPN ) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation. All the other cell division proteins that we tested were localized in the divIVA null mutant, although the percentage of cells having constricted Z rings was significantly reduced. In agreement with its localization profile and consistent with its coiled-coil nature, DivIVA interacted with itself and with a number of known or putative S. pneumoniae cell division proteins. Finally, a missense divIVA mutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of the divIVA mutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA in S. pneumoniae seems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVA SPN raise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.A number of cell division proteins have been identified in Streptococcus pneumoniae and have been shown to localize at midcell to form the septal machinery (the septosome or divisome), consistent with what is known about the best-characterized rod-shaped model organisms, Escherichia coli and Bacillus subtilis (for recent reviews, see references 12, 16, 49, and 50).These proteins include the cell division initiator proteins FtsZ and FtsA, which are required at the early stages of the process (25,29,32), and some of the later proteins, DivIB/ FtsQ, DivIC/FtsB, FtsL, FtsW, PBP 2X, and PBP 1A (29,32,33,38), which are the septal markers for S. pneumoniae cells. Recent studies have confirmed that, overall, the major events in septation are conserved in S. pneumoniae. However, other aspects related to the division process, such as the associated morphological changes, the correct choice of the division site, and proper chromosome segregation, and the factors that regulate these aspects remain largely unknown.We have described characterization of a chromosome region in S. pneumoniae, downstream of the ftsZ gene, that is well conserved among gram-positive bacteria and is physically and transcriptionally related to the division and cell wall (dcw) cluster. We showed that functional inactivation of each of the five genes in the region resulted in defects in cell morphology, chromosome segregation, and/or cell division (13), and the importance of these genes in other species has been confirmed (18,23,30). In S. pneumonia...

We analyzed the chromosome region of Streptococcus pneumoniae located downstream of the division and cell wall (dcw) cluster that contains the homolog of the Bacillus subtilis cell division gene divIVA and some genes of unknown function. Inactivation of divIVA in S. pneumoniae resulted in severe growth inhibition and defects in cell shape, nucleoid segregation, and cell division. Inactivation of the ylm genes resulted in some morphological and/or division abnormalities, depending on the inactivated gene. Transcriptional analysis revealed a relationship between these genes and the ftsA and ftsZ cell division genes, also indicating that the connection between the dcw cluster and the divIVA region is more extensive than just chromosomal position and gene organization.Historically, most of the available information about bacterial cell division comes from the intensively studied gramnegative rod Escherichia coli and the gram-positive rod Bacillus subtilis (reviewed in references 1, 9, 10, 15, 17, and 24). This situation is changing, due to both the increasing interest in the field of cell division and the availability of genomic data. Most of the cell division genes have now been identified in a wide range of bacteria, and despite the differences observed among species, many of these genes are found organized in a chromosomal region corresponding to the 2-min region of the E. coli chromosome known as the dcw (for "division and cell wall") cluster.Notwithstanding the large amount of sequence information, very little is known about the molecular mechanism that regulates cell division in bacteria other than the model organisms and cell division in gram-positive cocci remains poorly understood. However, some extrapolations from the analysis of the Fts proteins that are essential for cell division, in particular the FtsZ protein, the major component of the septal ring structure (15), suggest that the basic mechanism involved in septum formation should not differ from cocci to rods.A more intriguing problem and one that shows significantly less conservation is how different bacteria operate the division site selection mechanism that ensures correct FtsZ positioning at midcell. In E. coli, the correct division site at the center is distinguished from the other potential division sites at the poles through the combined action of three proteins, MinC, MinD, and MinE, encoded by the minCDE genes (5, 6, 21).In B. subtilis, divIVB encodes the MinC and MinD homologs but not the MinE homolog (13, 26). The DivIVA protein has been proposed to play a role in the control of division site selection as the functional counterpart of the missing MinE in B. subtilis (3,7,18,19). Recently, we identified a Streptococcus pneumoniae gene encoding the B. subtilis DivIVA homolog (20). We noted that divIVA is the last gene of a region located downstream of the cell division genes ftsA to ftsZ and is conserved in the same position in a number of gram-positive cocci. In addition to divIVA, the region contains some open reading frames of unknown fu...

This analysis of a large European dataset confirms a role of occupational exposure to solvents in the aetiology of B-NHL, and particularly, CLL. It is suggested that benzene is most likely to be implicated, but we cannot exclude the possibility of a role for other solvents in relation to other lymphoma subtypes, such as follicular lymphoma. No association with risk of T-cell lymphoma and Hodgkin's lymphoma was shown.