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.

To clarify the function of DivIVA inStreptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found thatS. pneumoniaeDivIVA (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 thedivIVAnull 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 putativeS. pneumoniaecell division proteins. Finally, a missensedivIVAmutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of thedivIVAmutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA inS. pneumoniaeseems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVA_SPNraise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.

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. IMPORTANCE Bacterial cell division is a highly ordered process regulated in time and space. Recently, we reported that the Ser/Thr protein kinase StkP regulates cell division in Streptococcus pneumoniae, through phosphorylation of several key proteins. Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ. We show that LocZ is a new cell division protein important for proper septum placement and likely functions as a marker of the cell division site. Consistently, LocZ supports proper Z-ring positioning at midcell. LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.

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.