Streptococcus pneumoniaeDivIVA: Localization and Interactions in a MinCD-Free Context

Abstract

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...

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

“…Markerless deletions of the division genes, mapZ (alternative name, locZ) and divIVA, fused short (20-amino acid) stretches from the beginning and end of the genes to maintain possible translational coupling (see Tables S1 and S2). The phenotypes of the markerless ⌬mapZ and ⌬divIVA mutants were similar to those reported previously for other mapZ and divIVA mutants (41)(42)(43).…”

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

“…FtsA, together with FtsZ, is one of the earliest proteins to localize at the division sites, whereas DivIVA arrives somewhat later (43,44). Interestingly, we now have shown that StkP phosphorylates DivIVA and FtsA in vitro (Fig.…”

“…As in the model organisms E. coli and B. subtilis, FtsZ and FtsA are the first proteins to arrive at midcell in S. pneumoniae and form the Zring before cell division initiates (47,48). Once formed, the Zring recruits the later cell-division proteins such as the membrane connectors, DivIVA, and the enzymes required for septal cell-wall synthesis [PBPs, likely PBP2x and PBP1a (4,43)]. However, before septation can proceed in oval cells such as S. pneumoniae, peripheral cell-wall synthesis must occur (1).…”

“…Where relevant, Nile red (Invitrogen) was added to a final concentration of 8 ng/mL Phase contrast images were segmented automatically and analyzed using Microbetracker (63), and cell-length distributions were plotted using MATLAB. Immunofluorescence experiments were carried out as previously described (43). Van-FL staining was performed as recently described (58).…”

Pneumococcal meningitis is promoted by single cocci expressing pilus adhesin RrgA

“…The pneumococcal protein DivIVA is one of a number of membrane proteins that orchestrate and fine tune cell wall synthesis during septal growth and cell separation and localize to the division septum and to the poles of cocci (21,22). It has been proposed that DivIVA tunes the 2 modes of peptidoglycan (peripheral and septal) synthesis in pneumococci and is responsible for the cell's ovoid shape (22).…”