Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division
[['Bacterial cell growth and division are intimately linked .', 'Complex webs of proteins interacting with each other temporally and spatially control the cellular events leading to the production of two identical daughter cells [1]–[3] .', 'Most of the proteins required for cell division and elongation have been characterized in rod-shaped bacterial models like the Gram-negative bacteria Escherichia coli and Caulobacter crescentus or the Gram-positive bacterium Bacillus subtilis , and robust models depicting their division process are proposed .', 'This knowledge has been beneficial for characterizing and understanding cell division of other bacteria .', 'However , some aspects related to cell division , including the achievement of cell shape , are often hardly transposable and species-specific mechanisms exist to allow cells to divide , assume a given shape and/or cope with their environment [4] , [5] .', 'In the Gram-positive human pathogen Streptococcus pneumoniae ( the pneumococcus ) , some conserved division proteins have been studied , but overall , little is known about the mechanisms governing cell division and those responsible for peptidoglycan ( PG ) synthesis , as well as how this species achieves its characteristic ellipsoid ( rugby-ball like ) shape [6]–[10] .', 'Early studies have suggested that S . pneumoniae utilizes a combination of two PG synthesis modes , namely septal and peripheral [11] .', 'Due to the absence of the actin-like protein MreB and any homologues in the pneumococcus , it is speculated that these two modes of PG synthesis are coordinated with and organized by FtsZ-ring formation [12] .', 'A two-state model in which two different PG synthesis machineries are responsible for either septal or peripheral synthesis has been proposed .', 'In this model , the PG transpeptidase PBP2x , a penicillin binding protein ( PBP ) that catalyzes PG cross-linking , and the lipid-flippase FtsW , that transports lipid-linked PG precursors from the inner to the outer leaflet of the cytoplasmic membrane , belong to a septal machinery and are exclusively required for septal PG synthesis and cell separation .', 'On the other hand , the transpeptidase PBP2b and the lipid-flippase RodA would be exclusively associated with a peripheral machinery , and required for peripheral PG synthesis and cell elongation .', 'However , it is unclear how S . pneumoniae would coordinate peripheral and septal synthesis .', 'An interesting possibility comes from work in B . subtilis showing that cell elongation-division cycles are controlled by shuttling of PBP1 , a transpeptidase/transglycosidase class A PBP involved in peptidoglycan polymerization [13] .', 'PG synthesis could thus be fine-tuned by a yet uncharacterized process to allow the alternate synthesis of septal and peripheral PG in pneumococcus .', 'StkP , a membrane eukaryotic-type serine/threonine kinase , represents an attractive candidate to regulate septal and peripheral PG synthesis in S . pneumoniae .', 'This kinase has been recently shown to play an important role in pneumococcal cell division and growth [14]–[16] .', 'In vivo , only a few proteins appear specifically phosphorylated by StkP [14] , [17] , [18] .', 'Among them , it is noteworthy to find the division protein DivIVA , which was shown to be phosphorylated by StkP on Thr-201 .', 'Expression of the non-phosphorylatable form of DivIVA ( i . e . , a mutant in which Thr-201 is substituted for an alanine ) induced severe defects in cell shape , possibly by affecting pole maturation [14] .', 'Interestingly , a DivIVA paralog , named GpsB [19] , was identified in B . subtilis and shown to be involved together with EzrA in PBP1 shuttling between elongation and division sites [13] .', 'Global phosphoproteome analyses of B . subtilis and Streptococcus agalactiae indicated that GpsB is phosphorylated in these species [20] , [21] .', 'GpsB is also found in S . pneumoniae , as in most Firmicutes .', 'This situation prompted us to investigate the role of the two paralogs , GpsB and DivIVA , in PG synthesis and cell morphogenesis of S . pneumoniae , and to examine whether their phosphorylation by StkP could affect their role .', 'Here , we establish that GpsB and DivIVA are both crucial for cell morphogenesis , and demonstrate that DivIVA is necessary for cell elongation whereas GpsB acts as a negative regulator of DivIVA to prevent cell elongation .', 'Moreover , we show that GpsB is not phosphorylated , but required for StkP septal localization and subsequent phosphorylation of DivIVA .', 'In light of these observations , we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of PG synthesis to achieve the ovoid shape of pneumococci and we discuss the relevance of this process in other bacteria .', 'Our observation of similar localization patterns for the transpeptidases PBP2x and PBP2b as well as for the lipid-flippases FtsW and RodA in cells deficient for GpsB and/or DivIVA questions the existence of two distinct PG biosynthesis machineries .'], ['To analyze the potential role of DivIVA in pneumococcal morphogenesis , we constructed a nonpolar , markerless divIVA-null mutant and investigated its cell morphology .', 'As previously reported for the S . pneumoniae RX1 strain [6] , [22] , 99 . 8% of ΔdivIVA R800 cells exhibited a striking chain phenotype ( Figure 1A and Table S1 ) .', 'When the divIVA mutation was repaired back to wild type ( WT ) by transformation , the morphology of the resulting strain was similar to that of the WT strain with a typical diplo-ovococcal shape ( compare Figure 1A with Figure S1 ) indicating that the chain phenotype is due to the deletion of divIVA .', 'ΔdivIVA chains contained up to several dozen of tightly joined cells separated by well-defined membranes ( Figures 1A and S2A ) .', 'Cells were clearly not ovoid but flattened at the poles , exhibiting a rounded shape .', 'Analysis of individual cell parameters further confirmed this visual impression and showed that divIVA deletion resulted in reduced pneumococcal cell length ( Figure S2B and Table S1 ) .', 'We also examined ΔdivIVA cells by scanning- and transmission-electron microscopy ( SEM and TEM ) .', 'Using SEM , cells seemed to be interlocked into the neighboring ones ( Figure 1B ) .', 'Nevertheless , TEM indicated that cells were clearly separated by membranes , consistent with efficient Z-ring constriction and closure , and suggesting that septal PG is efficiently produced ( Figure 1C ) .', 'To confirm the latter , we applied the strategy described by Kuru and co-workers [23] and PG synthesis was visualized using Bodipy-FL containing fluorescent D-amino acid , namely Bodipy-FL-amino-D-alanine , or BADA [24] .', 'More specifically , the exponentially growing cells are pulsed with BADA for 4 min corresponding to ca .', '10–12% of the generation times of the WT and the mutants .', 'As a control , we checked that BADA labeled the division site in WT cells as previously described using fluorescent vancomycin [14] ( Figure 2 ) .', 'BADA labeling of ΔdivIVA cells revealed PG synthesis localizing exclusively as bands across the cells at the division septa in 99 . 4% of cells ( Figure 2 and Table S2 ) .', 'Altogether , these results suggest that in the absence of DivIVA , cell elongation is hampered while septum closure still occurs .', 'On the other hand , the last step of cell division allowing the final separation of daughter cells is affected .', 'To unravel the role of GpsB in S . pneumoniae , we first constructed a nonpolar markerless deletion mutant of gpsB in strain R800 .', 'In contrast to the deletion of divIVA , the deletion of gpsB severely affected growth ( Figure S3A ) and cell viability decreased to 60% suggesting that GpsB is crucial for the pneumococcus .', 'Microscopy analysis revealed a striking phenotype characterized by the presence of very elongated cells ( Figures 1A , S3B and Table S1 ) .', 'Morphometric measurements indicated that the length was below 1 . 4 µm for 90% of wild-type cells ( WT ) , whereas nearly 90% of ΔgpsB cells exhibited length greater than 1 . 3 µm .', 'ΔgpsB cells seemed irregularly shaped and septal membranes across cells were lacking , indicating that cell constriction was seriously hampered ( Figure 1A ) .', 'Examination of the ultrastructure of ΔgpsB cells by TEM and SEM confirmed that mutant cells displayed a strongly affected morphology with irregular width ( Figures 1B–C ) .', 'The presence of several septal initiations positioned asymmetrically on each side of the long axis of the cells was detected by TEM ( Figure 1C ) .', 'SEM images confirmed an irregular elongation of ΔgpsB cells which displayed a “twisted-towel” shape ( Figure 1B ) .', 'One could further observe the presence of a helical groove at the surface of the cells that seemed to correspond to the asymmetric septal initiations detected by TEM , suggesting that the divisome is stretched upon cell elongation .', 'Importantly , a wild-type diplo-ovococcal shape and normal growth were restored when the ΔgpsB strain was transformed back to gpsB+ confirming that the observed phenotype resulted from the inactivation of gpsB ( compare Figure 1A with Figure S1 ) .', 'Deletion of gpsB was also attempted into four other well-characterized and widely used S . pneumoniae strains , the encapsulated D39 and TIGR4 , and the unencapsulated R6 and RX1 .', 'The same elongated phenotype was observed with gpsB− derivatives of the unencapsulated strains , while our efforts to delete gpsB failed with both encapsulated strains ( Figure S3C ) .', 'The latter observation is consistent with the recent report of Land and co-workers [25] .', 'Altogether , these data show that deletion of gpsB triggers cell elongation and prevents proper pneumococcal cell division .', 'We then analyzed the effect of the deletion of gpsB on FtsZ localization .', 'For this purpose , we first constructed a C-terminal GFP fusion to FtsZ .', 'Throughout this study and unless otherwise indicated , C-terminal and N-terminal fusions ( denoted respectively Protein-GFP and GFP-Protein ) were constructed at each native chromosomal locus , expressed under the control of the native promoter and represented the only source of protein .', 'The FtsZ-GFP fusion seemed fully functional as cells grew as rapidly as WT cells and did not display any shape defect ( Figures 3A and S4A ) .', 'As expected , FtsZ-GFP localized at midcell in exponentially grown WT cells as well as in ΔdivIVA rounded cells ( Figure 3A and Table S2 ) .', 'FtsZ-GFP also appeared as several transversal bands in a majority of ΔgpsB cells .', 'Interestingly however , a number of cells ( 25 . 2% ) displayed a zig-zag localization of FtsZ ( Figures 3A , S5 and Table S2 ) .', 'To clarify the dimensional nature of this zig-zag structure , we carried out deconvolution microscopy ( Movie S1 ) that revealed a continuous FtsZ-helical organization .', 'This unexpected helical localization of FtsZ in ΔgpsB cells was confirmed by immunostaining using anti-FtsZ antibodies ( Figure 3B ) indicating that the helical pattern is not an artifact due to the GFP tag on FtsZ .', 'Control experiments also confirmed that FtsZ levels were unaffected in the absence of GpsB ( Figure S4B ) .', 'To reconcile the dual FtsZ localization ( spirals vs . bands ) in ΔgpsB elongated cells , time-lapse microscopy was performed .', 'During cell elongation , some Z-rings were replaced by helical structures which eventually split to generate several new Z-rings , whereas others continued to stretch ( Figure 3C and Movie S2 ) .', 'However , in both cases cells ended up bursting and dying ( e . g . , cells marked with arrows in Figure 3C ) .', 'We conclude from these observations that the Z-ring is replaced by a helical structure during elongation of ΔgpsB cells .', 'To test whether cell elongation in ΔgpsB cells was accompanied by altered localization of PG synthesis , the latter was labeled using BADA .', 'BADA labeling revealed a helical organization of neosynthesized PG in 28 . 7% of ΔgpsB elongated cells ( Figure 2 and Table S2 ) , which is comparable to the percentage of cells exhibiting GFP-FtsZ spirals .', 'This prompted us to examine the localization of PBP2x , PBP2b , FtsW and RodA , which are involved in PG synthesis , using GFP-PBP2x , GFP-PBP2b , FtsW-GFP or RodA-GFP .', 'All four GFP-fused proteins were functional as cells grew normally and displayed WT shape ( Figures 4A and S6A ) .', 'Fluorescence microscopy indicated that PBP2x , PBP2b , FtsW , and RodA localize at midcell in WT cells as well as in ΔdivIVA cells ( Figures 4A and 4C ) .', 'Strikingly , they all appeared mislocalized in elongated ΔgpsB cells , exhibiting a helical pattern ( Figure 4B and Table S2 ) reminiscent of that observed for FtsZ ( Figure 3A ) and PG synthesis ( Figure 2 ) .', 'Western blot control experiments confirmed that the four GFP-fusions were produced at similar levels in WT , ΔdivIVA and ΔgpsB cells , excluding any artifact due to aberrant protein expression ( Figure S7 ) .', 'These helical patterns suggested that the four GFP-proteins could co-localize with FtsZ .', 'To directly assess this , we constructed double-labeled strains containing FtsZ fused to RFP and either PBP2x , PBP2b , FtsW , or RodA fused to GFP .', 'Cells containing a pair of fusion proteins in an otherwise WT background exhibited a growth delay ( Figure S6B ) indicating that the combination of FtsZ-RFP with GFP-fused PBP2x , PBP2b , FtsW , or RodA is somehow detrimental .', 'Nevertheless , microscopy analyses indicated that cell shapes were normal and that each pair of RFP/GFP-fusions co-localized properly at midcell ( Figure 5A ) .', 'On the other hand , when we detected helical RFP-FtsZ in ΔgpsB elongated cells , PBP2x , PBP2b , FtsW and RodA also displayed an helical organization co-localizing with helical FtsZ ( Figure 5B ) .', 'To examine DivIVA localization in ΔgpsB elongated cells , we first generated a WT strain producing DivIVA-GFP .', 'WT cells expressing DivIVA-GFP grew normally and displayed a classic ovoid-shape ( Figures 6A and S8A ) establishing that the DivIVA-GFP fusion is functional .', 'In agreement with a previous report [15] , DivIVA-GFP localized at both midcell and the cell poles ( Figure 6A ) .', 'By contrast , DivIVA-GFP exhibited a helical organization in 20 . 1% of elongated ΔgpsB cells ( Figure 6A and Table S2 ) .', 'This phenotype was not due to an aberrant expression of DivIVA-GFP since western blot analyses confirmed that the fusion protein was synthesized at similar levels in WT and ΔgpsB cells ( Figure S8B ) .', 'DivIVA localization was thus comparable to that of FtsZ in ΔgpsB cells ( Figure 3A ) suggesting that the two proteins co-localize during cell elongation .', 'To confirm this , we constructed WT and ΔgpsB strains expressing both DivIVA-GFP and FtsZ-RFP .', 'As expected , FtsZ and DivIVA displayed respectively septal and septal/polar localization in WT cells ( Figure 6B ) .', 'In ΔgpsB cells , DivIVA co-localized with FtsZ in a helical pattern ( Figure 6B ) .', 'To further investigate the role of GpsB and DivIVA , we introduced the divIVA deletion in cells deficient for gpsB .', 'The double mutant was readily obtained .', 'FM4–64 membrane staining showed that ΔdivIVAΔgpsB cells exhibited the same cell shape and chain phenotype as ΔdivIVA cells ( 97 . 9% of cells ) , although a few cells were irregularly shaped ( Figure 1A and Table S1 ) , indicating that inactivation of divIVA suppressed the elongated cell phenotype typical of ΔgpsB cells ( the same was observed with a double mutant constructed by introducing the ΔgpsB mutation into ΔdivIVA cells; data not shown ) .', 'However , cell viability decreased to 60% and was comparable to that of ΔgpsB cells .', 'As controls , FtsZ-GFP still localized at the division septa in the absence of DivIVA and GpsB , and was produced to WT levels ( Figure 3A , Table S2 and compare Figures S4B with S9B ) .', 'These observations suggest that GpsB interplays with DivIVA to coordinate cell elongation and cell division , and that GpsB is dispensable for septal PG synthesis when DivIVA is absent .', 'To further test this hypothesis , we analyzed BADA labeling of PG in ΔdivIVAΔgpsB cells .', 'We observed that PG is produced properly at the division site ( Figure 2 and Table S2 ) .', 'Likewise , we analyzed the localization of PBP2x , PBP2b , FtsW and RodA in ΔdivIVAΔgpsB cells ( Figure 4D and Table S2 ) .', 'All of them still localized to the division septa as observed in ΔdivIVA cells ( Figure 4C and Table S2 ) .', 'Western blot control experiments confirmed that the four GFP-fused proteins were produced at similar levels in WT and ΔdivIVAΔgpsB cells ( Figure S7 ) .', 'These observations indicate that deletion of gpsB is tolerated in a ΔdivIVA mutant without inducing further detectable cell shape and septum closure defects and does not impair PG synthesis at the division site .', 'To gain an insight into a possible connection between GpsB , DivIVA and cell division , we first looked for physical interactions between these proteins and FtsZ using a bacterial two-hybrid screen [26] .', 'Neither GpsB nor DivIVA were found to interact with FtsZ ( Figure S10A ) .', 'However , as the cell division protein EzrA was found to bridge GpsB with the Z-ring in B . subtilis [13] , we also analyzed EzrA interactions with GpsB , DivIVA and FtsZ .', 'Reproducible interactions were detected between EzrA and either GpsB , DivIVA or FtsZ by bacterial two-hybrid assays ( Figure S10A ) .', 'These interactions were further analyzed by surface plasmon resonance ( SPR ) , which confirmed that EzrA interacts with GpsB ( KD\u200a=\u200a770±230 nM ) , DivIVA ( KD\u200a=\u200a530±75 nM ) and FtsZ ( KD\u200a=\u200a295±60 nM ) ( Figures S10B and S10C–E ) .', 'We also tested whether GpsB interacts with DivIVA .', 'Reproducible interactions were first detected with the two-hybrid screen ( Figure S10A ) and SPR confirmed that GpsB interacts with DivIVA ( KD\u200a=\u200a85±14 nM ) ( Figures S10B and S10F ) .', 'We then analyzed the localization of EzrA and GpsB fused to GFP .', 'WT cells producing GFP-GpsB or GpsB-GFP appeared elongated and displayed aberrant cell shapes indicating that both fusions were not fully functional ( Figure S11 ) .', 'We therefore constructed a merodiploid strain carrying an ectopic gfp-gpsB fusion under the control of the zinc-inducible PZn promoter at the non-essential bgaA locus .', 'Fluorescence microscopy indicated that GFP-GpsB localizes as bands across the short axis of the cells at the division septum in WT cells and the same observation was made in ΔdivIVA rounded cells ( Figure 7A and Table S2 ) .', 'By contrast , while EzrA-GFP localized at midcell in exponentially grown WT cells as well as in ΔdivIVA and ΔdivIVAΔgpsB cells , EzrA-GFP formed helical structures that extended across the long axis of the cell in 19 . 9% of ΔgpsB elongated cells ( Figure 7B and Table S2 ) as was found for FtsZ-GFP and DivIVA-GFP ( Figures 3A and 6 ) .', 'Western blot control experiments confirmed that GFP-GpsB and EzrA-GFP were produced at similar levels in WT and ΔdivIVA ( and ΔgpsB and ΔdivIVAΔgpsB cells for EzrA ) ( Figures S8B and S9 ) .', 'These observations are consistent with EzrA serving as a connector between FtsZ and GpsB and/or DivIVA .', 'The elongated phenotype with incomplete septa displayed by ΔgpsB cells was reminiscent of that reported for cells expressing the kinase-dead form of StkP and suggested a relationship between these two proteins [14] , [15] .', 'Hence , we hypothesized that GpsB could be phosphorylated by StkP in S . pneumoniae .', 'The general phosphorylation pattern of crude extracts of pneumococcal cells was thus analyzed using anti-phosphothreonine antibodies .', 'We detected an intense phosphorylation signal around 15 kDa , which could be compatible with the phosphorylation of GpsB ( 13 kDa ) ( Figure 8A ) .', 'Therefore , GpsB from S . pneumoniae cells was purified to examine its in vivo phosphorylation state using high-resolution based mass spectrometry ( Figures S12A–B ) .', 'No phosphorylated sites were detected suggesting that GpsB is not phosphorylated in vivo .', 'To confirm this result , we analyzed the phosphorylation pattern of cells expressing only GFP-GpsB .', 'An intense phosphorylation signal at 15 kDa was still detected and no new phosphorylation signal appeared around 45 kDa , the predicted mass of GFP-GpsB ( Figure 8A ) , though GFP-GpsB was efficiently stained with anti-GFP antibodies ( Figure 8B ) .', 'Altogether , these data show that GpsB is not phosphorylated in vivo and that the 15-kDa phosphorylation signal corresponds to another unidentified protein .', 'In parallel , we analyzed the phosphorylation pattern of ΔgpsB cells using anti-phosphothreonine antibodies .', 'Surprisingly , the deletion of gpsB abolished not only the phosphorylation of all the substrates of StkP , including DivIVA , but also StkP autophosphorylation itself ( Figure 8A ) .', 'Nevertheless , StkP was expressed at similar levels in WT and ΔgpsB cells ( Figure S12C ) .', 'Furthermore , SPR analysis showed that GpsB was able to interact with the inactive cytoplasmic domain of StkP-K42R ( KD\u200a=\u200a500±60 nM ) ( Figure S10G ) .', 'These data raised the question of whether GpsB could affect StkP septal localization [14] , [15] ( Figure 8C ) .', 'Therefore , we constructed a ΔgpsB mutant harboring a GFP-StkP fusion [14] .', 'Fluorescence microscopy revealed an intense signal distributed all around the cell , consistent with a diffuse membrane localization of GFP-StkP ( Figure 8C ) .', 'StkP localization and general phosphorylation patterns of ΔdivIVA and ΔdivIVAΔgpsB cells were also analyzed .', 'While StkP localized to midcell and was able to phosphorylate its targets in the absence of DivIVA , a deficient phosphorylation pattern as well as a StkP diffuse membrane localization were observed in the double mutant ( Figures 8A and 8C ) .', 'In both mutant strains , StkP was produced to similar levels as in WT cells ( Figure S12C ) .', 'These data establish that , although GpsB is not phosphorylated , it is crucial for StkP septal localization and its capacity to autophosphorylate and phosphorylate its substrates , notably DivIVA .', 'By contrast , DivIVA is not required for StkP kinase activity and localization .', 'Because GpsB is required for StkP septal localization and thus phosphorylation of DivIVA , we investigated the impact of either gpsB or divIVA deletion on the elongated morphology of cells producing the kinase-dead form of StkP , StkP-K42M [14] .', 'As shown in Figure 8D , ΔgpsB-stkP-K42M cells still displayed an elongated phenotype supporting the idea that the main function of GpsB is to allow StkP to phosphorylate its targets .', 'By contrast , the deletion of divIVA was found to abrogate stkP-K42M cell elongation .', 'Indeed , ΔdivIVA-stkP-K42M cells formed chains of rounded cells similar to those of the ΔdivIVAΔgpsB mutant ( compare Figure 8D with Figure 1A ) .', 'This observation confirms that DivIVA is crucial for cell elongation and the late step of cell separation , and further suggests that its non-phosphorylation likely results in aberrant elongation of stkP-K42M and ΔgpsB cells .'], ['According to the current model of PG synthesis in pneumococcus , cell elongation is due to the peripheral PG synthesis .', 'Our observations suggest that peripheral PG is impaired in ΔdivIVA cells ( Figures 1 and S2 ) .', 'In addition , we show that DivIVA co-localizes with FtsZ in ΔgpsB elongated cells and that divIVA deletion suppresses ΔgpsB cell elongation ( Figures 1A and 6B ) .', 'Therefore , we propose that one function of DivIVA is to switch from septal to peripheral PG synthesis to trigger cell elongation .', 'DivIVA performs quite different functions in B . subtilis and Staphylococcus aureus [27] , but it has already been shown to participate in cell wall biosynthesis in bacteria such as Streptomyces coelicolor , Mycobacterium tuberculosis and Corynebacterium glutamicum that either lack or do not require MreB for their vegetative growth .', 'In these bacteria , DivIVA is required for polar growth allowing tip extension and cell elongation [28]–[30] .', 'Pneumococcus is devoid of MreB and any identifiable homologues .', 'Altogether , these observations raise the possibility that DivIVA is crucial for cell elongation in those species in which vegetative growth is not dependent on MreB .', 'The chaining displayed by ΔdivIVA cells also suggests that while septum closure leading to the separation of the daughter cell cytoplasms is normal , their final separation is somehow affected , as previously reported for the RX1 strain [6] .', 'DivIVA has been previously found to interact or to contribute to the positioning of some PG hydrolases in the pneumococcus [31] , [32] or in autolysin secretion in other bacteria as Listeria monocytogenes [33] .', 'The chain phenotype displayed by the ΔdivIVA mutant is consistent with impairment of PG hydrolysis and remodeling required for final separation of daughter cells .', 'Previous studies using high-throughput gene disruption approaches have suggested that gpsB could be essential in pneumococcus [34]–[36] .', 'In this study , we show that GpsB is actually not essential for pneumococcal laboratory strains ( Figures 1 and S3 ) .', 'However , and in agreement with the previous observations , no ΔgpsB transformants could be obtained with the pathogenic strains D39 and TIGR4 indicating that the requirement for GpsB depends on the genetic background .', 'The recent work of Land and co-workers also suggests that suppressive mutations are required for growth of unencapsulated derivatives of pathogenic strains expressing low level of GpsB [25] .', 'The conditional essentiality of GpsB is reminiscent of the situation with MreC and MreD .', 'These proteins are essential in D39 and TIGR4 pathogenic strains but not in the R6 laboratory strain due to suppressive mutations in PBP1a and in proteins of unknown function in the latter [9] .', 'Inactivation of gpsB resulted in severely impaired cell division , with a large fraction of the population appearing as elongated cells with incomplete septa similar to cells producing the kinase-dead form of StkP [14] .', 'This phenotype is accompanied by helical patterns for PG synthesis and FtsZ , along the long axis of the cell ( Figures 2 and 3 ) .', 'Interestingly , Z-spiraling was not observed in the work published by Land and co-workers [25] .', 'Rather , the authors detected multiple non-constricted rings of FtsZ in elongated cells .', 'Because GpsB expression was under the control of an inducible fucose promoter , we tentatively attribute the absence of Z-spiraling to low level of gpsB expression in absence of fucose , likely preserving Z-ring formation in elongated cells .', 'This hypothesis is consistent with our time-lapse analysis showing Z-spiraling upon cell elongation , eventually splitting to generate several new Z-rings ( Figure 3C and Movie S2 ) .', 'The stimulation of cell elongation and aberrant helical organization of the divisome observed in our study in ΔgpsB cells suggest that GpsB is a negative regulator of cell elongation in WT cells .', 'On the other hand , the deletion of gpsB has no effect on cell shape and septum closure in the absence of DivIVA ( Figure 1A ) , and septal localizations of PG synthesis , PBP2x , PBP2b , FtsW and RodA are not affected ( Figures 2 and 4D ) .', 'However , the deletion of both divIVA and gpsB genes has a detrimental effect on cell viability .', 'It could thus be proposed that in absence of DivIVA , GpsB is dispensable for septal PG synthesis but required for optimal cell survival .', 'We also show that GpsB interacts with StkP and is crucial for both StkP localization at the division site and its ability to phosphorylate its targets , including DivIVA ( Figure 8 ) .', 'This represents the first evidence of STPKs regulation by a cell division protein .', 'Consequently , GpsB becomes the major determinant of pneumococcal cell division .', 'Most of StkP targets remain to be identified but their phosphorylation is crucial for cell division [14] .', 'On this basis , one cannot exclude that deficient phosphorylation of StkP targets in the absence of GpsB could favor FtsZ-ring spiraling .', 'In other words , FtsZ could be prone to move in a spiral driving peripheral PG synthesis in the absence of GpsB , yet preventing septal PG synthesis and cell division .', 'The function of GpsB could be more complex than promoting septal PG synthesis during septum closure and also involved in mediating proper condensation of the divisome at midcell .', 'Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division EzrA , which itself interacts with FtsZ ( Figure S10 ) .', 'Together with the helical organization of EzrA and the co-localization of DivIVA with FtsZ in ΔgpsB elongated cells , we propose that septal and peripheral PG synthesis are coordinated with and organized by FtsZ via EzrA , GpsB and DivIVA .', 'Considering the opposing function of DivIVA and GpsB in cell elongation , and the finding that inactivation of divIVA in ΔgpsB cells results in the disappearance of elongated cells , we propose that GpsB is required to confine PG synthesis at the division site and to negatively control cell elongation promoted by DivIVA .', 'GpsB and DivIVA are also found to interact .', 'Therefore , we propose that GpsB and DivIVA constitute a molecular switch , connected to FtsZ via EzrA , that orchestrates the production of peripheral ( cell elongation ) and septal ( cell division ) PG to confer to the pneumococcus its characteristic ovoid shape .', 'How could this switch operate ?', 'The finding that inactivation of gpsB affects both StkP septal localization and kinase activity , and thus DivIVA phosphorylation , leads us to propose that cell elongation is stimulated by non-phosphorylated DivIVA and that DivIVA phosphorylation by StkP abolishes its ability to promote cell elongation .', 'Suppression of the elongated cell shape of the stkP-K42M mutant upon divIVA deletion is consistent with this hypothesis ( Figure 8D ) .', 'The current model of PG synthesis in S . pneumoniae , and more generally in ovococci , proposes that the two modes of PG synthesis depend on the action of two distinct machineries [12] , as described for rod-shaped bacteria .', 'Recently , Land and co-workers have analyzed the localization of PBP2x and PBP1a over the cell cycle [25] .', 'These two enzymes display similar localization patterns in pre- and mid-divisional cells , but not during septum closure .', 'Indeed , PBP1a localized as a ring larger than that of PBP2x .', 'This observation was interpreted as supporting the existence of two distinct PG synthesis machineries .', 'While PBP2x is essential and participates in cell constriction ( septal PG synthesis ) , the role of PBP1a in PG synthesis remains elusive .', 'A pbp1a mutant is affected both in length and width but cells grow normally with no viability defects and cells remain ovoid rather than being elongated or rounded [9] , [37] , [38] .', 'In the two-machinery model , PBP1a would be involved in both elongation and constriction .', 'That PBP2x and PBP1a display different localization dynamics during septum closure does not necessarily imply that they belong to two distinct machineries .', 'Here we have analyzed the localization of PBP2x and FtsW as well as PBP2b and RodA , which are proposed to be specific for septal and peripheral PG synthesis , respectively , in the two-machinery model .', 'We show that they all co-localize with helix-shaped FtsZ in elongated ΔgpsB cells ( Figure 5B ) .', 'In addition , we failed to delete the genes encoding PBP2x , PBP2b , FtsW or RodA in ΔgpsB , ΔdivIVA or ΔdivIVAΔgpsB cells indicating that all these proteins remain essential even when septal or peripheral PG synthesis is impaired .', 'Therefore , our data hardly fit with ( and challenge ) the two-machinery model .', 'An exciting and promising alternative conciliating the data reported by Land and co-workers with ours would be that the four proteins are present in a same unique complex , ensuring both septal and peripheral PG synthesis , whose composition varies in the course of the cell cycle .', 'A previous study has demonstrated that a first short step is dedicated to cell elongation ( around 300 nm ) ( peripheral PG synthesis ) [39] .', 'This is followed by a second step , in which cell constriction ( septal PG synthesis )
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Despite years of intensive research , much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division .', 'The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown .', 'In this study , we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP .', 'We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding .', 'By contrast , the absence of GpsB resulted in hampered cell division and triggered cell elongation .', 'Remarkably , ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring , accompanied by helical patterns for DivIVA and peptidoglycan synthesis .', 'Strikingly , divIVA deletion suppressed the elongated phenotype of ΔgpsB cells .', 'These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it .', 'Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA , which itself interacts with FtsZ .', 'In addition , GpsB interacts directly with DivIVA .', 'These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA .', 'The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex .', 'Finally , we show that GpsB is required for septal localization and kinase activity of StkP , and therefore for StkP-dependent phosphorylation of DivIVA .', 'Altogether , we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan ( peripheral and septal ) synthesis responsible for the pneumococcal ellipsoid cell shape .']
['Over the last decade , bacterial genomics have revealed the presence of eukaryotic-type serine/threonine protein kinases ( STKPs ) in many bacteria .', 'However , their role and mode of action is still elusive .', 'Recent studies have suggested that STKPs could play an important role in regulating cell division of some bacterial species but the underlying regulatory mechanisms are largely unknown .', 'Considering that much remains to be discovered about the mechanisms by which the cell division machinery is assembled at the cell center and how the diversity of bacterial cell shapes is achieved and maintained , studying the role of STKPs represents a promising approach to decipher the inner workings of bacterial cell division .', 'In this article , we show that the ser/thr-kinase StkP and the two cell division paralogs GpsB and DivIVA of Streptococcus pneumoniae ( the pneumococcus ) work together to finely tune peptidoglycan synthesis and achieve proper cell shape and division .', 'We discuss the likelihood that similar mechanisms occur in other bacteria requiring protein-kinases for the cell division process .', 'We propose that the interplay between protein-kinases and cell-division proteins like GpsB or DivIVA is of crucial importance to satisfy the modes of cell division and the cell shape displayed by streptococci and other bacteria .']
['bacteriology', 'bacterial physiology', 'medical microbiology', 'streptococcus', 'microbial pathogens', 'biology and life sciences', 'microbiology', 'bacterial pathogens']