Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae. Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

Competence for genetic transformation in the genus Streptococcus depends on an alternative sigma factor, X , for coordinated synthesis of 23 proteins, which together establish the X state by permitting lysis of incompetent streptococci, uptake of DNA fragments, and integration of strands of that DNA into the resident genome. Initiation of transient accumulation of high levels of X is coordinated between cells by transcription factors linked to peptide pheromone signals. In Streptococcus pneumoniae, elevated X is insufficient for development of full competence without coexpression of a second competence-specific protein, ComW. ComW, shared by eight species in the Streptococcus mitis and Streptococcus anginosus groups, is regulated by the same pheromone circuit that controls X , but its role in expression of the X regulon is unknown. Using the strong, but not absolute, dependence of transformation on comW as a selective tool, we collected 27 independent comW bypass mutations and mapped them to 10 single-base transitions, all within rpoD, encoding the primary sigma factor subunit of RNA polymerase, A . Eight mapped to sites in rpoD region 4 that are implicated in interaction with the core ␤ subunit, indicating that ComW may act to facilitate competition of the alternative sigma factor X for access to core polymerase.

Streptococcus pneumoniae is able to integrate exogenous DNA into its genome by natural genetic transformation. Transient accumulation of high levels of the only S. pneumoniae alternative factor is insufficient for development of full competence without expression of a second competence-specific protein, ComW. The ⌬comW mutant is 10 4 -fold deficient in the yield of recombinants, 10-fold deficient in the amount of X activity, and 10-fold deficient in the amount of X protein. The critical role of ComW during transformation can be partially obviated by A mutations clustered on surfaces controlling affinity for core RNA polymerase (RNAP). While strains harboring A mutations in the comW mutant background were transforming at higher rates, the mechanism of transformation restoration was not clear. To investigate the mechanism of transformation restoration, we measured late gene expression in A * suppressor strains. Restoration of late gene expression was observed in ⌬comW A * mutants, indicating that a consequence of the A * mutations is, at least, to restore X activity. Competence kinetics were normal in ⌬comW A * strains, indicating that strains with restored competence exhibit the same pattern of transience as wild-type (WT) strains. We also identified a direct interaction between ComW and X using the yeast two-hybrid (Y2H) assay. Taken together, these data are consistent with the idea that ComW increases X access to core RNAP, pointing to a direct role of ComW in factor exchange during genetic transformation. However, the lack of late gene shutoff in ⌬comW mutants also points to a potential new role for ComW in competence shutoff. IMPORTANCEThe sole alternative sigma factor of the streptococci, SigX, regulates development of competence for genetic transformation, a widespread mechanism of adaptation by horizontal gene transfer in this genus. The transient appearance of this sigma factor is strictly controlled at the levels of transcription and stability. This report shows that it is also controlled at the point of its substitution for SigA by a second transient competence-specific protein, ComW. Streptococcus pneumoniae, a Gram-positive opportunistic pathogen found in the human nasopharynx, causes diseases such as pneumonia and meningitis. S. pneumoniae's natural competence, or ability to integrate exogenous DNA into its chromosome, provides a major mechanism of rapidly overcoming selective pressure (1, 2). The ability to take up and exchange DNA depends on development of the competent state, which is prompted by quorum sensing (QS) mediated by a small peptide, via a signal transduction pathway that is incompletely understood.Cells in the exponential growth phase are first primed for transformation by a QS mechanism encoded by two genetic loci initially transcribed at a basal level by core RNA polymerase (RNAP) bound to the primary sigma factor, A (3). The loci are comAB (4, 5) and comCDE (3). ComC is a propeptide cleaved and exported by ComAB, as the mature peptide called CSP (competence-stimulating pepti...

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae

27Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the 28 coordinated dynamic movement of essential protein complexes. Recent studies show 29 that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting 30 for both septal PG synthesis and septum closure in some bacteria, but not in others. 31 Here we report the dynamics of FtsZ movement leading to septal and equatorial ring 32 formation in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn). 33 Conventional and single-molecule total internal reflection fluorescence microscopy 34 (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins 35 FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in 36 cell division. This mode of continuous nascent ring movement contrasts with a failsafe 37 streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another 38 Streptococcus species. This analysis also provides several parameters of FtsZ 39 treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells 40 and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ 41 filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. 42 In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW 43 glycosyl transferase binding partner relative to FtsZ treadmilling in Spn cells. Five lines 44 of evidence support the conclusion that movement of the bPBP2x:FtsW complex in 45 septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results 46 support a model in which FtsZ dynamics and associations organize and distribute septal 47 PG synthesis, but do not control its rate in Spn. 48 3 SIGNIFICANCE 49 This study answers two long-standing questions about FtsZ dynamics and its 50 relationship to septal PG synthesis in Spn for the first time. In previous models, FtsZ 51 concertedly moves from midcell septa to MapZ rings that have reached the equators of 52 daughter cells. Instead, the results presented here show that FtsZ, FtsA, and EzrA 53 filaments/bundles move continuously out from early septa as part of MapZ rings. In 54 addition, this study establishes that the movement of bPBP2x:FtsW complexes in septal 55 PG synthesis depends on and likely mirrors new PG synthesis and is not correlated with 56 the treadmilling of FtsZ filaments/bundles. These findings are consistent with a 57 mechanism where septal FtsZ rings organize directional movement of bPBP2x:FtsW 58 complexes dependent on PG substrate availability.59 60 Cell division in most bacteria is mediated by the tubulin homolog, FtsZ, which 61 polymerizes into dynamic filaments and bundles at the middle or toward the pole of 62 dividing cells (1, 2). Polymerization of FtsZ filaments/bundles initiates sequential binding 63 of a series of proteins that ultimately assemble into a controlled divisome machine for 64 septal peptidoglycan (PG) synthesis l...