Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39

Abstract

SUMMARY In ellipsoid-shaped ovococcus bacteria, such as the pathogen Streptococcus pneumoniae (pneumococcus), side-wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin-binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG-domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. The “synthetic viable” genetic relationship between Δpbp1a and ΔmltG mutations extends to essential ΔmreCD and ΔrodZ mutations that misregulate peripheral PG synthesis. Remarkably, the single MltG(Y488D) change suppresses the requirement for PBP2b, MreCD, RodZ, and RodA. Structural modeling and comparisons, catalytic-site changes, and an interspecies chimera indicate that pneumococcal MltG is the functional homologue of the recently reported MltG endo-lytic transglycosylase of Escherichia coli. Depletion of pneumococcal MltG or mltG(Y488D) increases sphericity of cells, and MltG localizes with peripheral PG synthesis proteins during division. Finally, growth of Δpbp1a ΔmltG or mltG(Y488D) mutants depends on induction of expression of the WalRK TCS regulon of PG hydrolases. These results fit a model in which MltG releases anchored PG glycan strands synthesized by PBP1a for crosslinking by a PBP2b:RodA complex in peripheral PG synthesis.

Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae

“…Similar to what we observed for Rx1, HADA staining of the Rx1 ΔftsA//P Zn gfp-ftsA ϩ cells grown in ZnCl 2 concentrations of 0.15 or 0.10 mM revealed a pattern of new PG synthesis at division sites that colocalized with GFP-FtsA for most of the cell cycle (83.3% and 82.7%, respectively). This pattern is consistent with the localization profile and timing reported for FtsZ, FtsA and the respective PBPs involved in peripheral and septal PG synthesis (20,33,37,40,41,44,45). In cells grown in 0.05 mM ZnCl 2 , 71.4% of the division sites still showed colocalization of GFP-FtsA and HADA signals.…”

“…In particular, this medium was used in gene expression experiments from the P czcD (P Zn ) promoter to allow more precise control of ZnCl 2 induction, as well as better visualization of the GFP fluorescence (39,54). D39 Δcps strains were grown statically in brain heart infusion broth (BHI) or on TSBA plates at 37°C or 30°C in an atmosphere of 5% CO 2 , as described previously (40,44,45). When appropriate, chloramphenicol (4.5 g/ml), tetracycline (2.5 g/ml), and/or erythromycin (0.25 g/ml) were added to the growth medium.…”

“…For induction of the P Zn promoter in Rx1 strains, ZnCl 2 (Sigma-Aldrich) was added to liquid medium and blood agar plates at different concentrations, depending on the medium used (39). The P Zn promoter was induced in D39 Δcps strains by the addition of different concentrations of ZnCl 2 and 0.1ϫ the concentration of MnSO 4 (45). Competent S. pneumoniae cells were prepared and transformed by the addition of competence-inducing peptide 1 (CSP-1), as previously described (40,60).…”

“…Growth was monitored turbidimetrically every 30 min with an Ultrospec 3100 (Amersham Pharmacia Biotech) or with a DU 730 spectrophotom-eter (Beckman Coulter) at 650 nm (TSB) or 600 nm (CϩY). Growth determinations were performed for D39 Δcps strains, as described previously (40,44,45).…”

“…However, genetic evidence for the essential role of the Fts proteins involved in the initial steps of cell division is presently lacking. In particular, it is not yet known how the PG synthetic complex involved in septation (the divisome) is assembled and coordinated with the protein complex involved in peripheral elongation (the elongasome) (7,42), which in S. pneumoniae is also located at midcell (35,36,(43)(44)(45).…”