Fig. 5

Depletion of SpPBP2a does not phenocopy depletion of SpGpsB. a Mutations in SpGpsB differentially affect interactions with SpPBP2a and SpMreC. BACTH analysis of the interactions of SpGpsB-T18 variants with wild-type SpGpsB, SpPBP2a and SpMreC. pKT25/pUT18C and pKT25-zip/pUT18C-zip plasmid pairs were used as negative (−ve) and positive (+ve) controls, respectively. The agar plates were photographed after 40h incubation at 30 °C. b, c SpGpsB variants that have lost SpPBP2a binding have a gpsB null growth and morphology phenotype. Representative growth curve of S. pneumoniae strains with ectopic expression of gpsB⁺ under a Zn²⁺-dependent promoter. GpsB variants of Y23A, V28A, L32A and D33A showed a gpsB null growth phenotype (b) and elongated cell morphology (c) on gpsB depletion. The D29A variant showed an intermediate growth phenotype, which was also obtained with an independent isogenic isolate and with a gpsB^D29A-FLAG labelled strain. The I36A strain has a reduced elongation phenotype. All phase‐contrast micrographs are at the same magnification (scale bar = 1 µm). d, e SpPBP2a depletion does not phenocopy SpGpsB. Representative growth curves (d) and phase-contrast micrographs (e) of parent IU1824 (WT, D39 ∆cps rpsL1), IU13444 (∆pbp1a), IU14381 (∆pbp2a//ΔbgaA::P_Zn-pbp2a⁺ ∆pbp1a) and IU14383 (∆gpsB//ΔbgaA::P_Zn-gpsB⁺ ∆pbp1a). Similar to the depletion of SpGpsB in S. pneumoniae pbp1a⁺ strains (see panel b), depletion of SpGpsB in IU14383 leads to extremely elongated cells, a growth cessation and lysis phenotype. By contrast, depletion of SpPBP2a in the ∆pbp1a background (right hand panels) leads to small but mostly ovococcal cells that do not lyse during the time course examined. All phase-contrast micrographs were taken at OD₆₂₀ ≈ 0.15 or at the time point marked by arrows in a for IU14381 and IU14383 under zinc depletion and are at the same magnification (scale bar = 1 µm)