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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Mol Microbiol. 2016 Apr 15;100(6):1039–1065. doi: 10.1111/mmi.13366

Fig. 3.

Fig. 3

Mutations in pbp1a suppress the ΔmltG mutations. (A) Arrangement of genes surrounding pbp1a in Streptococcus pneumoniae D39 chromosome. recU encodes recombination protein U. (B) Domain architecture of PBP1a, TP active site motifs, and mapped mutations in pbp1a in ΔmltG suppressor strains. M, transmembrane domain (aa 13 to 35); TG, transglycosylase domain (aa 59 to 237); TP, transpeptidase domain aa (332 to 622). All pbp1a mutants, except pbp1a(S89F), could not be transformed with a Δpbp2a deletion and showed the small-cell phenotype characteristic of Δpbp1a mutants, whereas pbp1a(S89F) mutants showed an intermediate size (data not shown). Western blots of FLAG-tagged PBP1a(S89F) or PBP1a(G494E) showed a wild-type or significantly reduced (≈33%) amount of PBP1a, respectively (data not shown). (C) Appearance and number of colonies obtained after transformation with a ΔmltG amplicon into S. pneumoniae D39 Δcps strains. 50 ng of ΔmltG::Pc-erm amplicon obtained from strain IU7260 was transformed into the strains as described in Experimental procedures and examined at 20 h of incubation at 37°C in an atmosphere of 5% CO2. Strains and genotypes, upper half of table: IU1945 wild-type parent (D39 Δcps); K180 (Δpbp1b::Pc-[kan-rpsL+]); IU6680 (Δpbp2a::Pc-[kan-rpsL+]); IU8872 (pbp1a+ mltG+//ΔbgaA::tet-PZn-RBSmltG-mltG); and IU6662 (Δpbp1a::Pc-[kan-rpsL+]). MltG expression in merodiploid strain IU8872 was induced by 0.2 mM ZnCl2 + 0.02 mM MnSO4 (to counter zinc toxicity), which was added where indicated to growing cultures 1h before transformation and to all subsequent steps in transformations, including plates. Strains and genotypes, lower half of table: IU1824 wild-type parent (D39 Δcps rpsL1); IU6741 (Δpbp1a); IU7845 (pbp1a (T insertion at Phe33); IU7840 (pbp1a (S89F)); IU7843 (pbp1a (A deletion at Lys160); IU7839 (pbp1a (G deletion at Gly451); and IU7837 (pbp1a (G494E). Construction of strains is described in Supplemental experimental procedures and Table S1. (TG) or (TP), aa substitution in transglycosylase or transpeptidase domain; (FS), frameshift mutation.