Mutational analysis of the Streptococcus pneumoniae bimodular class A penicillin-binding proteins

Abstract

One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding-transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.

FIG. 1

S. pneumoniae class A PBPs and mutant PBPs used in this study. The structures of PBP1a, PBP1b, and PBP2a are shown schematically, and their length (in amino acid residues) is indicated on the right. The small black box indicates the putative membrane-spanning domain. The conserved motif at the putative transition between the N-terminal glycosyltransferase domain (hatched area) and the penicillin-binding–transpeptidase domain, as well as the active-site serine, are indicated by black triangles. The length of the potentially produced peptide after insertion-duplication mutagenesis is represented by the black bar.

FIG. 2

Profiles of single and double PBP mutants. PBPs were visualized on fluorograms after labeling of cell lysates with [³H]propionylampicillin. The disrupted PBP(s) of the mutants or of the parent strain (R6) is indicated above the lanes. 1a∗ refers to transformants isolated after the attempt to disrupt pbp2a in a pbp1a mutant. Gels with 10% acrylamide (acrylamide-bisacrylamide [30:0.8]) (A and C) or 7.5% acrylamide (acrylamide-bisacrylamide [30:1.1]) (B) were used. The positions of PBPs are indicated to the left of the fluorograms.

FIG. 3

Growth of S. pneumoniae R6 and class A PBP double mutants. Cells of an exponentially growing culture were diluted in prewarmed C medium supplemented with erythromycin (1 μg/ml), and growth was monitored by nephelometry (in nephelometry units [N]) over time (in hours). Symbols: ●, S. pneumoniae R6; ■, pbp1a pbp1b mutant; ▴, pbp1b pbp2a mutant.

FIG. 4

Electron microscopy of S. pneumoniae class A PBP double mutants. Cells are shown after negative staining of exponentially grown cultures of the parent strain S. pneumoniae R6 (A) and the pbp1a pbp1b (B) and pbp1b pbp2a (C) double mutants. Cells were grown in C medium with (+) or without (−) the addition of 2% choline. Arrowheads indicate odd division septa. Bars, 2 μm.

FIG. 4

Electron microscopy of S. pneumoniae class A PBP double mutants. Cells are shown after negative staining of exponentially grown cultures of the parent strain S. pneumoniae R6 (A) and the pbp1a pbp1b (B) and pbp1b pbp2a (C) double mutants. Cells were grown in C medium with (+) or without (−) the addition of 2% choline. Arrowheads indicate odd division septa. Bars, 2 μm.

FIG. 4

Electron microscopy of S. pneumoniae class A PBP double mutants. Cells are shown after negative staining of exponentially grown cultures of the parent strain S. pneumoniae R6 (A) and the pbp1a pbp1b (B) and pbp1b pbp2a (C) double mutants. Cells were grown in C medium with (+) or without (−) the addition of 2% choline. Arrowheads indicate odd division septa. Bars, 2 μm.