Mutational analysis of class A and class B penicillin-binding proteins in Streptococcus gordonii

Abstract

High-molecular-weight (HMW) penicillin-binding proteins (PBPs) are divided into class A and class B PBPs, which are bifunctional transpeptidases/transglycosylases and monofunctional transpeptidases, respectively. We determined the sequences for the HMW PBP genes of Streptococcus gordonii, a gingivo-dental commensal related to Streptococcus pneumoniae. Five HMW PBPs were identified, including three class A (PBPs 1A, 1B, and 2A) and two class B (PBPs 2B and 2X) PBPs, by homology with those of S. pneumoniae and by radiolabeling with [3H]penicillin. Single and double deletions of each of them were achieved by allelic replacement. All could be deleted, except for PBP 2X, which was essential. Morphological alterations occurred after deletion of PBP 1A (lozenge shape), PBP 2A (separation defect and chaining), and PBP 2B (aberrant septation and premature lysis) but not PBP 1B. The muropeptide cross-link patterns remained similar in all strains, indicating that cross-linkage for one missing PBP could be replaced by others. However, PBP 1A mutants presented shorter glycan chains (by 30%) and a relative decrease (25%) in one monomer stem peptide. Growth rate and viability under aeration, hyperosmolarity, and penicillin exposure were affected primarily in PBP 2B-deleted mutants. In contrast, chain-forming PBP 2A-deleted mutants withstood better aeration, probably because they formed clusters that impaired oxygen diffusion. Double deletion could be generated with any PBP combination and resulted in more-altered mutants. Thus, single deletion of four of the five HMW genes had a detectable effect on the bacterial morphology and/or physiology, and only PBP 1B seemed redundant a priori.

FIG. 1.

Patterns of radiolabeled PBPs from the parent and PBP-deleted mutants. PBPs were visualized on fluorograms after labeling membrane proteins with [³H]benzylpenicillin and separation on 6% SDS-PAGE gels. The profiles of single PBP mutants (A) and double PBP mutants (B) are presented. The parent (wt) and the deleted PBP mutants are indicated at the tops of the lanes. Positions of PBPs are listed on the left of the fluorograms.

FIG. 2.

Diagrams of S. gordonii PBPs. Domains were determined in silico by homology with previously characterized PBPs in S. pneumoniae (11-13, 19, 31, 33). GT and TP stand for transglycosylase and transpeptidase domains, respectively. Annotations above the transpeptidase domains indicate their conserved motifs. Conserved motifs of the transglycosylase domains are not presented but are identical to those of S. pneumoniae. Annotations below the diagrams define the limits of the various protein domains. Solid, hatched, and shaded boxes represent the N-terminal cytoplasmic regions, the membrane anchors, and the C-terminal extensions, respectively.

FIG. 3.

Cell shape modifications detected by optical microscopy and FACS analysis. The left panel presents exponentially growing cells observed under phase contrast microscopy. Bars, 16 μm. The right panel presents bacteria from the same cultures analyzed by cell sorting. Bacteria were injected into a FACSCalibur and the results analyzed with the CELLQUEST PRO software. To compare populations, the data for each strain were plotted on a two-dimensional graph (x axis, forward scatter; y axis, side scatter). The wild type (wt) and the PBP-deleted mutants are indicated beside the panels.

FIG. 4.

Electron microscopy of individually deleted PBPs in S. gordonii. The parent strain (A) and the PBP 1A (B), PBP 2A (C), and PBP 2B (D to F) mutants were examined in the mid-log phase of growth in rich medium. Bars, 0.5 μm.

FIG. 5.

Penicillin-induced early lysis (A) and time kill curves (B). The S. gordonii wild type (♦) and the deletion mutant PBP 1A (Δ), 1B (▴), 2A (□), or 2B (▪) was treated with penicillin G (50-fold the MIC) at an OD₆₂₀ of 0.15 (arrow). The data points represent the means for ≥3 independent experiments, with an interexperimental variation of ≤10%.