Effects of low PBP2b levels on cell morphology and peptidoglycan composition in Streptococcus pneumoniae R6

Abstract

Streptococcus pneumoniae produces two class B penicillin-binding proteins, PBP2x and PBP2b, both of which are essential. It is generally assumed that PBP2x is specifically involved in septum formation, while PBP2b is dedicated to peripheral cell wall synthesis. However, little experimental evidence exists to substantiate this belief. In the present study, we obtained evidence that strongly supports the view that PBP2b is essential for peripheral peptidoglycan synthesis. Depletion of PBP2b expression gave rise to long chains of cells in which individual cells were compressed in the direction of the long axis and looked lentil shaped. This morphological change is consistent with a role for pneumococcal PBP2b in the synthesis of the lateral cell wall. Depletion of PBP2x, on the other hand, resulted in lemon-shaped and some elongated cells with a thickened midcell region. Low PBP2b levels gave rise to changes in the peptidoglycan layer that made pneumococci sensitive to exogenously added LytA during logarithmic growth and refractory to chain dispersion upon addition of LytB. Interestingly, analysis of the cell wall composition of PBP2b-depleted pneumococci revealed that they had a larger proportion of branched stem peptides in their peptidoglycan than the corresponding undepleted cells. Furthermore, MurM-deficient mutants, i.e., mutants lacking the ability to synthesize branched muropeptides, were found to require much higher levels of PBP2b to sustain growth than those required by MurM-proficient strains. These findings might help to explain why increased incorporation of branched muropeptides is required for high-level beta-lactam resistance in S. pneumoniae.

Fig 1

Effects of PBP2x (A) and PBP2b (B) depletion on pneumococcal growth. SPH165 (A) and SPH158 (B) cultures were grown in microtiter plates in a Fluostar Optima luminometer at 37°C in the presence and absence of the ComS* inducer. The OD₄₉₂ was read automatically every 10 min. Washed cells, pregrown in C medium containing 0.2 μM ComS*, were diluted to an OD₄₉₂ of ∼0.05. After splitting of each of the SPH165 and SPH158 cultures in two, one parallel was diluted 2-fold in C medium containing 0.2 μM ComS* (black symbols), and the other was diluted in plain C medium (white symbols). In the latter parallel, the cell-associated ComS* peptide was slowly metabolized by the bacteria, resulting in a gradual reduction in target gene expression with time. This was verified in the accompanying gels, in which Bocillin FL-labeled PBP2x (A) and PBP2b (B) are shown to decrease with increasing dilutions of the cultures (fold dilutions are indicated above the lanes). Samples from wild-type cells (RH1) and cells grown in the presence of 0.2 μM ComS* are included for comparison. Symbols: △ and ▲, 2-fold dilutions; ○ and ●, 32-fold dilutions; □ and ■, 128-fold dilutions; ▽ and ▼, 512-fold dilutions; black and white circles with dots, 2,048-fold dilutions. All experiments were repeated several times with almost identical results.

Fig 2

Scanning electron micrographs showing the morphology of PBP2b-depleted and LytB-deficient pneumococci. (A) Strain SPH158 (LytA⁻) grown in the presence of 0.2 μM ComS* inducer. (B) Strain SPH158 (LytA⁻) grown in the absence of ComS*. (C) Strain SPH157 (LytA⁺) grown in the presence of 0.2 μM ComS*. (D) Strain SPH157 (LytA⁺) grown in the absence of ComS*. The samples used were taken at an OD₄₉₂ of ∼0.3 from 512-fold-diluted cultures (A and B) and 256-fold-diluted cultures (C and D) in a depletion experiment corresponding to the one shown in Fig. 1B. (E) Strain RH426. (F) Strain SPH184, a ΔlytB derivative of strain RH426. Bars = 1 μm.

Fig 3

Scanning electron micrographs comparing the morphology of PBP2x-depleted pneumococci (B and D) to that of the corresponding undepleted controls (A and C). (A) Strain SPH165 (LytA⁻) grown in the presence of 0.2 μM ComS* inducer. (B) Strain SPH165 (LytA⁻) grown in the absence of ComS*. (C) Strain SPH164 (LytA⁺) grown in the presence of 0.2 μM ComS*. (D) Strain SPH164 (LytA⁺) grown in the absence of ComS*. The samples used were taken at an OD₄₉₂ of ∼0.3 from 128-fold-diluted cultures in a depletion experiment corresponding to the one shown in Fig. 1A. Bars = 1 μm.

Fig 4

Depletion of pbp2b induces LytA sensitivity in growing S. pneumoniae cells. In panels A, B, C, and D, the SPH158 strain was cultivated in the presence of 16, 8, 4, and 2 nM ComS*, respectively. Reduced amounts of ComS* in the growth medium resulted in reduced expression of pbp2b. Open symbols represent cultures subjected to purified LytA (10 μg ml⁻¹), while filled symbols represent untreated cultures. LytA was added when the cells reached an OD₄₉₂ of ∼0.25, as indicated by the arrows. As the expression of pbp2b reached a critically low level, exponentially growing SPH158 cells became susceptible to exogenous LytA. Data from one representative experiment of several are shown.

Fig 5

Comparison of growth rates of strains SPH157 (murM⁺ lytA⁺), SPH188 (murM lytA⁺), SPH158 (murM⁺ lytA), and SPH182 (murM lytA) during depletion of PBP2b. (A) ●, SPH157 cells grown in the presence of 0.2 μM ComS*; ▼, SPH188 cells grown in the presence of 0.2 μM ComS*; ○, SPH157 cells grown in the absence of ComS*; ▽, SPH188 cells grown in the absence of ComS*. (B) ●, SPH158 cells grown in the presence of 0.2 μM ComS*; ▼, SPH182 cells grown in the presence of 0.2 μM ComS*; ○, SPH158 cells grown in the absence of ComS*; ▽, SPH182 cells grown in the absence of ComS*. The depletion experiment was carried out as described in the legend to Fig. 1. The growth curves shown correspond to 64-fold, 256-fold, and 1,024-fold dilutions of a starting culture with an OD₄₉₂ of ∼0.05. Data from a representative experiment of several independent experiments are shown.

Fig 6

Effect of low PBP2b levels on the stem peptide composition of peptidoglycan isolated from S. pneumoniae. (A) Stem peptide profiles of the wild-type reference strain RH1 grown in plain C medium and of SPH158 cells grown in C medium containing 0, 0.02, or 2 μM ComS*. The profiles show that the peptidoglycan of PBP2b-depleted cells contains fewer directly linked (peak 1) and more branched (peaks 2, 3, and 4) stem peptides than the case in undepleted cells. (B) Structural assignments of materials eluting in peaks 1 to 4. The experiment was repeated three times with similar results.