Complementation of the essential peptidoglycan transpeptidase function of penicillin-binding protein 2 (PBP2) by the drug resistance protein PBP2A in Staphylococcus aureus

Abstract

The essential function of penicillin-binding protein 2 (PBP2) in methicillin-susceptible Staphylococcus aureus RN4220 was clearly established by placing the pbp2 gene under control of the inducible P(spac) promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by inhibition of transcription of the pbp2 gene did not block growth of a methicillin-resistant S. aureus strain expressing the extra penicillin-binding protein PBP2A, a protein of extraspecies origin that is central to the mechanism of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.

FIG. 1

Effect of suppression of pbp2 transcription on growth of MRSA strain COL and MSSA strain RN4220. (a) Bacterial cultures grown on solid medium (TSA) supplemented or not supplemented with IPTG. Sector A, COLpPBP2iII-1; Sector B, COLpPBP2iII-5; Sector C, RNpPBP2iII-1; Sector D, RNpPBP2iII-5. (b) Northern blot hybridization with probe for pbp2. Lanes A, COLpPBP2iII-1 lanes B, COLpPBP2iII-5. Lane COL contained wild-type strain COL. RNA was prepared from cultures grown with or without 0.5 mM IPTG.

FIG. 2

Effect of suppression of pbp2 transcription on growth of MRSA strain COL and MSSA strain RN4220. Growth of the cultures in liquid medium was monitored by determining OD₆₂₀. Symbols: ■ and □, COLpPBP2iII-5 grown with and without IPTG, respectively; ▴ and ▵, RNpPBP2iII-5 grown with and without IPTG, respectively. Cultures were grown until the OD₆₂₀ was approximately 0.2. At that point (indicated by grey and black arrows for RNpPBP2iII-5 and COLpPBP2iII-5, respectively), the IPTG was removed by washing, the cells were resuspended in fresh medium without inducer, and each culture was divided into two portions, only one of which was supplemented with 0.5 mM IPTG. We continued to monitor the OD₆₂₀ after this.

FIG. 3

Effect of suppression of pbp2 transcription on growth of MRSA strain COL and its isogenic derivative RUSA4 with inactivated mecA gene. Bacterial cultures were grown on solid medium (TSA) supplemented or not supplemented with IPTG. Sector A, COLpPBP2iC-3 construct; Sector B, RUSA4pPBP2iC-3 construct.

FIG. 4

Effect of suppression of pbp2 transcription on growth of MRSA strain COL and its isogenic derivative RUSA4 with inactivated mecA gene. Growth of the cultures in liquid medium was monitored by determining the OD₆₂₀. Symbols: ■ and □, COLpPBP2iC-2 grown with and without IPTG, respectively; ▴ and ▵, RUSA4pPBP2iC-2 grown with and without IPTG, respectively. Cultures were allowed to grow until the OD₆₂₀ was approximately 0.2. At that point (indicated by grey and black arrows for RUSA4pPBP2iC-2 and COL pPBP2iC-2, respectively) the IPTG was removed by washing, the cells were resuspended in fresh medium without inducer, and each culture was divided into two portions, only one of which was supplemented with 0.5 mM IPTG. We continued to monitor the OD₆₂₀ after this.

FIG. 5

Effect of PBP2 deficit on the muropeptide composition of the cell walls of MRSA strain COL. We determined the HPLC profiles of COLpPBP2iII-5 grown in the presence (A) and in the absence (B) of IPTG, as well as the HPLC profile of strain COL grown in the presence of 1 μg of oxacillin per ml (C). Muropeptides were purified and separated by HPLC as described in the text.

FIG. 6

Morphological abnormalities in MRSA strain COL grown with a deficit of PBP2. Strain COL was grown in TSB (A and B), and construct COLpPBP2iII-5 was grown in medium without ITPG (C and D). Thin sections of bacteria were analyzed by electron microscopy. Bars = 1 μm.