Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 20 Dec 2018 21:01:57 A link in transcription between the native pbpB and the acquired mecA gene in a strain of Staphylococcus aureus Susana Gardete, 1,2 Hermı ´nia de Lencastre 1,2 and Alexander Tomasz 2 Correspondence Alexander Tomasz tomasz@mail.rockefeller.edu 1 Molecular Genetics Laboratory, Instituto de Tecnologia Quı ´mica e Biolo´ gica da Universidade Nova de Lisboa, 2780 Oeiras, Portugal 2 Laboratory of Microbiology, The Rockefeller University, 1230 York Avenue, NY 10021, USA Received 20 April 2006 Revised 23 May 2006 Accepted 24 May 2006 Conditional mutants of pbpB with an IPTG-inducible promoter were used to compare the effects of interrupted transcription of this gene in a meticillin-sensitive (MSSA) and a meticillin-resistant (MRSA) strain of Staphylococcus aureus. After 3 h growth following the removal of IPTG, multiplication of the MSSA strain stopped abruptly, cells began to lyse, and membrane preparations showed greatly decreased quantities of penicillin-binding protein (PBP) 2. In contrast, the MRSA strain continued to grow for at least 20 h in the IPTG-free medium, but with gradually increasing doubling times, which eventually reached 180 min. The peptidoglycan produced during this period of extremely slow growth showed only minor alterations, but cells with abnormal morphology accumulated in the culture, the abundance of mecA transcript gradually declined, and the cellular amounts of PBP2A were significantly decreased. Adding back the IPTG inducer caused rapid resumption in the transcription of pbpB, followed by an increase in the transcription of mecA. No changes were detected in the transcription of pbpA, C and D, the determinant of 16S rRNA or the housekeeping gene pta. Promoter fusion experiments suggested that the transcription of the resistance gene mecA may respond to some regulatory signal generated in the bacteria during changes in the transcription of pbpB. INTRODUCTION Meticillin (formerly methicillin)-resistant Staphylococcus aureus (MRSA) strains carry the acquired resistance deter- minant mecA encoding the low-affinity penicillin-binding protein (PBP) 2A. The primary function of PBP2A is to take over the cross-linking of cell wall building blocks from the native PBPs when these become inactivated by b-lactam antibiotics (De Jonge & Tomasz, 1993). Recent experiments have indicated that the resistance protein PBP2A may also function in contexts other than drug resistance, and in co- operation with one of the native PBPs, namely PBP2. The first evidence for this cooperation was the finding that inhibition of transcription of pbpB, which is the struc- tural gene of PBP2, was lethal in a meticillin-sensitive S. aureus (MSSA), but not in the MRSA strain COL, which expresses PBP2A in a constitutive manner (Pinho et al., 2001b). PBP2 is a bifunctional cell wall synthetic enzyme containing both transglycosylase (TGase) and transpepti- dase (TPase) domains (Murakami et al., 1994), and genetic experiments have demonstrated that the essential function of this protein, successfully replaced by PBP2A, is that of the TPase domain (Pinho et al., 2001b). Cooperative function- ing of PBP2 and PBP2A has been further documented by the observation that growth of MRSA in the presence of high concentrations of antibiotic requires not only PBP2A, but the functioning of the TGase domain of the native PBP2 as well (Pinho et al., 2001a). Additional evidence for coope- rative functioning has come from the recent observation that the presence of PBP2A is able to prevent dislocation of PBP2 from the cell wall growth zone, a phenomenon that occurs in MSSA strains exposed to b-lactam antibiotics (Pinho & Errington, 2005). The purpose of the studies described here was to examine in more detail these intriguing and unusual phenomena, which involve cooperative functioning between two proteins: a cell wall synthetic enzyme native to S. aureus, and an antibiotic- resistance factor acquired by S. aureus from an extra-species source. In a conditional mutant, we followed the effects of turning off and turning on the transcription of pbpB on the growth, oxacillin resistance, cell structure and cell wall composition and gene expression profiles, and also the production of PBPs 2 (in strain RN4220 spac :: pbpB ) and 2A in the MRSA Abbreviations: C T , threshold cycle; MRSA, meticillin-resistant Staphy- lococcus aureus; MSSA, meticillin-sensitive S. aureus; PAP, population analysis profile; PBP, penicillin-binding protein; Q-PCR, quantitative real-time PCR; TGase, transglycosylase; TPase, transpeptidase. 0002-9078 G 2006 SGM Printed in Great Britain 2549 Microbiology (2006), 152, 2549–2558 DOI 10.1099/mic.0.29078-0