FEMS Microbiology Letters 14 (1982) 295-298 295 Published by Elsevier BiomedicalPress Interaction of fl-lactam antibiotics with penicillin-binding proteins from Pseudomonas aeruginosa Alfredo Rodriguez-Teb~, Fernando Rojo, Juan C. Montilla and David Vfizquez Centro de Biologia Moleculardel C.S.L C., Facultadde Ciencias, UAM, Canto Blanco, Madrid-34, Spain Received29 March 1982 Accepted 30 March 1982 1. INTRODUCTION 2.2. Preparation of cell envelopes The clinical treatment of Pseudomonas aeru- ginosa infections has become an important priority in recent years, since this bacterium displays high resistance to a large number of antibiotics effective against many other bacterial species [1]. The im- portance of the fact that some cell structures form penetration barriers, as a cause of antibiotic resis- tance, has recently been stressed [2]. Moreover, a decreased affinity of fl-lactams for penicillin-bind- ing proteins (PBPs), and an insensitivity towards fl-lactams of bacterial peptidoglycan biosynthetic enzymes have been observed in isolates of P. aeruginosa, shown previously to be resistant to these antibiotics [3]. In view of this problem, we have studied both the binding of a number of fl-lactams to the PBPs of a wild-type strain of P. aeruginosa and the effectiveness of bacterial bar- riers in controlling the penetration of carbenicillin, one of the most active fl-lactams against P. aeru- ginosa. 2. MATERIALS AND METHODS Envelopes were prepared essentially as de- scribed in [5] and resuspended in 20 mM sodium phosphate buffer, pH 7.0, containing 10 mM MgC12 (standard buffer), to give a final concentra- tion of 10 mg protein/ml. 2.3. Binding of fl-lactam antibiotics to penicillin binding proteins (PBPs) using isolated membrane preparations Competition experiments were carried out be- tween non-radioactive fl-lactams and either benzyl [14C]penicillin (Amersham International, spec. act. 51 Ci/mol) or the derivative of ampicillin N[3- (4-hydroxy 5-125 I-iodophenyl)propionyl] ampicil- lin (we will refer to it here as [125I]ampicillin) obtained by reacting the Bolton and Hunter rea- gent (2000 Ci/mmol, Amersham International) with ampicillin, following the method described by Schwarz et al. [6]. The procedure of fl-lactam binding to membrane PBPs was essentially the same as that described by Spratt [7] with the modifications introduced by Zimmermann [5]. 2.1. Strain and growth conditions P. aeruginosa NCTC 10662 was used throughout. Cells were grown in L medium [4] at 37°C under vigorous shaking. 2.4. Kinetics of carbenicillin binding to either iso- lated cell envelopes or intact cells of P. aeruginosa P. aeruginosa cultures were grown as indicated above, and when the bacteria had reached a den- 0378-1097/82/0000 0000/$02.75 © 1982 Federation of European MicrobiologicalSocieties Downloaded from https://academic.oup.com/femsle/article-abstract/14/4/295/528359 by guest on 13 June 2020