Molecular Microbiology (1989) 3(1). 95-102 Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae C. G. Dowson, A. Hutchison and B. G. Spratt* Microbial Genetios Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK. Summary Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (>1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin- binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin- binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin- resistant South African strain. The amino acid sequences ofthe transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substi- tution of seven consecutive residues in a region that is predicted to form a loop at the bottom ofthe penicillin- binding site. Introduction Clinical isolates of Streptococous pneumoniae are typically highly sensitive to penicillin with minimal inhibi- tory concentrations (MICs) of about 0.008 jig benzylpeni- cillin per ml. Strains with moderate levels of resistance to penicillin were isolated in Australia in 1967 (Hansman and Bullen, 1967) and highly penicillin-resistant strains, with MICs of 2-12 M-g ml" \ were reported subsequently, firstly Received 29 July, 1988; revised 15 September. 1988. 'For corres- pondence. from South Africa (Jacobs et al., 1978), and more recently from other locations (Appelbaum, 1987). Infections with penicillin-resistant strains are associated with a high morbidity and mortality and often fail to respond to penicillin therapy (Jacobs et ai, 1978; Paliares et al., 1987). In recent surveys of patients hospitalized with pneumococcal infections, the proportion of penicillin- resistant strains has increased dramatically in some regions and a parallel increase has also been reported in the proportion of penicillin-resistant pneumococci iso- lated from healthy carriers in the community (Feldman et al., 1985; Appelbaum, 1987; Perez efa/.. 1987). Penicillin resistance in S. pneumoniae is entirely due to alterations of the properties of the penicillin-binding proteins (PBPs) (Zighelboim and Tomasz, 1980; Tomasz ef a/.. 1984). In the most penicillin-resistant strains (e.g. those isolated in South Africa) there have been very large reductions in the affinities of each of the high molecular weight, physiologically-important PBPs (Hakenbeck etal., 1980; Zighelboim and Tomasz, 1980; Hakenbeck ef al., 1986). Laboratory studies have suggested that the development of PBPs with greatly decreased affinity for p-lactam antibiotics requires the introduction of multiple amino acid substitutions that allow the enzyme to re- model its active centre so that It can exclude the antibiotic without impairing its ability to recognize its structurally analogous peptide substrate (Hedge and Spratt, 1985a,b). Recent work on non-p-lactamase-producing, penicillin- resistant clinical isolates of Neisseria gonorrhoeae has confirmed these laboratory studies since the altered forms of PBP2 with decreased affinity for penicillin possess multiple amino acid substitutions and insertions that are found exclusively within the penicillin-sensitive transpep- tidase domain of the enzyme (Spratt, 1988). The reduction in the affinities for penicillin of the PBPs of penicillin-resistant pneumococci Is presumed to have occurred since the introduction of penicillin in the 1940s and is one of the most extreme examples of the ability of an enzyme to evolve (under intense selective pressures) to discriminate between its substrate and a structurally analogous inhibitor. We have therefore examined the extent to which the structure of PBP2B of a penicillin- resistant South African strain of S. pneumoniae has altered from that of typical penicillin-sensitive strains