Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term

Since around 2000—earlier in Poland and Spain and later in France and the UK—dramatic shifts have occurred in the prevalence and types of extended-spectrum β-lactamases (ESBLs) in Europe. Before this watershed, most producers were nosocomial isolates, often Klebsiella spp. or Enterobacter spp. from specialist care units, and had mutant TEM or SHV ESBLs. Subsequently, CTX-M ESBLs have become dominant, with much greater penetration into Escherichia coli, and with many infections in ‘complicated community’ patients, usually with underlying disease, recent antibiotic usage, or healthcare contact. The degree of clonality among producers varies with the country, as does the enzyme type produced, with group 9 (CTX-M-9 and -14) enzymes dominant in Spain and group 1 enzymes (particularly CTX-M-3 and -15) dominant elsewhere. Irrespective of the particular enzyme, most producers are multiresistant. These changing patterns present major therapeutic and infection control challenges, with the public health intervention points unclear.

For growth and division of rod-shaped bacteria, the cylindrical part of the sacculus has to be elongated and two new cell poles have to be synthesize

Twenty blood isolates of Acinetobacter baumannii were studied, representing eight pulsed-field gel electrophoresis patterns and all different antimicrobial susceptibility patterns observed during 1995–97 at the University Hospital Virgen Macarena, Seville, Spain. The MIC₉₀s (mg/L) of imipenem and meropenem decreased from 16 to 0.5 and from 8 to 4, respectively, in the presence of BRL 42715 (BRL) but not clavulanic acid. Hydrolysing activity (nmol/min/mg) of bacterial supernatants against cefaloridine ranged from 8.8 to 552.3 for A. baumannii type I (imipenem MICs ≤ 2), which expressed only a β-lactamase of pI ≥ 9, and from 12.3 to 1543.5 for A. baumannii type II (imipenem MICs ≥ 4), which expressed a β-lactamase of pI ≥ 9 and two others of pI 6.3 and 7. The hydrolysing activities of A. baumannii type II against imipenem, meropenem and oxacillin were higher than those observed for A. baumannii type I. Ten outer membrane protein (OMP) profiles (A. baumannii types I and II) were visualized on 10% SDS–PAGE gels with 6 M urea, whereas only five OMP profiles (A. baumannii types I and II) were differentiated in 12% SDS–PAGE gels. Five A. baumannii with OMP profile type B, characterized by the absence of a 22.5 kDa OMP, were resistant to meropenem and/or imipenem. Twelve penicillin-binding protein (PBP) patterns were observed. PBP patterns of A. baumannii type II were characterized by the absence of a 73.2 kDa band (PBP 2). We concluded that production of β-lactamases of pI 6.3 and 7.0 and reduced expression of PBP 2 are the most frequently observed mechanisms of resistance to carbapenems. In some isolates, loss of a 22.5 kDa OMP is also related to resistance to carbapenems.

The penicillin‐binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid‐transported N‐acetyl glucosaminyl‐β‐1,4‐N‐acetylmuramoyl‐l‐alanyl‐γ‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l)‐d‐alanyl‐d‐alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46‐N844) PBP1bγ. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46‐N844) PBP1bγ possesses an amino‐terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an ≅ 100‐amino‐acid insert, to a D198‐G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of ≅ 39 000 M⁻¹ s⁻¹. Glu‐233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu‐233 γ‐COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4‐OH group of a nucleophile N‐acetylglucosamine. Asp‐234 of motif 1 or Glu‐290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4‐OH group of the N‐acetylglucosamine. In turn, Tyr‐310 of motif 4 is an important component of the amino acid sequence‐folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447‐N844 acyl transferase module, which possesses the catalytic centre‐defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active‐site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser‐510‐assisted double‐proton shuttle are glycan strands substituted by cross‐linked tetrapeptide–pentapeptide and tetrapeptide–tetrapeptide dimers and uncross‐linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on d‐alanyl‐d‐alanine‐terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross‐linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.