Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

We describe a new, sensitive, rapid, and nonradioactive method involving the use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding proteins (PBPs). This method allowed rapid detection of 30 ng of a purified PBP protein under UV light and of 2 to 4 ng of the protein with the aid of a FluorImager. This method also allowed rapid determination of the PBP profiles of Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae. The PBP profiles obtained are virtually identical to those reported previously with 3H-,14C-, or 125I-labeled penicillin. Using this method enabled us to determine the 50% inhibitory concentrations of the penicillin-sensitive and -resistant PBP2x proteins of S. pneumoniae for penicillin G, thereby allowing a direct evaluation of their relative affinities for penicillin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for different β-lactam antibiotics with the aid of fluorescence polarization technology and to monitor a PBP2x protein during purification.

Group I β-lactamases are major resistance determinants to β-lactam antibiotics. Despite intense study, the identity of the catalytic base, the direction of hydrolytic attack, and the functional difference between β-lactam substrates and β-lactam inhibitors remain controversial. To explore these questions, we determined the X-ray crystal structures of several representative β-lactams in their acyl−adduct complexes with the group I β-lactamase AmpC. A complex with the substrate loracarbef and a deacylation-deficient mutant enzyme reveals an ordered water molecule that is consistent with β-face attack of the catalytic water in group I β-lactamases. The ring nitrogen of the substrate is placed to hydrogen-bond with this water in the position it is thought to adopt in the deacylation transition state. In complexes with the inhibitors cloxacillin and moxalactam, conformational restrictions displace the equivalent ring nitrogens, sterically blocking the formation of the presumed deacylation transition-state structure. In conjunction with earlier studies, these acyl−enzyme structures suggest that both Tyr150 and the ring nitrogen of the substrate itself stabilize the hydrolytic transition state, and that β-lactam inhibitors of group I β-lactamases can act by physically blocking this activation.

The structures of AmpC beta-lactamase from Escherichia coli, alone and in complex with a transition-state analogue, have been determined by X-ray crystallography. The native enzyme was determined to 2.0 A resolution, and the structure with the transition-state analogue m-aminophenylboronic acid was determined to 2.3 A resolution. The structure of AmpC from E. coli resembles those previously determined for the class C enzymes from Enterobacter cloacae and Citrobacter freundii. The transition-state analogue, m-aminophenylboronic acid, makes several interactions with AmpC that were unexpected. Perhaps most surprisingly, the putative "oxyanion" of the boronic acid forms what appears to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated. Although this interaction has not previously been discussed, a carbonyl oxygen contact with the putative oxyanion or ligand carbonyl oxygen appears in most complexes involving a beta-lactam recognizing enzyme. These observations may suggest that the high-energy intermediate for amide hydrolysis by beta-lactamases and related enzymes involves a hydroxyl and not an oxyanion, although the oxyanion form certainly cannot be discounted. The involvement of the main-chain carbonyl in ligand and transition-state recognition is a distinguishing feature between serine beta-lactamases and serine proteases, to which they are often compared. AmpC may use the interaction between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabilization energy might be relieved in the transition state by a hydroxyl hydrogen bond. The structure of the m-aminophenylboronic acid adduct also suggests several ways to improve the affinity of this class of inhibitor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.

Bacterial peptidoglycan is composed of a network of beta-[1,4]-linked glyan strands that are cross-linked through pendant peptide chains. The final product, the murein sacculus, is a single, covalently closed macromolecule that precisely defines the size and shape of the bacterial cell. The recent increase in bacterial resistance to cell wall active agents has led to a resurgence of activity directed toward improving our understanding of the resistance mechanisms at the molecular level. The biosynthetic enzymes and their natural substrates can be invaluable tools in this endeavor. While modern experimental techniques have led to isolation and purification of the biosynthetic enzymes utilized in peptidoglycan biosynthesis, securing useful quantities of their requisite substrates from natural substrates has remained problematic. In an effort to address this issue, we report the first total synthesis of lipid II (4), the final monomeric intermediate utilized by Gram positive bacteria for peptidoglycan biosynthesis.

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field effect transistors through pyrene linker molecules. The substrate driven, hinge bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibits processive slow turnover rates of 20 – 50 s−1 and rapid 200 – 400 s−1 non-productive motions. The latter, nonproductive binding events occupy 43% of the enzyme’s time in the presence of the cross-linked peptidoglycan, but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate, but appears to sidestep the peptide cross-links to zigzag through the wild-type substrate.

No abstract

A total synthesis of lipid I (4), a membrane-associated intermediate in the bacterial cell wall (peptidoglycan) biosynthesis pathway, is reported. This highly convergent synthesis will enable further studies on bacterial resistance mechanisms and may provide insight toward the development of new chemotherapeutic agents with novel modes of action.