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.

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The first step in oral absorption of many medically important peptide-based drugs is mediated by an intestinal proton-dependent peptide transporter. This transporter facilitates the oral absorption of beta-lactam antibiotics and angiotensin-converting enzyme inhibitors from the intestine into enterocytes lining the luminal wall. A monoclonal antibody that blocked uptake of cephalexin was used to identify and clone a gene that encodes an approximately 92-kilodalton membrane protein that was associated with the acquisition of peptide transport activity by transport-deficient cells. The amino acid sequence deduced from the complementary DNA sequence of the cloned gene indicated that this transport-associated protein shares several conserved structural elements with the cadherin superfamily of calcium-dependent, cell-cell adhesion proteins.

The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N, which is a central reaction in the pathway to clinically important penicillins and cephalosporins. Here we report the cloning, characterization and expression in Escherichia coli of the gene encoding the IPS protein in Cephalosporium acremonium. The IPS gene was identified by purifying IPS protein, determining the first 23 amino-terminal amino acids, preparing a set of synthetic oligonucleotides encoding a portion of the determined amino-acid sequence, and probing a cosmid genome library with the mixed oligonucleotides. A cosmid hybridizing with the probe was isolated and the IPS gene was localized and sequenced. The IPS gene encodes a polypeptide of relative molecular mass (Mr) 38,416. When this open reading frame was cloned into an E. coli expression vector and inserted into E. coli, the recombinant E. coli produced a new protein co-migrating with authentic IPS as the major protein of the cell (approximately 20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.

Era, an essential GTPase, is present in many bacteria and Mycoplasma spp. and appears to play a major role in the cell cycle and other cellular processes. To further understand its function, an era gene from Streptococcus pneumoniae was identified and cloned, and a mutant era gene with a deletion of 68 codons from its 3'4erminus was constructed. The truncated Era protein was then purified and characterized, and the ability of the truncated era gene to complement an Escherichia coli mutant strain defective in Era production was examined. Like the full-length Era protein, the truncated Era protein was able to bind and hydrolyse GTP, but its binding activity was increased twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era protein, the truncated Era protein lost its ability to bind to the E. coli cytoplasmic membrane. The fulllength era gene was able to complement the €. coli mutant deficient in Era production when carried on pACYCl84, while the truncated era gene failed to do so when carried on pACYCl84, pBR322 or pUC18. The cellular amounts of the truncated Era and the full-length Era proteins in €. coli and S. pneumoniae, respectively, were then determined by Western blot analysis. In addition, the minimal amount of the S. pneumoniae Era protein needed for complementation of the E. coli mutant was also measured. Taken together, these results suggest that the C-terminus of the Era protein might be responsible for the binding of the protein to the cytoplasmic membrane and be essential for function.