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.

Cloned glycosyltransferases from glycopeptide antibiotic producers can be used to produce novel hybrid antibiotics, both in vitro and in vivo. Because similar enzymes have differing degrees of substrate specificity, it is advantageous to characterize the substrate specificity with enzymes expressed in E. coli prior to constructing recombinant actinomycetes for production.

Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

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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.