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.

A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHBlOl also transduced many other Streptomyges species, including -several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB10l DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.Polyethylene glycol-induced plasmid transformation of protoplasts has provided the basis for gene cloning in Streptomyces spp. (1,8,26,32) and related actinomycete genera (34,38,48 (11,16,19,20,21,28,45 (32,34,48), and subtle procedural details often need to be worked out for different species. Second, most streptomycetes produce restriction endonucleases (5,11,15,27) that can restrict bacteriophage infection and inhibit plasmid transformation (12-14, 32, 33). This problem may be compounded by procedural requirements for efficient transformation (3,7,32,34). Thus, physiological conditions for cell growth and for protoplast regeneration that might minimize the expression of restriction may not coincide with conditions for efficient uptake of DNA, establishment of plasmld replication, or regeneration of protoplasts.One way to bypass protoplast transformation, particularly for rapid subcloning of DNA into many different streptomycete strains, might be by transduction of plasmid DNA. Generalized transduction of streptomycete chromosomal genes has been demonstrated with two phages, SV1 in Streptomyces venezuelae and (SF1 in the neomycin-producing Streptomyces fradiae (11). The host range of SV1 is restricted to S. venezuelae, whereas the host range of ,SF1 has not been reported. It has not been determined whether either of these phages will transduce plasmid DNA. Bacteriophage C31 will transduce segments of DNA cloned into its genome, but the insert size is limited to about 8 kilobases (kb). Bacteriophage R4, on the other hand, will transduce plasmid DNA containing the cos site from R4 (35, 36) between Streptomyces lividans and Streptomyces parvulus, but this system may be somewhat constrained by the requirement of cos sites for packaging of DNA ipto phage heads. * Corresponding author.In this report, we describe the cloning of a segment of DNA from the streptomycete bacteriophage FP43 that mediates high-frequency transduction of plasmid DNA by FP43. The transducing particles contain linear concatemers of plasmid DNA. The transduction system has a very wide host range, including numerous Streptomyces species and some species of the ...

Conditions for efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmid cloning vectors were identified. Three streptomycete plasmid origins of replication function in A. orientalis, as do the apramycin resistance gene from Escherichia coli, the thiostrepton resistance gene from Streptomyces azureus, and the tyrosinase gene from Streptomyces antibioticus. A. orientalis appears to express some restriction and modification, because highest transformation frequencies (10(6)/micrograms of DNA) were obtained when plasmid pIJ702 was modified by passage in A. orientalis.

Streptomyces roseosporus, the producer of the cyclic lipopeptide antibiotic daptomycin, was shown to be a suitable host for molecular genetic manipulation. S. roseosporus does not appear to express significant restriction barriers based upon bacteriophage plaque formation studies. Plasmid DNA can be introduced into 5. roseosporus by bacteriophage-FP43-mediated transduction and by conjugat ion from Escherichia coli. The streptomycete transposons Tn5096 and Tn5099, derived from 15493, transpose in 5.roseosporus, and Tn5099-induced transposition mutants altered in the production of daptomycin, red pigment or black pigment were identified, and mapped to Dral and Asnl fragments. Three auxotrophic mutations (argB1, ade-7 and metB7) were identified among 100 individual TnS096 insertions. Alignment and physical mapping of several Tn5099 insertions in DraI-E and Asnl-B fragments was facilitated by the presence of DraI and Asnl cleavage sites in Tn5099. T r a n s p o s it i o n in Strep to0mycc.r rascosp o m s -_ described previously (Baltz, 1978). Antibiotic resistance levels were determined by incorporating the antibiotic into 30 nil AS-1 agar (Baltz, 19XOj or modified R2 bottom agar (Baltz, 1978) and by adding dilutions of sonicated mycelia in 4 ml nutrient broth (NB) or modified R2 soft top agar overlays (Matsushima & Baltz, 1985 give final bottom agar concentrations of 20 pg ml-' for Nal, 400 pg ml-' for Hm and 100 pg ml-' for Am to select transconjugants. Excon jugants were grown for 6 d then individual colonies were inoculated into 10 ml TS broth containing the same antibiotics, homogenized, and the cells grown at 29 "C with shaking fur 16 h. The mycelia were homogenized, sonicated, diluted and plated onto H agar or AS-1 agar containing the appropriate antibiotic for selection of the transposon marker.

We initiated a survey of the Streptococcus pneumoniae genome by DNA sequence sampling. More than 9,500 random DNA sequences of approximately 500 bases average length were determined. Partial sequences sufficient to identify approximately 95% of the aminoacyl tRNA synthetase genes and ribosomal protein (rps) genes were found by comparing the database of partial sequences to known sequences from other organisms. Many genes involved in DNA replication, repair, and mutagenesis are present in S. pneumoniae. Genes for the major subunits of RNA polymerase are also present, as are genes for two alternative sigma factors, rpoD and rpoN. Many genes necessary for amino acid or cofactor biosynthesis and aerobic energy metabolism in other bacteria appear to be absent from the S. pneumoniae genome. A number of genes involved in cell wall biosynthesis and septation were identified, including six homologs to different penicillin binding proteins. Interestingly, four genes involved in the addition of D-alanine to lipoteicoic acid in other gram positive bacteria were found, even though the lipoteicoic acid in S. pneumoniae has not been shown to contain D-alanine. The S. pneumoniae genome contains a number of chaperonin genes similar to those found in other bacteria, but apparently does not contain genes involved in the type III secretion commonly observed in gram negative pathogens. The G+C content of S. pneumoniae genomic DNA is approximately 43 mole percent and the size of the genome is approximately 2.0 Mb as determined by pulsed-field gel electrophoresis. Many of the genes identified by sequence sampling have been physically mapped to the 19 different SmaI fragments derived from the S. pneumoniae genome. The database of random genome sequence tags (GSTs) provides the starting material for determining the complete genome sequence, gene disruption analysis, and comparative genomics to identify novel targets for antibiotic development.