Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae

Abstract

A physical map of the Streptococcus (Diplococcus ) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.Transformation of Streptococcus (Diplococcus) pneumoniae was the first method found to transfer genetic information in bacteria (1, 14), and it has had a monumental impact on the development of molecular biology. Although the pneumococcal transformation system has been extensively investigated for the past 45 years (6,17,40,41), understanding of its genetics and biochemistry, compared with that of Escherichia coli (2, 44) and/or Bacillus subtilis (16), is still relatively primitive. The absence of a genetic map has been a major impediment to the investigation of the molecular genetics of S. pneumoniae.The ability to separate large fragments of DNA by pulsedfield gel electrophoresis has provided the technology to map the chromosomes of bacteria (10, 19, 20, 25-27, 43, 44), and this technology has gained wide acceptance for analyzing the chromosomes of more complex organisms as well. The map of the S. pneumoniae chromosome should provide a useful framework for further molecular and genetic investigations. MATERIALS AND METHODSBacterial strains and growth conditions. All bacteria used in this study are derived from pneumococcus strain R6. A subculture of S. pneumoniae 800 or 801 was used in this study (28), along with a multiply marked strain (strain 119) resistant to 200 ,ug of streptomycin (str41) per ml, 75 ,ug of fusidic acid (fus-rA) per ml, 4 ,ug of novobiocin (nov-rl) per ml, 2 ,ug of optochin (opt-r2) per ml, 1 ,ug of rifampin (rif-rF) per ml, and 5 ,ug of streptolydigin (stg-rF) per ml (47). The uvr resistance gene (48) was located with the use of the cloned gene (42). To map the amiA gene, strain 801 bearing amiA3 was used (12). The methotrexate-sensitive bacteria in the resistant population were selected in synthetic medium containing an excess of isoleucine (40). The strain resistant to 0.1 ,ug of cefotaxime per ml was obtained by transforma- tion of strain 801 with DNA extracted from strain C506, which is resistant to a higher level of cefotaxime (1 ,ug/ml) (23). A second level of resistance (0.4 ,ug/ml) was obtained by transformation of a strain resistant to 0.1 ,ug of cefotaxime per ...

Chromosome organization of Streptococcus mutans GS-5

“…The S . pneumoniae chromosome, which also has a low GC content, was estimated to have a size of 2270 kb (Gasc et al, 1991), and lactococcal genomes have been reported to have genome sizes in a range from 1750 to 2700 kb (LeBourgeois et al, 1989;Tanskanen et al, 1990).…”

Physical and genetic map of Streptococcus thermophilus A054

“…Likewise, the ldh gene was localized in the region opposite the inn locus region in the two species, but its chromosomal location was not exactly conserved (33). Physical and genetic maps of other streptococcal strains, S. pneumoniae (18) and S. mutans (24,36), have also been established with PFGE. In the S. pneumoniae chromosome, six nn loci were also found, and three of them have been located on the chromosomal map.…”

Molecular characterization of cap3A, a gene from the operon required for the synthesis of the capsule of Streptococcus pneumoniae type 3: sequencing of mutations responsible for the unencapsulated phenotype and localization of the capsular cluster on the pneumococcal chromosome

“…Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA. DNA embedded in agarose plugs were prepared from S. pneumoniae and S. oralis as previously described (20). DNAs were digested with either SmaI, SacIl, or ApaI before electrophoresis.…”