A B S T R A C T A procedure has been developed for obtainingHem0philus influenzae of such competence that 1 to 10 per cent transform to any of several genetic factors by utilizing a period of aerobic growth followed by a non-aerobic period. Differences in levels of competence were not due to differences in genetic background. Competence was due to at least one factor intrinsic to the cell or site on the cell and was not transferable to non-competent cells. Competence was affected by salt concentration, pH, and temperature. Washing competent cells reduces their ability to transform, but not their capacity to bind DNA reversibly. The irreversible step could be restored with little or no accompanying growth. These facts suggest that reversible and irreversible binding represent separate biochemical steps. DNA initiates a reaction in cells leading to a loss of competence. In the absence of DNA the cells remain competent for at least an hour. Competence correlates quantitatively with predictability of multiple transformations. The observed and calculated values of multiple transformations are in closer agreement, the higher the frequency of transformation for single markers. The correction needed to bring the two figures into agreement is a measure of the fraction of non-competent ceils.

/mSTRAC¢H~mopkilus influen~--transforming DNA, which has been inactivated by ultraviolet radiation, is reactivated by visible light in the presence of a cell-free extract of Eschcric~ia coli B.The time rate of reactivation is increased by increasing the E. coli extract concentration, the temperature, and the intensity of illumination.Only DNA containing an ultraviolet-damaged genetic marker exhibits increased transforming activity after treatment with the photoreactivatlng system.The reactivating capacity of the extract remains in the top supernatant after centrifugation at 110,000 X g for 1 hour and is not present in the pellet. This capacity is destroyed by heating to 90°C. for 1 minute.The active system of the E. cdi extract is separable into dialyzable, heat-stable and non-dialyzable, heat-labile fractions. The dialyzable fraction contains at least one component which limits the max-imum degree of recovery attained.Photoreactivation, the reversal of short wave length ultraviolet effects on organisms by subsequent treatment with visible light, was first specifically described by Keiner (1, 2) for survival of the ultraviolet-irradiated conidia of Streptomyces griseus. It has since been recognized for a variety of other ultraviolet effects in a large number of species and tissues (3), including interruption of DNA synthesis (4), production of mutations (5), induction of vegetative phage in lysogenic bacteria (6), inhibition of adaptive enzyme formation in yeast (7), spheration of nudeoli in the grasshopper neuroblast (8), delaying of cleavage in eggs (9), and delaying of division in protozoa (10). Its detailed mechanism is unknown. A possible outline of the process, however, is suggested by existing evidence. * Present address:

During the process of transformation in Hemophilus infiuenzae integration of donor DNA, i.e. the formation of recombinant DNA, involves the incorporation of single-stranded DNA. Evidence was obtained from cesium chloride density gradient centrifugation of DNA from donor-recipient complexes that integration was accompanied by the formation of hybrid DNA with a density intermediate with respect to heavy, 2 H, 15

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 ...

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