The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6-8 to 8.0) all developed competence, as measured by [3H]DNA uptake, [ 3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.

Several transformation-deficient mutants of Streptococcus pneumoniae were isolated after insertionduplication mutagenesis. Mutagenesis was accomplished by transformation of competent cells with chimeric DNA formed by the ligation of Taql fragments of pneumococcal DNA to the erythromycin resistance determinant of the streptococcal plasmid pAM,31. The two mutants described were characterized as defective in the control of competence induction, possibly due to a block in the production of the intercellular competence-inducing protein.

Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partiallypurified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several maLt mutations revertible by nitrous acid showed them to correspond to A -T-*G C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G C-*A * T mutations. Examination of maIM mutant reversion frequencies in ung strains indicated that G. C-*A T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malMl mutation corresponding to an A. T->G -C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This "hot spot" was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.Uracil-DNA glycosylases hydrolyze the N-glycosidic bond between uracil and deoxyribose in DNA. First observed in Escherichia coli (33), these enzymes are found in many different procaryotic and eucaryotic cells. Genes encoding the enzyme from several sources-including E. coli (11,54), Saccharomyces cerevisiae (42), and human placenta (41)-have been cloned and sequenced. The results show the enzyme proteins from these diverse sources to be homologous and highly conserved, with >50% of the amino acid residues identical within a protein segment the size of the E. coli enzyme.One function of uracil-DNA glycosylase is the repair of DNA in which cytosine residues were spontaneously converted to uracil by deamination. Measurement in vitro of the rate of cytosine deamination in DNA at elevated temperatures (35) indicated that such deamination under physiological conditions could contribute significantly to spontaneous mutations in vivo. However, in E. coli these mutations only appear in ung mutants that have lost the glycosylase activity and therefore cannot remove uracil, which is the first step in restoring the cytosine residue (12, 13).The glycosylase can also remove uracil residues that are incorporated into DNA from dUTP precursors during replication. Although such incorporation occurs in wild-type cells, the extent of incorporation of uracil is greatly increased in dut mutants of E. coli, and its removal is blocked in ung dut double mutants (53). These cells are viable even though as much as 15% of the thymine residues in their DNA are substituted by uracil (55). The function, therefore, of the incorporation of ur...