Table 6.

Transfer of a silent point mutation at thecomCDE locus: use of Janus-containing strain R1036

Donor DNAGrowth (CFU/ml) with selection on:Smr frequency per 10,000 cellsFold increase in Smr frequency
NoneSmRifa
R304 chromosomal DNA1.56 × 108 2.44 × 106 5.8 × 105 156246.5
cat3-BM52 (UP) PCR fragment2.02 × 104 1.32.04b
None9.9 × 103 0.63c 1
  • a The ratio Rifr/Smr is normally close to 0.15 in ahex + recipient because Hex-dependent mismatch repair affects integration of the rif23 marker more severely than of rpsL41. The value (0.24) obtained here was expected, as Smr transformants do not result from substitution of the genuine copy of rpsL by therpsL41 allele but from excisional replacement of thekan-rpsL cassette by comC +.

  • b Four Smr clones were further analyzed by PCR. Three corresponded to substitution of thekan-rpsL + cassette by thecomC + segment (excision events) and harbored the UP mutation (see Fig. 2); the fourth clone resulted from a spontaneous conversion event and had retained the kan-rpsL cassette.

  • c A lower spontaneous conversion frequency (0.12 per 10,000 cells) was measured in four independent cultures of strain R1036. This value is totally consistent with spontaneous conversion frequencies measured at the cbp3 locus using therpsL1-rpsL + mismatched combination and ahex + recipient (see Table 5).