Streptomycin-induced Oversuppression in E. Coli.

Any model for the action of extracistronic suppressors'-3 that involves the mistaken reading of a codon appearing often in the genome, predicts that if the efficiency of suppression is high, the suppressor would be lethal. We have found strains in which this "oversuppression" can be brought about by an external agent, streptomycin (Sm). This Sm-induced oversuppression appears to be the combined result of two suppressors of the same defect. These strains result from a second suppressor mutation in a Sm-suppressible (conditional Sm-dependent, CSD) parent. The CSD strains require either a growth factor or Sm for growth and have been demonstrated4 to contain a competent Smr mutation and a defect in a structural gene, whose suppression is Sm-dependent. In vitro experiments5 have provided support for the suggestion that Sm acts in the CSD strains to cause specific mistakes in the genetic code. We shall describe here the strains that contain the two suppressor functions, and show that in the presence of Sm their combined effect, although increasing the level of the suppressible defective enzyme, decreases the levels of other enzymes in the cell and inhibits growth. We propose to call this phenomenon "oversuppression." Isolation and Characterization of the Mutants.B4S-7SD is a CSD-arginine mutant containing a suppressible defect in the ornithine transcarbamylase structural gene (OTCsD) and a streptomycin-activated suppressor.4 Spontaneous revertants to arg+ were isolated (frequency 10-7) from a histidine-requiring derivative of this strain by spreading the cells (about 108 cells/plate) on solid minimal medium A6 + his + glucose. It was expected that the revertants would be Smr like the original Smr parent of these strains. However, 4 out of 38, when tested with the standard concentration of Sm (500 jig/ml) appeared sensitive. One of these mutants is B4S-71. The sensitivity of these mutants is different from the wild-type sensitivity. In minimal medium + histidine, 75 per cent of a growing culture of B4S-71 survives a one-hr exposure to 500 ,g/ml Sm at 37°. In rich medium the survival is 10 per cent, whereas that of the standard Sms strain is less than 0. 001 per cent in both media. B4S-71 is slow-growing in minimal medium + histidine as compared with the original prototrophic parent, B4S-7 (OTC+ Smr) (Table 1). Addition of arginine to the medium restores wild-type growth rate. When small amounts of Sm are added, the growth rate decreases sharply. The upper limit of Sm concentration permitting a measurable growth depends on the composition of the medium: in minimal medium, with or without arginine, this limit is 12 Ag/ml; in enriched medium, 50 ,4g/ml (Table 1). Mutants of B4S-71 resistant to high levels of Sm were isolated (approximate frequency 10-6) by spreading the cells on solid enriched medium + Sm (500 jig/ml). All 358 mutants examined were CSD mutants indistinguishable from the original B4S-7SD his-. This suggests that the revertant phenotype had been the result of a suppressor mutation affecting both the OTCSD and Smr characteristics.