BACTERIA WITH HIGH LEVELS OF SPECIFIC ENZYMES 95 



has reported that ^-galactosidase can comprise as much as 6.6 per cent 

 of the bacterial protein in fully induced Escherichia coli bacteria. And 

 Garen and Levinthal ( 1959) have found that during phosphate starva- 

 tion, where there is complete removal of repression of alkaline plios- 

 phatase fonnation (Horiuchi, Horiuchi, and Mizuno, 1959; Torriani, 

 1960), this enzyme amounts to as much as 5 per cent of the total pro- 

 tein produced. Levinthal (personal communication) suggested that this 

 represented the maximum rate at which any enzyme is made when its 

 synthesis is completely unrepressed. However, it remained possible 

 that there were mutants which could make a specific enzyme at an even 

 higher rate, and we decided to search for such mutants. 



It was realized that the chemostat (Novick and Szilard, 1950a) 

 might be employed to establish conditions which would select for 

 strains of bacteria that make large amounts of a specific enzyme. In the 

 chemostat, bacteria are limited in their growth rate by the low concen- 

 tration of some required nutrilite. Bacterial mutants able to grow 

 faster at these low concentrations would outgrow the original bacteria 

 and thus replace them as the prevailing population. In the chemostat, 

 then, one may observe a succession of bacterial populations, each suc- 

 cessive one being able to grow faster at a given concentration of the 

 substrate (or at the same rate at a lower concentration of the sub- 

 strate). For example, a case was observed in which a tryptophan- 

 requiring E. coh strain was grown for several hundred generations on a 

 medium limited in tryptophan (Novick and Szilard, 1950b; Novick, 

 1958). During this time there emerged some five or six successive pop- 

 ulations, each of which was able to grow more rapidly than its prede- 

 cessor. It was possible to establish the fact that the succeeding strains 

 were able to grow more rapidly at Ion^' concentrations of tryptophan, 

 but it was never discovered in what other way the faster growing bac- 

 teria differed from the slower. Presumably they were able to capture 

 tryptophan more effectively than the slower strains because they had 

 either a better tryptophan permease system or a better system for con- 

 verting tryptophan to some product which could not escape from the 

 cell. 



In thinking about what direction such an evolution would take in a 

 chemostat limited in the sugar lactose, Milton Weiner (personal com- 

 munication) realized that, among other things, the conditions would 

 favor the selection of strains which could make ^-galactosidase in the 

 absence of an inducer. He argued that for the wild-type strain, which 

 requires the presence of an inducer for the formation of both ^-galacto- 

 sidase and galactoside permease, the effect of the lactose concentration 

 in the chemostat in controlling the growth rate would depend upon the 

 ability of lactose to act as an inducer for formation of /5-galactosidase 

 and galactoside permease. A mutant capable of forming the enzymes 



