100 MOLECULES, VIRUSES, AND BACTERIA 



^-galactosidase* level was observed. After about 20 generations the 

 enzyme level rose to 115, which corresponds to the usual maximum 

 levels foimd in constitutive mutants. Samples of these bacteria, when 

 subcidtured outside the chemostat in a lactate or succinate medium, 

 continued to form /?-galactosidase at this rate, thus establishing that the 

 strain is constitutive for the synthesis of this enzyme. Presumably, with 

 the appearance of this strain there is a fall in the concentration of lac- 

 tose in the growth tube of the chemostat, because this strain— having a 

 higher /?-galactosidase level ( and probably a higher level of galactoside 

 permease as well ) grows at the rate defined by the chemostat flow rate 

 at a lower concentration of lactose than the original inducible strain. 



At this point some account of the role of galactoside permease in 

 this selection must be taken. It is known that simultaneously with the 

 induction of ^-galactosidase there is formation of a mechanism ( galac- 

 toside permease ) which increases the cell's permeability to galactosides 

 and can concentrate galactosides within the cell (Cohen and Monod, 

 1957). Unfortunately, no significant measurements of permease have 

 yet been made with the present strains. It is quite likely that the con- 

 stitutive strain described above is also constitutive for galactoside 

 permease, since the induction of the enzyme and the permease almost 

 always occur together (Jacob, Perrin, Sanchez, and Monod, 1960). In 

 fact, it is probable that the presence of high levels of the permease is 

 what gives the constitutive strain the advantage here over the inducible 

 one; thus the appearance of strains constitutive for /3-galactosidase re- 

 flects the selection of strains constitutive for the permease. 



Continued operation of the chemostat led to the appearance of 

 bacteria with higher and higher levels of /?-galactosidase. These high 

 rates of enzyme formation occurred constitutively: high enzyme levels 

 were maintained when the bacteria were subcultured in the absence 

 of lactose. 



Continued growth at a limiting concentration of lactose finally 

 gives rise to strains which seem to have some maximum level of 

 /?-galactosidase, because extensive further growth does not yield higher 

 levels. This "maximum" is at a specific activity of over 500, or about five 

 times that usually found in constitutive strains. Even after more than 

 two years ( <—' 1500 generations ) of growth in a chemostat with lactose 



* ^-galactosidase activity in toluene-treated bacteria was determined by the 

 hydrolysis of ortho-nitro-phenyl-^-D-galactoside (ONPG) by the procedure de- 

 scribed by Novick and Weiner ( 1957), except that 0.5 mg/ml of sarcosyl (at the 

 suggestion of Arthur Pardee) was added instead of sodium desoxycholate with 

 the toluene. The enzyme level is expressed in imits of milhmicromoles of ONPG 

 hydrolyzed per minute per microgram bacterial nitrogen at 28° C. at pH7 in M/10 

 sodium phosphate buffer. 



