102 MOLECULES, VIRUSES, AND BACTERIA 



Cohn (1957): namely, 2,100 millimicromoles of ONPG hydrolyzed per 

 minute (pHT in M/10 sodium phosphate bufiFer) per microgram of 

 nitrogen. The nitrogen content of the bacteria was determined by the 

 Kjeldahl procedure, and the enzyme was assayed by the procedure 

 described earher, which is identical to that employed by Cohn. The 

 maximum activities observed in the high-producing strains is about 

 500 millimicromoles per minute per microgram of Kjeldahl nitrogen, 

 although one strain was observed to have an activity of 750 millimicro- 

 moles per minute per microgram of nitrogen. Hence the usual maximum 

 level indicates that 24 per cent of the bacterial nitrogen is ^S-galacto- 

 sidase; the one exceptional case has 36 per cent. 



This estimate is almost certainly an upper limit, since Cohn's 

 value for the specific activity of ^-galactosidase must be a minimum 

 value. But it cannot be far from the correct value: Cohn's preparations 

 cannot contain very much inactive material, because of the high yields 

 he obtained in his enzyme isolations and because he demonstrated that 

 his preparations contained very little protein other than /?-galactosidase. 



As has been mentioned earlier, a feature of the strains with high 

 activity is their relative instability. Some strains are so easily lost that 

 often one subculture (20 generations) leads to a strain of much lower 

 activity. Some of the strains are more stable, one having been sub- 

 cultured for more than 30 generations with no apparent loss in activity. 



The relative instability of the high-activity strains makes for 

 serious difficulty in their use, but some help is found when the cul- 

 tures are grown under conditions in which there is some inhibition of 

 enzyme formation. The presence of glucose (0.5 per cent) in the cul- 

 ture medium will lower the rate of production of ^-galactosidase by 

 constitutive strains ( Monod and Cohen-Bazire, 1953; Cohn and Hori- 

 bata, 1959) and will cut the highly active strains' production of the 

 enzyme to less than one-half. This reduction in the rate of /?-galacto- 

 sidase formation, no doubt relieving the highly active strains' burden 

 of synthesizing protein, may explain why they are not so quickly out- 

 grown under these conditions by bacteria with lower levels of the 

 enzyme. 



A curious feature of the high-activity strains, first observed by 

 Arthur Dalby, is the extremely toxic eflFect that lactose and other 

 galactosides have on them. When aliquots of a suspension of bacteria 

 possessing high levels of ^-galactosidase are plated on a nutrient broth 

 medium and on an EMB lactose medium, the number of colonies on 

 the lactose-containing plate varies, depending on the strain, from one- 

 tenth down to one-ten millionth of the number found on the nutrient 

 broth plate. A number of other galactose derivatives, including thi- 

 omethyl-/3-D-galactoside (TMG) and galactose, also are effective in 



