BACTERIA WITH HIGH LEVELS OF SPECIFIC ENZYMES 101 



limitation, the yS-galactosidase level in the E. coli strain remains at 

 about this value. 



Properties of bacteria with high /3-gaIactosidase activity 



We believe that in these strains with high /i-galactosidase activity 

 there is actually a corresponding high level of enzyme in the cell rather 

 than an altered enzyme with a higher turnover number. The principal 

 reason for believing this to be the case comes from ultra-centrifugal 

 studies being made by Burton Guttman. He has examined the sedimen- 

 tation pattern in the Spinco analytical ultra-centrifuge of cell extracts 

 made by breaking bacteria suspended in cold buffer containing 10"^ M 

 Mg++ in a French press. A series of strains varying from high to low 

 yS-galactosidase levels were used, and he detected a sedimentation 

 boundary at about 16 S which he ascribed to /?-galactosidase. He 

 found that the amount of material present, computed from the Schlie- 

 ren peak, is proportional to the measured ^-galactosidase activity for 

 strains having different levels of /?-galactosidase, thus showing that 

 more enzyme activity results from more enzyme. 



Less convincing but plausible evidence for this conclusion comes 

 from studies of the "stabihty" of cultures with high specific activities. 

 Subculture of the very active strains away from the highly selective 

 conditions imposed by the chcmostat usually leads to the appearance of 

 strains of lower /?-galactosidase activity. In this sense many of the high- 

 level strains are unstable. For example, subculture for some 50 genera- 

 tions in nutrient broth medium, or in synthetic medium with lactate or 

 succinate as the carbon source, frequently yields cultures which have a 

 /?-galactosidase activity equal to or less than that of the normal con- 

 stitutive strain. This change occurs, it appears, because bacteria with 

 unusually high enzyme acti\'ity have a lower growth rate than bacteria 

 having less enzyme, and are thus outgrown by them. The lower growth 

 rate might result from the burden of producing the substantial— but in 

 these conditions useless— amounts of protein represented by the yS-ga- 

 lactosidase in the high-activity strains. It would not be expected that a 

 bacterium producing an altered enzyme not needed for growth would 

 necessarily have a lower growth rate. 



Measurement of the Michaelis constant for the substrate ortho- 

 nitro-phenyl galactoside of the enzyme in extracts from bacteria of 

 high activity invariably yields a value around 1.3 x 10"* M, which is the 

 value normally observed for /^-galactosidase (Lederberg, 1950). This 

 is another reason to believe that there is nothing unusual about the 

 /?-galactosidase of strains with high levels of the enzyme. 



To estimate the actual relative amount of /3-galactosidase in the 

 bacteria, we used the specific activity- for /8-galactosidase reported by 



