Reduced Oxidative Activities in Escherichia coli and Bacillus megaterium in Relation to Other Changes during Inhibition of Growth by Streptomycin

Abstract

SUMMARY When the growth of cultures of Bacillus megaterium KM, Escherichia coli B orBacillus subtilis ceased following addition of streptomycin during the exponential phase of growth, harvested organisms showed oxidative activities on glucose, lactate, pyruvate and succinate which were between 20 and 60 yo of those of normal organisms. With E . coli, this reduction in oxidative activity was closely parallel to the decrease in growth rate. During the period in which growth became fully inhibited by streptomycin, the total quantities of amino acids and 260 mpabsorbing compounds in hot water extracts of E . coli and B. rnegaterium did not differ significantly from those in normal organisms, Protoplasts could be prepared in normal yield from streptomycin-inhibited organisms of both these species ; such protoplasts showed normal stability and had internal osmotic pressures which differed only slightly from normal. From these results it appears unlikely that there was any general breakdown of the permeability barrier of these organisms during inhibition of growth by streptomycin. Lysates of protoplasts from streptomycininhibited Bacillus megaterium showed oxidative activities which were also decreased to between 20 yo and 45 yo of those of lysates from normal organisms. With SUCcinate and malate as substrates this difference appeared to be due predominantly to a considerable decrease in the activity of the sedimentable 'ghost' fraction of the lysed protoplasts.

EARLY EFFECTS OF STREPTOMYCIN ON BACILLUS MEGATERIUM

“…The present experiments show clearly that the effect of Sm on respiratory activity in B. megaterium, as in E. coli (Dubin et al, 1963), does not begin until after killing has commenced; previous experiments (Hancock, 1961) had suggested that this effect is more nearly concurrent with reduction in growth rate. Since nucleic acid is synthesized at the normal rate after viability is lost in medium AP1, it seemed probable that ATP synthesis must continue, and direct estimations show that the cellular pool of ATP does remain nearly normal until after killing has begun.…”

“…The earliest detectable changes were an increase in the rate of efflux of cellular K ions coinciding with a stimulation of the rate of ribonucleic acid (RNA) synthesis, followed by a specific inhibition of protein synthesis coinciding with loss of viability. To see whether a similar picture of the action of Sm is observed in other organisms, and to extend earlier observations (Hancock, 1961(Hancock, , 1962) which indicated some differences in the effect of Sm on Bacillus megaterium and E. coli, similar experiments were carried out with B. megaterium. This organism is highly sensitive to Sm, and low concentrations of the drug (4 to 5,ug/ml) were used to spread out the effects in time.…”

“…Respiration rate and ATP level. Sm-treated bacteria frequently show deficient respiratory activities (Henry, Housewright, and Berkman, 1949;Jackson, 1958;Hancock, 1960Hancock, , 1961, and changes in the quantities of excreted metabolites have suggested that intermediate metabolism of glucose may be altered (Barkulis, 1953;Rosanoff and Sevag, 1953;Bragg and Polglase, 1962). In the present experiments, careful comparison with viability showed that reduction of respiration rate does not occur until well after killing and inhibition of protein synthesis has begun (Fig.…”

Uptake of 14G-Streptomycin by Bacillus megaterium

“…Bacillus mgaterium KM was grown in CGY medium (Hancock, 1962) adjusted to pH 7.2, except in one experiment where the pH value was deliberately changed. The conditions of growth were the same as R. HANCOCK those used earlier for this organism (Hancock, 1961). Streptomycin was added to cultures in the exponential phase of growth when the optical density (at 700 mp) was 0-6-0-7.…”