The Assimilation of Amino-acids by Bacteria: 5. The Action of Penicillin in Preventing the Assimilation of Glutamic Acid by Staphylococcus aureus

SUMMARY: To see whether the initial damage caused by penicillin involved the osmotic barrier, certain characters of Staphylococcus aureus were examined at intervals during the first 2 hr. after addition of penicillin to growing cultures. In the 30 min. following addition of penicillin the cell appeared to expose nearly all its reserve penicillin-binding component (PBC), the penicillin uptake being double that which occurred without growth and the reserve PBC disappearing. The amount of PBC in the cell and its rate of exposure to penicillin in excess of normal synthesis (rate of turnover?) were both very small. Between 30 and 60 min. the uptake of Na, Mg, K, P and increase of total dry matter ceased abruptly. Continued increase in dry wt., while P, Nay Mg and K decreased slightly, resulted in the penicillintreated cells becoming relatively deficient in these elements. Synthesis of lipid phosphorus and some, but not all, large molecule phosphates still continued, and Fe and Co uptake were not affected. After 60-80 min. water entered and solutes began to leave the cell, and synthesis of large molecule P ceased. The primary site of action of penicillin is probably not concerned with gross assimilation of Na, K, Mg, P, Fe, Co, or any substance contributing more than 10% of the dry wt., or with gross synthesis of lipid P. It may, however, involve a reaction turning over 3000 times while the cell mass is doubled.To describe fully the mode of action of a drug one must account for all the changes which take place from the moment of first contact between cell and drug. Although this is clearly impossible to do in detail, some such attempt is necessary as it is very difficult to pin-point any particular defect as being the cause of death. The specific nature of the penicillin molecule and the small amounts bound by cells suggest that the changes caused by the drug are sequences of events branching from perhaps only one small and specific injury. Each event plays its part in a sequence which culminates in the inability of most of the population to divide, and the relative time of appearance of abnormalities is important.It was shown elsewhere (Cooper, 1954) that penicillin is firmly bound in a specific manner by a lipid-containing fraction near the cell wall of a sensitive staphylococcus. Believing that the penicillin-binding component (PBC) of sensitive cells (Rowley, Cooper, Roberts & Lester Smith, 1950) is likely to be the initial site of the bactericidal action of penicillin when acting a t its minimal concentration, it was suggested that its presence in this fraction might indicate that the first effect of penicillin is to disorganize some function specific to the cell wall or to the osmotic barrier. To obtain further information on this possibility, penicillin was added to growing cultures of sensitive staphylococci,

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Site of Action of Penicillin: some Changes in Staphylococcus aureus during the First Two Hours Growth in Penicillin Media

Cooper

1955

“…The statement that the metabolism of glutamic acid takes place inside the cell rests upon the facts that no assimilation of glutarnic takes place in the absence of glucose (Gale, 1947cc), and no disappearance of glutamic acid from combined internal and external environments occurs in the absence of glucose. Further evidence will be presented in the nest paper of this series (Gale & Taylor, 1947) that this metabolism is intracellular only. It follows that for a dyestuff to inhibit such metabolism it must first penetrate the cell wall, and this fact can probably be correlated with the variation of antibacterial efficiency of the triphenylmethane dyes and its relation, in turn, to the isobutanollwater partition coefficient.…”

Section: Discussionmentioning

confidence: 99%

The Assimilation of Amino-acids by Bacteria: 4. The Action of Triphenylmethane Dyes on Glutamic Acid Assimilation

Gale

Mitchell

1947

Self Cite

SUMMARY:The level of glutamic acid concentration measured inside the streptococcal cell during assimilation represents a balance between the rate a t which the amino-acid is withdrawn from the external medium and the rate a t which it is metabolized within the cell.Treatment of the cells, prior to or during assimilation, with dyes of the triphenylmethane series results in raising the level of free glutamic acid attained within the cell. Evidence is presented that the triphenylmethane dyes prevent the metabolism of glutamic acid within the cell. The activity of the triphenylmethane molecule as an antibacterial agent and in raising the level of glutamic acid assimilation is increased by alkyl substitution and can be correlated with the lipid solubility of the dye.Dyes of the triphenylmethane series have long been known as effective antibacterial agents. Kligler (1918) showed that the quinonoid structure of the dye molecule is important for antimicrobial activity and that the effectiveness of the dyes is increased by substitution of alkyl radicles in the amino-groups of triamino-and diamino-triphenylmethanes. He showed that bacteria differ widely in their sensitivity to these dyestuffs and that, in general, Gram-positive organisms are more sensitive than Gram-negative species. The dyes fell into the following order of increasing effectiveness : fuchsin (1 CH,), methyl violet ( 5 CH,), crystal violet (6 CH,), malachite green (4CH,), brilliant green (4C2H5).

“…There would appear to be no a priori reason for a decreased oxidative activity which occurs as a nonspecific accompaniment of a decreased growth rate; no such change occurred in S . aureus after inhibition of growth by penicillin (Gale & Taylor, 1947) or other antibiotics …”

Section: Discussionmentioning

confidence: 99%

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

Hancock

1961

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.