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Title: The biosynthesis of β-lactamase in Staphylococcus aureus
Author: Leggate, John
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1967
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beta-Lactamase is an inducible enzyme in Staphyloeoecus aureus C23/19. Until recently, the only available inducer were powerful antibacterials which were hydrolysed by the enzyme. Those which were not hydrolysed by the enzyme were either weak inducers or had antibacterial activity, CBAP, 2 (2'-carboxyphenyl) benzoyl-6-amino-penicillanie acid, combines insensitivity to beta-Lactamase with minimal antibacterial activity while still retaining activity as an inducer. Using CBAP, the differential rate of beta-Lactamase synthesis has been determined on cells growing under accurately reproducible conditions. The rate is affected by a wide variety of growth conditions. Among these were growth at different temperatures in both a defined medium and a complex medium, which produced a wide variation in growth rate; aeration by mixtures of oxygen and nitrogen where the percentage of oxygen varied up to 50% even when growth was slowed down by the high oxygen tension, the differential rate remained the same. Very little variation is scen in the differential rate when the culture is grown anaerobically in nutrient broth compared to an aerobic culture. Growth from different sizes of inecula in defined medium results in different growth rates but the differential rate of beta-Lactamase synthesis is unaltered. Concurrent induction of other enzymes does not affect the differential rate of enzyme synthesis. Those tested were beta-galactosidease, nitrate reductase, and those which are required for the metabolism of nineteen different carbon sources utilized by this organism. Addition of CBAP to growing cells causes an initial rapid rise in the level of beta-Lactamase before the steady state rate of doubling is reached. It is demonstrated, by consideration of a hypothetical culture synthesizing an inducible enzyme, that these kinetics are consistent with a constant differential rate of onzyme synthesis from soon after addition of inducer. These theoretical considerations demonstrate that the doubling time of enzyme and specific activity measurements lead to erroneous conclusions if they are used to deseribe the rate of synthesis of an inducible enzyme. The kinetics of induction are consistent with a constant differential rate, after a lag, which is dependent on the concentration of CBAP. However, the lag and aceeleration period are dependent on the growth medium. Discontinuous beta-Lactamase synthesis has been observed in this strain of s. aureus. Since the synthesis of the enzyme is completely gratuitoue, it cannot be subject to control by catabolite repression. It is suggested that the translation of the gene is dependent on DNA replication. Examination of the differential rate of beta-Lactamase synthesis on addition of inducer at different stages of growth reveals that there is an initial lag in the synthesis of enzyme relative to turbidity at high cell densities. A similar lag occurs at low inducer concentrations. Both observations could be consistent with the presence of a permease controlling entry of CBAP to the cells. The increase of activity obtained when the cells are disrupted by ultrasound, supports this hypothesis. However, on transfer of growing cells from high to low inducer concentration, a lag in enzyme synthesis is still evident. It is reasonable to assume that growth on high CBAP concentration would result in permease induction. On this evidence, therefore, it is concluded that the beta-Lactamase system does not depend on an induced permease. The kinetics of induction at low CBAP levels are consistent with a very low differential rate of synthecis from the moment of addition of inducer. After the logarithmic growth phase is complete, the differential rate increases. The increase in differential rate when growth slows occurs in several other circumstances but is most marked in low concentration of inducer. It is shown that the half life of beta-Lactamase mRNA is relatively long in this organism and is dependent on the growth medium. It is suggested that, as growth slows, mRNA decay slows and therefore the capacity to synthesise enzyme remains. Since the inducer, CBAP, is present at the same concentration at all times, enzyme synthesis continues unabated and the differential rate increases by virtue of diminution in growth rate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available