Penicillinase synthesis

Detailed X-ray crystallographic data of certain cephalosporin antibiotics was applied to obtaining insights into biological activity in this series. 2 The Cg-eplmer (trans-isomer) of benzylpenlclllin was found to be very stable to penicillinases prepared from aureus and coli carrying an amplclllln-resistant R-factor, and was a powerful Inducer of penicillinase synthesis in aureus and cephalosporlnase synthesis in P. vulgaris. 3... 

The Regulation of Penicillinase Synthesis in Gram-Positive Bacteria... 

Extensive studies of penicillinase induction in B. cereus NRRL B-569 (referred to here as 569) have been made by Pollock and co-workers since 1950 (see reference [1]) and by other workers. Cells grown in the absence of penicillin produce little penicillinase, but after the addition of penicillin there is a period of 60 to 90 minutes during which penicillinase synthesis accelerates to a new differential rate about 300 times the basal rate (Fig. 1). Magnoconstitutive mutants (e.g., strain 569/H) make up to 800 times the basal level of penicillinase. The induction process is continuous, and Imsande [2] claimed to detect the initial increase in penicillinase activity within 30 seconds of the addition of penicillin to a culture of strain 569. (Some confusion has been caused by different interpretations of the term lag period used by Pollock, which he defined as the time intercept of a line drawn tangent to the curve of penicillinase production at the point where the maximum rate of penicillinase synthesis was reached. Other workers, e.g., see Leggate and Holms [11], have used lag period to refer to a period after induction when no response was detectable—with consequent confusion in the discussion of experimental results). In induction experiments using 1 unit of... 

Pollock [12] discovered that the induced penicillinase synthesis lasted at least 5 hours, though by this time the specific activity of the culture (units of penicilUnase per milligram dry weight of bacteria) had passed its peak (at ca. 2.5 hours) and was almost back to the original basal level. The course of penicillinase synthesis was similar if, a short time after exposure to penicillin (even at 0°C), the cells were washed and regrown in fresh medium. This drew attention to the possibility of the inducer s remaining associated with the cells, and this was tested by penicillin binding studies. 

Fig. 2. Comparison of the fixation of penicillin to cells of Bacillus cereus 569 and the rate of penicillinase synthesis reached by the cells during subsequent growth. Data from Pollock and Perret [13]. = bound penicillin O = rate of penicillinase synthesis.

In a different approach, Csanyi et al. [15] attempted to show the importance of the bound penicillin to the continuing production of penicillinase, by removing the bound penicillin from cells by treatment at pH 9.5 in tris buffer. A correlation was found between loss of bound penicillin and decline in subsequent penicillinase production. The alkaline treatment also slowed the rate of penicillinase synthesis for about 20 minutes before a return to a steady rate occurred. The same alkaline treatment, applied for varying times to the cells before induction, affected the lag before penieillinase synthesis reached a steady state after the induction. In the extreme case quoted, 30 minutes of alkaline pretreatment delayed penicillinase synthesis for 60 minutes. The temperature of treatment was also critieal little effect was produced at 30°C or under. Reinduction by penicillin after an induced culture had been treated with alkaline tris was rapid, the rate of synthesis rising immediately to the new induced level. In view of the general disturbance to the cells caused by the alkaline treatment and the many factors that can affect penicillinase synthesis in B. cereus (see later sections), it cannot be concluded that the effect of the alkaline treatment demonstrates solely a link between the bound penicillin and penicillinase synthesis. 

Csanyi [29] investigated several factors that affect basal penicillinase synthesis in B. cereus 569 and concluded that almost any disturbance to normal growth produced a perturbation of penicillinase synthesis. These... 

In this case, where the messenger half-life is about 2 minutes (see Section III, A, 6), the long duration of the increased penicillinase synthesis must indicate prolonged penicillinase messenger synthesis and a lag in the production or action of new repressor. A key experiment is illustrated in Fig. 5. Csanyi [29] tested the specific activity of cells in-... 

Anaerobiosis (up to 100 minutes duration), once the maximum induction response was reached, halted growth and penicillinase synthesis immediately. When air was readmitted to the system, penicillinase synthesis resumed, often showing an enhanced rate for a few minutes before returning to the original rate. 

Anaerobiosis during the period of accelerating penicillinase synthesis has profound inhibitory effects on the induction process, the critical period being the first 20 minutes from the addition of the inducer. The earlier anaerobiosis was started, the greater the ultimate reduction in the response observed. Pollock contrasted this with the effects of a similar period at 0°C to halt metabolism temporarily, which did not afTect the final induction response. 

Day and Shah [43], Shah and Day [44], and Yip et al. [42] showed that some constitutive strains of B. cereus (569/H, 5, and 5/B) responded to the additions of penicillins and cephalosporins by altering their rate of penicillinase synthesis, sometimes less than the control level and more often above the control rate twice the control rate was the greatest effect observed. It is possible that some vestige of response to the inducer still remains in these strains. Day and his co-workers suggest that regulation at the level of translation is involved, but the evidence does not appear to be conclusive. 

Ferrous ions stimulated penicillinase synthesis in uninduced or pre-induced inducible strains of S. aureus but not in constitutive mutants. 

The increased rate of penicillinase synthesis was maintained for at least 4 hours, or 90 minutes after all the iron had been taken up from the medium into the cells. 

Cohen et al. [61] reported that inducible strains of S. aureus were derepressed by 10 minutes of treatment at 42°C, producing a burst of penicillinase synthesis on subsequent growth at 37°C. The increase in rate started immediately but had stopped after 10 minutes. In the presence of puromycin no protein was formed, but, when the puromycin was removed, the same burst of penicillinase synthesis occurred. Cohen et al. conclude that this behavior could be produced by the thermal inactivation of a repressor and that the restoration of the repressed state required protein synthesis. This thermal derepression is sensitive to certain metabolic conditions, for one particular auxotrophic mutant requiring both adenine and guanine was derepressed only if optimal concentrations of the purines were present at lower concentrations derepression was not observed. Constitutive mutants were not stimulated by the heat treatment. 

Penicillinase was discovered to be an inducible enzyme by Duthie [79] in 1947. Pollock [80] studied the induction kinetics of B. licheniformis 6346 and found that induction by 0.06 pg of benzylpenicillin/ml stimulated penicillinase synthesis for about 3 hours at this point the culture could be reinduced by further penicillin. Induction with cephalosporin C (almost undestroyed by the enzyme) gave more prolonged penicillinase synthesis. 

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