Abstract
The addition of phenoxymethylpenicillin (10 mg/ml) at any time during the penicillin fermentation inhibited further accumulation of the antibiotic in broth but had no effect on growth. Benzylpenicillin, 6-aminopenicillanic acid (6-APA), and some semisynthetic penicillins also showed this effect, but penicillin N, peniciuloic acid, cephalosporin C, and 7-aminocephalosporanic acid did not limit penicillin accretion. Incorporation of radioactive precursors (cysteine, valine, and sodium phenoxyacetate) into penicillin in the presence of inhibitory concentrations of the antibiotic indicated that penicillin synthesis continued despite the lack of accretion of the antibiotic in broth. The rates of penicillin synthesis in a 48-hr and a 136-hr culture were compared by short-term exposure to Na2"5SO4, and no significant difference in the biosynthetic rate was observed. Exogenous penicillin in the range of 1 to 15 mg/ml of culture broth had no effect on utilization of acetate or glucose by Penicillium chrysogenum. The antibiotic-synthesizing capacity of the organism was not irreversibly inhibited by exogenous penicillin. The degradation of penicillin during the fermentation was also studied. Penicillin V was stable in broth filtrate. Catabolic enzymes such as penicillinase and penicillin-acylase were not demonstrated in whole broth, nor was the accumulation of 6-APA, penicilloic acid, or other degradation products detected. An examination of the intracellular penicillin concentration and the amount of penicillin associated with the mycelium revealed that cells contained significantly more penicillin late in the fermentation than earlier in the cycle. This cell-associated antibiotic may be a regulatory factor in further penicillin synthesis.

The addition of phenoxymethylpenicillin (10 mg/ml) at any time during the penicillin fermentation inhibited further accumulation of the antibiotic in broth but had no effect on growth. Benzylpenicillin, 6-aminopenicillanic acid (6-APA), and some semisynthetic penicillins also showed this effect, but penicillin N, peniciuloic acid, cephalosporin C, and 7-aminocephalosporanic acid did not limit penicillin accretion. Incorporation of radioactive precursors (cysteine, valine, and sodium phenoxyacetate) into penicillin in the presence of inhibitory concentrations of the antibiotic indicated that penicillin synthesis continued despite the lack of accretion of the antibiotic in broth. The rates of penicillin synthesis in a 48-hr and a 136-hr culture were compared by short-term exposure to Na2"5SO4, and no significant difference in the biosynthetic rate was observed. Exogenous penicillin in the range of 1 to 15 mg/ml of culture broth had no effect on utilization of acetate or glucose by Penicillium chrysogenum. The antibiotic-synthesizing capacity of the organism was not irreversibly inhibited by exogenous penicillin. The degradation of penicillin during the fermentation was also studied. Penicillin V was stable in broth filtrate. Catabolic enzymes such as penicillinase and penicillin-acylase were not demonstrated in whole broth, nor was the accumulation of 6-APA, penicilloic acid, or other degradation products detected. An examination of the intracellular penicillin concentration and the amount of penicillin associated with the mycelium revealed that cells contained significantly more penicillin late in the fermentation than earlier in the cycle. This cell-associated antibiotic may be a regulatory factor in further penicillin synthesis.

The penicillin fermentation, like the synthesis of many other antibiotics and secondary metabolites, is characterized by two phases. After a period of rapid growth, strains of Penicillium chrysogenum steadily synthesize penicillin. The cultures, nutrients, and physical factors influencing penicillin production have been reviewed extensively (1, 4, 7-9, 13, 14) . The cause of the termination of penicillin accumulation-in spite of the maintenance of optimal conditions during fermentation has not been fully determined.

This investigation was undertaken to ascertain whether penicillin itself acts as an inhibitor or regulator of penicillin biosynthesis.
MATERIALS AND METHODS
Antibiotics. Potassium benzylpenicillin (penicillin G), potassium phenoxymethylpenicillin (penicillin V), p-methylpenicillin V, phenoxypropylpenicillin, penicillin N, 6-aminopenicillanic acid (6-APA), and cephalosporin C were products of Eli Lilly & Co., Antibiotic Manufacturing and Development. Methylpenicillin and 7-aminodeacetoxycephalosporanic acid were prepared at the Lilly Research Laboratories. a-Aminobenzylpenicillin (ampicillin), sodium 2,6-dimethoxyphenylpenicillin trihydrate (methicillin), and sodium 5-methyl-3-phenyl-4-isoxazolylpenicillin (oxacillin) were products of Bristol Laboratories, Syracuse, N.Y. Potassium phenoxyethylpenicillin (phenethicillin) was obtained from Pfizer, Inc., New York, N.Y., and 6-(2-ethoxy-l-naphthamide) penicillin (nafcillin) was obtained from Wyeth Laboratories, Inc., Philadelphia, Pa. Preparation of phenoxymethylpenicilloic acid was achieved by alkaline hydrolysis of potassium penicillin V. 35S-penicillin V was prepared by fermentation with Na235SO4, acid extraction into butyl acetate, and crystallization as the potassium salt.
MATERIALS AND METHODS
Cultures and fermentation conditions. A strain of P. chrysogenum, mutant E-15 (10), was used throughout this investigation with the exception of one study with P. chrysogenum Q176 and NRRL 1951. Spore inocula were prepared by growing cultures at 25 C on the agar medium of Somerson, Demain, and Nunheimer (19) . Spores were removed from the surface of a slant suspended in 10 ml of nutrient broth, and were used to inoculate a 2-liter Erlenmeyer flask containing 1 liter of the vegetative medium described ANTIMICROB. AG. CHEMOTHER.
MATERIALS AND METHODS
by Vanderhaeghe (24) except that Na2S203-5H20 was omitted. The flasks were incubated at 25 C on a rotary shaker operating at 250 rev/min. After 2 days, 10 ml of germinated culture was used to inoculate each 500-ml Erlenmeyer flask containing 100 ml of one of the following: (i) the chemically defined medium of Tardrew and Johnson (21) ; (ii) a lactose-corn steep liquor fermentation medium (15) ; or (iii) the same medium as ii above except that the lactose was omitted and 0.5% glucose was added to the medium during makeup (sterile glucose, 0.7 ml of a 70% glucose solution, was added aseptically with a syringe at 8-hr intervals starting at 16 hr after inoculation and continuing until 8 hr prior to harvest).
MATERIALS AND METHODS
Unless indicated, 0.3% and 0.5% sodium phenoxyacetate were added to the synthetic and complex fermentation media, respectively. Growth conditions were the same as described above, with the fermentation terminated at 6 days. At harvest, 10 ml of whole broth was centrifuged for 10 min at 2,000 X g, and from the volume of the wet pellet growth was calcalculated as the percentage of solids. All flasks were closed with two fiber-filter discs (Kendall Co., Walpole, Mass.) and were autoclaved at 120 C for 20 Chromatography. Penicillins and 6-APA were identified by applying samples and appropriate standards to Whatman no. 1 paper, which underwent descending development overnight in the upper phase of n-butanol-ethanol-water (4:1:5). After phenylacetylation (5) and drying, chromatograms were placed on agar plates seeded with Bacillus subtilis. After 15-min contact, chromatograms were removed and the plates were incubated overnight at 37 C. Duplicate chromatograms were prepared in those studies in which isotopic labeling was used. The position of radioactive compounds was detected with a Vanguard strip scanner (model 880), and the Rp of radioactive peaks was compared with the RF of biologically active zones.
MATERIALS AND METHODS
The RF values of 6-APA, penicilloic acid, and penicillin V were 0.19, 0.27, and 0.63. respectively, under these conditions. Penicilloic acid and other biologically inactive penicillins were detected by spraying chromatograms with iodine as described by Thomas (22) .
MATERIALS AND METHODS
Respiration studies. 
RESULTS
The effect of penicillin on growth of P. chrysogenum E-15 and on net antibiotic synthesis was evaluated by adding various levels ofpenicillin V throughout the fermentation. The results in Table 1 show that the addition of 10 mg of penicillin V/ml at inoculation (O hr) or at 66 hr postinoculation did not inhibit growth but exerted a limiting effect on net penicillin synthesis. The addition of penicillin in increments more closely resembling the accretion of penicillin in a fermentation did not lessen the inhibition. Penicillin G added to penicillin fermentations had a similar effect on antibiotic production without inhibiting growth ( Table 2) .
RESULTS
The penicillin nucleus, 6-APA, at concentra- tions of 10 mg/ml completely inhibited penicillin accumulation when added to fermentations either initially or throughout the synthetic phase ( Table  3 ). The acyl precursor of penicillin V, sodium phenoxyacetate, at levels beyond that normally incorporated into the medium, had no effect on growth or subsequent synthesis of penicillin V. Cysteine and valine, which have been shown to be precursors of the penicillin nucleus (2, 3), were added to fermentations but did not limit penicillin synthesis at concentrations equivalent to 10 mg of penicillin V/ml.
RESULTS
The effect of other penicillins and cephalosporins on penicillin V accumulation was examined ( Table 4 ). The effect of these ranged from no inhibition with p-methylpenicillin V, penicillin N, cephalosporin C, and 7-aminodeacetoxycephalosporanic acid to 100% inhibition with methicillin and penicillin V. The uptake of these compounds by the synthesizing organism was not studied.
RESULTS
The specificity of the intact j-lactam ring as an inhibitor of penicillin synthesis was further ex- amined by the addition of phenoxymethylpenicilloic acid to penicillin fermentations (Table 5) .
RESULTS
No inhibition of penicillin synthesis in the presence of penicilloic acid was observed, whereas the equivalent amount of penicillin V completely blocked antibiotic accumulation. Penicillin V as an inhibitor of penicillin synthesis was investigated by use of strains of P. chrysogenum with varying capacity to synthesize antibiotic. The addition of penicillin V at concentrations equal to or exceeding that accumulated by each culture under control conditions inhibited subsequent penicillin synthesis (Table  6 ). When less than total inhibitory concentrations of penicillin were added, the amount of penicillin ultimately synthesized usually equaled the difference between the concentration originally in the medium and that which would have been produced in its absence.
RESULTS
Exogenous penicillin V and 6-APA at levels of 10 mg/ml were each capable of preventing the accumulation of 6-APA in a fermentation from which the acyl precursor had been omitted (Table 7) . This inhibitory concentration was considerably greater than the amount of 6-APA normally produced in the absence of precursor. Table 8 showed little effect of penicillin on the utilization of these substrates.
RESULTS
The antibiotic-synthesizing capacity of the organism was not irreversibly inhibited by exogenous penicillin. Cells (72 and 96 hr) that were washed and transferred to 48-hr spent medium produced control levels of penicillin. In addition, Penicillium cells were removed from fermentation broth at 72 and 96 hr, washed, and transferred to 48-hr sterile penicillin broth. One sample of 72-hr cells was exposed to 10 mg of penicillin V/ml for 2 hr prior to transfer. The cell suspensions were incubated for 3 to 4 days, during which time penicillin synthesis was monitored. Penicillin production by all cell suspensions was comparable to that of the control in fresh lactose medium during the same period (Fig. 1) .
RESULTS
The incorporation of radioactive precursors into penicillin V was examined in the presence of exogenous penicillin V and 6-APA. DL-Cystine-1-14C, L-valine-U-14C, and phenoxyacetic-1-'4C acid were added to penicillin fermentations at 48 hr along with 10 mg of 6-APA or penicillin V per ml. Penicillin extracted and crystallized from harvested broth filtrates was assayed for purity and radioactivity. Net penicillin synthesis was determined. The micromoles of labeled precursor incorporated into penicillin in the presence and absence of inhibitory concentrations of 6-APA and penicillin V are given in Table 9 . Although net antibiotic synthesis was limited by exogenous penicillin and 6-APA, the incorporation of DL-cystine-1-_4C was not equally inhibited, 7.08 X 10-2 9.90 X 10-2 a Penicillin V (Pen V) and 6-aminopenicillanic acid (6-APA) were added at 10 mg/ml. L-valine-U-'4C incorporation was unaffected, and phenoxyacetic-1-'4C acid incorporation was enhanced.
RESULTS
The rates of penicillin synthesis early and late in the fermentation cycle were compared. Penicillin was extracted from 48-and 136-hr broths after 2 hr of incubation with Na235SO4. The specific activity of the crystallized penicillin indicated no difference in rate of penicillin synthesis at these times. The addition of penicillin to a 48-hr fermentation did not significantly affect the incorporation of Na235SO4 into penicillin (Table  10) .
RESULTS
Degradation of penicillin. The significant incorporation of "4C-labeled precursors into penicillin with little or no net increase in antibiotic accumulation suggested product turnover in the penicillin fermentation. Thus, the stability of penicillin in fermentation broth of various ages was examined by adding penicillin V to broths that had been sterilized by filtration. After 48 hr of incubation at 25 C, penicillin potencies dropped less than 10%o. Washed mycelium, cell extracts, and dialyzed broth filtrates were examined for penicillin acylase activity. No 6-APA was formed when these were incubated with penicillin V for 6 hr at pH 7.0 and 30 C.
RESULTS
Penicilloic acid was measured in broths to which penicillin had been added, as well as in control fermentations, and was found to be negligible. Four days after the addition of "Spenicillin V to 48-hr fermentation broths, no 8-hydroxypenillic acid, penicic acid, nor other IIS-containing degradation products of penicillin were detected by chromatographic techniques. All 35S activity was associated with penicillin V on the chromatographs, and the total radioactivity added initially was accounted for in the broth and mycelium.
RESULTS
Intracellular and cell-bound penicillin. The possibility that accumulation of penicillin on or within the cell during the fermentation caused apparent inhibition of penicillin synthesis was considered. Extracts of cells from a control penicillin fermentation and from one to which penicillin had been added were assayed for penicillin content. The data (Table 11) showed that the intracellular penicillin concentration increased during the middle of the fermentation cycle, but decreased toward the end. The penicillin content of control cells was lower than that of cells exposed to added penicillin. The amount of penicillin associated with older mycelium was twice that of younger cells.
RESULTS
An "equilibrium" between extracellular and intracellular penicillin in fermentation broths with high penicillin potencies was investigated. After a 2-hr exposure to "5S-penicillin V, cells were harvested from 48-and 136-hr whole broth and washed; cell-free extracts were prepared. Radioactivity of the concentrated extracts was determined and chromatographically shown to be associated with penicillin. Extracts obtained from cells late in the fermentation cycle contained about three times the "5S-penicillin V found in extracts of younger cells, whereas the radioactivity of the particulate fractions was equivalent.
RESULTS
Little antibiotic could be extracted or eluted from washed Penicillium cells taken at different times throughout a control fermentation (Table  12) . However, the amount of penicillin associated with 137-hr cells was considerably higher than with any other cells throughout the fermentation.
RESULTS
The adsorption of penicillin by mycelium in the presence of increasing levels of the antibiotic was estimated by incubating a constant quantity of washed cells with "5S-penicillin V. The amount of 35S-penicillin V adsorbed by intact cells was proportional to the increased exogenous penicillin concentration but was too small to account for the loss of penicillin late in the fermentation (Table 13 ).
DISCUSSION
The results of this investigation show that the cessation of penicillin accumulation during fermentation may be related to the end product itself. Exogenous penicillin added at any time throughout the fermentation cycle limited subsequent antibiotic accumulation without affecting growth.
DISCUSSION
Termination of penicillin production has been attributed to cessation of growth (17) . High con

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