KINETICS OF INCORPORATION OF p ... - Europe PMC

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corporation, this is not the case with the re- sistant strain, in which the differential rate of incorporation isan ever-decreasing one. If the incorporation of theĀ ...
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amino acid, whereas 70 per cent of the 70 strains which were not isolated from infections also attacked arginine. There was no correlation between arginine

degradation and growth. Turbidity readings of the group of cultures which failed to remove arginine averaged the same as cultures degrading the amino acid.

KINETICS OF INCORPORATION OF p-FLUOROPHENYLALANINE BY A MUTANT OF ESCHERICHIA COLI RESISTANT TO THIS ANALOGUE GEORGES N. COHEN AND EDWARD A. ADELBERG' Service de Biochimie Cellulaire, Institut Pasteur, Paris, France Received for publication April 7, 1958

Miunier and Cohen (Biochim. et Biophys. Acta, 21, 592, 1956; Ann. inst. Pasteur, in press) have shown that addition of p-fluorophenylalanine (FPA), 3-2-thienylalanine (TA), or norleucine, to an exponentially growing culture of Escherichia coli causes a shift from exponential to linear growth, which continues until there has been an increase in mass of 4- to 5-fold. During this linear growth, the analogues are extensively incorporated into the newly formed proteins. It has been possible to select mutants which are specifically resistant to each of these three analogues and to some others (Adelberg, J. Bacteriol., 76, 326, 1958). The FPA-resistant mutant has been studied in some detail for its capacity to incorporate phenylalanine and FPA into its proteins; we find that the wild-type and the FPA-resistant strain incorporate phenylalanine into their proteins at the same differential rate, as shown in figure 1. The rates of incorporation of radioactive FPA were then compared in the sensitive wildtype and in the resistant mutant. The results in figure 2 show that whereas the sensitive strain exhibits the usual linear differential rate of incorporation, this is not the case with the resistant strain, in which the differential rate of incorporation is an ever-decreasing one. If the incorporation of the analogue in the resistant strain is plotted against time, it is seen that the incorporation is linear with time (figure 3). We are thus witnessing a linear incorporation in an exponentially growing mass, the result of which is a constant reduction of I Fellow of the John Simon Guggenheim Memorial Foundation, 1956-1957. Permanent address: Department of Bacteriology, University of California, Berkeley, California.

the FPA content of the newly formed mass. This by itself could be sufficient to account for the resistance of the organism. The resistant mutant excretes a substance reversing the inhibition by FPA, as evidenced by the growth of the wild-type on analoguecontaining agar when streaked side by side with the resistant mutant. The excreted substance is inferred to be tyrosine, siince the tyrosinerequiring mutant M83-8 will grow on minimal agar when streaked side by side with the FPA-

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Figure 1. Incorporation of phenylalanine into the proteins of the sensitive wild-type and of the FPA-resistant mutant. Radioactive DL-phenylalanine was added to exponentially growing cultures of the sensitive wild-type and of the resistant mutant. At intervals, samples were taken and the radioactivity of the protein fraction was determined. DL-Phenylalanine concentration: 10-4 M. Specific activity: 1.7 X 10' cpm/,umole.

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Figure 2. Incorporation of p-fluorophenylalanine into the proteins of the sensitive wild-type and of the FPA-resistant mutant. Radioactive DL-pfluorophenylalanine was added to exponentially growing cultures of the two organisms. The sensitive strain grew linearly, while the exponential growth of the resistant mutant was unaffected. Samples taken at intervals were analyzed for radioactivity of the protein fractions. DL-pfluorophenylalanine concentration: 10-3 M. Specific activity: 1.7 X 104 cpm/,Amole.

resistant mutant.2 Control tests show that strain M83-8 is not cross-fed by the wild-type strain. This overproduction of tyrosine by the resistant mutant provides a satisfactory explanation of its resistance to FPA; increasing amounts of tyrosine may inhibit the incorporation of the analogue by interfering at the uptake level, at the activation level, and at the incorporation level. That tyrosine overproduction accounts for the ever-decreasing differential rate of FPA incorporation in the resistant mutant is also suggested by the following experiment: cells of the FPA-resistant strain were allowed to incorporate radioactive FPA until the differential rate of its incorporation had decreased markedly. The cells were then washed and resuspended in medium containing the same concentration of radioac2 Strain M83-8 responds to either tyrosine or its precursor, p-hydroxyphenylpyruvic acid. The excreted compound could thus be either; the amount produced is too small to permit a ready identification by chromatography. In cases where sufficient material is excreted, the product can be identified as the amino acid; e. g., a thienylalanineresistant mutant excretes phenylalanine.

Time (minutes)

Figure S. FPA-resistant strain. The incorporation of radio-p-fluorophenylalanine, plotted against time. The experiment was similar to the one reported in figure 2. DL-p-fluorophenylalanine concentration: 5.3 X 10-5 M. Specific activity: 1.5 X 105 cpm/,umole.

Optical density (arbitrary units)

Figure 4. Kinetics of incorporation of p-fluorophenylalanine by the FPA-resistant strain. The cells were grown on C'4-DL-p-fluorophenylalanine. Samples were taken at intervals for radioactivity analysis. At point A, a known volume of the culture was centrifuged, the cells were washed and resuspended in the same volume of fresh medium with the same concentration of the radioactive analogue. Samples were then taken both in the untreated and in the washed culture for radioactivity analysis. DL-p-fluorophenylalanine concentration: 10-4 M. Specific activity: 1.5 X 105 cpm/,umole.

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tive FPA. FPA incorporation began again at an increased differential rate, and once more decreased with time (figure 4). Thus, the washing process removed a substance from the cells

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which had accumulated and progressively interfered with FPA incorporation. It seems highly probable that the substance removed was tyrosine.

ISOLATION OF LEPTOTRICHIA BUCCALIS ARDEN HOWELL, JR., AND MORRISON ROGOSA National Institute of Dental Research, National Institutes of Health, Public Health Service, U. S. Department of Health, Education and Welfare, Bethesda, Maryland Received for publication April 14, 1958

Bibby and Berry (J. Bacteriol., 38, 263, 1939) isolated and described filamentous organisms from oral specimens which they designated as Leptotrichia buccalis. However, these organisms have not since been adequately studied and this genus is not recognized as valid (Bergey's Manual of Determinative Bacteriology 7th Ed., 1957). Nevertheless, organisms similar to the foregoing have been isolated repeatedly by the authors and also by Gilmour (J. Dental Research, 37, 10, 1958). Thirty-seven salivas and 233 other oral samples from various people have been cultured. Forty-two isolates were obtained in subculture from aerobic plates of beef extract agar, containing 0.1 per cent soluble starch, 1:1,000,000 basic fuchsin, and 10 per cent defibrinated sheep blood. Also, 9 were isolated from anaerobic plates and one from a bacteremia following dental extraction. Colonies in the original streak plates are minute,