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Nalidixic Acid-Induced Protein Alterations in Escherichia coli

Lee Chao Antimicrob. Agents Chemother. 1977, 11(1):167. DOI: 10.1128/AAC.11.1.167.

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ANTIMICROBIAL AGENT AND CHEMOTHERAPY, Jan. 1977, p. 167-170 Copyright 1977 American Society for Microbiology

Vol. 11, No. 1 Printed in U.S.A.

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Nalidixic Acid-Induced Protein Alterations in Escherichia coli
LEE CHAO Department ofBiochemistry, Medical University of South Carolina, Charleston, South Carolina 29401

Received for publication 15 July 1976

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The effect of nalidixic acid on pulse-labeled protein patterns was analyzed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The drug was found to alter protein patterns in a drug-sensitive strain but not in the isogenic drug-resistant strain of Escherichia coli. The drug-induced alteration of protein patterns was demonstrated by the appearance of some and the disappearance of other pulse-labeled peptides elicited by the drug.

Nalidixic acid is an extensively used antibacterial drug whose mode of action is poorly understood. It is known that nalidixic acid specifically inhibits bacterial deoxyribonucleic acid (DNA) synthesis but only partially inhibits the synthesis of ribonucleic acid (RNA) and proteins (2, 12). The primary target of inhibition is the semiconservative DNA replication (21), with a possible effect on the repair synthesis of DNA (10). The inhibition of DNA synthesis is not attributable to any interaction between nalidixic acid and the DNA template (3, 4).It has been shown (18) that the thermal susceptibility of a DNA ligase temperature-sensitive mutant is enhanced by nalidixic acid, but the search for a direct interference of the drug with enzymes involved in DNA metabolism has been so far unsuccessful (4, 19). A recent report by Crumplin and Smith (8) indicates that, in addition to inhibiting DNA synthesis, nalidixic acid may inhibit RNA synthesis as a second target site. However, the inhibition of bacterial RNA synthesis is only partial (12) and may or may not affect bacterial growth significantly, since the rate of bacterial RNA synthesis can also vary widely depending on the growth conditions. Studies have therefore been conducted to investigate the possibility that nalidixic acid may alter the expression of many gene products in a qualitative manner. Results of these studies, reported here, indicate that the protein pattern in bacteria is indeed altered by nalidixic acid treatment. The alteration is quite complex in that the synthesis of certain proteins is depressed whereas that of other proteins is induced by the drug.
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The bacterial strains used were Escherichia coli KL161 and KL166. These two strains are isogenic except that KL166 is a NalA derivative of KL161 (1). The growth of KL161 is totally inhibited by nalidixic acid at a concentration of 10 ,g/ml in minimum medium M9 supplemented with 100 gg of thymine per ml. (5). The growth of KL166 is unaffected by nalidixic acid at 500 gg/ml in the same medium. A direct analysis of the effect of nalidixic acid on the profiles of pulse-labeled RNA is clearly not feasible. An indirect approach of analyzing pulselabeled proteins on sodium dodecyl sulfatepolyacrylamide gel was used. This approach is based on the assumption that the profile of pulse-labeled proteins reflects that of the RNA and that nalidixic acid does not act on translation (8). Cells at a density of 3 x 108 per ml in M9 medium plus 100 ,g of thymine per ml were treated with nalidixic acid (Sigma Chemical Co.) at 100 gg/ml. Nalidixic acid-treated and untreated samples were then divided and were separately pulse labeled with [14C]thymidine (0.5 ,uCi/ml), [3H]uridine (1 ,uCi/ml), and 14Clabeled amino acid mixtures (2 ,Ci/ml) at predetermined time intervals. All labeled chemicals were purchased from New England Nuclear. ['4C]thymidine- and [3H]uridine-labeled samples of 1 ml each were stopped with 10% trichloroacetic acid and counted. Samples (1 ml each) labeled with '4C-labeled amino acids were stopped by adding 2 ml of ice-cold 2% unlabeled amino acid mixture and 10% glycerol. Labeled cells were pelleted and lysed in the extraction buffer (15). Portions of the lysate were precipi-

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tated with 5% trichloroacetic acicI and counted.

Another portion of each sample !was used for sodium dodecyl sulfate-polyacryleumide gel electrophoresis according to the staw ndard method (16). The results of the pulse-labelirag experiment are shown in Fig. 1. The rate of protein synthesis in KL161 and KL166 was re duced by nalidixic acid treatment by about 25i and 10%, respectively. The rate of RNA syntthesis in these two strains was also reduced proF)ortionately as reported by others (12). Thymid ine incorporation was inhibited in KL161 but not in KL166 (data not shown). Thus, the bact ericidal action of nalidixic acid does not seem to be related specifically to its action on bactterial RNA or protein synthesis. However, a close examination of individual peptides by s(xdium dodecyl sulfate-polyacrylamide gel anal,Lysis indicates

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FIG. 1. Effect of nalidixic acid on the rate ofRNA and protein synthesis. E. coli KL161 grown in M9 minimal medium sup 100 pg of thymine per ml at 30DoC to 3 x 108 cellslml. Nalidixic acid was added t o each culture at zero time to give a final concentratic n of100 g/Iml. Samples of 1 ml each were taken lat the indicated time and pulse labeled with either [3H]uridine (1 ,uCi/ml) for 1 min or 14C-labeled amiino acid mixture (2 uCi/ml) for 3 min. Labeled RB JA and proteins were determined by scintillation cou,nting. Acid-precipitable counts were plotted as the of the control which was pulse labeled beforre the drug addition. [3H]uridine incorporation: (I0) KL61; (0) KL166. "4C-labeled amino acid inc,orporation: (a) KL161; (E) KL166.

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otherwise. Certain major qualitative differences in protein patterns between KL161 and KL166 elicited by nalidixic acid are shown in Fig. 2. The pattern of pulse-labeled proteins was drastically altered by nalidixic acid in KL161 but not in KL166. Furthermore, in KL161 the synthesis of many proteins was depressed whereas that of others was induced by nalidixic acid. The most prominent protein bands whose synthesis was depressed by nalidixic acid were the 165-K (165,000 daltons), 155K, 95-K and 30-K peptides. Of these peptides, only the 165- and 155-K peptides have been identified. These are the (3' and (8 subunits of RNA polymerase, respectively (17; my unpublished data). The synthesis of these two subunits was depressed by about 10-fold in KL161 but only by about 30% in KL166 by nalidixic acid as revealed by direct scanning ofthe bands on radioautograms (data not shown). In KL161 the following peptides were induced by nalidixic acid: 135, 110, 90, 59, 55, 52, 40, and 18 K. The 135-K peptide has been identified as (3galactosidase (unpublished data). Shuman and Schwartz have shown (20) that nalidixic acid reduces the level of induced f-galactosidase in E. coli strain K-12. Unfortunately, they did not mention the effect of nalidixic acid on the level of 8-galactosidase in uninduced cultures. The relationship between the action of nalidixic acid and the induction of 83-galactosidase, and possibly the induction of other catabolite-repressible operons, appears to be complex in that f3-galactosidase is only poorly induced by isopropyl-,3-r-thiogalactoside in KL166 as compared with KL161 even in the absence of nalidixic acid (L. Chao, manuscript in preparation). The other readily identifiable peptide is the 40-K peptide, which corresponds to protein X previously reported by Inouye and Pardee (14). This protein has been implied to function in DNA repair (13). Note that certain peptides, such as the 59- and 52-K peptides, are only induced transiently in KL161 by nalidixic acid. The induction of other peptides in KL161 persists until the viability of the cells begin to drop significantly (data not shown). It is known that nalidixic acid treatment converts cellular DNA into acid-soluble fragments and that this solubilization is antagonized by conditions restricting RNA and protein synthesis (6). In addition, nalidixic acid, like other agents that cause lesions in DNA, induces prophages (7, 11, 22). It is therefore very attractive to implicate nalidixic acid in inducing a DNA lesion or inhibiting DNA repair, or both. The results presented here are very suggestive of such actions by nalidixic acid. First, nalidixic acid drastically alters the pattern of newly syn-

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FIG. 2. Effect of nalidixic acid on pulse-labeled protein patterns. KL161 and KL166 were treated with nalidixic acid and pulse labeled with '4C-labeled amino acid mixture as described in the legend to Fig. 1. Samples corresponding to 5 x 107 cells were lysed and analyzed on a 7.5 to 12.5% gradient sodium dodecyl sulfate-polyacrylamide slab gel. Pulse-labeled protein patterns were obtained by autoradiography. The following proteins were electrophoresed on the same gel for molecular weight determination: E. coli RNA polymerase subunits (165,155,90 and 39 K), bovine serum albumin (67 K), ovalbumin (45 K), uricase (33 K) and myoglobin (18 K). (1) Control; (2) 10 min, (3) 20 min, and (4) 30 min after drug addition. The molecular weights (in kilodaltons) of relevant peptides are indicated.

thesized proteins in sensitive but not in resistant strains of bacteria. This alteration is likely due to a direct or indirect effect of nalidixic acid on gene expression at the RNA level and not at the level of translation (8). However, a modification of the translational machinery by the drug cannot be totally excluded at the present time. Second, nalidixic acid elicits different effects on different gene products, as shown by the induction of certain proteins and the depression of others. Some of the induced proteins, such as 8-galactosidase, are apparently not related to the bactericidal effect of the drug. Other induced or depressed proteins may play a role in the drug action. One of the possibilities

is that some of the drug-depressed proteins may be involved in cellular DNA metabolism in a way similar to DNA repair enzymes or nucleases. Recently Crumplin and Smith reported (9) that the step blocked by nalidixic acid in DNA synthesis is the conversion of 38S singlestranded DNA fragments into high-molecularweight DNA in sensitive cells. The nalidixic acid-induced proteins may therefore directly interfere with the conversion of the 38S fragments into full-sized DNA or rapidly degrade the 388 intermediate fragments, and thus exert the bactericidal action of the drug. This notion is supported by the observation that competent RNA and protein syntheses are essential pre-

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thesis after ultraviolet irradiation in Escherichia coli. J. Bacteriol. 105:908-912. Geissler, E. 1967. Untersuchungen uber den Mechanismus der Induktion lysogener Bakterien. XI. Der Einfluss von DNA-Synthesehemmern auf lysogene Bakterien. Biol. Zentralbl. 86(Suppl.):55-67. Gross, W. A., W. H. Deitz, and T. M. Cook. 1965. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 89:1068-1074. Gudas, L. J., and A. B. Pardee. 1975. Model for regulation of Escherichia coli DNA repair functions. Proc. Natl. Acad. Sci. U.S.A. 72:2330-2334. Inouye, M. and A. B. Pardee. 1970. Changes of membrane proteins and their relation to deoxyribonucleic acid synthesis and cell division in Escherichia coli. J. Biol. Chem. 245:5813-5819. Karam, J. D., and M. G. Bowles. 1974. Mutation to overproduction of bacteriophage T4 gene products. J. Virol. 13:428-438. Laemmli, U. K. 1970. Cleavage of structural proteins during assembly of the head bacteriophage T4. Nature (London) 227:680-685. Matzura, H., S. Molin, and 0. Maaloe. 1971. Sequential biosynthesis of the P and ,3' subunits of the DNAdependent RNA polymerase from Escherichia coli. J. Mol. Biol. 59:17-25. Nishida, M., Y. Mishima, J. Kawada, and K. L. Yielding. 1975. Enhancement by nalidixic acid of the thermal susceptibility of the Ts-7 mutant of Escherichia coli TAU-bar. Antimicrob. Agents Chemother. 8:384386. Pedrini, A. M., D. Geroldi, A. Siccardi, and A. Falashi. 1972. Studies on the mode of action of nalidixic acid. Eur. J. Biochem. 25:359-365. Shuman, H., and M. Schwartz. 1975. The effect of nalidixic acid on the expression of some genes in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 64:204-209. Simon, T. J., W. E. Masker, and P. C. Hanawalt. 1974. Selective inhibition of semiconservative DNA synthesis by nalidixic acid in permeabilized bacteria. Biochim. Biophys. Acta 349:271-274. Taketo, A., and H. Watanbe. 1967. Effect of nalidixic acid on the growth of bacterial viruses. J. Biochem. 61:520-522.

requisites for both exonuclease action and the bactericidal action of nalidixic acid (6, 8).
I am grateful to J. D. Karam for his generosity in providing equipment and his patience in instructing in the use of slab gel techniques. I also wish to thank J. D. Karam and B. Baggett for their critical reading of the manuscript. The bacterial strains were kindly provided by Barbara Bachman. This work was supported by Public Health Service General Research Grant RR05420 (from the Division of Research Resources) to the Medical University and by the South Carolina State Appropriation for Biomedical Research.
LITERATURE CITED 1. Bachman, B. J. 1972. Pedigrees of some mutant strains ofEscherichia coli. J. Bacteriol. 36:525-557. 2. Bauernfeind, A., and G. Grummer. 1965. Biochemical effects of nalidixic acid on Escherichia coli. Chemotherapia 10:95-102. 3. Bourguignon, G. J., M. Levitt, and R. Sternglanz. 1973. Studies on the mechanism of action of nalidixic acid. Antimicrob. Agents Chemother. 4:479-486. 4. Boyle, J. V., T. M. Cook, and W. A. Goss. 1969. Mechanism of action of nalidixic acid on Escherichia coli. VI. Cell-free studies. J. Bacteriol. 97:230-236. 5. Champe, S. P., and S. Benzer. 1962. Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequences in mesienger-RNA. Proc. Natl. Acad. Sci. U.S.A. 48:532-546. 6. Cook, T. M., W. H. Deitz, and W. A. Gross. 1966. Mechanism of action of nalidixic acid on Escherichia coli. IV. Effect on the stability of cellular constituents. J. Bacteriol. 91:774-779. 7. Cowlishaw, J., and W. Ginoza. 1970. Induction of A prophage by nalidixic acid. Virology 41:244-255. 8. Crumplin, G. C., and J. T. Smith. 1975. Nalidixic acid: an antibacterial paradox. Antimicrob. Agents Chemother. 8:251-261. 9. Crumplin, G. C., and J. T. Smith. 1976. Nalidixic acid and bacterial chromosome replication. Nature (London) 260:643-645. 10. Eberle, H., and W. Masker. 1971. Effect of nalidixic acid on semiconservative replication and repair syn-

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