Vol. 129, No. 3 Printed in U.S.A.

JOURNAL OF BACTrRiOLOGY, Mar. 1977, P. 1639-1641 Copyright 0 1977 American Society for Microbiology

Inhibition of Iron Uptake and Deoxyribonucleic Acid Synthesis by Desferal in a Mutant Strain of Bacillus subtilis J. E. L. ARCENEAUX AND B. R. BYERS* Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216

Received for publication 13 October 1976

In the Bacillus subtilis mutant 1D-4, the hydroxamate Desferal inhibited growth, iron uptake, and deoxyribonucleic acid synthesis but did not quantitatively affect synthesis of ribonucleic acid and protein.

Iron acquisition by many microbial cells is accomplished by processes utilizing special iron-chelating molecules (siderochromes) that facilitate movement of the iron atom across the cytoplasmic membrane (B. R. Byers and J. E. L. Arceneaux, in E. D. Weinberg [ed.], Microorganisms and Minerals, in press). One major category of siderochromes is the secondary hydroxamic acids. Hydroxamates produced by different organisms vary in structure; some organisms are able to use a variety of structurally different natural hydroxamates. Mutation to resistance to one of the hydroxamate antibiotics sometimes changes the siderochrome utilization profile of an organism (1, 8, 13); for example, a Bacillus m.egaterium mutant that is resistant to the hydroxamate antibiotic A22765 also loses the capacity to utilize the structurally similar hydroxamate Desferal (1). In certain eucaryotic cell culture systems, Desferal prevents iron uptake. In HeLa cell cultures, addition of Desferal interrupted iron uptake and halted deoxyribonucleic acid (DNA) synthesis but had little quantitative effect on synthesis of ribonucleic acid (RNA) and protein (9, 10). Similar inhibition of iron uptake and early termination of DNA synthesis by Desferal were observed in Novikoff hepatoma cells (4). We wished to determine if iron starvation (induced by Desferal) blocked DNA synthesis but not overall synthesis of RNA and protein in procaryotic cells unable to utilize Desferal because of their resistance to the hydroxamate antibiotic A22765. For these studies, spontaneous mutants resistant to A22765 were selected from the thymine-requiring organism B. subtilis 23 thy (obtained from N. Sueoka) and screened for inability to grow in the presence of Desferal by previously used methods (5). Approximately 10% of the A22765-resistant colonies were unable to grow on a sucrose-mineral salts agar containing 1 ,ug of Desferal per ml.

One of these strains, designated B. subtilis 1D4, was selected for further study. Addition of 18 IuM Desferal to cultures of strain 1D-4 (in a Chelex-treated, sucrose-mineral salts medium containing 0.18 ,uM iron and 40 ,M thymine) reduced growth to 50 and 40% of controls at 5 and 7 h of incubation, respectively (Fig. 1). The residual growth noted in the presence of Desferal may have been due to carry-over of intracellular iron, because Desferal inhibited iron uptake in this organism. When assayed by the method of Arceneaux et al. (2) in medium containing 0.045 M 59Fe (added as ferric chloride; specific activity, 5 to 10 mCi/mg of Fe; Amersham/Searle Corp., Chicago) and 0.45 FM Desferal, total uptake of labeled iron from the Desferal-"9Fe chelate after 10 min was only 8% of that taken up by cells exposed to "FeCl3 as the iron source (Fig. 1). To ascertain if Desferal caused an early block in DNA synthesis but permitted continued overall synthesis of RNA and protein, we followed incorporation of radioactive precursors into cold 10% trichloroacetic acid-insoluble cell material for a 6-h incubation period. Test media contained 0.7 pM thymine, 0.7 FM uracil, and 0.04 AM [3H]leucine (5.0 Ci/mmol; New England Nuclear Corp., Boston). To estimate DNA synthesis, [14C]thymine (57.3 mCi/mmol; New England Nuclear Corp.) was substituted for nonradioactive thymine; to estimate RNA synthesis, ['4C]uracil (54.8 mCi/mmol; New England Nuclear Corp.) was substituted for nonradioactive uracil. Incorporation of radioactive leucine was used to estimate protein synthesis. All cultures also received 0.18 ZM iron. Desferal (18 WM) was added to appropriate cultures. Apparent synthesis of DNA was similar both with and without added Desferal for the first 2 h of incubation; however, after 2 h a sharp increase in DNA synthesis in controls without Desferal occurred (Fig. 1). After about 5 h,

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MINUTES HOURS FIG. 1. Effect of Desferal on growth, iron uptake, and DNA synthesis in B. subtilis 1D-4 (thymine requiring and resistant to hydroxamate antibiotic A22765). Medium for growth studies contained 0.18 PM iron and, where indicated, 18 p.M Desferal. Iron uptake assays contained 0.045 MM -59Fe and, where indicated, 0.45 uM Desferal. Assays for DNA synthesis contained 0.7 uM ['4CJthymine, 0.7 pM uracil, 0.04 pM [3H]leucine, 0.18 pM iron and, where indicated, 18 pM Desferal. Symbols: 0, no Desferal; 0, with Desferal. HOURS

radioactive thymine incorporation in the presence of Desferal ceased. The increased rate of DNA synthesis after 2 h in control cells corresponded to entrance of the cells into logarithmic growth (Fig. 1). Cessation of DNA synthesis at about 5 h in cells exposed to Desferal probably corresponded to termination of slow residual cell division in these cultures. Incorporation of both radioactive uracil and radioactive leucine into cold 10% trichloroacetic acid-insoluble material in the presence and absence of Desferal was identical during the 6-h testing period (data not shown). This suggests that inhibition of B. subtilis 1D-4 growth by Desferal did not quantitatively alter either RNA or protein synthesis during the test period. Reversal of B. subtilis 1D-4 growth inhibition by Desferal was achieved by adding sufficient iron to saturate Desferal and produce about 0.1 ,uM "non-chelated" iron in the medium. In the parent strain B. subtilis 23 thy, neither growth, iron uptake, nor DNA synthesis was altered by Desferal. Biosynthesis of DNA in both procaryotic and eucaryotic cells may be particularly sensitive to iron starvation. The conversion of ribonucleotide diphosphates to the corresponding deoxyribonucleotide diphosphates is a possible target for this effect. The enzyme responsible for this reaction in Escherichia coli contains iron as an essential component (3, 7). The present work did not establish that altered activity of this enzyme caused inhibition of DNA synthesis in the presence of Desferal. Iron starvation should lead to eventual decrease in the function of

many or all iron-requiring systems. However, the experiments did reveal the close association that exists between cellular iron levels, control of cell division, and DNA synthesis. Overall synthesis of RNA and protein appeared not to be directly linked to this control point(s). It should be emphasized that the present experiments measured only quantitative differences in RNA and protein syntheses. In bacteria, iron deficiency is known to cause derepression of synthesis of certain proteins (6, 12) and to alter the types of certain transfer RNA molecules (11; M. J. McCauley, J. L. Arceneaux, and B. R. Byers, Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, P227, p. 173). Examination for these qualitative differences was not the purpose of the studies reported here. This research was supported by Public Health Service Research Career Development Award 6K4-GM29366 (to B. R. B.) from the National Institute of General Medical Sciences and by Public Health Service Research Grant CA11886 from the National Cancer Institute. Desferal was supplied by Ciba-Geigy Pharmaceutical Co., and antibiotic A22765 was a gift from F. Knusel. LITERATURE CITED 1. Arceneaux, J. E. L., and B. R. Byers. 1976. Ferric hydroxamate transport without subsequent iron utilization in Bacillus megaterium. J. Bacteriol. 127:1324-1330. 2. Arceneaux, J. E. L., W. B. Davis, D. N. Downer, A. H. Haydon, and B. R. Byers. 1973. Fate of labeled hydroxamates during iron transport from hydroxamateiron chelates. J. Bacteriol. 115:919-927. 3. Brown, N. C., R. Eliasson, P. Reichard, and J. Thelander. 1969. Spectrum and iron content of protein B2 from ribonucleoside diphosphate reductase. Eur. J. Biochem. 9:512-518.

VOL. 129, 1977 4. Byers, B. R., J. E. L. Arceneaux, C. G. Gaines, and C. V. Sciortino. 1976. Isolation of microbial iron chelators: some possible effects of their chemotherapeutic use, p. 213-228. In W. F. Anderson and M. C. Hiller (ed.), Development of iron chelators for clinical use. Department of Health, Education, and Welfare Publication (NIH) 76-994. U.S. Government Printing Office, Washington, D.C. 5. Davis, W. B., and B. R. Byers. 1971. Active transport of iron in Bacillus megaterium: role of secondary hydroxamic acids. J. Bacteriol. 107:491-498. 6. Downer, D. N., W. B. Davis, and B. R. Byers. 1970. Repression of phenolic acid-synthesizing enzymes and its relation to iron uptake in Bacillus subtilis. J. Bacteriol. 101:181-187. 7. Ehrenberg, A., and P. Reichard. 1972. Electron spin resonance of the iron-containing protein B2 from ribonucleotide reductase. J. Biol. Chem. 247:3485-3488. 8. Luckey, M., J. R. Pollock, R. Wayne, B. N. Ames, and

NOTES

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10. 11.

12.

13.

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J. B. Neilands. 1972. Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers. J. Bacteriol. 111:731-738. Robbins, E., J. Fant, and W. Norton. 1972. Intracellular iron-binding macromolecules in HeLa Cells. Proc. Natl. Acad. Sci. U.S.A. 69:3708-3712. Robbins, E., and T. Pederson. 1970. Iron: its intracellular localization and possible role in cell division. Proc. Natl. Acad. Sci. U.S.A. 66:1244-1251. Wettstein, F. O., and G. S. Stent. 1968. Physiologically induced changes in the property of phenylalanine tRNA in Escherichia coli. J. Mol. Biol. 38:25-40. Young, I. G., and F. Gibson. 1969. Regulation of the enzymes involved in the biosynthesis of 2,3-dihydroxybenzoic acid in Aerobacter aerogenes and Escherichia coli. Biochim. Biophys. Acta 177:401-411. Zimmerman, W., and F. Knusel. 1969. Permeability of Staphylococcus aureus to the sideromycin antibiotic A22765. Arch. Microbiol. 68:107-112.