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* This work was supported in part by the Air Force Cambridge Research Center under Contract AF19(604)-1003. 1 H. H. Nielsen, J. Chem. Phys., 5, 818, 1937. 2 H. H. Nielsen, J. Opt. Soc. America, 34, 521, 1944. 3 S. Silver, J. Chem. Phys., 9, 565, 1941. 4H. Jahn, Phys. Rev., 56, 680, 1939. 5 E. S. Ebers and H. H. Nielsen, J. Chem. Phys., 5, 822, 1937. 6 H. W. Thompson and G. P. Harris, Trans. Faraday Soc., 40, 295, 1944. 7C. H. Miller and H. W. Thompson, Proc. Roy. Soc. London, A 200, 1, 1949. 8 R. C. Lord and P. Venkateswarlu, J. Chem. Phys., 20, 1237, 1952. 9 J. Overend and H. W. Thompson, Trans. Faraday Soc., 52, 1295, 1956. 10 K. N. Rao and E. D. Palik, J. Molecular Spectroscopy, 1, 24, 1957. 11 K. N. Rao, A. H. Nielsen, and W. H. Fletcher, J. Chem. Phys., 26, 1572, 1957. 12 H. H. Nielsen, Rev. Mod. Phys., 23, 90, 1951. 13 J. de Heer, J. Chem. Phys., 20, 637, 1952. 14 H. H. Nielsen, Disc. Faraday Soc., 9, 85, 1950. ]6 H, H. Nielsen, J. Chem. Phys., 21, 142, 1953. 16 V. M. McConaghie and H. H. Nielsen, J. Chem. Phys., 21, 1836, 1953. 17 W. H. Haynie and H. H. Nielsen, J. Chem. Phys., 21, 1839, 1953. 18 H. H. Nielsen, Trans. Inst. and Meas. Conf. (Stockholm), 1952. '9 T. J. Coburn, Ph.D. dissertation, Ohio State University, 1957. 20 E. F. Barker, Phys. Rev., 55, 657, 1939. 21 D. L. Wood, E. E. Bell, and H. H. Nielsen, these PROCEEDINGS, 36, 497, 1950. 22 C. Cummings, Can. J. Phys., 33, 635, 1955. 23 W. S. Benedict and E. K. Plyler, Can. J. Phys., 35, 1235, 1957.

ACETATE AS A CALCIUM-SPARING FACTOR IN NITROGEN FIXATION BY AZOTOBACTER VINELANDII* BY RAYMOND G. ESPOSITO AND P. W. WILSON DEPARTMENT OF BACTERIOLOGY, UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN Communicated March 25, 1958 Studies" 2, 1 on the function of calcium in nitrogen fixation by Azotobacter vine- landii 0 revealed that this ion is required for the synthesis of metaphosphate by whole cells. Of the ions tested, it is the most potent stimulator of metaphosphate formation from adenosine triphosphate by cell-free extracts of this organism. In the course of these studies, it was observed that the rate of oxygen uptake of cal- cium-deficient cells in Burk's medium was stimulated by the addition of this ion. This stimulation is relatively insensitive to inhibition by 0.001 M sodium azide as compared to the sensitivity of calcium-deficient control cells. Roberts and Rake- straw4 have reported a reversal of the azide inhibition of oxygen uptake by A. vine- landii 0 by the addition of ammonium acetate. This observation and that of Bulen and Frear5 concerning the reversal of phosphite inhibition of growth and oxy- gen uptake of A. vinelandii by ammonium acetate led us to test the effect of acetate ion on the calcium requirement of A. vinelandii for nitrogen fixation. Previous studies' have shown that low calcium ion concentrations in Burk's medium result in an extension of the lag phase of growth, as well as a decrease in the total growth of A. vinelandii 0 when using N2 but not when NH4+ is supplied. We have now found that the addition of acetate or ethyl alcohol to Burk's sucrose- Downloaded by guest on September 30, 2021 VOL. 44, 1958 PHYSIOLOGY AND BACTERIOLOGY: ESPOSITO AND WILSON 473

salts medium partially replaces this requirement of calcium when N2 is the source of nitrogen. Materials and Methods.-Cultures of A. vinelandii 0 were maintained on a modi- fied Burk's sucrose-salts liquid medium6 and studied in 500-ml. Erlenmeyer shake flasks containing 100 ml. of this medium. The flasks were incubated at 300 C. on a New Brunswick rotary shaker rotating at 400 rpm. Tests for purity of culture were made periodically with the Gram stain and nutrient peptone technique de- scribed by Burk and Burris.7 Analytical reagent-grade chemicals were used for the prep- LOG I I I aration of media. Magnesium K. S. 1 sulfate was prepared free of cal- 1.8 cium contamination by recrystal- lizing twice from ion-free water. Ion-free water was obtained and glassware cleaned by methods previously described.2 Growth curves were determined in 7 specially made shake flasks with glass side arms made to fit the Klett-Summerson calorimeter. All readings were taken at 600 m/u. 1.6 Test media were inoculated with a 2 per cent by volume of a 24-hour shake culture of A. vine- 2 landii in Burk's medium whose calcium concentration (as Ca- o S04 -2H20) is 0.6 X 10-3 M. At times the inoculum was brought 2 4 6 8 10 up on a medium containing only HOURS FIG. 1.Effect of acetate on the growth of Azoto- 20 per cent of the normal cal- bacter vinelandii 0 in a calcium-deficient medium. cium concentration (0.12 X 10-s Burk's sucrose, N-free medium modified as follows: 1-1.5 X 10-3 M phosphate; 1.2 X 10-5 M calcium M). The final concentration of sulfate; 1.5 X 10-2 M sodium acetate; 2-6 X 10-3 calcium in the deficient media is M phosphate; 1.2 X 10- M calcium sulfate; 3- 1.5 X 10-3 M 1.2 X 10- M calcium that carred over in the inoculum sulfate. The effectphosphate;of a decrease in phosphate on the and is specifically indicated in the lag in a calcium-deficient medium is discussed by Espo- legends of the figures. The pH sito567,1956.and Wilson, Proc. Soc. Exptl. Biol. Med., 93, 564- of the test media containing salts of organic acids was maintained at 7.0-7.2 by the addition of dilute HCl at hourly intervals. Results.-The effect of acetate ion on the calcium requirement for nitrogen fixa- tion is shown in Figure 1. Glucose, fructose, ribose, dihydroxyacetone, ethyl alcohol, sodium pyruvate, potassium glycolate, potassium glyoxylate, thioctic acid, and thiamine were all tested in the presence of sucrose; but, of these, only ethyl alcohol could be substituted for acetate. The failure of sodium pyruvate was not the result of the organism's inability to oxidize it, as the uptake and apparent oxida- Downloaded by guest on September 30, 2021 474 PHYSIOLOGY AND BACTERIOLOGY: ESPOSITO AND WILSON PROC. N. A. S.

tion of pyruvate as measured by pH change of the medium was essentially at the same rate as acetate. Most of the compounds tested are precursors of, or participants in reactions that form precursors of, acetate. The intermediates of the tricarboxylic acid cycle may be considered products of acetate metabolism, since the azotobacter contains a com- plete tricarboxylic acid cycle and means for the entry of acetate.8 Recrystallized malic acid neutralized with potassium hydroxide when added to Burk's sucrose medium, however, did replace the need for added calcium ion. However, this re- placement may be the result of a "jamming" of the metabolic pathways, with a

LOG l 1 1

______K.S . LOG I. K. S.H4CL AC+ Co 1.8 Mal-+ Cao2+C

1.6 1.6

AC-Ca~ ~ ~ ~ ~ ~ ~ p-C

Mal--Ca I l I 1 2 4 6 8 HOURS 2 4 6 8 FIG. 2.-Growth of Azotobacter vinelandii HOURS o in Burk's medium with acetate and malate FIG. 3.-Comparison of effect of calcium on as sole carbon sources and high and low levels ofof calcium._caliu. AC-:Ac: 1. X 102l1U-2 MM sodium.odu- aeace- freethe growthand combinedof Azotobacternitrogenvinelaudjiwith malate0 usingas tate; Mal-: 1.5 X 10-2 M sodium malate; sole source of carbon. Medium was Burk's + Ca: 0.5 X 10-3 M calcium sulfate; -Ca: mineral salts 1.5 X 10-3 M potassium 2.5 X 10 -6 M calcium sulfate. malate. NH4+:plus8 X 10-3 M ammonium sulfate; +Ca: 0.6 X 10-3M calcium sulfate; -Ca: 2.5 X 10-6 M calcium sulfate. resultant spillover to acetate or a compound formed from it. This explanation is indicated by the results contained in Figure 2. When malate is added as the sole carbon source for the organism, adequate calcium is necessary for nitrogen fixation to be initiated. However, if acetate is added as the sole carbon source, nitrogen fixation is initiated in the calcium-deficient medium. One possible explanation of the failure of malate added as the sole carbon source to replace calcium is a failure of sucrose-grown cells to adapt to the utilization of malate. This would necessarily result in an extension of the lag phase of growth and indicate an apparent lack of calcium-sparing activity. Figure 3 demonstrates that this is not so. The addition of either calcium or ammonium ions to a medium Downloaded by guest on September 30, 2021 VOL. 44, 1958 PHYSIOLOGY AND BACTERIOLOGY: ESPOSITO AND WILSON 475

of Burk's mineral salts plus potassium malate results in a normal growth curve. This indicates that the cells adapt to malate within a normal period of lag and that the utilization of malate in a calcium-deficient medium is adequate for the assimila- tion of ammonium ion but not for molecular nitrogen. An examination of Figure 2 reveals that, although the presence of acetate in the calcium-deficient medium suffices for the initiation of growth, the addition of cal- cium results in a higher rate of growth. If, however, sucrose is present in addition to acetate, the rate of growth is as high as that of cells in the presence of acetate and adequate calcium. Preliminary results suggest that acetate plus malate also results in a high rate of growth in the calcium-deficient medium. Discussion.-Studies9' 10, 11 on the kinetics of incorporation of molecular nitrogen into the organic pool of the cell indicate that ammonia is the major terminal inor- ganic form of the newly fixed ("juvenile") nitrogen prior to formation of amino acids. If so, obviously, the tricarboxylic acid cycle could play an important role as a source of acceptors for the ammonia-e.g., a-ketoglutaric acid. The data re- ported here, however, suggest that some other compound(s) apart from those of the tricarboxylic acid cycle may function in the trapping of the "juvenile" nitrogen or have some other role in the formation of ammonia from molecular nitrogen. When ammonium ion is supplied, malate functions satisfactorily as a source of carbon in the presence of limited calcium, (2.6 X 10-6M) but, if molecular nitrogen is the nitrogen source, adequate calcium, acetate, or sucrose must be available for the initiation of growth. It seems, then, that calcium functions in an indirect manner in nitrogen fixation by A. vinelandii 0, apparently being necessary for the formation of acetate or of a compound related to acetate that is required for the fixation reaction. Whether acetate is necessary for the formation of an acceptor of "juvenile" nitrogen at a level preceding ammonia or serves some other function is as yet unknown. One possibility that has been tested was directed toward providing evidence that hy- droxylamine is a precursor of ammonia. If so, it could combine with acetyl-CoA to form acetohydroxamic acid; attempts to demonstrate utilization of this com- pound by whole cells and by cell-free extracts were unsuccessful. Summary.-The requirement of calcium ion for nitrogen fixation by A. vinelandii 0 is relieved when sodium acetate is supplied the organism. For example, at a calcium ion level of 10-5-10-6 Ml the organism will not initiate growth on N2 but will on NH4+. Addition of sodium acetate to the calcium-deficient medium allows growth to occur in the absence of fixed nitrogen. Ethyl alcohol alone or potassium malate plus sucrose also can replace this calcium requirement. The following compounds could not substitute for acetate in this calcium-alleviating role: glu- cose, fructose, ribose, dihydroxyacetone, sodium pyruvate, potassium glycolate, potassium glyoxylate, thiamine, or thioctic acid.

* Supported in part by grants from the Rockefeller Foundation and the Research Committee of the Graduate School from funds provided by the Wisconsin Alumni Research Foundation. I R. G. Esposito and P. W. Wilson, Biochim. et Biophys. Acta, 22, 186-187, 1956. 2 R. G. Esposito and P. W. Wilson, Proc. Soc. Exptl. Biol. Med., 93, 564-567, 1956. 3 R. G. Esposito, Ph.D. thesis, University of Wisconsin, Madison, Wis., 1957. 4 E. R. Roberts and J. A. Rakestraw, in Inorganic Nitrogen Metabolism, ed. W. D. McElroy and B. Glass (Baltimore, Md.: Johns Hopkins Press, 1956), pp. 361-367. Downloaded by guest on September 30, 2021 476 PHYSIOLOGY AND BACTERIOLOGY: HUGHES ET AL. PROC. N. A. S.

6 W. A. Bulen and D. S. Frear, Arch. Biochem. Biophys., 66, 502-503, 1957. 6 P. W. Wilson and S. G. Knight, Experiments in Bacterial Physiology (Minneapolis, Minn.: Burgess Publishing Co., 1952). 7D. Burk and R. H. Burris, Ann. Rev. Biochem., 10, 587-618, 1941. 8 R. W. Stone and P. W. Wilson, J. Biol. Chem., 196, 221-225, 1952. 9 J. W. Newton, P. W. Wilson, and R. H. Burris, J. Biol. Chem., 204, 445-451, 1953. "' R. M. Allison and R. H. Burris, J. Biol. Chem., 224, 351-364, 1957. 11 D. P. Burma and R. H. Burris, J. Biol. Chem., 225, 287-295, 1957.

CELLULAR PROLIFERATION IN THE MOUSE AS REVEALED BY A UTORADIOGRAPH Y WITH TRITIA TED TH YMIDINE* BY W. L. HUGHES, V. P. BOND, G. BRECHER, E. P. CRONKITE, R. B. PAINTER, H. QUASTLER, AND F. G. SHERMAN BROOKHAVEN NATIONAL LABORATORY, NATIONAL INSTITUTES OF HEALTH, AND BROWN UNIVERSITY Communicated by Donald D. Van Slyke INTRODUCTION Many cell populations within the body undergo constant breakdown and re- newal.1 Classical histological techniques do not allow adequate study of dynamic processes, and autoradiography is proving increasingly valuable for following these processes at the cellular level. In order to distinguish cell renewal from the re- newal of cellular constituents, the label must be incorporated into a fixed component of the cell which is not lost during the cell's lifetime; and present knowledge sug- gests that a label incorporated into deoxyribonucleic acid (DNA) should be most useful for this purpose. DNA occurs within the nucleus of the cell associated with the chromosomes and appears, in fact, to be the bearer of the genetic information which each cell passes on to its descendants. Since this information must be handed down through countless generations, extreme immutability of the DNA molecules would seem desirable, and, in fact, evidence is accumulating that DNA in a cell is never replaced. This evidence, as reviewed2 by Thorell and Smellie, shows that DNA is metabolically inert in resting cells. The possibility of turnover during cellular division, which these authors considered, would appear to be denied by more recent reports. Thus Taylor, Woods, and Hughes3 used tritiated thymidine (thymine occurs uniquely in DNA, and thymidine is incorporated efficiently and exclusively into DNA4) to label the new DNA of bean roots. Autoradiography showed that chromosomes are transmitted to daughter cells as intact half-chromosomal units. This suggests that duplication of the genetic material involves the separation of each chromosome into complementary halves and the formation of new complements upon the existing halves as templates. Escherichia coli labeled with tritiated thymidine distributed their DNA among their progeny in a like fashion.5 In both these cases no loss of label from the total population was observed over several cell generations. Autoradiographic identification of cells should permit a more detailed analysis of cellular dynamics. The specific advantage6 of tritium for autoradiography lies in the very high resolution which can be obtained because of the very weak energy and consequently short range of its : radiation (Fig. 1). The maximum range in Downloaded by guest on September 30, 2021