Considerable quantities of iron were incorporated into the Mycoplasma capri- colum cell membrane. Mossbauer studies showed that the iron is in a form which.
JOURNAL OF BACTERIOLOGY, Jan. 1980, p. 378-381 0021-9193/80/01-0378/04$02.00/0
Vol. 141, No. 1
Iron Storage in Mycoplasma capricolum E. R. BAUMINGER,I* S. G. COHEN,' F. LABENSKI DE KANTER,t A. LEVY,' S. OFER,' M. KESSEL,' AND S. ROTTEM' Racah Institute of Physics, The Hebrew University,' and Biomembrane Research Laboratory, The Hebrew University-Hadassah Medical School,' Jerusalem, Israel
Considerable quantities of iron were incorporated into the Mycoplasma capricolum cell membrane. Mossbauer studies showed that the iron is in a form which becomes magnetically ordered at low temperatures. The iron-enriched cells contained membrane-bound electron-dense particles of about 6.0 nm in diameter. Mossbauer spectroscopic studies of Esche- horse serum. The total iron content of this merichia coli (1) and Proteus mirabilis (D. P. E. dium was about 0.05 ,ug/ml. FeSO4 enriched to Dickson and S. Rottem, Biochem. J., in press) about 90% in "7Fe was added to the growth cells grown in "7Fe-enriched media have shown medium as a solution in 9 N H2SO4 to a fmal that these organisms incorporated substantial concentration of a 4 ,ug/ml, and the pH of the quantities of iron from the growth media. The medium was adjusted to 7.5 with NaOH. The iron was found to be stored in the form of medium was inoculated at an inoculum level of aggregates of high-spin Fe3", magnetically or- 0.1% (vol/vol), and growth was followed by dered at low temperatures (1). Moreover, iron- measuring absorbance in a Spectronic 80 specrich proteins which show some similarities to trophotometer. The cells were harvested at the ferritin, the iron storage protein found in higher mid-exponential phase of growth (absorbance at organisms, have been recently isolated from the 640 nm = 0.17) by centrifugation at 10,000 x g soluble fraction of E. coli (J. Yariv, R. Sperling, for 15 min at 4°C, washed twice, and resusE. R. Bauminger, S. G. Cohen, and S. Ofer, pended in 0.25 M NaCl solution. Protein was submitted for publication) and from Azotobac- determined by the method of Lowry et al. (4). ter vinelandi (11). The iron cores of this protein About 0.6 ml of heavy cell suspensions containin E. coli were found to be of the same structure ing about 20 mg of cell protein was transferred as the iron aggregates in the whole cells of E. to perspex containers and stored in liquid nitrocoli, although the iron in the soluble protein gen. Spectra were measured with a conventional accounts for less than 1% of the cell iron. It Mossbauer drive with a 100 mCi of 57Co (Rh) seems likely that under conditions of iron suffi- source and a Harwell proportional counter, runciency procaryotic cells in general develop the ning at about 105 cps. Refrigeration below 1.5°K ability to synthesize iron storage material. The was attained with a 3He-4He dilution refrigerator role of the iron storage material in bacteria is (3). M. capricolum cells (under the conditions yet unknown. In A. vinelandi the iron storage described above) incorporated iron to the level protein contains iron in the form of a dense core of about 2 jig of s7Fe per mg of cell protein. and also heme, the latter giving rise to features The Mossbauer spectra of whole M. capriin the absorption spectra similar to those found colum cells below 4.1°K are shown in Fig. 1. All in cytochrome bi. Therefore, it was suggested of the spectra between 4 and 250°K have prethat this protein functions as an electron storage dominantly the form of an Fe3" quadrupole douprotein that supplies low potential redox equiv- blet, whereas at 0.07°K a six-line magnetically alents. The parasitic mycoplasmas represent the split spectrum of Fe3+ is the main component. smallest and simplest self-replicating procar- (In all spectra, analysis shows the presence of yotes (7). These cells have truncated respiratory another doublet of weaker intensity, correspondsystems lacking quinones and cytochromes (5). ing to Fe2+. The intensity of this doublet grows It was therefore of special interest to investigate when the sample is thawed and refrozen. We the presence of an iron storage material in a ascribe the Fe2' doublet to changes produced in representative species of the parasitic mycoplas- the main iron component by thermal recycling. mas. Its intensity is therefore larger in the 0.07°K Mycoplasma capricolum (California kid) was spectra, because the sample had to be thawed grown in Edward medium (8) containing 5% before inserting it in the dilution refrigerator. In the following we confine our discussion to the t Permanent address: Comision Nacional de Energia Atom- main Fe3" component.) In the magnetically split ica, Buenos Aires, Argentina. spectrum at 0.07°K, the outer lines are consid378
NOTES
VOL. 141, 1980
379
1.
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uj
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2.65 K
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FIG. 1. Mossbauer spectra of whole M. capricolum cells. The solid lines are least-squares computer fits to the spectra, assuming each spectrum to be a sum of a Fe3+ quadrupole doublet (subspectra a), a magnetically split spectrum (subspectra b), and an Fe 2' doublet. The position of the Fe 3+ and Fe 21 lines are shown on top of the figure. In fitting subspectra b an asymmetric Lorentzian distribution of magnetic hyperfine fields was assumed. The distribution is given by N(H) Nol (1 + [(H - Hc)AH]2), where lSH is different for H > Ho and H < Ho. The position of the lines for a magnetic field Ho are shown in the bottom of the figure, =
erably broader than the inner lines, indicating that the hyperfine fields at the iron nuclei are spread over a range of values (between 350 and 440 kilooersteds). In the transition region be-
tween 1.5 and 3.5°K, the spectra are composed mainly of two subspectra: (i) an Fe3+ quadrupole split doublet (with an isomer shift of 0.51 ± 0.01 mm/s relative to iron metal and a quadrupole
380
NOTES
splitting of 0.63 ± 0.01 mm/s) who.se relative intensity grows with increasing tempe,rature, until above 3.5°K when it is the only 4component that remains and (ii) an Fe3" magnettically split spectrum, which becomes more smesired out as the temperature is raised and which also shows that the magnetic hyperfine field decireases with temperature until it vanishes at 3.5i°K, where this magnetically split spectrum cotlapses into the above-mentioned quadrupole dooublet. The form of the magnetically split spect;ra and the way in which they depend on temp4erature are characteristic of a magnetically order ed phase of Fe3+ (e.g., ferromagnetic or antifenromagnetic ordering). Figure 2 shows the ratio between the intensity of the 3+ quadrupole spliit spectrum and the magnetically split spectrumLas a function of temperature. This temperatuare dependence and the observed spread in thea hyperfine fields show that the magnetic transiti on temperature is not uniquely defined: there iis a continuous distribution of transition tempe,ratures between 1.2 and 3.5°K. The observedI spread in hyperfme fields and magnetic transitiion temperatures indicates inhomogeneity in tiie Fe environment and can be explained by an;amorphous type of structure, or by small and rxonuniform particle sizes, or a combination of suich factors. To account for the presence of a mkagnetically ordered phase of iron, we must assu me the FeFe interatomic distances to be sma l1 within a structure containing many iron atom1S. These conclusions were further supported by electron microscopy studies. Figure 3,shows thin sections of M. capricolum cells grnDwn in the
100
,
80
-
60
-
S 4 2 3 TEMPERATURE (OK) FIG. 2. The percentage of the Fe3" mtagnetically unsplit part of the spectra as a function of tempera0
ture.
1
J. BACTERIOL.
presence of 57FeSO4. The cells were fixed in Ryter and Kellenberger osmium tetroxide (9) for 20 to 24 h at 40C, washed with cold buffer, poststained in bloc with 1% uranylacetate, dehydrated in alcohol, and embedded in Spurr Epon (10). Unstained sections were viewed in a Philips EM 400 electron microscope at 80 kV. The appearance of unstained M. capricolum cells grown with the iron shows a marked concentration of electron-dense particles bordering the cell membrane (see arrows). The particles appear to be preferentially associated with the outer face of the membrane. The particles seem to have an approximately uniform size between 6.0 to 7.0 nm. No such particles were present in thin sections of cells not enriched with iron. M. capricolum cells grown with 57FeSO4 were osmotically lysed, and the soluble fraction was separated from the cell membrane by centrifugation (6). The amounts of iron in the various fractions were estimated from the areas under their Mossbauer spectra at 80°K. More than 99% of the iron was found to be associated with the cell membrane fraction. The iron concentration in the membrane preparations was about 5.5 ,ug of "7Fe per mg of membrane protein. The membrane preparations retained all the iron even after a sucrose density gradient analysis with a linear 25 to 55% sucrose gradient centrifuged at 100,000 x g for 3.5 h in an SW41 rotor. The iron-containing cell membranes banded, as do normal cell membranes, at 1.16 g of sucrose per ml. The Mossbauer spectra obtained with the purified membranes were identical to those found in whole cells. No attempts were made to isolate an iron storage protein from the soluble part of the M. capricolum cells. M. capricolum cells were also grown without 57FeSO4 in the nutrition media. The iron concentrations in these cells were less than 0.1 ,ug/mg of cell protein. Because the iron wasnot enriched in 57Fe, the sizes of the effects in the Mossbauer spectra were less than 0.1% of those obtained with the cells grown in the medium containing 57FeSO4. No information concerning the exact shape of the spectra and the distribution of the iron between the various fractions could be obtained. The main features of the Mossbauer spectra observed in M. capricolum are very similar to those observed in E. coli and P. mirabilis, yet differ from those obtained in ferritin or hemosiderin. The magnetic ordering temperature of the iron in the procaryotes is about 3°K and has the characteristic of a true magnetic transition temperature, whereas the "blocking" temperature of ferritin or hemosiderin associated with superparamagnetism is about 40°K (2). Moreover, the magnetic hyperfine field acting on iron nuclei at
VOL. 141, 1980
381
NOTES ¼t
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FIG. 3. Thin section of unstained M. capricolum cells enriched with 57FeSO4, x172,000. Scale represents IWUU nm. ferritin. Austr. J. Phys. 19:573-576. magnetic saturation (at 0.08'K) in the procaryotes is 430 + 5 kilooersteds much smaller than 3. Hess, J., A. Levy, and U. Schmid. 1977. Fabrication of stainless steel dewars for experiments at low temperathe corresponding field in ferritin or hemosiderin ture. Cryogenics 17:501-507. 497 ± 3 kilooersteds (1, 2). 4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin The occurrence of very similar Mossbauer spectra in E. coli, P. mirabilis and M. capri- 5. phenol reagent. J. Biol. Chem. 193:265-275. Pollack, J. D. 1979. Respiratory pathways and energycolum point to the existence of a universal type yielding mechanisms, p. 188-209. In M. F. Barile and S. of iron storage material in procaryotes, which Razin (ed.), The mycoplasmas, vol. I. Academic Press Inc., New York. differs in some respects from the iron storage S. 1963. Osmotic lysis of mycoplasma. J. Gen. protein, ferritin, and its insoluble analog, hemo- 6. Razin, Microbiol. 33:471-475. siderin, found in higher forms of life. Whether 7. Razin, S. 1978. The mycoplasmas. Microbiol. Rev. 42: all or part of the membrane bound iron in M. 414-470. capricolum and in E. coli is associated with 8. Razin, S., and S. Rottem. 1976. Techniques for the manipulation of mycoplasma membranes, p. 3-26. In A. protein or whether it exists in some other form H. Maddy (ed.), Biochemical analysis of membranes. remains an open question. Chapman and Hall, London. I If
, This work was supported in part by the Stiftung Volkswagenwerk.
9. Ryter, A., and E. Kellenberger. 1958. Etude au microscope electronique de plasma contenant de l'acid desoxyribonucleique. I. Les nucleotides des bacteries en
LITERATURE CITED 1. Bauminger, E. R., S. G. Cohen, D. P. E. Dickson, A. Levy, S. Ofer, and J. Yariv. 1979. Observation of iron storage protein in E. coli by Mossbauer spectroscopy. J. Phys. (Paris) 40:523-525. 2. Boas, J. F., and X. Window. 1966. Mossbauer effect in
croissance active. Z. Naturforsch. 13:597-605. 10. Spurr, A. R. 1969. A low viscosity resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: 3143. 11. Steifel, E. I., and G. D. Watt. 1979. Azotobacter cytochrome b557.5 is a bacterioferritin. Nature (London) 279:81-83.
