RECOIL-FREE SPECTRA FROM 57Co-ENTEROCHELIN IN E. COLI CELLS

HAL Id: jpa-00215824

https://hal.archives-ouvertes.fr/jpa-00215824

Submitted on 1 Jan 1974

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

RECOIL-FREE SPECTRA FROM

57Co-ENTEROCHELIN IN E. COLI CELLS

E. Giberman, Y. Yariv, A. Kalb, E. Bauminger, S. Cohen, D. Froindlich, S.

Ofer

To cite this version:

E. Giberman, Y. Yariv, A. Kalb, E. Bauminger, S. Cohen, et al.. RECOIL-FREE SPECTRA FROM

57Co-ENTEROCHELIN IN E. COLI CELLS. Journal de Physique Colloques, 1974, 35 (C6), pp.C6-

371-C6-374. �10.1051/jphyscol:1974667�. �jpa-00215824�

JOURNAL

DE

Colloque C6, supplément au no 12, Tome 35, Décembre 1974, page C6-371

RECOIL-FREE SPECTRA FROM CO-ENTEROCHELIN IN E. COLI CELLS

E. GIBERMAN

Israel Institute for Biological Research, Ness-Ziona, Israel Y. YARIV and A. J. KALB

Department of Biophysics, Weizmann Institute, Rehovot, Israel and

E. R. BAUMINGER, S. G. COHEN, D . F R O I N D L I C H and S. O F E R The Racah Institute of Physics, The Hebrew University, Jerusalem, Israel

Résumé. - Le transport du fer dans les bactéries a lieu selon plusieurs modes impliquant des agents chélatants. Dans E. Coli, l'un des modes implique l'entérocheline, un trimère cyclique de la N-2,3, dihydrozybenzoylsérine, qui est un ligand hexadentate du Fe (III) et d'autres ions métal- liques. La présente étude a été réalisée à l'aide du complexe entérocheline marqué par S'Co et une souche de mutant (AN 272) de E. Coli. Des spectres Mossbauer par rapport à un absorbant d'inox enrichi en 57Fe ont été mesurés sur des échantillons compacts de ces bactéries contenant environ 0,5 mCi de 57C0. Les mesures ont été réalisées sur des sources de bactérie maintenues à environ 3 OC.

A cette température, on a observé durant les 24 premières heures un effet sans recul (0,03 f 0,005 %, largeur environ 3 mm/s) qui disparaissait ensuite. La différence des spectres d'échantillons frais et vieillis mesurés sur systèmes gelés à - 190 OC, a révélé la présence d'une faible composante sans recul dans la bactérie fraîche dans la même gamme spectrale que le spectre d'absorption relevé à 3 OC. On suggère que les 2 spectres correspondent aux mêmes sites particuliers présentant un environ- nement solide ou quasi solide à 3 OC et situés probablement dans la membrane de la cellule. On estime grossièrement la population relative des noyaux de 57C0 et le facteur sans recul à ces sites spéciaux. Assignant l'élargissement observé à 3 "C à une diffusion, on propose une estimation grossière des constantes de diffusion de 57C0 à ces sites.

Abstract. - Transport of iron into bacteria occurs by several routes which involve chelating agents. In E. coli, one of the routes involves enterochelin, a cyclic trimer of N-2,3, dihydrozy- benzoylserine, which is a hexadentate ligand of iron (III) and other metal ions. The present study was done with the s7Co-enterochelin complex and a mutant strain (AN 272) of E. coli. Mossbauer absorption measurements using a stainless steel absorber enriched in 57Fe were performed on a tightly packed sample of these bacteria which contained about 0.5 mC of carrier free s7C0. Measure- ments were carried out with the bacterial source maintained at approximately 3 OC. At this tempera- ture a recoil-free effect (0.03 + ^0.005 % ; width about 3 mm/s) was observed during the first 24 hours, but disappeared thereafter. Difference spectra between fresh and aged samples taken in frozen systems at - 190 OC showed the presence of a small recoil-free component in the fresh bacteria at the same spectral region as the absorption spectrum observed at 3 OC. It is suggested that both spectra originate at the same special sites, having a solid or quasi-solid environment at 3 OC, and which are probably situated in the ce11 membrane. Rough estimates are made of the relative population of 57C0 nuclei and the recoil-free efficiency at these special sites. If we ascribe the line broadening at 3

to diffusion broadening, rough estimates of the diffusion constants of S'Co at these sites are made.

1. Introduction. - Previous attempts t o study recoil- The authors were particularly alive t o the possibility free spectra in bacteria were confined t o the study of that recoil-free spectra from such components might frozen bacterial systems containing 57Fe nuclei [ l , 21. show diffusion broadening which could provide new I n this work a special effort was made t o look for insights, for example, into the dynamics of transport recoil-free effects in non-frozen bacterial cells which of metal ions through the ce11 membrane, about which might be attributed t o solid o r quasi-solid components little is known.

of the cell, including particularly, the ce11 membrane. I n this study we have investigated the recoil-free

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1974667

C6-372 E. GIBERMAN, Y. YARIV, A. J. KALB, E. R. BAUMINGER, S. G. COHEN AND COLL.

spectrum of 57Fe produced after the decay of s7Co nuclei concentrated in E. coli cells (mutant strain AN 272) at 3 OC and at - 190 OC.

Iron transport into bacteria occurs by several routes involving iron chelating agents [3]. In E. coli, one of the possible routes involves enterochelin, a cyclic trimer of N-2,3 dihydroxybenzoylserine, which is a hexadentate ligand of Fe(II1) and other metal ions [4]. Estimates based on the uptake of iron by E. coli from a surround- ing medium containing Fe-enterochelin complex, showed that the 57Fe content in the bacteria would be too small for recoil-free spectroscopy even using iron enriched in 57Fe. However, as described below, Co-enterochelin complex can also be made and incor- porated into bacteria. Recoil-free experiments were therefore carried out using bacterial sources containing very small concentrations of s7Co-enterochelin complex.

2. Accumulation of Co-enterochelin by E. coli using 60Co. - The bacterial strain (E. coli, AN 272) used in this study and metal-free enterochelin were gifts of Dr. J. G. Young of the Australian National University, Canberra.

Evidence that Co2+ complexes with metal-free enterochelin was obtained by potentiometric titration.

In an accumulation experiment Co-enterochelin complex was prepared by dissolving enterochelin in absolute ethanol and diluting with twice-distilled water in the ratio 1 : 4. To a one molar equivalent of enterochelin solution 0.9 molar equivalents of 60CoC12 in 0.1 HC1 were added and the solution was neutralized by addition of 1 M sodium phosphate, pH 7.0.

I;; coli, strain AN 272, was grown as described by Langman et al. [5]. Bacteria were harvested and washed, essentially, as advised by the above authors.

The density of bacteria used in a typical accumulation experiment was 2 x 109/ml. 60Co-enterochelin was added from a stock solution to the following final concentrations 2 x IO-' M, 2 x M and 1 x 1 0 - ~ M. The mixture was maintained at room temperature (approx. 25 OC). At predetermined times 100 pl samples were removed and the bacteria sepa- rated from supernatant fluid,by sedimentation through oil in a Beckman microfuge at 4 OC, according to a previously described general procedure [ 6 ] . Accumu- lation of Co-enterochelin by E. coli exhibits saturation kinetics and is governed by a single association constant Ka,, ⁼ 5.3 x 106 M-'. The maximal initial rate of entry for Co-enterochelin at 25 OC is calculated to be 4 000 molecules/bacteria/min.

3. Recoil-free experiments.

a) PREPARATION OF

57Co

- E. coli strain AN 272 was grown and washed as described above. In a typical experiment a suspension of 3 x 10'' bacterial cells was mixed with 57Co-enterochelin solution and main- tained at 4 OC for 1 hour. 57Co-enterochelin solution was prepared as described above, using carrier-free

57Co (0.2 pg Co11 mCi), and filtered (Millipore, 0.10 p) as a routine precaution. The bacteria were then sedimented in a plastic container at IO O00 rpm in a Beckman ultracentrifuge, the supernatant fluid removed and the container sealed. These samples contained about 3 x 10'' bacteria and 0.5 mCi 57Co ; the volume was approximately 0.3 ml. This corresponds to an average concentration of about 3 000 57Co nuclei per bacterium.

b) SOME

- The source strengths were such as to give maximal counting rates, taking into account the time resolution of the counting and recording equip- ment. A 1 mm lithium drifted silicon counter was used as detector for the 14 keV radiation. In the measure- ments at 3 OC the source was kept stationary and the absorber (2 mg/cm2 enriched stainless steel) moved.

c) EXPERIMENTAL

3 OC. - Figure 1 shows the absorption spectra obtained from the bac- teria at 3 OC. Figure 1A displays the spectrum obtained during the first 24 hours after preparation, whereas figure 1B displays the results obtained with the same

-4 -2

O

V E L O C I T Y

FIG. 1 . - Recoilless absorption spectra in stainless steel of the 14.4 keV gamma rays emitted from a s7Co-enterochelin source in E. coli cells at 3 OC. A ) Spectrum obtained during the first 24 hours after preparation. B ) Spectrum obtained during the

subsequent period of 72 hours.

samples during the subsequent period of 72 hours. The results in each case represent the sum of five separate measurements carried out on five independently prepared samples. From figure 1A we see that a very small but definite recoil-free effect (0.032 + ^{0.005 %)}

characterized by a broad absorption line (width

- 3 mm/s) centred at - 0.7 mm/s, obtains, during the

first 24 hours after preparation. A statistically signi-

ficant effect was obtained in al1 five samples. However,

figure 1B does not show any recoil-free absorption

(recoil-free effect is smaller than 0.004 %). The

disappearance of the effect after 24 hours can be

ascribed to damage which must occur to bacteria

obtaining in the conditions of the experiment. We

shall, for convenience, albeit somewhat naively and

picturesquely, describe the fresh bacteria as live

and those obtaining after 24 hours as dead. We

RECOIL-FREE SPECTRA FROM s''Co-ENTEROCHELIN IN E. COLI CELLS C6-373

shall use the term svecial sites to denote the sites of I

those 57Co nuclei giving rise to the recoil-free spectrum at 3 OC (Fig. 1A).

It is very probable that only a fraction of the 57Co nuclei present in the ce11 are at these special sites From the measurements at 3 OC alone it is impossible, even roughly, to estimate this fraction. Knowledge of this fraction is essential in order to estimate the recoil- free efficiency observed at 3 OC.

d ) EXPERIMENTS AT - 190 QC. - In a further series of experiments, using similar bacterial sources, frozen at - 190 OC, an attempt was made to locate a specific component of the recoil-free spectrum associated with live bacteria which may perhaps have the same environmental origin as the spectrum observed at 3 OC, in the non-frozen state.

In these measurements a fresh sample of bacteria containing 57Co-enterochelin, prepared as above, was divided into two equal portions - 1 and II. A measu- rement of the recoil-free spectrum of portion II was made during the first 24 hours at 3 OC, and a small recoil-free effect was detected as in the above measure- ments. This portion was then kept at 3 OC for a further 3 days and then frozen suddenly at 83 K - this portion will be denoted as the dead sample. Portion 1 - the

!ive sample, was suddenly frozen at - 190 OC, imme- diately after preparation.

The recoil-free spectra from these two frozen sam- ples, were then compared accurately under identical conditions at - 190 OC. Good counting statistics were accumulated, measuring during four days for each sample.

The spectra obtained were very nearly identical and visual inspection was insufficient to establish a diffe- rence. Figure 2 A shows the spectrum obtained with sample II (dead bacteria). It is in fact very similar to the spectrum obtained from a frozen aqueous solution containing 57~o-enterochelin at - 190 OC. In fact we must conclude from this similarity that the major component of 57Co in the bacterial ce11 exists in a chemical form very close to that of 57Co-enterochelin complex in aqueous solution.

In figure 2B we display the difference between the spectra obtained at - 190 OC subtracting that of the dead bacteria from the spectrum of the Eive bacteria.

This difference spectrum shows two important features, which stand out beyond the statistical errors : a) the presence of an absorption peak a centred at about

- 0.7 mm/s ; b ) the presence of two anti-peaks b.

The fact that the centroid of peak a coincides with the centroid of the absorption peak observed at 3 OC, and that both are observed in the live bacteria only, suggests that they are of common origin and produced by 57Co at the same special sites. The anti-peaks in figure 2B indicate that the dead bacterial sources give rise to an excess recoil-free absorption with respect to the live sources in certain regions of the velocity spectrum. Our interpretation of this is that in the transformation of /ive to dead bacteria, the 57Co at the

-4 -2 O

2

6

V E L O C I T Y

FIG. 2. - A ) Recoilless absorption spectrum in stainless steel of the 14.4 keV gamma rays emitted from sample II (dead bacteria) at - 190°C. 3) Difference between the recoilless absorption spectrum obtained with sample 1 (live bacteria) at - 190 OC and the spectrum obtained with sample II (dead

bacteria) at - 190 OC.

special sites are exchanged with sites possessing a new environment in the dead bacteria. Since the anti-peaks do not occur at the same velocities as the two main peaks in the frozen spectrum (Fig. 2 A ) , we conclude that the new environment of 57Co in the dead bacteria giving rise to the anti-peaks does not coincide with the environment of most of the 57Co.

4. Discussion and conclusions. - We first discuss the relative intensities of peaks a and b in figure 2B. Since the peak a and the anti-peaks b are very close, the true absorption spectra corresponding to these lines may appreciably overlap. This prevents an accurate comparison of intensities. However, we make compa- risons on the basis of two extreme assumptions :

(1) No overlap : intensities can be compared by measuring areas up to the base-line.

(2) Maximal overlap : i. e. peak a sits on a single large and wide anti-peak with peak and anti-peak centred at about the same velocity.

On assumption (1) the width of peak a is about 1 mmls and is 2 mm/s according to assumption (2).

According to ( 1 ) the area of peak a is about 0.6 % of

the total area under the absorption-free spectrum

observed at - 190 OC and the ratio of area of anti-

peaks b to that of a is about 4. If we now assume, as a

very rough hypothesis, that the recoil-free efficiency at

the sites corresponding to the anii-peaks is equal to

that of the majority sites at - 190 OC, we obtain from

these figures that the relative population of special sites

is about 2.4 %. A similar analysis based on assump-

tion (2) leads to a value of 5.5 %. The above values for

the fraction of special sites must be regarded as rather

crude. However, we believe Our results are consistent

C6-374 E. GIBERMAN, Y. YARIV, A. J. KALB, E. R. BAUMINGER, S. G. COHEN AND COLL.

with the assumption that the number of 57Co nuclei at the special sites is about a few per cent of the total number. Assuming a value of 4 % for the relative population of the special sites and taking the total recoil-free fraction at - 190 OC to be 0.4 (obtained by calibrating against a standard source) we can estimate the recoil-free fractions for the special sites. These are : f (3 OC) = 0.04 and f (- 190 OC) = 0.15.

What can we say about the origin of the special sites of the 57Co in the bacterial cells ? It is clear that a large part of the cell consists of aqueous media and it seems extremely unlikely that Co ions isolated or bound in small molecules in such a medium could give measurable recoil-free effects at 3 OC. There seem to us to be two possibilities open :

a) The 57Co nuclei at the special sites aie situated in the ce11 membrane and participate in the dynamic transport through the memb~ane. We find support for this possibility in noting that Our estimate of the frac- tion of special sites (a few per cent) is close to the relative volume of the membrane to that of the total ce11 in a typical E. coli bacterium. (The E. coli bacterium is a rod-like structure about 5 ^p long and 0.6 y diame- ter. The thicltness of the ce11 membrane is about 50 A.) We estimate on the present assumption that there are about 100 57Co nuclei in the membrane of a single cell.

This number seems reasonable and is in accord with an estimate of H. Rosenberg (private communication) of the number of enterochelin binding sites in the membrane of E. coli.

b) The 57Co is bound to rather large macromolecules situated in aqueous media in the cell. In this case the viscosity of the medium would have to be sufficiently large to prevent excessive diffusion broadening from smearing out completely any observable recoil-free effects. A rough estimate based on previous Mossbauer experiments on diffusion of colloidal systems in glycerol at high temperatures [7,8, (*)] suggests that a viscosity of at least 10 poise would be necessary assuming molecules of dimension - ^{50 A.} This would correspond, for example, to the viscosity of glycerol at 25 OC. We know of no quoted value for the viscosity

(*)

COHEN, S. G. and IGNER, D.

Unpublished work.

of fluid in a live bacterial cell, but we consider such a high viscosity unlikely, in view of the high water content. Smaller molecules would of course imply even higher viscosities.

We, therefore, tentatively suggest that the former hypothesis is correct and that the special 57Co sites are located in the membrane.

If we ascribe the broadening of the line at 3 OC to diffusion through the membrane, one can reach conclu- sions concerning the dynamics of the diffusion process.

Such conclusions are, however, model dependent [9].

As an example, if we assume a model of continuous diffusion, the observed broadening leads to a value of diffusion constant of about 5 x cm2/s. (The line broadening î is equal to 2 hk2 D, where D is the diKusion constant and k is the wave number of the gamma rays). On this estimate the time for Co to diffuse through the ce11 membrane would be of the order of 12/D where 1 is the membrane thickness ; this gives for E. coli a time of about 0.1 milliseconds. On an alternative jump model, the maximum jump frequency will be given by ï/2h, where ï is the diffusion broaden- ing measured in units of energy. This model yields a jump frequency of about 2 x 107 s- '.

5. Concluding remarks.

We wish to stress the preliminary nature of these conclusions which must await further work for clarification. However, it would seem that the present experiments open up a profitable field of investigation in ce11 biology which may yield important information on membranes not yet attai- nable by other methods. Unfortunately, experiments in non-frozen bacteria in the present systems are beset by severe difficulties limiting the statistical accuracy obtainable. These are principally a) the small effects, b) the fact that the recoil-free effects do not last more than a day at 3 OC, this precludes accumulation of good statistics during a long period.

Acknowledgement. - The authors wish to thank Dr. H. Rosenberg and his colleagues in the Department of Biochemistry, Institute of Advanced Studies, Australian National University, Canberra, whose help and advice made this work possible.

References

[Il MOSHKOVSKI, YU, Sh., Chemical Applications of Mossbauer Spectroscopy Chapter 10, p. 524, Edited by V. 1. Gol- danskii and R. H. Herber, Academic Press 1968.

[2] MOSHKOVSKI, et al., Biojizika 11 (1966) 317.

[31 LANKFORD, Ch. E., « Bacterial Assimilation of Iron », Critical Reviews in Microbiology 2 (1973) 273.

141 O'BRIEN, 1. G. and GIBSON, F., Biochern. Biophys. Acta 215 (1970) 393.

[5] LANGMAN, L., YOUNG, 1. G., FROST, G. E., ROSENBERG, H.

and GIBSON, F., J. Bacteriology 112 (1972) 1142.

[6] YARIV, J., KALB, A. J. and GIBERMAN, E., J. MOI. Biology 85 (1974) 183.

[7] BUNBURY D. St., ELLIOTT, T. A., HALL, H. E. and WIL- LIAMS, J. M., Phys. Lett. 6 (1963) 34.

[SI CRAIG, P. P. and SUTIN, N., Phys. Rev. Lett. 11 (1963) 460.

[9] SINGWI, K. S. and SJOLANDER, A., Phys. Rev. 120 (1960) 1093.