scintillation and autoradiographic studies on … · 63ni2+ uptake in pseudomonas tabaci 89...

J. Cell Sci. 69, 87-105 (1984) 87Printed in Great Britain © The Company of Biologists Limited 1984

SCINTILLATION AND AUTORADIOGRAPHICSTUDIES ON 63NICKEL UPTAKE IN PSEUDOMONAS

TABACI

R. H. AL-RABAEE AND D. C. SIGEE*Cytology Unit, Departments of Botany and Zoology, University of Manchester,Manchester, U.K.

SUMMARY

Scintillation studies on the uptake of 63Ni2+ by Pseudomonas tabaci demonstrate an incorporationof approximately 2-5 nmol per 10'° bacterial cells, in medium containing 12nmol (8-3 /iCi) per ml.Over 80% of the incorporated Ni2+ is lost from the cells during washing, fixation and dehydrationwith ethanol. The remaining insoluble (bound) 63Ni2+ has the highest level in cells fixed in aceticacid/ethanol (0-4 nmol/1010 cells), with smaller amounts in paraformaldehyde- and glutaraldehyde-fixed cells. The radioactive level in aldehyde-fixed cells represents a total Ni2+ uptake of aboutlCTl8g or 104 atoms per cell.

Light- and electron-microscope autoradiography corroborated the scintillation studies in demon-strating a higher retention of label by cells fixed in acetic acid/ethanol, possibly reflecting a higherretention of medium M, proteins with this type of fixation. High-resolution electron-microscopeautoradiography involving gold latensification with physical development demonstrated a clearlocalization of silver grains to the central nucleoid region (seen most clearly over the discretenucleoid of aldehyde-fixed cells) and within this to the chromatin (seen most clearly over thecondensed chromatin of acetic acid/ethanol-fixed cells). It is suggested that the incorporated63Ni2+ labels mainly central, genetically inactive DNA, while peripheral, actively transcribing DNAhas little associated radioactivity. The pattern of cation association seen in this bacterium shows anumber of close similarities to the situation seen in dinoflagellate cells.

INTRODUCTION

The uptake of nickel from the environment, and its biological effects on wholeorganisms, tissues and cells, has increasingly occupied the attentions of researchworkers in recent years (Sunderman, 1978; Brown & Sunderman, 1980; Hutchinson,1981). Parallel to this development has been the need for improved methods of nickeldetection in biological and environmental samples (Dulka & Risby, 1976; Sunder-man, 1980a,b; Jaworski, 1974), with particular emphasis being given to the deter-mination of nickel levels in liquids — including water samples, body fluids, tissue andcell homogenates and solutions. This chemical analytical approach gives an overallpicture of nickel levels, but provides little information about the location of nickelwithin tissues and within individual cells. Various techniques are available, however,at the levels of the light and electron microscopes for intracellular nickel quantitation,including X-ray microanalysis (Kearns & Sigee, 1980; Sigee & Kearns, 1980) and63Ni2+ autoradiography (Sigee, 1982).

• Requests for reprints should be sent to this author.

88 R. H. Al-Rabaee and D. C. Sigee

The purpose of the work reported here was to investigate the uptake of 63Ni2+ intobacterial cells cultured in the laboratory, with particular reference to the localizationof incorporated label at the level of the electron microscope. The use of 63Ni2+

(Kasprzak & Sunderman, 1979) provides a highly sensitive measure for monitoringrelatively low levels of Ni2+ uptake, for which both liquid scintillation counting andautoradiography can be used. The use of these techniques, on cells processed in avariety of ways, gives information on the presence and location of insoluble nickel inbacterial cells. The results obtained are of general interest in relation to the metabolicrole and toxicology of Ni2+ in bacteria. More particularly, they give direct informationon the association of Ni ions with bacterial chromatin, and in this respect haverelevance to previous studies carried out on dinoflagellate (eucaryotic) cells (Sigee,1983a,6).

MATERIALS AND METHODS

Culture of bacteriaCultures of Pseudomonas tabaci, originally obtained from Professor R. N. Goodman, University

of Missouri, were grown in nutrient broth in a shaker at 23 °C to log phase. A sample (1 ml) ofbacterial culture was added to 99 ml of nutrient broth (Oxoid), and the bacterial population wasdetermined every 4h by making direct counts using a haemocytometer. Atomic absorptionspectrophotometric studies on the nutrient broth showed that the level of Ni2+ in normal growthmedium was below the limits of detection (1 part per million) of the technique.

Labelling procedureRadioactive label was added to the bacterial culture 5 h after inoculation, at the beginning of the

logarithmic phase of growth (Fig. 1); 10ml of "nickel chloride (Amersham International Ltd) in0-1 M-HC1 was adjusted to pH 7-2 (the pH of the bacterial culture) by addition of 0-5 M-Tris buffer,then added to 50 ml of cell culture to give an overall concentration of 8 3 fiCi/ml (sp. act. of addedNi2+, l l-6mCi/mg). Samples were taken after labelling periods of 1 —16h (Fig. 1), washed insodium cacodylate buffer (01 M, pH 7-2), and used for scintillation counting and autoradiography.

Cell processingThe level of incorporated Ni2"1" was determined either in fresh, unfixed cells (soluble and insoluble

cations: scintillation counting) or in fixed, chemically dehydrated cells (insoluble cations: scintilla-tion counting and autoradiography). Fixation was carried out at 20CC using the following fixatives,(a) Glutaraldehyde, 2-5 % in 0-1 M-sodium cacodylate buffer (pH 7-2), for 2-3 h. Some cells werealso postfixed (2h) in 2% buffered osmium tetroxide for autoradiography. (b) Paraformaldehyde,4 % freshly-prepared solution in 0-1 M-sodium cacodylate buffer, for 2-3 h. (c) Acetic acid/ethanol(1:3, v/v), for 2-3 h. Cells fixed in acetic acid/ethanol were transferred directly to 100% ethanol(Fig. 2), while aldehyde-fixed cells were dehydrated in an ethanol series (Fig. 3).

Scintillation countingScintillation studies were carried out both on fluids used to process the cells (supernatants) and

the final cell suspension. For each washing, fixation or dehydration stage 10 ml of fluid was addedto the cell pellet, and 1 ml of liquid taken from the resulting supernatant and used for scintillationcounting. This was added to 10 ml of Packard scintillation fluid (type 299 TM) and allowed to standin the dark for 48 h to eliminate errors due to chemiluminescence (Kasprzak & Sunderman, 1979).Counts were made in a Packard liquid scintillation spectrometer, model 300CD, over an interval oflOmin, and were converted to radioactive concentrations (/iCi/ml) by reference to a standardcalibration curve. The overall counting efficiency (c.p.m.X 100/disints per min) was 47%. Bac-terial population counts (cells/ml) were made from the final samples.

63Ni2+ uptake in Pseudomonas tabaci 89

AutoradiographyLight- and electron-microscope autoradiography was carried out on separate samples from those

used for scintillation counting. Fixed, dehydrated cells were embedded in Spurr resin and sectionedat a thickness of Zfim (light microscopy) or 60nm (electron microscopy). Light-microscopepreparations were coated with a layer of Ilford G-5 emulsion, using a dipping technique. For theelectron microscope, ultrathin sections were coated with a monolayer of Ilford L4 emulsion usinga loop method (Williams, 1977). Preparations were incubated in a light-tight box for periods of 7days (light microscopy) and up to 6 months (electron microscopy), and were then processed usingD19 developer and 10% Hypam fixative. Some electron-microscope preparations were alsoprocessed by fine-grain physical developer (paraphenylenediamine) preceded by gold latensifica-tion, according to the method of Salpeter & Bachmann (1964).

RESULTS

Scintillation counts

In separate experiments, scintillation studies were carried out to determine theeffect of varying: (a) the type of cell processing (fixation experiment), and (b) thelabelling period (time-course experiment).

Fixation experiment. Cells labelled for 2h (Fig. 1) were washed three times inbuffer and then either sampled directly or fixed and dehydrated. For each processingschedule, scintillation counts were converted to ^Ci, and expressed as radioactivityper 1010 bacterial cells (Figs 2, 3). These data provide a direct comparison betweendifferent treatments in the form of a 'balance sheet', from the second wash to the finalcell suspension. The cumulative totals for each schedule were constant, at about2-1 jUCi/1010 cells, indicating that overall comparison between treatments was valid.Supernatant from the first wash was not included since this would contain a highproportion of original radioactive medium.

With each treatment, approximately half the 63Ni2+ contained in the cells after thefirst wash was lost in the second and third washes. This represents a pool of water-soluble label that is readily lost from the cells by simple diffusion. The remaininglabel, totalling about O-9^Ci/lO10 cells in the final suspension (Fig. 2A), representsbound and soluble 63Ni2+ retained during the washing. Fixation in acetic acid/ethanoland subsequent dehydration in 100 % ethanol (Fig. 2B) removes much of the soluble63Ni2+ by direct extraction, leaving a largely insoluble component at about 0-3 Ci/1010 cells in the final suspension. Fixation in paraformaldehyde (Fig. 3A) and glutaral-dehyde (Fig. 3B), followed by dehydration, results in a more complete extraction ofsoluble 63Ni2+, leaving about 0-2^Ci/1010 cells in the final suspension. Theprogressive drop in radioactive content of the processing liquids during thesedehydration series (Fig. 3A,B), with very low levels in the 90% and 100% ethanol,suggests that very little, if any, of the extracted 63Ni2+ is lipid-associated.

Analysis of the final bacterial suspensions is presented in Table 1, for unfixed cellsin buffer and for fixed cells in 100 % ethanol. On the basis that all the label is containedin the suspended bacterial cells, values for the mass of incorporated Ni2+ vary from3-0x10"" to 12-3xlO~unmol/cell. These are equivalent to a retention of 1-8X104

to 7-4X104 Ni2+ atoms/cell. The level of nickel retained by cells fixed in acetic

CEL69


Time (h)

Fig. 1. Growth curve of P. tabaci and associated radiolabelling periods. Mean values ofpopulation count (each derived from three separate readings made using a haemocytometer)are given at various times after inoculation of high nutrient broth with dense culture.

acid/ethanol amounts to about 32% of that in unfixed cells, and in the case of thealdehyde fixations the level falls to 24%.

Time-course experiment. Cells were labelled for periods of 1, 2, 8 and 16 h, com-mencing during the early log phase of bacterial growth (Fig. 1). Although the radio-active content of the final suspension shows a considerable increase (from 0-4x 10~3 to12-6xlO~3;uCi/ml), the level of incorporated 63Ni2+ per bacterium (Table 2) remainsfairly constant at about 1-4X 10" " nmol/cellfrom 1-8 h, dropping to 0-8 XlO~" nmol/

aNi2+ uptake in Pseudomonas tabaci 91

1 0 -

0-8

0-6

0-2 -

1j? 1 0

I °'8toa:

0-4

0-2

Wash Cells

Wash Wash AA fix Ethanol Ethanol2 3 (100%) (100%)

Sample

Cells

Fig. 2. Extraction of 63Ni2+ from P. tabaci during cell processing in unfixed (A) and aceticacid/ethanol-fixed (AA fix) preparations (B). Radioactive levels (derived from scintillationcounts of 1 ml samples) are expressed in relation to 1010 processed cells, and are given forwashing solutions (cross-hatched columns), fixation/dehydration liquids (open columns)and cell suspensions (solid columns). For each schedule, the cumulative totals (2-11 ^Ci /10'° bacteria in A; 2-32/iCi/lO10 bacteria in B) represent the overall levels of radioactivitypresent in the samples after the first wash, and are derived by addition of all the separatesupernatant levels plus the cell suspension.

Table 1. Retention of63Ni2+ by cells of P. tabaci after different fixation procedures

Mass of Ni/ No. of Ni atoms/Treatment ^iCi/ml* Total bacteria/ml bacterium (nmol)f bacterium};

UntreatedAcetic acid/ethanolParaformaldehydeGlutaraldehyde

3-9XKT3

1-2X10"3

0-9XKT3

0-9XKT3

4-7X107

4-6X107

4-3X107

4-5X107

12-3X10-"4-OXlfr"3-0XKT"3-Oxlfr"

7-4X104

2-4X104

1-8X104

1-8X104

* Values (derived from scintillation counts of 1 ml samples) are shown for suspensions of cells inbuffer (unfixed) and 100% ethanol (fixed).

f The mass of Niz+ per bacterium is calculated from the known specific activity of the radioisotope.JThe number of atoms is derived using Avogadro's constant.

cell at 16 h. The lower value in the 16 h sample may result from a reduced availabilityof 63Ni2+ in the incubation medium, or from a reduction in cell uptake as the culturepasses from log to stationary phase.

Eth

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. 3.

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ract

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Table 2. Retention of63Ni2+ by cells of P. tabaci after different labelling periods

Treatment Mass of Ni/ No. of Ni atoms/time (h) /iCi/ml* Total bacteria/ml bacterium (nmol) bacterium

128

16

0-4X10"3

1-3X10"3

8-9X10"3

12-6X10"3

S-3X107

12-0X107

86-0X107

240-OX107

1-lxlO"11

1-6X10"11

l-SxlO""0-8X10"11

6-7X103

9-7X103

91X103

4-8X103

• Values for /id/ml and derived parameters are calculated as for Table 1, for cells labelled over1-16 h periods. All samples were fixed for 2h in glutaraldehyde, and scintillation counting wascarried out on cell suspensions in 100% ethanol.

Autoradiography

Light- and electron-microscope autoradiography was carried out on 2-h labelledbacteria fixed separately in acetic acid/ethanol, paraformaldehyde, glutaraldehydeand glutaraldehyde with osmium tetroxide postfixation.

Light microscopy. Phase-contrast microscopy of 2/im thick sections revealed bac-terial aggregations of varying size and packing (Fig. 4A) with each fixation. Theautoradiographic preparations invariably had numerous silver grains over the separatemasses of bacteria, with an appreciable scatter from these aggregates over thesurrounding background (Fig. 4B). Although problems in making reliable counts ofbacteria per unit area made autoradiographic quantitation impossible with thesepreparations, simple visual inspection indicated a considerable higher frequency ofsilver grains over acetic acid/ethanol preparations (Fig. 4B) compared with thosefixed in either paraformaldehyde (Fig. 5) or glutaraldehyde.

Electron microscopy. Low-power views of bacterial masses in ultrathin section(Figs 6, 7) confirmed the irregular close-packing seen under the light microscope. Atthis magnification, cells fixed in acetic acid/ethanol appeared to have rather feature-less, diffuse contents (Fig. 6), while aldehyde-fixed cells (Fig. 7) had a clear centralnucleoid area containing coarse strands and aggregations of electron-dense chromatin.

Autoradiographs processed with D19 developer showed a clear labellingthroughout the bacterial populations, with silver grains (coiled filaments) localized to

Fig. 4. Light-microscope preparations, acetic acid/ethanol-fixed cells, A. Non-autoradiograph. Phase-contrast view of bacterial group, B. Autoradiograph; adjacent sec-tion to A. Bright-field view showing dense accumulation of silver grains over bacterialgroup, and scatter over adjacent resin area. Bar, 10/im.

Fig. 5. Light microscope autoradiograph (phase-contrast) of paraformaldehyde-fixedbacteria. The bacterial group is well-labelled, but less heavily than 4B. Bar, 10 (im.

Figs 6—7. Electron-microscope autoradiographs. D19 development. Silver grains arepresent as coiled filaments. Both preparations are stained with lead citrate.

Fig. 6. Acetic acid/ethanol preparation. Bar, 0-5/im.Fig. 7. Glutaraldehyde (3 h) fixation. Strands and accumulations of condensed DNA

are prominent within the central nucleoid (n). Bar, 0-5

R. H. Al-Rabaee and D. C. Sigee

Figs 4-5. For legend see p. 93.

63\r;2 +Ni uptake in Pseudomonas tabaci 95

\

I-

s

*>if¥fj i*"

s--• Kf-

>i

n%

•JM*

mFigs 6-7. For legend see p. 93.


Table 3. Mean grain counts over electron microscope autoradiographs

Incubation period Acetic acid/ Glutaraldehyde/(months) ethanol Paraformaldehyde Glutaraldehyde osmium tetroxide

26

7-22-

6±2-6±6-

59

2-2 ±0-710-2 ±2-8 3

0-Z ± 1-1

01-7 ±0-5

Mean grain counts (per 10 bacterial profiles) are given for D19-developed autoradiographs,processed in two batches at 2 and 6 months after coating with emulsion. Each mean count is derivedfrom a sample of at least 200 profiles. The grain counts over glutaraldehyde and glutaraldehyde/osmium tetroxide-fixed cells after 2 months incubation were so low that they did not exceed back-ground. Confidence limits are at 95% level.

individual bacterial profiles (Figs 6, 7). Background labelling (over pure resin) wasnormally low, amounting to no more than 1-2 grains per SOOjUm2. Analysis ofautoradiographs processed after exposure periods ranging from 2-6 months showeda consistent relationship between extent of labelling and type of fixation. Table 3shows the mean grain count per 10 bacteria in autoradiographs processed at differentperiods. Preparations developed after a short (2-month) interval typically had a highgrain frequency over cells fixed in acetic acid/ethanol, fewer grains overparaformaldehyde-fixed preparations and no appreciable label over glutaraldehyde-fixed cells. Significant levels of silver grains over the latter were seen only after long(6-month) periods, when they were considerably less than in the paraformaldehydeand acetic acid/ethanol preparations. Postfixation in osmium tetroxide (Table 3)appears to reduce the level of retained 63Ni2+ even further. The level of labelling canalso be considered in terms of the percentages of bacteria with 0, 1, 2 etc. associatedsilver grains. The histograms shown in Fig. 8 for a 6-month batch of grids show cleardifferences between methods of fixation, with 80% labelled profiles in the case ofacetic acid/ethanol fixation, compared to 56% for paraformaldehyde, 32% forglutaraldehyde and 11 % for glutaraldehyde/osmium tetroxide.

Electron-microscope autoradiographs processed by gold latensification and physi-cal developer (which gives increased emulsion sensitivity; Langford, 1974; Kopriwa,1975) had more silver grains per bacterial cell with smaller, often clustered silvergrains (Figs 9—11). The silver grains in these preparations were identified as such bytheir regular shape and complete electron opacity. They were observed in both stained(Figs 9, 11) and unstained (Fig. 10) preparations, and some of the larger grains gavea clear characteristic silver peak when checked by X-ray microanalysis. Although theoccurrence of silver grains outside bacteria was very infrequent (low scatter andbackground), non-localized grains over pure resin were occasionally observed. Aswith the D19 preparations, highest grain frequencies occurred over the aceticacid/ethanol preparations (Fig. 9). The uptake of 63Ni2+ did not vary with thedivision state of the bacteria, since heavy labelling occurred with both dividing andnon-dividing cells (Fig. 9). In all of the preparations observed, the silver grainsappeared to be largely restricted to the centre of the cells. This was tested statistically

30-

20-

10-

63Ni2+ uptake in Pseudomonas tabaci

Acetic acid/ethanol

97

gIS3a.oa.

cCD3CTCD

roCO

40-

30-

20-

10-

0 1 2 3 4

— ^

5 6 7 8 9 10

Paraformaldehyde

1

70-

60-

50-

40-

30-

20-

10-

80-

70-

60-

50-

40-

30-

20-

10-

0

1

1

1

2 3 4 5 6 7 8 9 10

Glutaraldehyde

i i i i i t i i (

2 3 4 5 6 7 8 9 10

Glutaraldehyde/osmium tetroxide

1 2 3 4 5 6 7 8 9Number of silver grains per bacterium

10

Fig. 8. Bacterial labelling (EM autoradiographs). The frequency of bacterial profiles with0, 1, 2 or more silver grains are shown for D19-processed autoradiographs of cells fixed infour ways. All the preparations were processed at the same time, so had identical con-ditions of development and photographic fixation. Each histogram is derived from asample of at least 200 profiles.

R. H. Al-Rabaee and D. C. Sigee

%h.—t

. • - l <

* «*

Figs 9-10


in the acetic acid/ethanol-fixed preparations by comparing the distances betweenindividual silver grains and the nearest point on the bacterial boundary withequivalent measurements in an equal sample of random points (Fig. 12). The twohistograms seen in Fig. 12 appear to be quite distinct, and comparison of the meanson a null hypothesis confirms that the distributions of silver grains and random pointsare significantly different.

High-power examination of acetic acid/ethanol-preserved cells reveals a surprisingamount of detail for a fixative that is generally regarded as unsuitable for electronmicroscopy. The central nucleoid appears as a system of interconnecting spacesramifying throughout the ribosomal groundplasm, and contains diffuse chromatin,which is condensed at various sites in the bacterium as electron-dense patches (Fig.11). A high proportion of the silver grains shows clear localization to these regions ofcondensed chromatin (Fig. 11). Silver grains were also observed over apparently clearareas of nucleoid and over ribosomal groundplasm, but were hardly ever seen over thebacterial cell wall.

In aldehyde-fixed cells, with a more clearly defined central nucleoid space, the finesilver grains showed an even clearer localization to the chromatin-containing regionof the cell (Fig. 10). Very few grains occurred over surrounding ribosomal ground-plasm or cell wall.

DISCUSSION

Addition of 63Ni2+ to the bacterial cultures used in these experiments resulted inan overall nickel concentration of 1 -2x 10~5 M (no detectable Ni in the original growthmedium). This level of Ni had no limiting effect on the multiplication of bacteria(population increase similar to control without nickel), so that the uptake of 63Ni2+

in this investigation was occurring in non-toxic conditions.The scintillation counts of the processing solutions indicate that most of the incor-

porated 63Ni2+ is water-soluble. This soluble Ni is present partly as a readily diffusibleform (with approximately 50 % being lost during washing) and partly as an extract-able form (with a further 30% being lost during fixation and dehydration). These

Figs 9—11. Electron-microscope autoradiographs. Gold latensification and physicaldevelopment (1-2min). Silver grains (Ag) are present as small electron-dense granules.

Fig. 9. Acetic acid/ethanol preparation. Stained with alkaline lead citrate afterphotographic processing. The mean grain size in this preparation is 0-04 /^m. Silver grainsare present over both dividing (d) and non-dividing bacteria, and over all angles of section.t, transverse section; /, longitudinal section. Bar, 0-5 fan.

Fig. 10. Glutaraldehyde preparation. Unstained. These cells were labelled with MNifor 8h, and fixed for 2h. n, central electron transparent nucleoid containing chromatinreticulum; r, peripheral ribosomal groundplasm. The silver grains (Ag) have a meandiameter of 0-03 fun. Bar, 0-2 fim.

Fig. 11. Acetic acid/ethanol preparation. Stained. High-power view showing bacteriawith regions of condensed chromatin (arrows), surrounded by electron-transparentnucleoid spaces (s) and ribosomal groundplasm (r). Silver grains (Ag) show a clear localiza-tion to the condensed chromatin. Bar, 0-2 fim.

m R. H. Al-Rabaee and D. C. Sigee

Fig. 11. For legend see p. 99.

aNi2+ uptake in Pseudomonas tabaci 101

2 0 -

CO

§ 15

a

|

Z

5 -

_D •_005 0-1 0-15

i0-2

_n0-25 0-3 0-35

15 -

3

Io 10-Ca

005i

0-1 0-15 0-2 0-25 0-3 0-35

Distance to edge of bacterium

Fig. 12. Distribution of silver grains and random points over bacteria, A. Autoradiograph(EM). Acetic acid/ethanol-fixation. Physical developer. The position of 221 silvergrains over 40 bacteria was measured individually as the distance to the nearest point onthe cell boundary, and expressed cumulatively as a histogram. x = 0-213; i = 0-05;n = 221;

= 1 6 - 3z =

B. Random point distribution; 221 random points were plotted over tracings of the bac-terial profiles used in A, and a corresponding histogram was plotted. /io = 0'132;s' = 0-074; n' = 221.

The two histograms appear quite distinct. Comparison of the means (x,fio) on a nullhypothesis confirms this. With standard deviations (s, s') and sample size (n) as shown,the resulting value forz of 16-3 exceeds the theoretical value of zoo\ = 2-33.


experiments do not give any indication of the location of this soluble nickel in the cell,though adsorption of cations at the cell'surface (Corpe, 1975) or cell wall (Beveridge& Murray, 1976; Haavik, 1976), as well as uptake into the protoplast cytosol, arepossibilities. The rapid and continued labelling of bacterial cells during the log growthphase is consistent both with a continuous adsorption onto newly formed cell-wallmaterial, and with an active cation-transport system into the bacterial protoplast,similar to that observed in Bacillus megaterium (Schneider, 1977) and Escherichiacolt (Jasper & Silver, 1977).

The Ni2+ that remains in fixed, dehydrated cells represents the bound, insolublenickel, and amounts to roughly 10—20% of the total nickel initially present. The useof coagulative (acetic acid/ethanol) and additive (aldehyde) fixatives in determininginsoluble Ni content was important in terms of both possible fixation artefacts and theexact level retained. The possibility of artifactual binding of radioactive molecules hasbeen shown, for example, by Peters & Ashley (1967), where radioactive amino acidswere bound to cell proteins by glutaraldehyde but not paraformaldehyde fixation. Theretention of 63Ni2+ in bacteria fixed by all three types of fixative suggests that thebound (insoluble) nickel is not simply present as a fixation artefact.

Both the scintillation and autoradiographic studies show that higher levels of63Ni2+ were retained with acetic acid/ethanol fixation than with aldehyde fixation.Previous work with other cells (Sigee & Kearns, 1982) has suggested that coagulativefixation preserves more medium MT proteins than does aldehyde fixation, and theadditional "Ni2"1" seen in bacterial cells after acetic acid/ethanol treatment may reflectits association with these proteins.

The comparison between glutaraldehyde and paraformaldehyde fixationsrepresents a major difference between the scintillation and autoradiographic results.In the scintillation counts, these two fixatives led to equal retention of the 63Ni2+,while in the autoradiographs the level of label in the paraformaldehyde preparationswas considerably greater than with glutaraldehyde. There is no immediate explana-tion for these results, though the difference may be due to differences in fixation time,which was 2h in the scintillation studies and 3 h in the autoradiography. The extrafixation time does lead to a greater condensation of the bacterial chromatin (compareFigs 7 and 10), and there is a possible extra, and differential, loss of 63Ni2+ with thetwo fixations during the extra hour.

Both scintillation counting and autoradiography are highly sensitive methods forthe detection of 63Ni2+. It is interesting to note in this context that X-ray microanalysiswas not sufficiently sensitive to detect incorporated Ni in any of the sections. Scintilla-tion methods have been used to determine the uptake of Ni in a variety of organisms,with a substantial amount of work being carried out on bacteria (Tabillion & Kalt-wasser, 1977; Diekert, Weber & Thauer, 1980; Friedrich, Schneider & Friedrich,1982; Jarrell & Sprott, 1982). Autoradiography, on the other hand, has been muchless frequently used. Previous studies include light-microscope work by Oskarsson &Tjalve (1979, 1980) on ^NiCh uptake in mice, and light- and electron-microscopestudies by Sigee (1982) on 63Ni2+ uptake in dinoflagellates, but there appears to be nopreviously published work involving bacteria. This is surprising in view of the


apparent suitability of this isotope for autoradiography, since 63Ni2+ has a long half-life (92 years), the radiodecay results entirely in soft beta emission (Kirby, 1961),and the label can be obtained at high specific activity. The higher mean energy ofemission compared to tritium (67keV compared to 18keV) implies a lowerresolution than normally obtained with this technique, though this was only appar-ent at the level of the light microscope, where a wide scatter of silver grains occurredaround heavily labelled bacterial groups. At the level of the electron microscope,the use of gold latensification followed by physical development (Salpeter & Bach-mann, 1964; Kopriwa, 1975) provides a very powerful technique for determininglocalization of label within intact cells, resulting in high emulsion sensitivity, smallsilver grains (high resolution) and low background. The multiple silver grainsobtained with this technique probably arise by partial development of several latentimages within a single silver halide crystal (Mees, 1954; Langford, 1974), arisingfrom a single beta particle sensitization. This is in contrast to chemical (D19)development, where normally not more than one silver grain arises from a singlesensitization. For this reason, grain counts were made from the D19-processedautoradiographs, while studies on localization were made in the physically developedpreparations.

In both the acetic acid/ethanol- and aldehyde-fixed cells, there was a high degreeof localization of bound 63Ni2+ to the central nucleoid region of the cell. Silver grainswere conspicuously absent from the cell surface, and the cell wall, and were in-frequently seen over peripheral ribosomal groundplasm (aldehyde preparations). Inthe centre of the cell the extent of the nucleoid space and the appearance of thechromatin varied considerably with fixation. This variation proved quite useful, sincethe discrete central nucleoid in aldehyde-fixed cells permitted a defined localizationof 63Ni2+ uptake to this part of the cell, while the precipitated masses of chromatin inthe acetic acid/ethanol preparations allowed clear localization of label to this regionwithin the nucleoid.

It seems likely (Haggis & Bond, 1981) that the nucleoid regions seen in ultrathinsections of fixed cells (Ryter & Kellenberger, 1958) and in whole living cells (Bin-nerts, Woldringh & Brakenhoff, 1982) contain only part of the bacterial DNA. Ryter& Chang (1975) have suggested from autoradiographic studies that the nucleoid D NAcontains only inactive genes, while active genes involved in transcription extend intothe ribosomal groundplasm. Studies of isolatedE. coli nucleoids (Kavenoff & Bowen,1976; Pettijohn, 1976) confirm the presence of a core region with lateral loops spread-ing out, possibly corresponding to central and peripheral (ribosomal) regions in theintact cell. In view of these observations, the studies on63Ni2+ uptake presented heresuggest that incorporation of label occurs mainly into the core (genetically inactive)DNA, and that the genetically active DNA fibrils in the peripheral part of the cell havelittle associated radioactivity. The association of divalent cations with nucleic acidshas been widely documented (for discussion, see Kearns & Sigee, 1980; Sigee,19836). It is proposed that, among other functions, these cations are important instabilizing the in vivo structure of the DNA, particularly in the absence of associatedhistones (cationic non-histone stabilization; Sigee, 1983a,6).


The distinction between genetically active and inactive DNA seen in bacteria showsa remarkable resemblance to the situation in dinoflagellates, the only eucaryotic groupwith a complete lack of chromatin histones (Rizzo & Nooden, 1974). Indinoflagellates, as in the bacteria studied here, divalent cations are associated par-ticularly with the genetically inactive DNA (in permanently condensedchromosomes) (Kearns & Sigee, 1979, 1980; Sigee & Kearns, 1980, 1981), while thegenetically active extrachromosomal filaments appear to have much lower levels(Sigee, 1984). The results obtained in this investigation thus lend some support to thesuggestion (Sigee, 1983a,fc) that cationic non-histone stabilization of dinoflagellateDNA resembles the situation in bacteria, and might therefore be regarded asprimitive.

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(Received 19 October 1983-Accepted 10 February 1984)

scintillation and autoradiographic studies on … · 63ni2+ uptake in pseudomonas tabaci 89...

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