Journal of General Microbiology (1982), 128, 1849-1862.
Printed in Great Britain
1849
Magnesium-limited Growth of the C yanobacterium Anacystis nidulans
By H A N S C H R I S T I A N U T K I L E N
Botanical Laboratory, University of Oslo, PO Box 1045, Blindern, Oslo 3, Norway
(Received 15 July 1981; revised 22 September 1981)
Mg2+-limitedgrowth of the cyanobacterium Anacystis nidulans was investigated in batch and
chemostat cultures. In batch cultures the growth rate of the organism depended on the Mg2+
concentration up to 5 p ~ Although
.
the maximum growth rate was achieved at this
concentration, the organism formed aseptate filaments of three to four times the ‘normal’ cell
length. About 90 min after increasing the Mg2+concentration from 5 p~ to 1 m M the cell size
decreased, followed by an increase in the division rate, which lasted for about 60 min and
resulted in a 6 6 % increase in cell number. The rates of DNA, R N A and protein synthesis
were not altered during these Mg2+shift-up experiments, showing that the control by Mg2+of
growth had been separated from its control of cell division, In Mg2+-limited chemostat
cultures, the mean cell volume decreased from about 2.0 to 0.6 pm3 when the Mg2+
concentration was increased from 2.5 to 10 PM. This increase in Mg2+ also resulted in an
increase in the calculated intracellular Mg2+concentration from 27 to 78 m M , and the amount
of cellular Mg2+bound in chlorophyll increased from 17 to 22%. A comparison of Mg2+-and
SOi--limited chemostat cultures showed that the mean cell volume decreased with increasing
dilution rate when Mg2+ was the limiting factor, whereas it increased with dilution rate when
SO;- was limiting. Only small differences in the rates of R N A and protein synthesis were
found in the two cultures, although the synthesis of R N A was Mg2+-dependent.The ratio of
total R N A to protein, which gives the amount of R N A necessary to synthesize one protein
unit (RNA efficiency), was independent of the growth rate in both SO:-- and Mg2+limited chemostat cultures showing that the efficiency of culture R N A was variable in both
cases. The efficiency was higher under SO:-- than Mg2+-limitedconditions.
INTRODUCTION
The importance of Mg2+ for normal growth and cell division of bacteria was demonstrated
by Webb (1949), who showed that a deficiency or excess of Mg2+inhibited growth and cell
division, causing the formation of filaments. Tempest et al. (1965) compared the cell size of
Klebsiella aerogenes (Aerobacter aerogenes) in Mg2+-and C-limited chemostat cultures and
found that at corresponding growth rates, the Mg2+-limited bacteria were the larger.
Schizosaccharomyces pombe (Ahluwalia et al., 1978) and Chlorella (Finkel & Appleman,
1953; Retovsky & Klasterska, 1961) also became enlarged when deprived of Mg2+.
Enlargement of single-celled organisms therefore seems to be a general feature of
Mg2+-limitation.
Several other processes and structures are Mg’+-dependent: MgzS is an integral part of
ribosome structure (Tissieres & Watson, 1958) and affects R N A synthesis in Klebsiella
aerogenes (Tempest et al., 1965). It is a cofactor for many enzyme systems (Dixon & Webb,
1958; Garrett, 1969) and influences bacterial permeability (Brock, 1962), presumably
because it maintains structural and functional integrity of bacterial membranes (Weibull,
;
0022-1287/82/0001-0062 $02.00 @ 1982 SGM
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1850
H . C. UTKILEN
1956; Rogers et al., 1967). In photosynthetic organisms, Mg2+ is a critical component of
chlorophyll.
The effect of Mg2+-limitation has been thoroughly studied in the heterotrophic bacterium
Klebsiella aerogenes (Tempest et al., 1965; Tempest & Hunter, 1965; Tempest & Strange,
1966; Kennell, 1967; Strange & Hunter, 1967; Sykes, 1967; Tempest & Dicks, 1967). The
aim of the present work was to compare these results with those obtained for a photosynthetic
prokaryote: the non-filamentous cyanobacterium Anacystis nidulans was chosen for this
purpose since it has simple nutritional requirements, is robust and has been studied by others
(see Carr & Whitton, 1973), who have provided much of the background knowledge of the
physiology of this organism.
METHODS
Organism. Anacystis nidulans (strain no. UTEX 625 of the Culture Collection of Algae, Department of Botany,
University of Texas) was used. The organism was maintained in pure culture on agar slopes (Allen, 1968).
Growth conditions. Batch cultures were grown on medium C (Kratz & Myers, 1955) modified by adding
NaHCO, (1 g 1-I), replacing MgSO, by Na,SO,, reducing the FeSO, concentration to 2 mg 1-’ and adding MgCl,
to the desired concentration. Cultures in 1 1 Roux flasks containing about 800 ml medium were incubated in a
waterbath at 40 “C. Continuous illumination (106 pE m-’ s-l, 10 klx) was provided by a combination of three
Warm de Luxe T L 40 W/32 and two Daylight T L 40 WI/55 fluorescent tubes, and the cultures were aerated with
sterile air/CO, (95 :5, v/v; 0.3 1 min-’), which also provided mixing.
Inocula for the growth experiments were prepared by transferring organisms from agar slopes to small conical
flasks containing medium (20 ml) with 5 pM-Mg2+.These were incubated for about 24 h at 40 *C and aerated with
the air/CO, mixture. The flask contents were then transferred to the Roux flasks containing medium with the
desired MgZf concentration. Growth rate constants were calculated from the exponential part of the growth curves
obtained. There was no significant difference in growth rates whether or not the cells from the inocula were
washed with ‘MgZt-free’ medium before transfer. The Mg’+ shift-up experiments were carried out by adding MgCI,
to give a final concentration of 1 mM.
The chemostats consisted of a I 1 Quickfit culture vessel FV lL, with multisocket/flat flange lid M A F 2/2. The
culture vessel was placed in a waterbath at 40 OC. Medium and air/CO, (as described above) were supplied
through a glass tube. The aeration (0.6 1 min-’) together with magnetic stirring of the culture ensured a rapid
mixing of the feed medium with the culture. The pH of the autoclaved medium was 8-8, but the CO, in the gas
phase caused this to drop to 8-0& 0.1, which was the pH used in all the experiments. Continuous illumination was
provided by eight Philips TL AK 40/W33 fluorescent tubes, four at each side, at a distance of 3.5 cm. This gave a
light intensity of 365 pE mP2 sP1 (26 klx) from each side, measured inside a water-filled culture vessel with a LI
1854 Quantum/Radiometer/Photometer(Lambda Instruments Corporation, U.S.A.). The medium flow was regulated by a Varioperpex 12000 peristaltic pump (LKB). The culture volume was maintained at 1 1 by an internally
placed overflow tube. Samples were taken by closing the overflow, thereby forcing culture to flow through a submerged tube, connected to a 100 ml glass cylinder, from which the sample was drained. Samples of 100 ml were
drained (morning and evening) from the chemostat without significant influence on the steady state. Larger samples
(600-800 ml) were also drained directly from the chemostat and a new steady state was established after about
5 d.
The medium was as described above for batch cultures when Mg2+ was the limiting factor. When SO:- was
limiting, the Mg2+ concentration was increased to 1 mM by adding MgCl,, and FeSO,, ZnSO, and CuSO, were
replaced by FeCl,, ZnC1, and Cu(NO,),. The SO:- concentration in the SO:--limited chemostat was 5 p~ (added
as Na,SO,), which was chosen after growth experiments at different concentrations in batch cultures. The media
were prepared with distilled water in 20 1 bottles and autoclaved; K,HPO, was autoclaved separately to avoid
precipitation.
Mg2+-deficientbatch cultures. For Mg2+-deficient growth of Mg2+- and SOP-limited cells, the procedure of
Tempest et al. (1965) was used. The modified medium C (Kratz & Myers, 1955) as described above, but without
added Mg’+ (‘Mg2+-free’ medium) was used. Cells were harvested by centrifugation (5000 g, 10 min) from
Mg2+-limited ( 5 pM-Mg2+,D = 0.1 17 hk’) and SO:--limited ( 5 p~-S0:-, D = 0.120 h-l) chemostats, washed
once with sterile ‘Mg2+-free’medium, resuspended in 10 ml medium, transferred to sterile Roux flasks containing
800 ml of the same medium?and incubated as described above.
Estimation of cell number and volume. Cell number and volume were estimated by means of an electronic
particle counter (Coulter counter model ZB 1 ; Coulter Electronics, Dunstable, U.K.). A sample of the culture was
diluted in 0.9% (w/v) NaCl containing 4 ml 25% (v/v) glutaraldehyde 1-’ which had been filtered through a
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Magnesium limitation of Anacystis nidulans
1851
membrane filter (0-2 pm) before use. The counting tube aperture was 70 pm. The mean cell volume was estimated
with a Coulter Channelyzer calibrated by means of latex particles with a diameter of 2-08 pm. The standard error
for the mean cell volume estimation was 12% and for the cell number estimation was 3%, and the standard
deviation from the mean cell volume within one population was 25-40%. The method used for cell size
determination by the electronic particle counter was considered to be satisfactory from the following observations.
Cells with a mean cell volume of 2-0 & 0.2 pm3 (measured electronically) had a mean length of 3.2 -t 0 . 7 ym and
a width of 0.7 k 0 - 1 pm (50 cells were randomly chosen from electron micrographs of thin sections), whereas
those with a mean cell volume of 0.9 t 0.1 pm3had a length of 1 . 7 ? 0.5 pm and a width of 0.7 -t 0- 1 pm. Thus,
the smaller cells in both cases had a volume of about one-half of the larger ones.
Dry weight estimations. Organisms from 50 ml of culture were harvested by centrifugation (15 000 g, 10 min),
washed with distilled water, and transferred to small dried and preweighed glass tubes. These were dried overnzht
at 95 OC, and then placed in a desiccator for 8 h before weighing. Cell mass was also measured as turbidity at
560 nm.
Estimation of macromolecules. Samples for protein, RNA and DNA determinations were harvested by
centrifugation (15 000 g, 10 min), washed once with distilled water and stored at -20 OC until the experiment was
completed. Before assay of the individual components, chlorophyll was extracted with 5 ml methanol. For protein
estimation the pellet was resuspended in 0.5 ml distilled water, and protein was assayed by the Lowry method. For
RNA estimation the pellet was resuspended in 0.2 M-NaCl (2-5 ml), then perchloric acid (0.1 ml; 60%' v/v) was
added and after incubation at 70 OC for 80 min the suspension was chilled on ice and centrifuged; 1 ml of the
supernatant was assayed for D-ribose by the orcinol method of Herbert et al. (197 l), using RNA from yeast as the
standard. DNA was measured by the method of Burton (1956), with rat testicular DNA as the standard.
Chlorophyll estimation. Organisms from 50 ml of culture were harvested by centrifugation (15 000 g, 10 min),
washed once with distilled water and resuspended in 5 ml acetone. The suspension was sonicated using a
'Sonorode' 100 W ultrasonic drill (Kerry's Ultrasonics, London, U.K.) for three periods of 1 min, while cooling in
ice. The suspension was then placed in the dark at room temperature for 1 h. After centrifuging, the A,,, of the
supernatant was measured (Pye Unicam SP 1800 ultraviolet spectrophotometer) and the chlorophyll a
concentration was calculated by the method of Parsons & Strickland (1963).
Magnesium measurements. Attempts were made to measure Mg2+ in the supernatants from steady-state
Mgz+-limited chemostats by atomic absorption (EEL atomic absorption spectrophotometer). By using an
air/acetylene burner, determinations could be carried out over the range 0 to 2p.p.m.; however, the readings
obtained with culture supernatants were in all cases virtually 0. This indicates that almost all the Mg2+ was
completely removed by the organism.
Scanning electron microscopy. Samples were fixed with 2 96 (v/v) glutaraldehyde in 0.2 M-sodium cacodylate
buffer pH 7.0 for 1 h at 4 "C. After washing with cacodylate buffer, the samples were mounted on
polylysine-covered microscope slides and dehydrated in a graded series of ethanol/water mixtures. Thereafter, the
samples were critical-point dried and coated with gold/palladium before examination in a JEOL scanning electron
microscope (JEM 100C).
Chemicals. All chemicals and reagents were of analytical grade and obtained commercially.
RESULTS
Influence of Mg2+concentration on growth and cell size of A . nidulans grown in batch
cultures
To examine the influence of low Mg2+ concentrations on the growth of A. nidulans, the
increase in biomass (As6&at initial Mg2+ concentrations from 2 to 100 p~ was measured.
After transfer from 5 pM-Mg2+(see Methods), there was a lag of 4 to 6 h followed by a period
of exponential growth which, even at the lowest initial Mg2+concentrations tested, lasted for
at least 10 h. The results (Fig. 1) showed that the specific growth rate of A . nidulans under
these conditions was dependent on Mg2+when the concentration of this ion was below 5 p ~ .
The maximum specific growth rate achieved under the conditions used was about 0.2 h-l,
which gave a generation time of 3.5 h. The K , for Mg2+ for growth of A . nidulans was about
2.5 p~ (Fig. 1); thus, the organism had a high affinity for Mg2+.
Webb (1 949) showed that under conditions of Mg2+-deficiency, the division of various
bacterial species was inhibited and filamentous cells were formed. Microscopic examination
of A . nidulans grown at the different Mg2+concentrations showed a similar effect. The cells
used as inoculum were all filamentous, but after transfer the organisms growing at
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1852
H. C. UTKILEN
2
4
6
8
Initial Mg” (PM)
10’100
Fig. 1. Variation in specific growth rate of A . nidulans with initial Mg2+concentration in batch cultures.
100 pM-Mg2 reverted to ‘normal’ size, whereas those growing at concentrations below
10 pM-Mg2+ remained in a filamentous form. Figure 2 shows scanning electron micrographs
of A . nidulans at 15 h after transfer to media containing 2.5 and 100 pM-Mg2+. The cell
surface appeared similar at low and high M g 2 +concentrations even at an increased (60 OOOx)
magnification (not shown), and no invaginations of the cell surface were observed in the
filamentous forms. This suggests that the filaments did not contain transverse walls, as was
confirmed by transmission electron microscopy of thin sections (not shown).
Injluence of Mg2+concentration on mean cell size and macromolecular synthesis in
chemostat cultures
To avoid the possible effects of a continuous decrease in Mg2+ concentration during
growth in batch cultures, chemostat cultures were used for further studies of growth and
macromolecular synthesis in A . nidulans during Mg’+-limited growth.
The effect of increasing the Mg2+ concentration in the feed medium on the steady-state
bacterial biomass (dry weight) at D = 0.085 h-’ (Fig. 3 a ) revealed that Mg2+was the only
limiting factor below 7.5 pM-Mg2+. Plots of dry weight against Mg2+ concentration
extrapolated to zero indicating that the culture had an absolute Mg2+requirement for growth
in the medium used. However, when biomass was measured as A5607 the relation between
biomass and Mg2+in the feed medium extrapolated to a positive intercept on the y-axis (Fig.
3 a ) suggesting that biomass could be produced without Mg2+.This observed relation between
A,,, and Mg2+ was probably affected by the decrease in particle size with increasing Mg2+
concentration, and therefore shows only that A,,, cannot be used as a measure of biomass in
this case.
The effect of increasing, but still limiting, MgZt concentration on cell number and mean cell
volume (Fig. 3b) showed that cell number followed a similar pattern to dry weight. On the
other hand, the mean cell volume decreased with increasing Mg2+ concentration thus
verifying the observations on cell size in batch cultures with different MgZs concentrations.
From the results in Fig. 3 , the dry weight per cell was calculated to be almost constant
(1.19 & 0.02 pg per cell at 2.5 yM-MgZt and 0 - 9 5 & 0.18 pg per cell at 10 pM-MgZt). This
indicates that the cells could be highly vacuolated at the low Mg2+concentrations or that the
small cells could contain a large amount of cellular reserve material.
Tempest et al. (1965) suggested that the availability of Mg2+controls R N A synthesis in K.
aerogenes. Therefore, the total R N A in the A. nidulans culture was measured using different
concentrations of Mg2+ in the feed medium. The results (Fig. 4) showed there was a linear
relationship between Mg2+ concentration and culture RNA. The positive intercept on the
y-axis indicates that the function of Mg2+in R N A synthesis could, to some extent, be fulfilled
by some other component(s) of the medium, since R N A was produced without Mg2+,or that
the synthesis of some species of R N A is not associated with Mg2+.
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Fig. 2. Scanning electron micrographs of A . niduluns grown in batch cultures with initial Mgz+ concentrations of ( a ) 2-5 W M and (b) 100 p~.
The bar markers
represent 1 pm.
1854
H . C. UTKILEN
Mg2+in feed medium ( p ~ )
Mg2+in feed medium (FM)
Fig. 3. Effects of altering the Mg2+concentration in the feed medium for a Mgz+-limited chemostat on
( a ) dry weight (0)and A,,,, (0)and (b) mean cell volume ( 0 )and cell number (a),at D = 0.085 h-.’.
0.20
-
h
2
4
6
8
1
0
Mg2+in feed medium ( p ~ )
Fig. 4. Effects of altering the Mg2+ concentration in the feed medium for a Mg2+-limited chemostat on
chlorophyll (0)and RNA (O), at D = 0.085 h-I.
The relationship between culture chlorophyll and Mg2+ was not linear (Fig. 4); thus, the
synthesis of chlorophyll in these experiments was controlled by factor(s) other than Mg2+.
Variation in cell number and mean cell volume with growth rate
Tempest et al. (1965) showed that the mean cell size increased with growth rate in both
Mg2+-and C-limited cultures of K. aerogenes. The same relationship between growth rate and
cell size was found for Salmonella typhimurium (Maaloe & Kjeldgaard, 1966) and for
SOi--limited (5 p ~A). nidulans (Table 1). In contrast, the mean cell volume of A . nidulans
grown with 5 pM-Mg2+in the feed medium decreased with increasing growth rate (Table 1).
At steady state, cell numbers (Table 1) increased slightly with dilution rate (D)in the
Mg2+-limitedcultures, while they decreased slightly for the SO:--limited cultures. Tempest et
al. (1965) found a decrease in dry weight with increasing D for Mg2+-limitedK . aerogenes.
This could also be the case for A . nidulans, since the A560,which reflects total cell mass,
tended to decrease with D when Mg2+was limiting (Table 1).
Influence of dilution rate on macromolecular composition of Mg2+-and SOi--Eimited cultures
Tempest et al. (1965) reported that culture RNA was independent of growth rate when
Mg2+ was limiting, but not when carbon was the limiting factor for K . aerogenes. Table 1
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Magnesium limitation of A nacystis nidulans
1855
Table 1. Changes in bacterial concentration, RNA, protein and chlorophyll with changes in
dilution rate for Anacystis nidulans, grown in MgZf-and SOi--limited chemostat cultures
All the analytical data in this table are average values obtained from two samples (harvested and
processed on different days) at each steady state.
D
10-7 x
Cell no.
ml-I
Cell volume
(pm-7
Protein
(pg m1-l)
RNA
(pg rnI-')
Chlorophyll
(Pg m1-I)
W')
A 560
0.089
0.094
0.100
0.111
0.124
0.135
0.136
0.168
0.176
0.199
0.475
0.388
0-457
0.485
0.4 10
0-418
0.384
0-394
0-414
0.35 1
Mgz+-limited ( 5 PM) chemostat cultures
6.5
1-73
67
9.5
5.2
1-85
59
8.7
8.5
1.38
67
9.0
8.6
1.45
68
9.5
12.2
0.9 1
65
8-5
10.7
1.20
66
9.1
9.9
1.24
62
8.5
11.9
0.92
68
8-5
10.3
1.19
70
10.5
8.6
1.09
58
10.8
0.66
0-64
0.71
0.80
0.79
0.73
0-84
0.86
0-82
0.69
0.093
0.634
0-600
0,781
0-735
0.590
0.605
0-594
0.65 1
0.673
0.610
0-300
)
cultures
SO:--limited ( 5 p ~ chemostat
21.4
0.66
121
19.5
0.54
115
26.6
0.8 1
160
27.1
0.98
140
13.9
1-25
140
13.6
1.32
152
15.0
1-25
165
16.1
1.26
160
14.4
1*48
158
12.8
1.30
160
7.1
1.54
52
1.28
1.35
1.46
1.41
1.32
1.82
1.75
1.95
2-07
1.90
0.46
0*100
0.110
0.111
0.135
0.147
0.150
0.174
0.200
0.2 10
0.230
10.5
11.1
12.3
11.0
12.8
11.7
12.6
13.5
13.8
13.0
4-8
shows that RNA of A . nidulans was independent of the growth rate under Mg2+-limited
conditions, but increased slightly with growth rate when SO;- was limiting. Protein (Table 1)
showed the same pattern as RNA, indicating a constant relationship between them and
growth rate. Chlorophyll (ml culture)-' was also independent of the growth rate when growth
was limited by Mg*+, but it increased slightly with growth rate when SOi- was limiting
(Table 1).
Macromolecular synthesis by washed suspensions incubated in Mg2+-deficientmedium
The results presented so far show there was little difference in the effect of Mg2+- or
SO:--limitation on macromolecular synthesis in A . nidulans under the culture conditions
used. Further information on how growth and macromolecular synthesis responded to
Mg2+-deficiencywas obtained from observations on washed cells transferred from Mg2+-or
SO:--limited chemostats to Mg2+-deficientbatch cultures. These experiments showed that the
cells from Mg2+-and SO&-limited cultures continued to multiply for about 8 h after transfer
(Fig. 5 a). During this period the mean cell volume was constant (1.33pm3) for the cells from
the Mg2+-limitedchemostat, while it increased from 0.84 to 1.45 pm3 for the cells transferred
from the SOi--limited chemostats. The rates of chlorophyll (Fig. 5c) and D N A synthesis
(Fig. 5b) were independent of the preceding limitation. RNA synthesis, on the other hand,
clearly depended on Mg2+(Fig. 5 a), since its synthesis was slower in cells from Mg2+-limited
than from SOf--limited cultures.
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H. C. UTKILEN
2
4
6
8
1
0
2
4
6 8 1
Time (h)
0
2
4
6
8
1
0
Fig. 5 . Effect of transferring A . niduluns from SO:--limited ( 0 , O ) and Mg2+-limited (e,.> chemostats
to Mg2+-deficient batch cultures on growth and macromolecular synthesis: (a) RNA, (b) DNA and (c)
cell number (0,0 )and chlorophyll (0,
M). Each component is normalized to a value of one at time
zero.
Efect of a Mg2+shift-up on cell size, growth and macromolecular synthesis
Figures 2 and 3 (b) show that cell size depended on the Mg2+concentration of the medium,
and Fig. 1 indicates that a Mg2+ shift-up from 5 p ~ at, which concentration the cells are
filamentous, would not influence the growth rate of A . nidulans. Thus, it appeared possible to
investigate the effect of Mg2+ on cell division under conditions which were not inhibitory to
growth. With this background, Mg2+ shift-up experiments from 5 p~ to 1 mM in batch
cultures were performed.
Balanced growth with an initial Mg2+concentration of 5 p~ resulted in a mean cell volume
of about 2 pm3 (Fig. 6a). After a Mg2+ shift-up to 1 mM the mean cell volume began to
decrease 90 min later (Fig. 6a). Together with the decrease in cell volume there was a
pronounced rise in division rate (Fig. 6a), which lasted for about 60 min, suggesting that
synchronized division of the filamentous cells occurred. There was no change in the rate of
DNA, protein or cell mass (As6,-,)synthesis after the Mg2+ shift-up, while there was a slight
increase in RNA synthesis (Fig. 6b). When the Mg2+ concentration was shifted from 2.5 p 4
to 1 mM, there was an increase in the rate of synthesis of all these macromolecules. These
results show that it is possible to separate the effect of Mg2+ on the cell size control
mechanism from its effect on macromolecular synthesis by using an initial Mg2+
.
6 also shows that macromolecular synthesis (growth) has a
concentration of p ~ Figure
higher affinity for Mg2+ than the processes leading to transverse wall formation and cell
division, since cell division is inhibited before growth. RNA synthesis was also followed by
incorporation of L3H1uracil. Uracil uptake ceased after the increase in Mg2+ concentration
and incorporation of [3Hluracilinto macromolecules, measured as material insoluble in cold
trichloroacetic acid ( 10%, w/v), also ceased.
Since there was no detectable alteration in DNA or protein synthesis after the Mg2+
shift-up, the division taking place 90 min after the addition of Mg2+might be independent of
macromolecular synthesis. However, on addition of mitomycin C or chloramphenicol (final
concentrations 1 and 20 pg ml-’, respectively) together with Mg2+(final concentration 1 mM)
cell division stopped inmediately (Fig. 7). In contrast, on addition of actinomycin D (0.25 pg
ml-’) cell division followed the same pattern as in the control, suggesting that it was
independent of RNA synthesis, although this result could be due to the impermeability of the
inhibitor. Thus, DNA and protein synthesis were necessary for the division of the filaments to
occur, while the cell division might be independent of RNA synthesis.
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Magnesium limitation of Anacystis nidulans
E
1857
3 ,
-5
t-
W
p-----o-o-o--aso-o-a
2
I
l
l
2
6
4
l
l
4
l
l
1 0 1 2
8
l
l
6
8
Time (h)
l
l
l
l
1 0 1 2
j
Fig. 6. Effects of a Mg2+shift-up from 5 ,UM to 1 m M on mean cell volume. growth and macromolecular
and cell number ( 0 )and (b)RNA (0,log pg ml-!),
synthesis of A . nidufans:( a ) mean cell volume (0)
DNA (a, log [pg ml-' x lo]), protein (0, log pg ml-I) and A560 (H, log I A - x lo]). The Mg2+
concentration was shifted-up at the time indicated by the arrows.
Time (h)
Fig. 7. Effect of antibiotics on the increase in cell number during a Mg2+shift-up from 5 p~ to I mM in
batch cultures: chloramphenicol (O),mitomycin ( 0 )and control and actinomycin D (0).See text for
concentrations of antibiotics, which were added together with Mg2'. The Mgz+ concentration was
shifted-up at the time indicated by the arrow.
DISCUSSION
In a chemostat culture operated at a constant dilution rate the 'steady-state' cell mass
should increase linearly with the input concentration of limiting nutrient, and should
extrapolate to zero at zero input if this nutrient is the only limiting factor (Herbert et al.,
1956). This must be examined very carefully when working with photosynthetic organisms in
nutrient-limited chemostats, since light might easily become an additional limitation. Figure
3 ( a ) shows that the Mg2+-limitedchemostat cultures used in this work were controlled by a
single limitation (Mg2+) below a concentration of 7.5 pM-Mg2+ in the feed medium. On the
other hand, the putative SOi--limited cultures might have been light-limited, since these
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1858
H. C. UTKILEN
cultures have a cell mass, measured as AS6,,,close to that obtained at 10 pM-Mg2+ (Fig. 3a),
where light might be an additional limitation. However, the cell mass of a light-limited culture
would decrease with increasing growth rate, owing to an increased energy demand. Since this
did not occur (Table l), it is probable that these cultures were indeed limited by SO:-.
Since RNA synthesis seems to be influenced by Mg2+(Tempest et al., 1965; Fig. 5 a), and
the kinetics of association and dissociation of Escherichia coli 30s and 50s subunits are
strongly Mg2+-dependent (Grunberg-Manago et al., 1978), inhibition of RNA synthesis or
alteration of ribosome efficiency could be the mechanism through which Mg2+concentration,
,
the growth rate in A . nidulans (Fig. 1).
below 5 p ~affected
Figure 3 (a) shows that Mg2+is almost completely utilized by A . nidulans in a Mg2+-limited
chemostat culture. On this assumption the cellular Mg2+ concentration can be calculated
from the data in Fig. 3 (b). The values obtained (Fig. 8) range from 27 to 78 mM for
concentrations in the feed medium from 2.5 to 10 p ~ If. a large fraction of the cellular Mg2+
is associated with the ribosomes, as is found for other micro-organisms (Jasper & Silver,
1977), then according to the results obtained for E. coli (Grunberg-Manago et al., 1978),
association of ribosomal units would be favoured. Lack of stable ribosomes might therefore
not be the growth-limiting factor in the chemostat cultures, while it might provide an
explanation for the results obtained with batch cultures. The extrapolation of the relation
between cellular Mg2+and Mg2+in the feed medium (Fig. 8) indicates that A . nidulans has to
maintain its cellular Mg2+concentration above 9 mM to prevent washout under the conditions
used.
Although RNA synthesis is slowed down in a Mg2+-limited chemostat culture of A .
nidulans (Fig. 5), this does not appear to affect the growth rate, suggesting that the organism
contains surplus RNA. This is supported by the finding that the rate of RNA synthesis is
altered during a Mg2+ shift-up while the rate of protein synthesis is not. According to Koch
(197 1) it is possible to distinguish between constant RNA efficiency, as postulated by Maaloe
& Kjeldgaard (1966), and variable RNA efficiency by examining total RNA/total protein as
a function of growth rate. Using the results in Table 1, this relation is shown (Table 2) to be
independent of the growth rate, both in Mg2+-and SOi--limited cultures. Thus, the efficiency
of RNA in A . nidulans is variable, and since the ratio of RNA to protein is lower in the
SO:--limited cultures than in the Mg2+-limitedcultures, the results also indicate that Mg2+is
involved in the RNA efficiency.
The non-linear relation between culture chlorophyll and Mg2+in the feed medium (Fig. 4)
is caused by an increase in specific chlorophyll content (Table 3) resulting from increased
self-shading. Since the culture probably also became energy (light)-limited at 10 pM-Mg2+in
2
4
6
8
1
Mg2+ in feed medium ( p ~ )
0
Fig. 8. Variation in the cellular Mg2+ concentrations with the Mgz+ concentration in the feed medium
for A . nidulans grown in Mgz+-limitedchemostats at D = 0-085 h-l.
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Magnesium limitation of Anacystis nidulans
1859
Table 2. Changes in RNA to protein and chlorophyll to protein ratios with changes in
dilution rate, in Mg2+-and SOi--lirnited chemostat cultures
D (h-')
RNA (pg)
Chlorophyll (pg)
Protein (pg)
Protein (pg)
Mg?+-limited( 5 p ~ chemostat
)
0-089
0 - 14
0.094
0.15
0.100
0.14
0.111
0.14
0.124
0.13
0.135
0.14
0,136
0.14
0.168
0.13
0.176
0.15
0-199
0.19
cultures
0.010
0.01 1
0.01 1
0.012
0.012
0.01 1
0.014
0-013
0.0 I2
0.012
SO:--limited ( 5 p ~ chemostat
)
cultures
0.093
0.09
0.01 1
0.100
0.10
0.012
0.110
0.08
0.009
0.111
0.08
0.009
0.135
0.09
0.009
0.147
0-08
0.012
0.150
0.08
0.01 1
0.174
0.08
0.012
0.200
0.09
0.0 13
0.210
0.08
0.012
0.230
0.09
0.009
Table 3. Spec$c chlorophyll content and percentage of culture Mg2+bound in chlorophyll
at diflerent Mg2+concentrations in thefeed medium, during Mg2+-limitedgrowth at
D = 0.085 h-'
The analytical data in this table are average values obtained from two samples at each steady state.
Mg2+in
feed medium ( p ~ )
2-5
5.0
7.5
10.0
Chlorophyll
(% dry wt)
Cellular Mg2+
bound in chlorophyll (%)
0.8
0.6
1.0
1.1
17
14
22
22
the feed medium (Fig. 3a), the results obtained in Table 3 indicate that the maximal level of
chlorophyll in A . nidulans is about 1% of the dry weight. However, Myers & Kratz (1956)
showed that nearly 3 % of the dry weight in A . nidulans might be chlorophyll. The different
maximal chlorophyll contents obtained make it likely that Mg2+ interfered with chlorophyll
synthesis under the conditions used in this work.
The percentage of cellular Mg2+ present in chlorophyll can be calculated from the data in
Figs 3 (b) and 4. The results (Table 3) show that as much as 22% of the cellular Mg2+could
be present in chlorophyll. The energy generating system in photosynthetic organisms is
therefore an important Mg2+-bindingcomponent in addition to ribosomes and cell wall, which
are the main Mg2+-binding components in non-photosynthetic organisms (Jasper & Silver,
1971).
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1860
H . C . UTKILEN
The results presented in this work show that the most pronounced effect of Mg2+ is its
control of cell size. Severai workers (Webb, 1949; Brock, 1962; Tempest et al., 1965) have
shown that bacteria become enlarged or filamentous during Mg2+-limitedgrowth. Chlorella
(Retovsky & Klasterska, 196 1) and Schizosaccharomyces pombe (Ahluwalia el al., 1978) are
also enlarged when deprived of Mg2+.The cell size of bacteria usually increases with growth
rate (Maaloe & Kjeldgaard, 1966; Tempest et al., 1965) and this has been shown to be true
for Mg2+-limited K. aerogenes (Tempest et al., 1965). The difference in the relationship
between cell size and growth rate for Mg2+-limitedA . nidulans (Table 1) and K . aerogenes
(Tempest et al., 1965) is probably due to the fact that the Mg2+ concentration used in the
present work was 100 times less than that used by the other workers. All these observations
indicate that cell enlargement is a general feature of Mg*+-limitedgrowth and that A . nidulans
responded like other organisms to a limited availability of this ion.
Low Mg2+ concentrations may control cell division in A . nidulans by inhibiting the
synthesis of a particular protein. Protein synthesis is necessary for the Mg2+-induced cell
division to occur (Fig. 7). Moreover, Mann & Carr (1977) showed that protein synthesis
occurring at the termination of DNA replication is necessary for cell division to take place.
Another possibility is that the low Mg2+ concentration could have perturbed the cell
membrane such that one of the normal control mechanisms for cell size (i.e. initiation of the
transverse septum) was disturbed. The results obtained with L3HIuraciluptake in A . nidulans
indicate that the membrane is altered during a Mg2+shift-up from 5 p~ to 1 mM.
Ahluwalia et al. (1978) have shown that Schizosaccharomyces pornbe can be
synchronized by reducing the availability of Mg2+, and Duffus & Paterson (1974) have
indicated that a continuous fall in intracellular Mg2+concentration may play a crucial part in
the volume regulation and cell division of S . pombe. Although the cellular organization in S.
pombe is quite different from that in A . nidulans, the effect of Mg2+ on cell division and cell
size is very similar for these organisms. A certain period after the division-disturbing events a
synchronized division occurs. In A . nidulans this synchronized division takes place over a
period of 60 rnin (while the generation time is 3.5 h) resulting in a 66% increase in cell
number. The synchronized division of S. pombe takes place during a period of 15 min with an
increase in cell number of 45% (Ahluwalia et al., 1978). In both organisms only division is
affected (at a start concentration of 5 p~ for A . nidulans), while DNA, protein and cell mass
synthesis are not. The reduction in cell volume observed for A . nidulans during the
synchronized division may also occur in S. pombe (Ahluwalia et al., 1978). However, since
there is no change in the synthesis of macromolecules when the synchronized division occurs,
the concentration of macromolecules per cell decreases while their ratio to unit cell volume
might remain constant.
The results presented here for A . nidulans together with those of others (Webb, 1949;
Retovsky & Klasterska, 1961; Finkel & Appleman, 1953; Ahluwalia et al., 1978) indicate
that the cell volume of prokaryotic and eukaryotic micro-organisms is regulated by Mg2$ at
low concentrations and that these organisms respond in the same way to changes in Mg2+
concentrations. Mg2$ might therefore control cell size through a universal mechanism. Cell
enlargement caused by Mg’+-limitation could, on the other hand, be due to a different
mechanism in each group of organisms. For S. pornbe, Walker & Duffus (1980) concluded
that the Mg2+ concentration is the transducer for cell size. Through a fall in cellular Mg2+
concentration, which results in an increase of cell size, the cellular concentration of this ion
reaches a level which permits tubulin polymerization and spindle formation. When
chromosome separation is completed, a rapid influx of Mg2+ is necessary for breakdown of
the spindle and for nuclear and cell division to occur. Cell elongation in Bacillus subtilis could
be a result of an inhibition of peptidoglycan synthesis, since its synthesis depends on Mg2+
(Garrett, 1969). In Gram-negative organisms cell elongation could occur by inhibiting the
synthesis of the cell wall lipoprotein characterized by Braun (1 975), since deficiency of this
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Magnesium limitation of Anacystis nidulans
1861
lipoprotein results in filamentous forms of E. coli (Torti & Park, 1976). This lipoprotein has
also been demonstrated in A . nidulans (Golecki, 1977).
I am grateful to Dr J. G. Ormerod for helpful advice and discussion during the course of this work and for his
critical reading of the manuscript.
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