J. Cell Sci. 49, 341-352 (1981)
Printed in Great Britain © Company of Biologists Limited 1981
341
HETEROCYST DIFFERENTIATION AND CELL
DIVISION IN THE CYANOBACTERIUM
ANABAENA CYLINDRICA: EFFECT OF HIGH
LIGHT INTENSITY
DAVID G. ADAMS AND NOEL G. CARR
Department of Biochemistry, University of Liverpool,
P.O. Box 147, Liverpool L69 3BX, England
SUMMARY
Heterocyst differentiation in the cyanobacterium Anabaena cylindrica is initiated by the
removal of fixed nitrogen from the medium. These specialized cells occur singly at regular
intervals within filaments of vegetative cells. Incubation of cultures for periods of up to 12 h
immediately prior to or following removal of fixed nitrogen, at a light intensity (500 /iEinsteins
cm~ 2 s"1) approximately 10-fold higher than that required for optimum growth, resulted in
the differentiation of pairs of adjacent (double) heterocysts. The frequency of double heterocysts
was proportional to the length of the period of high light intensity. During growth at normal
light intensity approximately 5 % of cell divisions were symmetrical, but this increased more
than 3-fold during 10-h incubation at high light intensity. The frequency of dividing cells
remained constant during this period, but increased rapidly on return to normal light. T h e
frequency of double heterocysts was reduced if a period of incubation at normal light intensity
was interposed between the 12-h period at high light intensity and transfer to nitrogen-free
medium. A period of 8 h normal light was required to reduce the frequency of double heterocysts
to control values, and this corresponded to the length of time needed for the frequency of
symmetrical divisions to return to control levels following 12 h at high light intensity. We
confirm that cell division in Anabaena cylindrica is asymmetrical and conclude that the presence
of double heterocysts results from an increase in the symmetry of cell division during incubation
at high light intensity. The results also support the finding of previous workers that a cell is
only susceptible to differentiation during a short period following its formation. During the
period of high light the rate of doubling of the absorbance of the culture at 750 mn increased
from 24 h to approximately 10 h and decreased to more than 100 h on return to normal light.
The very high rate could be explained by increases in the volume and granular content of cells
during incubation at high light intensity and did not represent an equivalent increase in the
rate of cell division.
INTRODUCTION
When grown in the presence of a source of fixed nitrogen such as ammonium
chloride, the cyanobacterium Anabaena cylindrica consists of long filaments of
vegetative cells. Following transfer to nitrogen-free medium a regular, spaced pattern
of specialized cells known as heterocysts develops within each filament. These cells
are regarded as the major if not exclusive site of aerobic nitrogen fixation in heterocystous cyanobacteria (see Stanier & Cohen-Bazire, 1977; Haselkorn, 1978; Carr,
1979). Little is known about the mechanisms that control the regular spacing of
single heterocysts, although a limited number of chemicals are known to modify the
342
D. G. Adams and N. G. Can
pattern. ./V-methyl-./V'-nitro-./V-nitrosoguanidine mutagenesis has yielded a number
of mutants that show alterations in heterocyst spacing (Wilcox, Mitchison & Smith,
1975 a). Rifampin (Wolk, 1975) and 7-azatryptophan (Mitchison & Wilcox, 1973)
cause a reduction in the number of vegetative cells between heterocysts (intervalwidth) and the production of adjacent (multiple) heterocysts.
Fogg (X949) suggested that the diffusion from heterocysts of ammonia, or some
simple derivative, inhibited the differentiation of neighbouring vegetative cells.
Although it is now clear that ammonia itself cannot inhibit heterocyst development
(Carr & Bradley, 1973; Stewart & Rowell, 1975; Bradley & Carr, 1976), the concept
of a diffusible inhibitor establishing a decreasing concentration gradient away from the
heterocyst is still an accepted model for heterocyst pattern control. Mitchison &
Wilcox (1972) demonstrated a relationship between cell division and heterocyst
differentiation in Anabaena species. They found that cell division was asymmetrical
and heterocysts developed only from the smaller daughter cell of a division arising
outside the inhibitory zone of an existing heterocyst. This paper reports the production
of double heterocysts following incubation of A. cylindrica, for short periods, at high
light intensities and relates this to changes in cell division.
MATERIALS AND METHODS
Organism and growth conditions
Anabaena cylindrica no. 1403/28 was obtained from the Culture Centre of Algae and Protozoa,
Cambridge, U.K. Cultures were grown on the medium of Allen & Arnon (1955) supplemented
with ammonium chloride (41HM; A2 N 4 medium) in 1-litre Quickfit glass reaction vessels,
maintained at 29 ± 0-5 deg. C in a thermostatically controlled water bath. The organism was kept
in suspension by magnetic stirring and was gassed with sterile 5 % CO 2 in air. For experimental
purposes 30-ml volumes of culture were incubated in boiling tubes at 29 ± 1 deg. C in a warm
room and gassed with sterile 5 % CO 2 in air.
Illumination
Cultures in boiling tubes were placed 30 cm from three 40 W warm white fluorescent tubes
(Cryselco Ltd) and a single layer of tracing paper (90 g. m~2, Wiggins Teape, London, England)
was placed mid-way between to reduce the incident light intensity to approximately 45 /tEinsteins
cm" 2 s - 1 (normal light intensity, NL). For incubation at high light intensity (HL) the boiling
tubes were placed mid-way between 2 banks of similar fluorescent tubes, 30 cm apart, one
consisting of three 40 W tubes, the other of six 20 W tubes. This produced a total incident light
intensity of approximately 500 /iEinsteins cm~2 s" 1 . An electric fan was used during incubation
in boiling tubes at H L to ensure adequate circulation of air and maintain the temperature at
2 9 + 1 deg. C. For cultures grown in 1-litre reaction vessels, normal light was provided by one
60 W incandescent light-bulb 15 cm from the centre of the growth vessel and high light
intensity by four 75 W reflector bulbs (Cryselco Ltd), each 15 cm from the centre of the vessel.
These conditions produced incident light intensities of approximately 45 and 1200 /<Einsteins
cm~2 s" 1 , respectively. Light intensities were estimated using a model 3000 photometer/
radiometer (Macam Photometries Ltd, Livingstone, Scotland, U.K.), and distances were
measured to the front surface of the bulb or fluorescent tube.
Initiation of heterocyst differentiation
A2 N 4 -grown cultures of A. cylindrica were transferred to 1-litre sterile polypropylene
centrifuge pots and filaments pelleted by centrifugation at 3000 rev./min and 29 °C for 15 min
in a Mistral 6L centrifuge (Measuring and Scientific Equipment Ltd, Crawley, England).
Heterocyst differentiation in A. cylindrica
343
The supernatant was removed and thefilamentsresuspended in sterile medium lacking ammonium chloride (A2 medium). Removal of ammonium chloride from 30-ml cultures in boiling
tubes was achieved as follows. The gassing tube and cotton-wool plug were replaced by a
square of sterile aluminium foil, which was pressed over the top of the tube. The cells were
sedimented by centrifugation at 1500 rev./min and 29 °C for 20 min in the Mistral centrifuge.
The old medium was removed, the cells resuspended in A2 medium and the foil cap replaced
by a sterile cotton-wool plug and gassing tube.
High light intensity and heterocyst differentiation
Cultures were incubated at HL either before transfer to ammonia-free medium (preincubation) or following transfer (post-incubation). For pre-incubation, samples (30 ml) were
removed from a 1-litre A2 N4-grown culture and transferred to sterile boiling tubes. These
were exposed to HL for increasing periods before transfer to ammonia-free medium, as described
above, and return to NL. For post-incubation a 1-litre ammonia-grown culture was transferred
to A2 medium and 30-ml samples incubated in boiling tubes for increasing periods at HL
before being returned to NL. Cell frequencies were estimated 48 h following ammonia removal.
Estimation of the duration of effects produced by incubation at high light intensity
To investigate the duration of any effects produced by HL, samples (30 ml) were removed
from a 1-litre ammonia-grown culture of A. cylindrica. These were incubated in boiling tubes,
the first being left at NL throughout the experiment (control). All subsequent tubes were
exposed to HL for 8 h, followed by increasing periods at NL prior to transfer to ammonia-free
medium. The experiment was arranged in such a way that all samples were transferred to
minimal medium at the same time and incubated at NL for the remainder of the experiment.
Cells were counted 33 h after the removal of ammonia.
The effect of high light intensity on growth and cell division of cultures grown in the
presence of ammonium chloride
Cultures were grown on A2 N4 medium in 1-litre glass reaction vessels. The culture was
grown at normal light intensity for 23-5 h, transferred to the higher light for 12 h and then
returned to normal light. Changes in optical density and cell division were measured, as
described below, throughout the experiment. Growth was estimated turbidimetrically at 750 nm
in a Pye Unicam SP600 spectrophotometer. Estimation of cell division was done under phasecontrast microscopy at a magnification of x 2500. A cell was regarded as being in division if it
had a partially formed septum at its centre. A division was counted as equal if the 2 developing
daughter cells could not be said with certainty to be of different sizes. Heterocyst frequency was
estimated at a magnification of x 600. For all cell counts a minimum of 1000 vegetative cells
was counted for each determination.
RESULTS
High light intensity and heterocyst differentiation
Incubation of A. cylindrica at high light intensity (see Materials and methods)
resulted in an increase in the frequency of double heterocysts (DHC), which are the
pairs of adjacent heterocysts that can be seen in Fig. IA-C. Pre-incubation of A.
cylindrica at HL for short periods resulted in the production of a greatly increased
frequency of double heterocysts when ammonia was later removed. A linear relationship was found between the frequency of double heterocysts and the length of
exposure to HL, such that after 10 h almost 20% of the heterocysts that had formed
following ammonia removal were double (Fig. 2 A). Exposure to HL after transfer to
344
D. G. Adams and N. G. Carr
Fig. i. Phase-contrast photomicrograph of A. cylindrica showing double heterocysts
induced by incubation at high light intensity. A, x 750; B, c, x 480.
ammonia-free medium produced a similar linear increase in the frequency of double
heterocysts (Fig. 2B). In an untreated control culture, the frequency of double
heterocysts was less than 0-2 %. Growth of cultures at HL for periods in excess of
15 h resulted in extensive granulation and fragmentation of filaments, which did not
recover on return to normal light. However, following HL periods of less than 15 b,
cultures showed apparently normal and healthy growth after return to normal light
intensity.
Estimation of the duration of effects produced by incubation at high light intensity
Exposure of A. cylindrica to HL for 8 h immediately prior to transfer to ammoniafree medium resulted in about 7-5 % of the mature heterocysts that developed follow-
Heterocyst differentiation in A. cylindrica
+N
30
345
-N
B
A
20 -
D
10
0
A
/
:
Piii
i
ii
12
Time at HL (h)
Fig. 2. Relationship between double heterocyst (DHC) frequency and period of
incubation at high light intensity (HL) in A. cylindrica. Cultures were incubated at
HL for the periods indicated, either immediately prior to (A) or following (B) removal
of ammonium chloride (+ N or —N) from the medium. DHC frequency was estimated
48 h after ammonia removal.
10
Time at NL (h)
Fig. 3. Estimation of the duration of effects produced by high light intensity (HL) in
A. cylindrica. Samples of culture were incubated at HL for 8 h, followed by a different
period at normal light intensity (NL) for each sample (abscissa) prior to ammonia
removal. Following ammonia removal samples were incubated at NL. Frequencies
of double heterocysts ( • ) , heterocysts (•), proheterocysts ( • ) and total heterocysts
(O) were estimated 33 h after ammonia removal. To estimate total heterocyst (HC)
frequency, double heterocysts were counted as one cell, c, control culture.
12
CEL
49
346
D. G. Adams and N. G. Carr
ing transfer being in the double form (Fig. 3 A). A 2-h period at normal light before
the removal of ammonia had little effect on this percentage. Incubation at normal Ugh
for 4 h, however, resulted in more than a 50 % decrease in the frequency of double
heterocysts. Following 8 h at normal light, there was little difference between the
control and treated cultures (Fig. 3 A). The final level of heterocysts and proheterocysts
(not including double heterocysts) in the cultures described above can be seen in
Fig. 3 B. In the control there was about 2-5 % of both cell types. Exposure to high light
intensity for 8 h immediately prior to transfer to minimal medium, resulted in an
increase in mature heterocyst frequency to approximately 8% and a decrease in
NL
i
HL
|
NL
0-50 0-40 0-30 -
0-20 -
>
t
010
008
006
i
10
20
1
1
30
40
1
50
60
Time (h)
Fig. 4. Effect of alterations in light intensity on the growth of A. cylindrica in the
presence of ammonium chloride. The culture was transferred from normal light
intensity (NL) to high light intensity (HL) and back to NL at the times indicated.
The ordinate is a logarithmic scale.
proheterocysts to about 0-5 %. The total percentage of differentiated cells (counting
double heterocysts as 1 cell) also increased from 5% to over 9%. Subsequent increasing incubation times at normal light, following the 8 h at high light intensity,
resulted in an almost complete return to control values.
Effect of high light intensity on growth and cell division of cultures grown in the presence
of ammonium chloride
Under normal light conditions A. cylindrica grew with a mean generation time of
24 h. During exposure to the higher light intensity the doubling rate of culture
absorbance at 750 nm increased to 9-10 h and decreased to more than 100 h on
return to normal light (Fig. 4). Microscopic examination of filaments from the 2 light
regimes revealed 2 obvious differences. There was a great increase in the granular content of the cells during the period of high light intensity (compare Fig. 5 A, D with B, E).
Heterocyst differentiation in A. cylindrica
347
This appearance was gradually lost on return to normal light conditions (Fig. 5 c).
The nature of these refractile granules is not known, although microscopically they
resemble the cyanophycin granules present in akinetes (Miller & Lang, 1968; Simon,
1971; Wildman, Loescher & Winger, 1975). The second major change to occur during
exposure to HL was a considerable increase in cell volume. Measurement of cell size
from photographs showed an approximately 3-fold increase in volume after 11 h
Fig. 5. Phase-constrast micrographs of the same culture of A. cylindrica grown on
A2 N4 medium. A, D, normal light intensity ( x 480 and 750, respectively); B, E, 11 h
after transfer to high light intensity ( x 480 and 750, respectively); c, 13 h after return
to normal light intensity ( x 480). Bars, 10 fim.
exposure, assuming each cell to be a cylinder of uniform diameter. Cultures grown in
the presence of ammonium chloride often contained a low frequency (< 1 %) of
proheterocysts and a small increase in this frequency was usually observed during and
after exposure to high light intensity. Double proheterocysts, however, were not
detected. Even small increases in light intensity induced transient increases in the
frequency of proheterocysts in such cultures.
The changes in cell division during incubation at HL are shown in Fig. 6. During
D. G. Adams and N. G. Carr
348
NL
HL
NL
100
A
80
-
60
/
40
20
-
1
0
1
B
15
10
/
\
5
1
n
0
i
1
20
1
60
40
Time (h)
Fig. 6. Effect of high light intensity (HL) on cell division in A. cylindrica grown on
Aa N4 medium. The culture was transferred from normal light intensity (NL) to
HL and back to NL at the times indicated. The frequencies of: A, number of cells in
division; and B, equal divisions, are indicated.
20
10
A
- [l|
Pi
"
0
B
10
• A
-
JI
n
10
20
30
Interval-width (cells)
Fig. 7. Histograms illustrating the range and frequency of intervals bounded by a
single and a double heterocyst (A) or by 2 single heterocysts (B) in the same culture
of A. cylindrica. The culture was transferred to ammonia-free medium and incubated
at high light intensity for 12 h. It was then returned to normal light intensity and
interval-widths counted 36 h later.
Heterocyst differentiation in A. cylindrica
349
growth at NL the frequency of cells in division (50 %) was relatively constant. This
was maintained during the HL period, but increased steadily to about 90 % during the
following 15-20 h at normal light (Fig. 6 A). This considerable increase in the number
of cells in division can be clearly seen by comparison of Fig. 5 A and c. Equal divisions
constituted approximately 5 % of the total during normal growth. This increased by
about 3-fold after 10 h at HL and quickly decreased to slightly less than the original
level after 15-20 h normal light (Fig. 6 B). Very similar results were obtained when the
high light intensity was reduced by half (two 75 W bulbs each 15 cm from the centre of
the growth vessel, producing an incident light intensity of approximately 600 fi Einsteins
cm"2 s"1), when a greater than 4-fold increase in the frequency of equal divisions was
observed.
DISCUSSION
We have examined the influence of high light intensity on the development of
heterocysts immediately following removal of fixed nitrogen from the medium. This
period was chosen since the full complement of proheterocysts are selected within less
than half the generation time and any effects caused by high light intensity would be
enhanced. In cultures growing in the absence of fixed nitrogen heterocysts are
continually developing, but over the full generation time, which would reduce the
influence of a relatively short period of incubation at high light intensity. We have no
reason to believe, however, that the effects reported here would not apply to such
cultures, and certainly nitrogen-fixing cultures grown for prolonged periods at light
intensities slightly above normal, possess elevated frequencies of double heterocysts,
although the effect is small (data not shown).
Implicit in the model of Mitchison & Wilcox (1972; see also Wilcox, Mitchison &
Smith, 19756) is the asymmetric division of cells and the differentiation of only the
smaller daughter of such divisions, arising outside the inhibitory zone of neighbouring
heterocysts. A minor reduction in the inhibitory zone of pro- or mature heterocysts
would result in the production of more closely spaced heterocysts. Indeed, an increase
in heterocyst frequency and a consequent reduction in mean interval-widths, was
observed when A. cylindrica was allowed to differentiate following incubation at high
light intensity (see Fig. 3B). The reason for this is not clear, although it may result
from changes in cell division induced by incubation at HL. The observed increase in
cell volume during exposure to high light intensity might also have been a contributory factor. If inhibitor production by developing heterocysts did not increase in a
similar manner, then its concentration within vegetative cells would decrease, resulting
in diminished zones of inhibition during the early stages of differentiation and the
formation of a closer pattern of heterocysts.
Such small changes in inhibitory zones cannot, however, explain the presence of
double heterocysts. While a small decrease in inhibitor concentration within a pair of
daughter cells might induce their premature development, it would not be expected to
affect the competition between them. It should still be possible to select a single cell
for continued development. Proheterocysts can normally divide only during the early
stages of development and they will then regress and resume vegetative growth. It
35°
D. G. Adam and N. G. Can
might be argued that incubation at high light intensity alters this process and permits a
proheterocyst that divides to continue to differentiate and form a double heterocyst.
We feel, however, that there is a more likely and attractive explanation. In common
with 7-azatryptophan and rifampin, incubation at high light intensity resulted in an
increase in the frequency of single heterocysts. However, unlike these 2 chemicals,
which cause the production of groups of 2, 3, 4 or more adjacent heterocysts, the only
multiple heterocysts resulting from exposure to HL were doubles. This suggests that
the effects might originate in cell division and the rapid increase in equal divisions
during the HL period may help explain the development of these heterocyst pairs.
Should such an equal division occur outside the zone of inhibition of a proheterocyst or
heterocyst, then the daughter cells would be able to differentiate. Since the 2 cells are
identical, neither would have a selective advantage and both could develop into
heterocysts. This effect might be enhanced by the increased rate of maturation of
proheterocysts observed after exposure to high light intensity. (Fig. 3 B). This would
further distrupt the balance between cell division and differentiation, which normally
ensures the development of only single heterocysts.
The plausibility of this explanation is strengthened by a close observation of the
shape of double heterocysts. The individual cells of many such pairs were altered in
shape, the cell wall common to both being rather flattened and the 2 outer walls being
more pointed than is usual for single heterocysts. This is clearly seen in Fig. 1 A-C.
This effect would be unlikely to occur if the cells had developed at different times,
when 2 normally shaped heterocysts would be expected. A more likely explanation is
that they differentiated from the equal daughters of a very recent division. The shape
of the double heterocyst would depend on the timing of its development in relation
to the division. The greater the period of time between a cell's division and its
differentiation, the more normal in shape the resulting pair of heterocysts would be
expected to be.
The characteristic shape of many double heterocysts may provide one explanation
for the reports of mature heterocyst division (Kumar, 1963; Ladha & Kumar, 1975).
Such double heterocysts could be considered to be a single heterocyst in the process of
division and the combination of photographs of variously shaped double heterocysts
may give the appearance of sequential stages of division. In fact, the division occurred
before differentiation, not after. The authentication of claims for heterocyst division
can only arise from the use of time-lapse photography of individual heterocysts.
Double heterocysts could, in principle, be produced from pairs of identical daughter
cells that are formed prior to the induction of differentiation. There must, however,
be a maximum period after the formation of such identical pairs when they can no
longer produce a double heterocyst. This can indeed be seen in Fig. 3 in which this
maximum time is 6-8 h. This may represent the time following division during which
any cell remains able to differentiate when induced to do so. It may also be the time
required for a pair of identical daughter cells to become sufficiently dissimilar, perhaps
due to small differences in growth rate, to prevent them forming a double heterocyst.
Indeed, the length of this period correlates with the time required for the frequency of
equal cell divisions to return to normal following the HL period.
Heterocyst differentiation in A. cylindrica
351
Incubation at HL resulted in a decrease in the size of all interval-widths, there
being little difference in intervals bounded by 2 single heterocysts or by a single and a
double heterocyst. This is illustrated in Fig. 7 in which the 2 histograms represent the
intervals between single heterocysts (Fig. 7B), and between single and double heterocysts (Fig. 7 A) in the same culture. There was very little difference between the 2
ranges of interval-width and this can be explained as follows. If the heterocyst
inhibitor was lost or destroyed at a rate proportional to its concentration, then an
exponential gradient would be established (Wilcox, Mitchison & Smith, 1973; Wolk,
1975). If the gradient was a steep one, then even a doubling of the concentration of the
source inhibitor (i.e. a double heterocyst instead of a single) would have little effect
on the inhibitory zone generated and the interval to the next heterocyst would be
relatively unchanged.
It is clear that the rapid rate of increase in culture absorbance obtained during
exposure to HL was largely due to increases in both the volume of cells and their
granular content, rather than an increase in cell division rate. Changes in cell numbers
are difficult to measure in A. cylindrica since it is a filamentous organism and grows
slowly. However, a rapid increase in the number of cells in division was observed
following return to normal light (Fig. 6 B) and this permitted the organism to reduce
both its cell volume and granular content to pre-exposure values. Thus, although
growth, measured by changes in A750, appeared to have almost ceased on return to
normal light, cell division was occurring with the result that the cells had almost
returned to normal after about 13 h (see Fig. 5 c).
The work described here confirms the observations of Mitchison & Wilcox (1972)
that cell division in A. cylindrica is asymmetrical. It also illustrates the crucial importance of such asymmetry in ensuring that only one of two daughter cells, which
commence their cell cycles together, will develop into a heterocyst. This is an essential
prerequisite for the attainment of a spaced pattern of single heterocysts. The results
also indicate that a cell is susceptible to differentiation only during the period approximately 0-8 h following its formation. This corresponds to the candidate cell described
by Wilcox et al. (1973)-'a smaller daughter in the period immediately after a
division' - and confirms the conclusion of Bradley & Carr (1977), that the response of
any cell to the stimulus to differentiate depends on its stage within its cell cycle. Thus,
a further constraint is placed upon a population of cells, limiting the number that can
differentiate. The attainment of the final regular pattern of heterocysts, however,
requires more complex intercellular interactions, the nature of which are as yet
poorly understood.
This work was supported by the Science Research Council.
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