J. Cell Sci. 49, 341-352 (1981) 341Printed in Great Britain © Company of Biologists Limited 1981

HETEROCYST DIFFERENTIATION AND CELL

DIVISION IN THE CYANOBACTERIUM

ANABAENA CYLINDRICA: EFFECT OF HIGH

LIGHT INTENSITY

DAVID G. ADAMS AND NOEL G. CARRDepartment of Biochemistry, University of Liverpool,P.O. Box 147, Liverpool L69 3BX, England

SUMMARY

Heterocyst differentiation in the cyanobacterium Anabaena cylindrica is initiated by theremoval of fixed nitrogen from the medium. These specialized cells occur singly at regularintervals within filaments of vegetative cells. Incubation of cultures for periods of up to 12 himmediately prior to or following removal of fixed nitrogen, at a light intensity (500 /iEinsteinscm~2 s"1) approximately 10-fold higher than that required for optimum growth, resulted inthe differentiation of pairs of adjacent (double) heterocysts. The frequency of double heterocystswas proportional to the length of the period of high light intensity. During growth at normallight intensity approximately 5 % of cell divisions were symmetrical, but this increased morethan 3-fold during 10-h incubation at high light intensity. The frequency of dividing cellsremained constant during this period, but increased rapidly on return to normal light. Thefrequency of double heterocysts was reduced if a period of incubation at normal light intensitywas interposed between the 12-h period at high light intensity and transfer to nitrogen-freemedium. A period of 8 h normal light was required to reduce the frequency of double heterocyststo control values, and this corresponded to the length of time needed for the frequency ofsymmetrical divisions to return to control levels following 12 h at high light intensity. Weconfirm that cell division in Anabaena cylindrica is asymmetrical and conclude that the presenceof double heterocysts results from an increase in the symmetry of cell division during incubationat high light intensity. The results also support the finding of previous workers that a cell isonly susceptible to differentiation during a short period following its formation. During theperiod of high light the rate of doubling of the absorbance of the culture at 750 mn increasedfrom 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 cellsduring incubation at high light intensity and did not represent an equivalent increase in therate of cell division.

INTRODUCTION

When grown in the presence of a source of fixed nitrogen such as ammoniumchloride, the cyanobacterium Anabaena cylindrica consists of long filaments ofvegetative cells. Following transfer to nitrogen-free medium a regular, spaced patternof specialized cells known as heterocysts develops within each filament. These cellsare regarded as the major if not exclusive site of aerobic nitrogen fixation in hetero-cystous cyanobacteria (see Stanier & Cohen-Bazire, 1977; Haselkorn, 1978; Carr,1979). Little is known about the mechanisms that control the regular spacing ofsingle 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 numberof 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 (interval-width) and the production of adjacent (multiple) heterocysts.

Fogg (X949) suggested that the diffusion from heterocysts of ammonia, or somesimple 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 conceptof a diffusible inhibitor establishing a decreasing concentration gradient away from theheterocyst is still an accepted model for heterocyst pattern control. Mitchison &Wilcox (1972) demonstrated a relationship between cell division and heterocystdifferentiation in Anabaena species. They found that cell division was asymmetricaland heterocysts developed only from the smaller daughter cell of a division arisingoutside the inhibitory zone of an existing heterocyst. This paper reports the productionof double heterocysts following incubation of A. cylindrica, for short periods, at highlight intensities and relates this to changes in cell division.

MATERIALS AND METHODS

Organism and growth conditionsAnabaena 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) supplementedwith ammonium chloride (41HM; A2 N4 medium) in 1-litre Quickfit glass reaction vessels,maintained at 29 ± 0-5 deg. C in a thermostatically controlled water bath. The organism was keptin suspension by magnetic stirring and was gassed with sterile 5 % CO2 in air. For experimentalpurposes 30-ml volumes of culture were incubated in boiling tubes at 29 ± 1 deg. C in a warmroom and gassed with sterile 5 % CO2 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 /tEinsteinscm"2 s - 1 (normal light intensity, NL). For incubation at high light intensity (HL) the boilingtubes were placed mid-way between 2 banks of similar fluorescent tubes, 30 cm apart, oneconsisting of three 40 W tubes, the other of six 20 W tubes. This produced a total incident lightintensity of approximately 500 /iEinsteins cm~2 s"1. An electric fan was used during incubationin boiling tubes at HL to ensure adequate circulation of air and maintain the temperature at29+1 deg. C. For cultures grown in 1-litre reaction vessels, normal light was provided by one60 W incandescent light-bulb 15 cm from the centre of the growth vessel and high lightintensity 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 /<Einsteinscm~2 s"1, respectively. Light intensities were estimated using a model 3000 photometer/radiometer (Macam Photometries Ltd, Livingstone, Scotland, U.K.), and distances weremeasured to the front surface of the bulb or fluorescent tube.

Initiation of heterocyst differentiation

A2 N4-grown cultures of A. cylindrica were transferred to 1-litre sterile polypropylenecentrifuge pots and filaments pelleted by centrifugation at 3000 rev./min and 29 °C for 15 minin a Mistral 6L centrifuge (Measuring and Scientific Equipment Ltd, Crawley, England).

Heterocyst differentiation in A. cylindrica 343

The supernatant was removed and the filaments resuspended in sterile medium lacking ammo-nium chloride (A2 medium). Removal of ammonium chloride from 30-ml cultures in boilingtubes was achieved as follows. The gassing tube and cotton-wool plug were replaced by asquare of sterile aluminium foil, which was pressed over the top of the tube. The cells weresedimented 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 replacedby 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 (pre-incubation) or following transfer (post-incubation). For pre-incubation, samples (30 ml) wereremoved from a 1-litre A2 N4-grown culture and transferred to sterile boiling tubes. Thesewere exposed to HL for increasing periods before transfer to ammonia-free medium, as describedabove, and return to NL. For post-incubation a 1-litre ammonia-grown culture was transferredto A2 medium and 30-ml samples incubated in boiling tubes for increasing periods at HLbefore 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 removedfrom 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 wereexposed to HL for 8 h, followed by increasing periods at NL prior to transfer to ammonia-freemedium. The experiment was arranged in such a way that all samples were transferred tominimal 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 thepresence of ammonium chloride

Cultures were grown on A2 N4 medium in 1-litre glass reaction vessels. The culture wasgrown at normal light intensity for 23-5 h, transferred to the higher light for 12 h and thenreturned to normal light. Changes in optical density and cell division were measured, asdescribed below, throughout the experiment. Growth was estimated turbidimetrically at 750 nmin a Pye Unicam SP600 spectrophotometer. Estimation of cell division was done under phase-contrast microscopy at a magnification of x 2500. A cell was regarded as being in division if ithad a partially formed septum at its centre. A division was counted as equal if the 2 developingdaughter cells could not be said with certainty to be of different sizes. Heterocyst frequency wasestimated at a magnification of x 600. For all cell counts a minimum of 1000 vegetative cellswas 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 thepairs 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 increasedfrequency of double heterocysts when ammonia was later removed. A linear relation-ship was found between the frequency of double heterocysts and the length ofexposure to HL, such that after 10 h almost 20% of the heterocysts that had formedfollowing 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 heterocystsinduced 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 doubleheterocysts (Fig. 2B). In an untreated control culture, the frequency of doubleheterocysts was less than 0-2 %. Growth of cultures at HL for periods in excess of15 h resulted in extensive granulation and fragmentation of filaments, which did notrecover 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 lightintensity.

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 ammonia-free medium resulted in about 7-5 % of the mature heterocysts that developed follow-

Heterocyst differentiation in A. cylindrica 345

30

20 -

D 10

0

+N

A

A

- N

B

/ :

P i i i i i i

12

Time at HL (h)

Fig. 2. Relationship between double heterocyst (DHC) frequency and period ofincubation at high light intensity (HL) in A. cylindrica. Cultures were incubated atHL for the periods indicated, either immediately prior to (A) or following (B) removalof ammonium chloride (+ N or —N) from the medium. DHC frequency was estimated48 h after ammonia removal.

10

Time at NL (h)

Fig. 3. Estimation of the duration of effects produced by high light intensity (HL) inA. cylindrica. Samples of culture were incubated at HL for 8 h, followed by a differentperiod at normal light intensity (NL) for each sample (abscissa) prior to ammoniaremoval. Following ammonia removal samples were incubated at NL. Frequenciesof 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 beforethe removal of ammonia had little effect on this percentage. Incubation at normal Ughfor 4 h, however, resulted in more than a 50 % decrease in the frequency of doubleheterocysts. Following 8 h at normal light, there was little difference between thecontrol 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 inFig. 3 B. In the control there was about 2-5 % of both cell types. Exposure to high lightintensity for 8 h immediately prior to transfer to minimal medium, resulted in anincrease in mature heterocyst frequency to approximately 8% and a decrease in

0-50

0-40

0-30

0-20

010

008

006

NL

-

-

-

-

i

i

>t

HL | NL

1 1 1

10 20 30 40 50 60

Time (h)

Fig. 4. Effect of alterations in light intensity on the growth of A. cylindrica in thepresence of ammonium chloride. The culture was transferred from normal lightintensity (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 (countingdouble heterocysts as 1 cell) also increased from 5% to over 9%. Subsequent in-creasing 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 presenceof ammonium chloride

Under normal light conditions A. cylindrica grew with a mean generation time of24 h. During exposure to the higher light intensity the doubling rate of cultureabsorbance at 750 nm increased to 9-10 h and decreased to more than 100 h onreturn to normal light (Fig. 4). Microscopic examination of filaments from the 2 lightregimes revealed 2 obvious differences. There was a great increase in the granular con-tent 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 theyresemble the cyanophycin granules present in akinetes (Miller & Lang, 1968; Simon,1971; Wildman, Loescher & Winger, 1975). The second major change to occur duringexposure to HL was a considerable increase in cell volume. Measurement of cell sizefrom 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 onA2 N4 medium. A, D, normal light intensity ( x 480 and 750, respectively); B, E, 11 hafter transfer to high light intensity ( x 480 and 750, respectively); c, 13 h after returnto normal light intensity ( x 480). Bars, 10 fim.

exposure, assuming each cell to be a cylinder of uniform diameter. Cultures grown inthe presence of ammonium chloride often contained a low frequency (< 1 %) ofproheterocysts and a small increase in this frequency was usually observed during andafter exposure to high light intensity. Double proheterocysts, however, were notdetected. Even small increases in light intensity induced transient increases in thefrequency of proheterocysts in such cultures.

The changes in cell division during incubation at HL are shown in Fig. 6. During

348 D. G. Adams and N. G. Carr

100

80

60

40

20

0

15

10

5

n

NL

A

-

-

1 1

B

1 i

HL

/

NL

/

\

1 1

0 20 40 60

Time (h)

Fig. 6. Effect of high light intensity (HL) on cell division in A. cylindrica grown onAa N4 medium. The culture was transferred from normal light intensity (NL) toHL and back to NL at the times indicated. The frequencies of: A, number of cells indivision; and B, equal divisions, are indicated.

20

10

0

10

n

- [ l |

A

Pi "B

• A JI -

10 20 30

Interval-width (cells)

Fig. 7. Histograms illustrating the range and frequency of intervals bounded by asingle and a double heterocyst (A) or by 2 single heterocysts (B) in the same cultureof A. cylindrica. The culture was transferred to ammonia-free medium and incubatedat high light intensity for 12 h. It was then returned to normal light intensity andinterval-widths counted 36 h later.

Heterocyst differentiation in A. cylindrica 349

growth at NL the frequency of cells in division (50 %) was relatively constant. Thiswas maintained during the HL period, but increased steadily to about 90 % during thefollowing 15-20 h at normal light (Fig. 6 A). This considerable increase in the numberof cells in division can be clearly seen by comparison of Fig. 5 A and c. Equal divisionsconstituted approximately 5 % of the total during normal growth. This increased byabout 3-fold after 10 h at HL and quickly decreased to slightly less than the originallevel after 15-20 h normal light (Fig. 6 B). Very similar results were obtained when thehigh light intensity was reduced by half (two 75 W bulbs each 15 cm from the centre ofthe growth vessel, producing an incident light intensity of approximately 600 fi Einsteinscm"2 s"1), when a greater than 4-fold increase in the frequency of equal divisions wasobserved.

DISCUSSION

We have examined the influence of high light intensity on the development ofheterocysts immediately following removal of fixed nitrogen from the medium. Thisperiod was chosen since the full complement of proheterocysts are selected within lessthan half the generation time and any effects caused by high light intensity would beenhanced. In cultures growing in the absence of fixed nitrogen heterocysts arecontinually developing, but over the full generation time, which would reduce theinfluence of a relatively short period of incubation at high light intensity. We have noreason to believe, however, that the effects reported here would not apply to suchcultures, and certainly nitrogen-fixing cultures grown for prolonged periods at lightintensities 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 thesmaller daughter of such divisions, arising outside the inhibitory zone of neighbouringheterocysts. A minor reduction in the inhibitory zone of pro- or mature heterocystswould result in the production of more closely spaced heterocysts. Indeed, an increasein heterocyst frequency and a consequent reduction in mean interval-widths, wasobserved when A. cylindrica was allowed to differentiate following incubation at highlight intensity (see Fig. 3B). The reason for this is not clear, although it may resultfrom changes in cell division induced by incubation at HL. The observed increase incell volume during exposure to high light intensity might also have been a contribu-tory factor. If inhibitor production by developing heterocysts did not increase in asimilar manner, then its concentration within vegetative cells would decrease, resultingin diminished zones of inhibition during the early stages of differentiation and theformation of a closer pattern of heterocysts.

Such small changes in inhibitory zones cannot, however, explain the presence ofdouble heterocysts. While a small decrease in inhibitor concentration within a pair ofdaughter cells might induce their premature development, it would not be expected toaffect the competition between them. It should still be possible to select a single cellfor continued development. Proheterocysts can normally divide only during the earlystages 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 aproheterocyst that divides to continue to differentiate and form a double heterocyst.We feel, however, that there is a more likely and attractive explanation. In commonwith 7-azatryptophan and rifampin, incubation at high light intensity resulted in anincrease 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 onlymultiple heterocysts resulting from exposure to HL were doubles. This suggests thatthe effects might originate in cell division and the rapid increase in equal divisionsduring 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 orheterocyst, then the daughter cells would be able to differentiate. Since the 2 cells areidentical, neither would have a selective advantage and both could develop intoheterocysts. This effect might be enhanced by the increased rate of maturation ofproheterocysts observed after exposure to high light intensity. (Fig. 3 B). This wouldfurther distrupt the balance between cell division and differentiation, which normallyensures the development of only single heterocysts.

The plausibility of this explanation is strengthened by a close observation of theshape of double heterocysts. The individual cells of many such pairs were altered inshape, the cell wall common to both being rather flattened and the 2 outer walls beingmore 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 isthat they differentiated from the equal daughters of a very recent division. The shapeof the double heterocyst would depend on the timing of its development in relationto the division. The greater the period of time between a cell's division and itsdifferentiation, the more normal in shape the resulting pair of heterocysts would beexpected to be.

The characteristic shape of many double heterocysts may provide one explanationfor 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 ofdivision and the combination of photographs of variously shaped double heterocystsmay give the appearance of sequential stages of division. In fact, the division occurredbefore differentiation, not after. The authentication of claims for heterocyst divisioncan only arise from the use of time-lapse photography of individual heterocysts.

Double heterocysts could, in principle, be produced from pairs of identical daughtercells 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 nolonger produce a double heterocyst. This can indeed be seen in Fig. 3 in which thismaximum time is 6-8 h. This may represent the time following division during whichany cell remains able to differentiate when induced to do so. It may also be the timerequired for a pair of identical daughter cells to become sufficiently dissimilar, perhapsdue 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 ofequal 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, therebeing little difference in intervals bounded by 2 single heterocysts or by a single and adouble heterocyst. This is illustrated in Fig. 7 in which the 2 histograms represent theintervals between single heterocysts (Fig. 7B), and between single and double hetero-cysts (Fig. 7 A) in the same culture. There was very little difference between the 2ranges of interval-width and this can be explained as follows. If the heterocystinhibitor was lost or destroyed at a rate proportional to its concentration, then anexponential 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 thesource inhibitor (i.e. a double heterocyst instead of a single) would have little effecton the inhibitory zone generated and the interval to the next heterocyst would berelatively unchanged.

It is clear that the rapid rate of increase in culture absorbance obtained duringexposure to HL was largely due to increases in both the volume of cells and theirgranular content, rather than an increase in cell division rate. Changes in cell numbersare difficult to measure in A. cylindrica since it is a filamentous organism and growsslowly. However, a rapid increase in the number of cells in division was observedfollowing return to normal light (Fig. 6 B) and this permitted the organism to reduceboth its cell volume and granular content to pre-exposure values. Thus, althoughgrowth, measured by changes in A750, appeared to have almost ceased on return tonormal light, cell division was occurring with the result that the cells had almostreturned 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 im-portance of such asymmetry in ensuring that only one of two daughter cells, whichcommence their cell cycles together, will develop into a heterocyst. This is an essentialprerequisite for the attainment of a spaced pattern of single heterocysts. The resultsalso indicate that a cell is susceptible to differentiation only during the period approxi-mately 0-8 h following its formation. This corresponds to the candidate cell describedby Wilcox et al. (1973)-'a smaller daughter in the period immediately after adivision' - and confirms the conclusion of Bradley & Carr (1977), that the response ofany 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 candifferentiate. The attainment of the final regular pattern of heterocysts, however,requires more complex intercellular interactions, the nature of which are as yetpoorly understood.

This work was supported by the Science Research Council.

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(Received 13 October 1980)