The Plant Cell, Vol. 3, 191-201, February 1991 O 1991 American Society of Plant Physiologists

Hormogonium Differentiation in the Cyanobacterium Calothrix: A Photoregulated Developmental Process

Thierry Damerval, Gérard Guglielmi, Jean Houmard, and Nicole Tandeau de Marsac'

Unité de Physiologie Microbienne, Centre National de Ia Recherche Scientifique, Unité de Recherche Associée 11 29, lnstitut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France

Hormogonium differentiation is part of the developmental cycle in many heterocystous cyanobacteria. Hormogonia are involved in the dispersa1 and survival of the species in its natural habitat. The formation of these differentiated filaments has been shown to depend on severa1 environmental conditions, including spectral light quality. We report here morphological and ultrastructural changes associated with the formation of hormogonia, as well as optimal light conditions required for their differentiation in the cyanobacterium Calothrix sp PCC 7601. The action spectrum for hormogonium differentiation is similar to that which triggers complementary chromatic adaptation because red and green radiation display antagonistic effects in both cases. However, these two photoregulated processes also show major differences. Transcription analyses of genes that are specifically expressed during hormogonium differentiation, as well as of genes encoding phycobiliproteins, suggest that two different photoregulatory pathways may exist in this cyanobacterium.

INTRODUCTION

Cyanobacteria constitute one of the most diverse groups of the prokaryote kingdom. They present an ubiquitous distribution and show great capacity to withstand adverse environmental conditions (Stanier and Cohen-Bazire, 1977). They are all photoautotrophic organisms capable of oxygenic photosynthesis, like phototrophic eukaryotes, and there now exist convergent lines of evidence support- ing the endosymbiotic theory of the cyanobacterial origin of chloroplasts (Gray and Doolittle, 1982; Woese, 1987; Giovannoni et al., 1988; Turner et al., 1989). As in chloro- phyta, the light-dependent activities of cyanobacteria are strongly wavelength dependent because they are based on photochemical reactions driven by pigments that ab- sorb in discrete regions of the spectrum. Although chlo- rophyll a-protein complexes constitute their photosynthetic reaction centers, cyanobacteria, in contrast to higher plants, contain no chlorophyll b. The light-harvesting an- tennae are composed of phycobiliproteins, which are or- ganized into supramolecular structures called phycobili- somes, arrayed perpendicular on the stromal surface of the thylakoid membranes (for reviews, see Cohen-Bazire and Bryant, 1982; Gantt, 1984; Glazer, 1989). The phy- cobilisome is composed of two domains: the core, which is mainly made of allophycocyanin, and the rods, which are composed of hexameric complexes of either constitu-

' To whom correspondence should be addressed.

tive phycocyanin and inducible phycocyanin under red light or darkness or constitutive phycocyanin and phycoerythrin under green light (for reviews, see Tandeau de Marsac, 1983; Grossman et al., 1986, 1988; Tandeau de Marsac et al., 1988). A number of cyanobacterial species are thus able to modulate their pigment content to optimize their performance in lights of different irradiance and spectral quality. This phenomenon, named complementary chro- matic adaptation (CCA), results in a synthesis of pigments whose absorption spectra are complementary to the spec- tral characteristics of the incident light. CCA is probably governed by at least one photoreversible pigment, pre- senting some analogy to the phytochrome of plants, al- though the most efficient wavelengths for its interconver- sion are different (green versus red radiation, instead of red versus far-red) (for reviews, see Bogorad et al., 1983; Tandeau de Marsac, 1983; Grossman et al., 1988; Tan- deau de Marsac et al., 1988).

In addition, some multicellular cyanobacteria possess a variety of life cycles quite without parallel in bacteria. Many of the filamentous species produce specialized structures in response to environmental factors. These structures may promote survival and reproduction or have specific metabolic functions. The best known example is the de- velopment of heterocysts, which differentiate from vege- tative cells under conditions of nitrogen starvation (see Wolk, 1982). During this irreversible differentiation proc- ess, the transcription of some genes is activated, whereas

192 The Plant Cell

that of others is repressed, and specific DNA re- arrangements occur (Haselkorn et al., 1983; Golden et al., 1985, 1987; Haselkorn, 1986, 1989).

In some filamentous strains, vegetative reproduction occurs by fragmentation of the trichome into short fila- ments called hormogonia. These are distinguishable from mature trichomes by cell size, cell shape, gas vacuolation, and the absence of heterocysts (Rippka et al., 1979). Hormogonium differentiation has not been fully investi- gated. It has been shown that these filaments may be formed abundantly after transfer of cultures to fresh me- dium and, for some strains, this differentiation process has been reported to be dependent upon the spectral light quality (Lazaroff, 1966; Tandeau de Marsac, 1983; Rippka and Herdman, 1985; Herdman and Rippka, 1988; Tandeau de Marsac et al., 1988).

CCA has been analyzed in detail, and many light-regu- lated genes have been isolated and characterized for the cyanobacterium Calothrix sp PCC 7601, which is also able to differentiate hormogonia (Tandeau de Marsac, 1983; Grossman et al., 1986, 1988; Tandeau de Marsac et al., 1988). Our aims were to understand photoregulation from signal perception to gene expression through the trans- duction chain(s) and to analyze the correlation between these two photoregulated processes. We describe the different aspects of the differentiation process, i.e., mor- phological and ultrastructural changes, as well as the physiological conditions that lead to the formation of hor- mogonia. Then we present and discuss the regulation of the expression of light-regulated genes during this differ- entiation process.

RESULTS

Morphological and Ultrastructural Changes

Figure 1 shows the major morphological and ultrastructural changes that occur during hormogonium differentiation in the cyanobacterium Calothrix sp PCC 7601. Hormogonium differentiation may begin in any cell within a filament and then progresses to neighboring cells along the trichome (data not shown). Under light microscopy, the differentia- tion process is characterized by a reduction in cell size, which starts to be detectable at 6 hr, and a fragmentation of the trichomes into short chains of cells that occurs between 12 hr and 24 hr of differentiation. Reduction in cell size results when one to three cell divisions take place without elongation. As a consequence, because little DNA replication takes place during hormogonium differentiation, the DNA that occurs as multiple copies of the genome in vegetative cells is reduced to about five genome copies in hormogonial cells (Herdman and Rippka, 1988). In Figure 1, we can also see that hormogonial cells are filled with

refractile granules that appear as aggregates of gas vesi- cles on the electron micrograph. Gas vesicle formation begins between 3 hr to 6 hr and aggregates increase in size until 24 hr, the average length of individual vesicles being about 200 nm at 6 hr and 800 nm to 1000 nm at 24 hr (Damerval et al., 1988).

An electron micrograph of an isolated gas vesicle is shown in Figure 2A. Comparison of Figures 26 and 2C reveals that pores are formed within the peptidoglycan layer of the cell envelopes during the course of hormogon- ium differentiation. These pores are localized close to the newly formed septa. On the other hand, as shown in Figure 2D, the hormogonial cell wall possesses fibrilar substruc- tures (hereafter termed pili) that radiate from the cell sur- face. These substructures are absent from the mature trichomes.

Chromatic Control of Hormogonium Differentiation

Hormogonium differentiation can be initiated when cells are transferred into a fresh culture medium (Rippka and Herdman, 1985; Herdman and Rippka, 1988; Tandeau de Marsac et al., 1988). However, the succession of events leading to fully differentiated hormogonia is strongly de- pendent on the light conditions during the first 12 hr of induction. As shown in Table 1, hormogonia can be pro- duced after a shift from white or green light to red light. A low number of differentiated cells can also be produced when exponentially growing cells are transferred into a fresh culture medium under white light. However, whatever the spectral quality of the light received during the precul- ture, 100% of the filaments differentiate into hormogonia when cells are transferred into fresh culture medium and further incubated under red light.

Using colored filters, we have defined light-dependent conditions required to optimize hormogonium differentia- tion. Cells were grown under white, yellow, red, purple, green, or blue light, under the same photosynthetic photon flux density (PPFD): 1 O pmol . m-*. sec-’, and then trans- ferred during the exponential phase of growth into fresh culture medium. To avoid changes in PPFD during this transfer, we did not dilute the cultures, but transferred them at the same cell density. As shown in Figure 3, 100°/~ of hormogonium differentiation was obtained when cells were incubated under red light during the differentiation process. However, no differentiation was observed when only green light was provided. When both green and red light were present (i.e., under white or yellow illumination), the rate of differentiation was greatly reduced. Further- more, we observed that the rate of differentiation increased with the ratio of red light to green light when both wave- lengths were provided (data not shown). It thus appears that red light activates hormogonium differentiation, whereas green light has an inhibitory effect. As shown in

Cell Differentiation in a Cyanobacterium 193

Ohr 6hr 24 hr

Figure 1. Morphological and Ultrastructural Changes during Hormogonium Differentiation.

(A) Light micrographs of Calothrix sp PCC 7601 vegetative trichomes (0 hr), filaments after 6 hr of hormogonium differentiation, and fullydifferentiated hormogonia after 24 hr. Arrows show cell divisions without elongation that occur during the differentiation of hormogonia.Bars = 20 urn.(B) Electron micrographs of a thin section of cells collected before (0 hr) and after (6 hr and 24 hr) the induction of hormogoniumdifferentiation. Arrows show gas vesicles (gv). Bars = 1 ̂ m.

194 The Plant Cell

Figure 2. Gas Vesicle, Pores, and Pili.

(A) Electron micrograph of an isolated gas vesicle from Calothrixsp PCC 7601.(B) Electron micrograph of a thin section of vegetative cells. Bar= 0.1 /jm.(C) Electron micrograph of a thin section of hormogonial cellsshowing pores (Po) that are formed in the peptidoglycan layerduring the differentiation process. Bar = 0.1 /im.(D) Electron micrograph of a negatively stained hormogoniumshowing the fibrilar or "pill-like" structures (Pi) formed duringdifferentiation. Bar = 0.1 urn.

Figure 3, blue light did not promote hormogonium forma-tion. However, a red light effect, leading to 100% differ-entiation, was obtained when red radiation was providedtogether with blue (purple light), indicating that blue radia-tion does not inhibit the process. For all of the lightconditions tested during hormogonium differentiation, thesame results were obtained whatever the spectral qualityof the light received by the cells during their preculture.

As shown in Table 2, no differentiation was observedafter transfer of the cells to darkness in the presence orabsence of 10 mM glucose, a utilizable energy and carbonsource for this strain. Similar results were obtained whencells were precultured under heterotrophic conditions(darkness + glucose), under photoheterotrophic conditions(white light + DCMU + glucose), or under white light in thepresence or absence of glucose. Tables 3 and 4 show theeffects of different PPFD received during hormogoniumdifferentiation and the effect of several times of exposureto red light. A photon flux of 1 jumol-nv2-sec~1 wassufficient to fully induce formation of hormogonia underred light. When cells were transferred to red light fordifferent periods of time and then incubated in the dark,the percentage of differentiation increased with the time ofexposure to red light; 80% was observed when cells wereincubated under red radiation during the first 12 hr of thedifferentiation process and 100% was obtained only whencells were continuously incubated under red light for 24hr. It thus appears that a continuous red illumination, dimor bright, received by the cell during the differentiationprocess is necessary to obtain maximal differentiationwhen exponentially growing cells are transferred into freshculture medium.

Table 1. Effect of Transfer into Fresh Culture Medium onHormogonium Differentiation

% Differentiation

Transfer: Same Medium Fresh Medium

White Red Green White Red GreenLight Light Light Light Light Light

PrecultureWhite lightRed lightGreen

light

000

750

100

000

0-300-300-30

100100100

000

Cells were grown in BG-11 medium and transferred during theexponential phase of growth either into the same medium or intofresh medium. Counting for hormogonium differentiation was per-formed after 24 hr of induction. PPFD = 10 nmol-m~2-sec~1.

Cell Differentiation in a Cyanobacterium 195 - DlFFERENTl ATlON

WHlTE

% DlFFERENTlATlON

480nm YELLOW

J RED 590 nm

PURPLE 460nm 600 nm I

BLUE 475 nm 725 nm

GAEEN 475nm 570nm 725 nm

DARKNESS

30-50

30-50

1 O0

1 O0

O

O

O

400 800 I 500 ;y 7y *"Y'.lenglh ,nm, 300

INHlBlTlON ACTlVATlON

Figure 3. Chromatic Control of Hormogonium Differentiation.

White boxes correspond to transmission above 25%. Black boxes correspond to transmission below 25%. Exponentially growing cells were transferred into a fresh culture medium, and counting for hormogonium differentiation was performed after 24 hr. PPFD = 1 O pmol. m+. se&.

Expression of the Gas Vesicle Genes

RNA gel blot hybridization experiments have been per- formed between a DNA probe carrying the gvpA7 gene and total RNAs sampled during succeeding intervals of time after induction of hormogonium differentiation. Four gvp mRNAs and an antisense RNA could be revealed with

this probe (Csiszar et al., 1987). The profile of the tran- scripts detected during differentiation is shown in Figure 48. At the time of induction (O hr), no transcript corre- sponding to the expression of the gvp genes was present. Significant levels of mRNA were first detected between 1.5 hr and 3 hr, and transcription of both the gvp genes and the antisense RNA became maximal at 6 hr. Degra- dation of the larger gvp transcripts then rapidly occurred between 9 hr'and 12 hr. This time course was observed when the PPFD was greater than 10 pmol.m-2.sec-'. Under lower illumination, the time course of expression was delayed, but similar patterns were observed (data not shown). As shown in Figure 4A, transfer to green light after 3 hr or 6 hr of induction under red light inhibited the formation of hormogonia. The differentiation process was incomplete, and cells rapidly reverted to mature trichomes. In addition, as shown in Figure 4B, exposure to green light after 3 hr of induction under red light repressed the expres- sion of the gvp genes.

Expression of the Phycobiliprotein Genes

Because the action spectrum for hormogonium differentia- tion is similar to that for CCA, we examined the transcrip- tion of genes encoding major phycobilisome components during hormogonium differentiation. As shown in Figure 5, RNA gel blot hybridizations performed with DNA probes corresponding to the major phycobiliproteins revealed that the transcription of all of these genes was totally and specifically arrested when the genes involved in gas vesicle formation were expressed. However, it is worth noting that phycoerythrin transcripts could be detected for a longer time than the other phycobiliprotein mRNA species. Be- tween 9 hr and 12 hr, when the gvp genes were repressed, the expression of the genes encoding allophycocyanin, phycocyanin-1 , and phycocyanin-2 restarted. As expected, the genes encoding phycoerythrin were not activated be-

Table 2. Effect of a Carbon Source on Hormogonium Differentiation

70 Differentiation

Transfer: Darkness Red Light + Red Red Light + Glucose Red Light

Darkness Glucose Light + Glucose + DCMU + DCMU

Preculture Darkness + glucose O O 1 O0 1 O0 1 O0 1 O0 White light + DCMU + glucose O O 1 O0 1 O0 1 O0 1 O0 White light + glucose O O 1 O0 1 O0 100 1 O0 White light O O 1 O0 1 O0 1 O0 1 O0

Cells were grown under white, red, or green light or in the dark in BG-1 1 medium and transferred during the exponential phase of growth into fresh culture medium. Counting for hormogonium differentiation was performed after 24 hr of induction. When present, glucose and DCMU were added at a final concentration of 1 O mM and 10-5 M, respectively. PPFD = 10 Fmol. m-'.sec-'.

196 The Plant Cell

~

Table 3. Effect of PPFD under Red Liqht

YO Differentiation

Transfer: Darkness Red Lighta

Preculture Darkness O 1 O0 White light O 1 O0 Red light O 1 O0

Conditions were the same as in Table 2. a l , 10, 25, 50, or 100pmo1.m-2.sec-’.

cause cells are maintained under red light. In contrast, the transcription of the genes encoding ribulose-bisphosphate carboxylase/oxygenase subunits does not seem to be affected by the differentiation process (Figure 5).

DlSCUSSlON

Properties and Function of Hormogonia

The cellular events leading to the formation of hormogonia are summarized in Figure 6. This differentiation process is characterized by the synthesis of gas vesicles, the frag- mentation of the trichomes into short chains of cells, the formation of pores, and the synthesis of pili. Gas vesicles are multimolecular structures filled with gas that provide cells with buoyancy (for reviews, see Walsby, 1978, 1987; Tandeau de Marsac et al., 1988; Walsby and Hayes, 1989). These cell inclusions, therefore, provide floating capacities to differentiated hormogonia. On the other hand, the pores that are formed in the peptidoglycan layer might constitute weak points that could facilitate the fragmentation of the trichome. Such a function has been suggested for oscilla- torian cyanobacteria for which the peptidoglycan layer tears along a zipper-like circumferential set of junctional pores (Lamont, 1969). Coupled with the formation of gas vesicles, the reduction in the length of the filaments en- hances the floating capacities of hormogonia because it has been shown that the sinking velocity of cyanobacterial filaments is inversely correlated with their length (Davey

and Walsby, 1985). Although the mechanism of gliding movement remains unclear, hormogonium motility seems to be correlated with the occurrence of the pili at their cell surface. In fact, the presence of such structures has been observed in many cases of motile bacteria (Halfen and Castenholz, 1971 ; Kaiser, 1979).

The formation of hormogonia is a rather unique differ- entiation process in that it may affect all of the cells within a filament and is fully reversible. The hormogonial state is always transient; as growth proceeds, regeneration of vegetative filaments occurs with a concomitant loss of gas vesicles, pores, and pili. However, when placed under conditions inappropriate for growth, hormogonia can sur- vive for a longer time than vegetative filaments. For ex- ample, we observed that hormogonia can be maintained at 4OC for severa1 weeks with no loss of their regenerative capacities or viability, whereas vegetative filaments do not survive this treatment (data not shown). Therefore, regen- eration may wait until appropriate growth conditions be- come available. In natural environments, hormogonia may be released from a sessile parenta1 colony, glide and float away, or be washed by water currents to a new location (Wyatt et al., 1973). Consequently, hormogonia seem to be involved in the dispersa1 of the species in its natural habitat and likely allow its survival in some hostile environments.

Photoregulation of Gene Expression

From the results presented in Tables 2,3, and 4, it appears that red light promotes hormogonium differentiation, whereas green light has an inhibitory effect. These results suggest that, as already proposed for CCA, a photorev- ersible pigment might be involved in the control of hormo- gonium differentiation, the most efficient wavelengths for its interconversion being located in the same regions of the light spectrum. Furthermore, during the differentiation process under red light, the presence of DCMU, glucose, or both did not inhibit the formation of hormogonia, sug- gesting that this differentiation process is largely independ- ent of the photosystem II activity, as well as of the available carbon source. The response of the developmental phe- nomenon to much lower levels of light energy than those

Table 4. Effect of Transfer to Darkness after Different Periods of Time under Red Light

O/i Differentiation

Transfer: Red Light: 30 min 1 hr 3 hr 6 hr 12 hr 24 hr + Darkness: 23 hr, 30 min 23 hr 21 hr 18 hr 12 hr O hr

Preculture White liaht O O 30 30 80 1 O0

Conditions were the same as in Table 2.

Cell Differentiation in a Cyanobacterium 197

-A-

RL 3hr RL 6hr RL 24hr

t tGL 21hr GL 18hr

-B-

RL*0 1.5 3 6 9 12 24 48 hr

I I I I I I I I

AP RL PC10 1.5 3 6 912 24 hours 0 1.5 3 6 9 12 24 hours

RL GL

+ *0 1 5 3 6 9 12 24 48 hr

I I I I I I I I

0.6 —

04 _

0.3 —

r..RL PC2 (RL-specific) RL PE (GL-specific)

i *0 1.5 3 6 9 12 24 hours 0 1.5 3 6 9 12 24 hoursI I I I I I I I I I I I I I

Figure 4. gvp Gene Expression during Hormogonium Differentia-tion and Effect of a Transfer from Red to Green Light.

(A) Light micrographs of cells induced for differentiation under redlight (RL) and then transferred to green light (GL) after 3 hr (RL 3hr-»GL 21 hr) or 6 hr (RL 6 hr-.GL 18 hr) of induction. The control(RL 24 hr) shows fully differentiated hormogonia after 24 hr ofincubation under red light.(B) Top panel: RNA gel blot hybridization of Calothrix sp PCC7601 total RNAs extracted at various times during hormogoniumdifferentiation under continuous red light. The probe, described inMethods, carries most of the gvpA1 gene that encodes the majorgas vesicle structural protein. Bottom panel: RNA gel blot hybrid-ization of Calothrix sp PCC 7601 total RNA extracted at differenttimes after induction under red light followed, 3 hr later, by atransfer to green light. The probe, described in Methods, carriesmost of the gvpA1 gene that encodes the major gas vesiclestructural protein, kb, kilobase pair.

PL RuBPt0 1.5 3 6 9 12 24 hours

required for photoautrophic growth also indicates that thephotomorphogenetic effect is independent of the photo-synthetic activity.

It has been shown previously that the transcription ofthe Calothrix sp PCC 7601 gvpA1A2C operon resulted inthree mRNA species 0.3 kb, 0.8 kb, and 1.4 kb long. Afourth mRNA species, 0.6 kb long, corresponds to thetranscription of the gvpD gene, and an antisense RNA, 0.4kb long, starts in gyp/42 and ends in gvpA1 (Csiszar et al.,1987; Damerval et al., 1987). The expression of the gasvesicle genes is induced during the induction of hormo-

Figure 5. Gene Expression during Hormogonium Differentiation.

Cells growing in white light were collected in exponential growthphase and resuspended in fresh culture medium under red light(RL). Total RNA was extracted at different times during hormo-gonium differentiation. Probes are described in Methods. AP,allophycocyanin, constitutively expressed in vegetative cells; PC1,phycocyanin-1, constitutively expressed in vegetative cells; PC2,phycocyanin-2, red light inducible; PE, phycoerythrin, green lightinducible; RuBP, large subunit of ribulose-bisphosphate carbox-ylase/oxygenase. kb, kilobase pair.

198 The Plant Cell

Mature tric home Differentiation Hormogonium

O 1.5 3 6 9 12 24

I I I 1 I I - - - I_

cell division without elongation trichome fragmentation I- I I 1

formation of peptidoglycan pores: synthesis of pili

t I

formation of gas vesicles

Regeneration into mature trichome

72-96

I time (hr) * loss of pili and gas vesicles: cell elongation and division

gvp genes repressed degradation oí transcripts

gvp genes activated

-ame- PB genes repressed - PB genes activated

Figure 6. Timing of Hormogonium Differentiation in Calothrix sp PCC 7601.

This figure summarizes the major morphological and ultrastructural changes that occur during the differentiation of mature trichomes (O hr) into hormogonia (24 hr), as well as the time course for the expression of some genes (gvp, gas vesicle protein; PB, phycobiliprotein). The top line indicates the time scale after the beginning of induction for hormogonium differentiation.

gonia under red light, whereas it is arrested when cells are transferred to green light after a period of induction under red radiation. Unexpectedly, when the genes encoding gas vesicles are activated, the expression of the genes encod- ing phycobiliproteins is repressed. In vegetative cells, it has been demonstrated that the expression of allophyco- cyanin and phycocyanin-1 is wavelength independent (Houmard et al., 1988; Mazel et al., 1988), whereas the synthesis of phycocyanin-2 and phycoerythrin is depend- ent upon the spectral quality of the incident light. Whereas phycocyanin-2 is present in cells receiving red radiation or cultivated in the dark, the genes encoding phycoerythrin subunits are specifically expressed when incident light contains green radiation. In both cases, the regulation of gene expression has been shown to occur at the transcrip- tional leve1 (Conley et al., 1985; Mazel et al., 1986; Oel- müller et al., 1988). Taken together, these results confirm that a red light/green light antagonistic effect exists for both processes, and they raise the interesting question of the extent of the similarity between the mechanisms in- volved in gene regulation during CCA and during hormo- gonium differentiation.

It has been proposed that in Nostoc sp or Calothrix sp strains the same photoreceptor could be involved in the differentiation of hormogonia and in the control of phycob- iliprotein synthesis during CCA (for a review, see Tandeau

de Marsac, 1983). However, we have shown that major differences exist between these two biological phenom- ena. First, differentiation of hormogonia can only proceed under continuous red illumination, even though the PPFD required can be as low as 1 pmol . m-'. sec'. In contrast, CCA can be induced by short pulses of appropriate light wavelength (Oelmüller et al., 1988). Second, darkness is equivalent to red light for CCA (Tandeau de Marsac, 1983), whereas no hormogonium differentiation occurs in com- plete darkness, even if cells are preilluminated with red light and/or subsequently incubated under heterotrophic conditions. Third, transcription of each of the phycobilipro- tein genes tested is repressed during hormogonium differ- entiation. These results might suggest that two photorev- ersible systems presenting different photobiological fea- tures are involved in CCA and in hormogonium differentiation. Alternatively, different forms of a unique photoreceptor could regulate the expression of the differ- ent sets of genes through a sequence of events that constitute, at some stages, divergent signal transduction pathways in which at least a few steps are mutually exclusive.

In prokaryotic organisms, the most studied models for regulatory processes are found in bacterial sporulation and in the lytic development of bacteriophages. In both cases, as well as during heterocyst differentiation in cyanobac-

Cell Differentiation in a Cyanobacterium 199

teria, a major role has been assigned to the transcriptional apparatus, which appears to be modified to recognize different promoters at different times. Transcription speci- ficity most often resides in the interaction between the RNA polymerase holoenzyme and the promoter site of genes or operons. The RNA polymerase subunit that con- trols the specificity of this interaction has been shown to be the u factor because the polymerase core itself does not specifically recognize promoter sites (Doi and Wang, 1986). Because sequential induction and/or repression of sets of genes govern gene expression during hormogon- ium differentiation, transcription mechanisms are likely to differ from those that occur in cells from the mature trichomes. Therefore, it seems that either different u fac- tors and/or different auxiliary transcriptional effectors reg- ulate gene expression in Calorhrix sp PCC 7601. At pres- ent, only a limited number of promoter sequences have been characterized that are all rather different, and proofs for the existence of different c factors are likely but still await demonstration.

Strain PCC 7601 is a member of the Tolypothrix tenuis type of Calothrix strains (Rippka, 1988) that shows a complex life cycle involving green light-type and red light- type cells, differentiation of gas-vacuolated hormogonia as well as heterocyst differentiation under conditions of nitro- gen starvation. Preliminary observations indicate that some interactions exist in the regulatory mechanisms that govern the two differentiation processes, i.e., heterocyst formation and hormogonium differentiation. First, when cells are transferred into a medium lacking combined nitro- gen under red light, heterocyst formation follows hormo- gonium differentiation and only takes place when hormo- gonial cells return to the vegetative stage. Second, we observed that both heterocyst differentiation and their associated nitrogen-fixing ability are promoted by green light (T. Damerval and G. Guglielmi, unpublished data). This result is in full agreement with previous observations reported for another chromatically adapting strain, Nosroc sp (Wyman and Fay, 1987), and for the cyanobiont Ana- baena azollae, in which green light causes a dramatic increase in heterocyst frequency (Wu et al., 1982). It would be of interest to examine in more detail the chromatic regulation of heterocyst differentiation and nitrogen fixa- tion, as well as the correlations between nitrogen metab- olism and hormogonium differentiation, to establish the nature of the regulatory networks that occur in this cyano- bacterial genus.

METHODS

Materials

Restriction enzymes were purchased from either Boehringer or Genofit. CI-~’P-~CTP (3000 Ci/mmol) and multiprime DNA labeling

kit were from Amersham. Enzymes were used according to the manufacturer’s instructions. All chemicals were reagent grade.

Culture Conditions

The strain Calothrix sp PCC 7601 (Fremyella diplosiphon UTEX 481) was grown in BG-11 medium (Rippka et al., 1979) at 25OC. Cultures were continuously bubbled with 1 COz/990/o air and illuminated with cool-white fluorescent tubes or with chromatic light provided by colored cellulose acetate filters interposed be- tween the culture vessel and the fluorescent tubes. Photosyn- thetically active radiations between 400 nm and 700 nm were measured with a LI-COR LI-1 858 quantum/radiometer/photome- ter equipped with an LI-l90SB quantum sensor; the PPFD was expressed as micromoles per square meter per second. When present, glucose was added at 10 mM (final concentration) and DCMU (Sigma) at 10-5 M (final concentration). For induction of hormogonium differentiation, cells were harvested by filtration through glass microfiber filters (Whatman GF/A) and resuspended in the same volume of fresh culture medium.

RNA Purification and Hybridization with 32P-Labeled Probes

Total RNAs were isolated as described (Mazel et al., 1986). RNA gel blot transfer and hybridization were performed as previously described (Damerval et al., 1987). Hybridizations were done at 55°C in 50% formamide, conditions which allow no more than 20% mismatch (Casey and Davidson, 1977).

The gas vesicle protein probe was a 237-bp Hincll-Hindlll fragment containing 186 bp of the Calothrix sp PCC 7601 gvpA7 gene (formerly designated gvpA) encoding the major structural gas vesicle protein and 51 bp of its downstream region (Tandeau de Marsac et al., 1985). The allophycocyanin probe was a 1.2-kb- long Dral fragment carrying the Calothrix sp PCC 7601 apcA787 genes, encoding a-subunits and 0-subunits of allophycocyanin (Houmard et al., 1988). The phycocyanin-1 probe was a 1-kb Hindlll fragment covering the cpc67 gene and the 5’ end of the cpcA7 gene, encoding 0-subunits and a-subunits of phycocyanin- 1, respectively (Mazel et al., 1988). The phycocyanin-2 probe was a 640-bp-long Hpal DNA fragment internal to the cpcB2A2 gene cluster from Calothrix sp PCC 7601, encoding the P-subunits and a-subunits of phycocyanin-2 (Capuano et al., 1988). The phy- coerythrin probe was a 205-bp-long Hindlll-Xbal DNA fragment internal to the cpeB gene from Calothrix sp PCC 7601 that encodes the P-subunit of phycoerythrin (Mazel et al., 1986). The ribulose-bisphosphate carboxylase/oxygenase probe was a 1.5- kb Pstl DNA fragment carrying the rbcL gene from Synechococ- cus sp PCC 6301 encoding the large subunit of ribulose-bisphos- phate carboxylase/oxygenase (Shinozaki et al., 1983). Plasmid DNA purifications were performed as described previously (Tan- deau de Marsac et al., 1985). Restricted DNA fragments were eluted as described (Vogelstein and Gillepsie, 1979).

Estimation of Hormogonium Differentiation

After 24 hr of induction, cells were harvested by filtration through glass microfiber filters (Whatman GF/A) to prevent gas vesicle

200 The Plant Cell

collapsing. Filaments were then photographed at low magnifica- tion, five to 1 O fields being taken to provide representative sam- ples. About 500 cells were then counted and the percentage of differentiation was expressed as the ratio of hormogonial cells to the total number of cells.

Electron Microscopy and Gas Vesicle lsolation

Gas vesicles were isolated as described (Damerval et al., 1991). Cells and gas vesicles were prepared for electron microscopic studies as described previously (Guglielmi and Cohen-Bazire, 1982).

ACKNOWLEDGMENTS

We thank Michael Herdman for critical reading of the manuscript and Didier Mazel for helpful discussions. This work was supported by the Centre National de Ia Recherche Scientifique, Unite de Recherche Associée 11 29 and by the lnstitut Pasteur.

Received August 6, 1990; accepted December 11, 1990.

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DOI 10.1105/tpc.3.2.191 1991;3;191-201Plant Cell

T. Damerval, G. Guglielmi, J. Houmard and N. T. De MarsacDevelopmental Process.

Hormogonium Differentiation in the Cyanobacterium Calothrix: A Photoregulated

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