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Commentary 1

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Preventing accidental heterocyst development in cyanobacteria 3

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Wei-Yue Xing1,2

and Cheng-Cai Zhang1#

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1State Key Laboratory of Freshwater Ecology and Biotechnology and Key Laboratory of Algal 9

Biology, Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan, Hubei 430072, 10

People’s Republic of China 11

2University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China 12

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#Corresponding author. Institute of Hydrobiology, the Chinese Academy of Sciences, Donghu 15

Nan Road 7, 430072 Wuhan, Hubei, People’s Republic of China. Email: cczhang@ihb.ac.cn. 16

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JB Accepted Manuscript Posted Online 17 June 2019J. Bacteriol. doi:10.1128/JB.00349-19Copyright © 2019 American Society for Microbiology. All Rights Reserved.

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Abstract 20

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The filamentous cyanobacterium Anabaena can form heterocysts specialized in N2 fixation, 22

mostly through a cascade of transcriptional activation in response to the nitrogen-starvation signal 23

2-oxoglutarate. It is reported now that a transcription repressor, CalA, acts as a safety device to 24

prevent heterocyst development under certain conditions where 2-oxoglutarate level may touch 25

the threshold to trigger unnecessary initiation of heterocyst development. Such a control may 26

increase the fitness of Anabaena under a constantly changing environment. 27

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Labor division and cell-cell interaction are characteristic of certain cyanobacteria, making 30

these organisms a wonderful prokaryotic model for the understanding of multicellular behaviors, 31

from basic mechanism to evolutionary biology. The genetic model the most frequently used for 32

such studies, is Anabaena (Nostoc) PCC 7120 (Anabaena hereafter), a filamentous and 33

facultative diazotrophic cyanobacterium (1). Anabaena can use either combined nitrogen such as 34

ammonium or nitrate, or atmospheric N2 for its growth, with a hierarchy in the preference of the 35

nitrogen sources: ammonium being the most economic nitrogen source, followed by nitrate which 36

needs to be reduced to ammonium by nitrate reductase and nitrite reductase (Fig. 1). N2 is the 37

most costly nitrogen source since its reduction into ammonium catalyzed by nitrogenase 38

consumes ATP and requires a micro-oxic environment for the protection of nitrogenase against 39

oxygen inactivation. In the case of Anabaena, the micro-oxic environment is provided by 40

heterocyst, a cell type specialized in N2 fixation. Heterocyst differentiation is triggered by the 41

deprivation of combined nitrogen in the growth medium and the process takes 20-24 h. Thus far, 42

the model on the initiation of heterocyst differentiation relies mostly on transcriptional activation, 43

in particular a regulatory circuit composed of 2-oxoglutarate (2-OG) acting as a nitrogen 44

starvation signal, and two transcription factors NtcA (a receptor of 2-OG) and HetR (master 45

regulator of heterocyst differentiation), together activating heterocyst differentiation (2). Now, in 46

a study reported in this issue, Higo et al., propose that beyond the activating mechanism 47

summarized above, a repression mechanism may exist to prevent accidental development of 48

heterocyst under certain conditions (3). The authors found that calA, encoding a transcriptional 49

repressor essential in cyanobacteria, exercises a negative control over the initiation of heterocyst 50

development in the presence of nitrate that normally inhibits heterocyst differentiation. When 51

calA is knocked down, the inhibitory effect of nitrate on heterocyst differentiation is partially 52

relieved and this effect seems to be amplified with a carbon oversupply. Although, the data 53

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reported are short of providing all the necessary evidence for such a negative control mechanism 54

involved in the initiation of heterocyst differentiation, the concept itself and its significance 55

should prompt the scientific community to investigate into the matter. calA is also known in other 56

cyanobacteria as cyabrB, and in the paper in question, both names are used. For simplicity and 57

by respect to those authors who published previous works in Anabaena under the name of calA, 58

we suggest to keep the original name. 59

To understand what is happening, let us go briefly through the model of heterocyst 60

development and the coupling mechanism of carbon and nitrogen metabolism (Fig. 1). 61

Heterocysts constitute 5-10% of all cells of the filaments, and are semi-regularly intercalated 62

among vegetative cells. This distribution of heterocysts along Anabaena filaments represents a 63

simple, one-dimensional pattern in developmental biology (1, 4). After the perception of the 64

nitrogen-starvation signal 2-OG by NtcA (5, 6), heterocyst development is initiated through the 65

autoregulation of ntcA and hetR, as well as their mutual regulation (7-9). HetR then starts to 66

accumulate in developing cells, and activate, through a mechanism that still need to be clarified, 67

the expression of patS (10). HetR acts cell-autonomously, while, PatS- and PatX-derived signals 68

are supposed to act in a non cell-autonomous manner, diffusing from developing cell to 69

neighboring vegetative cells (10, 11). HetR forms a homodimer, and each monomer is composed 70

of a N-terminal DNA-binding domain (DBD), a central flap domain, and a C-terminal hood 71

domain (12, 13). Peptide signals containing the RGSGR motif bind to each HetR monomer at the 72

lateral cleft of the hood domain, triggering a conformational change of the flap domain and 73

destabilizing the HetR-DNA complex (13). Thus, cell-cell interaction under the joint action of 74

HetR and diffusible inhibitory signals leads to the formation of heterocyst pattern and the 75

determination of cell fate (Fig. 1). Two direct targets of the HetR transcriptional activator have 76

been confirmed, hetZ and hetP (14-18). How HetP and HetZ regulate heterocyst development 77

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remains unknown, but they have redundant functions since depending on the expression levels, 78

they can bypass, alone or together, the requirement of HetR in heterocyst development. HetP and 79

likely HetZ represent a transition step from initiation towards commitment during heterocyst 80

development (16-18) (Fig. 1). The commitment point occurs around 8 h after nitrogen stepdown, 81

and is defined as the non-return point in heterocyst development where addition of a combined 82

nitrogen source can no longer pushes a developing cell back to its vegetative state (19). After the 83

commitment, morphogenesis starts with the expression of genes involved in the synthesis of the 84

polysaccharide layer and the glycolipid layer added in heterocyst cell wall, contributing, together 85

with other physiological changes, to the formation of a micro-oxic environment so that 86

nitrogenase can fix N2 when filaments are grown under aerobic conditions (1). HepA is required 87

for the synthesis of the polysaccharide layer (20). 88

The nitrogen starvation signal 2-OG, a trigger of heterocyst differentiation derived from the 89

incomplete Krebs cycle in cyanobacteria, is also a carbon skeleton for ammonium assimilation 90

through the glutamine synthetase-glutamate synthase (GS-GOGAT) cycle. Thus, 2-OG stands at 91

the crossroad of carbon and nitrogen metabolism (4, 21, 22). Another metabolite 2-92

phosphoglycolate (2-PG), a product of the oxygenase activity of Rubisco under carbon limitation, 93

is considered as a carbon starvation signal in cyanobacteria (22). Because of the intimate 94

coupling of carbon and nitrogen metabolism, both carbon and nitrogen input can cause changes in 95

the ratio of 2-OG and 2-PG, which then act allosterically on transcriptional factors such as NtcA 96

(a receptor of 2-OG) and NdhR (a receptor of both 2-OG and 2-PG) in order to keep carbon and 97

nitrogen metabolic balance (6, 22). The carbon/nitrogen metabolic coupling also means that 98

nitrogen starvation corresponds to carbon oversupply and vice versa. The nature of the nitrogen 99

source also affects the nitrogen/carbon metabolic balance. Indeed, because of the complexity and 100

the cost linked to the nitrogen assimilation processes in cyanobacteria, 2-OG concentration of the 101

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cells is the lowest when ammonium is directly used as a nitrogen source, highest under N2 102

fixation, and at an intermediate level when nitrate is present in the growth medium (4, 23). 103

Consequently, the repressive effect of nitrate on heterocyst differentiation is less strong than 104

ammonium; similarly, a high CO2 supply can also boost heterocyst development (24). 105

calA is an AbrB-type transcription factor, highly conserved and considered as essential in 106

cyanobacteria (3). Lack of efficient genetic tools and the polyploid nature of freshwater 107

cyanobacteria hinder our ability to reveal the function of essential genes in these organisms. So 108

far, one approach used was the copper-inducible promoter of petE which in theory can turn on/off 109

gene expression, thus allowing to examine gene function (8). However, copper as a trace metal is 110

hard to deplete from the growth media and culture glassware, and therefore, such a system is not 111

stringent enough and sufficiently reliable to allow the autopsy of the essential gene function. This 112

may explain why no particulate phenotype was observed when the petE promoter replaced the 113

native promoter of calA in Anabaena (25). Two genetic tools have been recently developed to 114

strengthen our ability to manipulate essential genes in cyanobacteria. One used in the study by 115

Higo et al. is the dCas9-based interference system (CRISPRi) combined with a double control 116

with the copper-inducible petE promoter, and the anhydrotetracycline-inducible tetR promoter 117

(26). The second tool relies on CRISPR/Cpf1-assisted gene replacement on the chromosome and 118

the control of gene expression by a synthetic promoter under the control of theophylline alone or 119

with copper (27). In both cases, the expression of target genes can be turned down to a 120

sufficiently low level so that their function be examined. The availability of these genetic tools 121

should facilitate functional dissection of essential genes in cyanobacteria. 122

Higo et al. used CRISPRi approach to switch the expression of calA to a level low enough to 123

get some phenotypes (3). Unfortunately, the authors could not confirm the essential function of 124

calA, as one would expect, albeit the growth of the corresponding mutant under interference 125

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conditions was significantly inhibited. Thus, the control system still has room for further 126

improvement, and this is a major shortcoming of the approach. When the phenotypes were 127

examined, the results depended on the carbon and nitrogen regimes used in the culture. If nitrate 128

was used as a nitrogen source, together with 1% of CO2 bubbling, the mutant could form a few 129

heterocysts (0.6% as compared with less than 0.1% in the controls). When CO2 increased to 5%, 130

about 2.9% of heterocysts could be observed, relative to 0.4-0.6% in the controls. If ammonium 131

replaced nitrate, heterocyst differentiation is still repressed. Thus, CalA suppresses heterocyst 132

differentiation under conditions of carbon oversupply, but this effect is masked by ammonium, 133

but not by nitrate. It is already known that nitrate does not repress heterocyst differentiation 134

completely, since the wild type can develop a few heterocysts under nitrate regime, as also shown 135

in the study by Higo et al (3). It is also known that high CO2 input leads to heterocyst 136

differentiation as well (24). As already summarized above, the concentration of heterocyst 137

differentiation signal 2-OG is the lowest under ammonium regime, highest under nitrogen 138

starvation, and at an intermediate level in the presence of nitrate (4, 23). Thus, its level in cells of 139

Anabaena filaments cultured with nitrate is just high enough to activate genes involved in nitrate 140

uptake and assimilation through NtcA (2), yet just below the threshold concentration for 141

triggering heterocyst differentiation. However, under such a situation, accidental differentiation 142

may occur as a consequence of the NtcA activation, albeit at a lower level than that under 143

combined-nitrogen deprivation, accounting for the formation of a low percentage of heterocysts. 144

Due to the coupling mechanism of carbon/nitrogen metabolism, carbon oversupply should 145

exacerbate nitrogen starvation status of the cells, causing the 2-OG level to increase further. 146

Therefore, high CO2 input weakens the repressive effect of nitrate on heterocyst development, 147

which could explain why the phenotype of calA knockdown is more evident under such 148

conditions. When ammonium is used as a nitrogen source, it drains the 2-OG pool faster, keeping 149

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its level below the threshold necessary for the initiation of heterocyst development even under 150

carbon oversupply. Thus, the balance of carbon/nitrogen metabolism suffices to explain the 151

difference in the phenotypes observed under different nitrogen regimes. Unless additional data 152

are available, calling in an additional unknown mechanism as discussed by the authors may only 153

add an unnecessary level of complexity. 154

At least two direct targets of CalA are identified, hetP and hepA as CalA can bind to their 155

promoter regions (3). The expression of these two genes increased once calA expression is turned 156

down, and this regulation is independent of HetR. The expression levels of hetR and hetZ are not 157

affected. When calA is overexpressed, heterocyst development is inhibited, further confirming its 158

role in the negative regulation of heterocyst formation. Strangely, while hepA expression is 159

negatively affected by calA overexpression, that of hetP is little changed. This result is puzzling 160

since both hetP and hepA are reported to be under the dual control of HetR and CalA (3, 28). The 161

authors argue that HetR binds more strongly to the hetP promoter than that of hepA, thus 162

outcompetes CalA for the binding to the promoter region of hetP (3). Alternatively, direct control 163

of hepA by HetR itself is questionable, since the binding affinity of HetR towards the hepA 164

promoter in vitro is too low to be significant in vivo, and the specificity of such a binding in vitro 165

still needs to be tested (28). Another puzzling question is how heterocyst differentiation is 166

repressed by calA overexpression while both hetP and hetZ are still expressed. Indeed, these two 167

genes, as direct targets of HetR, can bypass the requirement of the latter for heterocyst 168

differentiation. When overexpressed alone or together, they can partly or completely rescue 169

heterocyst differentiation in the absence of hetR (14-18). One hypothesis would be that heterocyst 170

developed under calA overexpression progressed beyond the steps controlled by hetZ and hetP, 171

but blocked at the morphogenesis step through the action of CalA on hepA (Fig. 1). In summary, 172

CalA negatively controls two steps in heterocyst development, the commitment step through hetP, 173

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and the formation of the polysaccharide layer at the beginning of morphogenesis through hepA. 174

The regulation of calA expression in different cell types during the course of heterocyst is not 175

known. Higo et al suggest that changes in the amount of CalA examined by immunoblotting were 176

not significant relative to the strong induction of HetR during heterocyst differentiation, they thus 177

proposed a model according to which the accumulation of HetR after the induction of heterocyst 178

differentiation outcompetes the repressive effect of CalA that remains little changed, thus 179

allowing heterocyst differentiation to proceed. This may not be completely true. Since developing 180

cells or mature heterocysts account for only 5-10% of the cells on the filaments, immunoblotting 181

with total filaments could not tell the real story. Higo et al did observe a slightly low level of 182

CalA in isolated mature heterocysts; in contrast, Xu’s group reported a much dramatic decrease 183

in the level of CalA in heterocysts as compared to vegetative cells, by using immunoblotting as 184

well (25). Thus, despite certain discrepancy, both groups observed a downregulation of calA in 185

mature heterocysts. Taken together, a more likely scenario as a working model is the following: 186

once heterocyst development is initiated, the transcriptional activator HetR accumulates in 187

developing cells, followed by or concomitant with a downregulation of the transcriptional 188

repressor CalA. The combined effect of such regulations changes the relative ratio of HetR/CalA 189

sufficiently to relieve the repressive effect of CalA on heterocyst development. In the presence of 190

ammonium, the 2-OG levels is sufficiently low to prevent the initiation of heterocyst 191

development. When nitrate is used as a nitrogen source, or when carbon is oversupplied, a low 192

percentage of cells may cause 2-OG to reach the threshold level for heterocyst differentiation to 193

happen; the combination of both may even amplify the effect of relative nitrogen starvation, 194

leading to more heterocyst differentiation as observed by Higo et al (3). The existence of CalA 195

helps the filaments to limit the number of heterocyst formed when nitrate is still available, saving 196

filament resources from the costly formation of heterocysts. The concept proposed by the authors 197

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that CalA acts as a safety device preventing unnecessary heterocyst differentiation is thus very 198

attractive. 199

CalA is not the first player reported to suppress heterocyst differentiation in Anabaena. A trpE 200

mutant displayed a similar, and even much stronger phenotype than that resulted from a 201

knockdown of calA (29) (Fig. 1). Anabaena has two genes encoding anthranilate synthase in the 202

tryptophan biosynthesis pathway. One of the two, trpE, is a member of the hetR regulon (29). 203

The trpE mutant forms about 8% of heterocysts when cultured in the presence of nitrate, similar 204

to the wild type under combined-nitrogen deprivation; ammonium, on the other hand, can still 205

repress heterocyst differentiation. Tryptophan synthesis requires glutamine as a precursor. 206

According the proposed model (29), tryptophan can be transformed by tryptophan transaminase, 207

in a 2-OG dependent manner, to glutamate and 3-indole pyruvate. In the absence of TrpE, less 2-208

OG would be consumed, resulting in a transient accumulation of 2-OG. In this case, the filament 209

will perceive the signal of nitrogen starvation, leading to heterocyst differentiation. Therefore, the 210

mechanism of trpE and calA seems to be different. The hetY gene, when overexpressed, can also 211

partly suppress heterocyst development, but the mechanism is unknown (30). 212

The study by Higo et al. raised some interesting questions for further investigation. For 213

example, if calA expression could be turned down even lower, to a lethal level, could the 214

phenotype related to heterocyst differentiation become stronger? If calA is downregulated during 215

heterocyst development, what would be the mechanism? Could one test the hypothesis on the 216

ratio of HetR/CalA in the regulation of heterocyst development? Can we rule out the possibility 217

that CalA regulates nitrate uptake or assimilation, thus accounting for the difference in the 218

phenotypes observed with ammonium and nitrate as nitrogen source? 219

Despite all the questions raised, the shortcoming of CRISPRi, and several contradictions that 220

need to be clarified in the future, the significance of the study published by Higo et al. cannot be 221

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ignored. Except several RGSGR-motif containing peptide signals involved in heterocyst 222

patterning, heterocyst development is driven mostly by a cascade of transcriptional activation. A 223

negative control mechanism through CalA acting as a safety device to prevent unnecessary 224

heterocyst formation constitutes another layer of regulation for the developmental process. The 225

benefice of having such a control mechanism can be discussed in the ecological context. 226

Freshwater cyanobacteria such as Anabaena in the natural environment should encounter 227

constant changes in both the amount and the nature of nitrogen and carbon sources. A transition 228

from one source to another, or variation in their concentration may cause the level of 2-OG to 229

fluctuate constantly. Under certain conditions, the level of 2-OG may touch the threshold for the 230

initiation of heterocyst differentiation, even though a combined nitrogen such as nitrate is still 231

available. By controlling the expression of hetP at the commitment step, CalA can push the 232

developmental process back, therefore saving resources from further development. Under 233

combined nitrogen deprivation, 2-OG level may go beyond the threshold level, leading to even a 234

higher level of HetR, which, together with the decrease of CalA through un unknown mechanism, 235

will blow up the safety control of CalA, allowing heterocyst development to proceed. It is now 236

becoming increasingly evident that carbon supply affects also heterocyst development. In view of 237

our concept on the mechanism of carbon/nitrogen metabolic balance that starts to emerge in the 238

field of cyanobacterial studies, the initiation of heterocyst development should be conceived not 239

just as a consequence of combined-nitrogen deprivation, but rather a result of a disbalance in 240

carbon/nitrogen metabolism. Thus, more integrated approaches at the metabolic levels will be 241

required for a better understanding of the mechanism of heterocyst development. 242

243

Acknowledgements 244

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The work related to the topic described here is supported by the Chinese Academy of Sciences 245

(grant number QYZDJ-SSW-SMC016). We thank Qui-Min Lin for the fluorescent image used in 246

Fig. 1. 247

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Figure legend 341

342

Fig. 1. Mechanism of heterocyst development in Anabaena. A. Fluorescent image of Anabaena 343

filaments grown under diazotrophic conditions, with the presence of heterocysts (indicated by 344

arrow heads). For details of the fluorescent labeling, refer to 31. Only major steps most closely 345

related to the topic is depicted. Anabaena can use combined nitrogen sources such as nitrate or 346

ammonium, or N2 under combined-nitrogen deprivation. In the latter case, 2-oxoglutarate (2-OG) 347

as a carbon skeleton for ammonium assimilation accumulates and activates the transcription 348

factor NtcA. HetR is also a transcriptional activator and a master regulator specific for heterocyst 349

development. Inhibitory signals derived from PatS and PatX act on HetR to determine heterocyst 350

pattern along the filaments. hetP and hetZ are two direct targets of HetR, involved in the 351

commitment step, making heterocyst development irreversible. At the later stages, 352

morphogenesis takes place with the formation of the polysaccharide layer (HEP layer) and the 353

glycolipids layer (HGL layer) at the heterocyst cell wall. HepA regulates the formation of the 354

HEP layer. CalA negatively controls hetP and hepA. trpE is required for tryptophan synthesis 355

from glutamine, and it affects the 2-OG pool, and thus also heterocyst development. Dotted lines 356

represent steps that need further experimental confirmation. The binding of HetR on the hepA 357

promoter is weak in vitro, thus may not be significant in vivo (a question mark is added). 358

359

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2-OG NtcA HetR hetP

PatS

hetZ

CO2 N2

NH4+ NO3

-

amino acids Morphogenesis Patterning Commitment

CalA

HEP layer HGL layer

hepA

Initiation

trpE

?

PatX

A

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