Cyanobacteria are the only bacteria on Earth able to draw their energy directly from the sun in the same way that plants do. In addition, some strains are able to ‘fix’ the nitrogen present in the atmosphere: they can extract this gas and transform it into nitrogen-based compounds necessary for life. However, both processes cannot happen in a given cell at the same time.

A strain of cyanobacteria called Nostoc PCC 7120 can organise itself into long filaments of interconnected cells. Under certain conditions, one in every ten cells stops drawing its energy from the sun, and starts fixing atmospheric nitrogen instead. Exactly how the bacteria are able to ‘count to ten’ and organize themselves in such a precise pattern is still unclear.

Cells can communicate and establish patterns by exchanging molecular signals that switch on and off certain cell programs. For instance, a protein called HetR turns on the genetic program that allows cyanobacteria to fix nitrogen; on the other hand, a signal known as PatS binds to HetR and turns it off. Cells starting to specialise in fixing nitrogen produce both HetR and PatS, with the latter diffusing in surrounding cells and preventing them from extracting nitrogen.

However, it remained unclear how the nitrogen-fixing cell could ignore its own PatS signal and keep its HetR signal active. HetL – another protein produced by the future nitrogen-fixing cell – could potentially play this role, but how it acts was unknown.

Here, Xu et al. show that HetL cannot diffuse from one cell to the other, and that it binds to HetR at the same place than PatS does. When both PatS and HetL are present, they compete to attach to HetR, which stops PatS from turning off HetR and deactivating the nitrogen-fixing program.

Understanding how cyanobacteria fix nitrogen could help to develop new types of natural fertiliser. More generally, dissecting how these simple organisms can create patterns could help to grasp how patterning emerges in more complex creatures.

Periodic patterning of iterative forms is one of the most common features observed in developmental processes across the living kingdom. Unraveling the rules governing pattern formation helps to elucidate the genetic basis of cell differentiation. A key contribution in theoretical biology was made by Alan Turing in 1952 who demonstrated that the reaction of two molecules with different diffusion rates generates regular patterns (Turing, 1952). Later on, Turing equations were adapted in a new mathematical model that emphasized conspicuous features of biological development, in particular local activation linked to autocatalysis and long range inhibition (Gierer and Meinhardt, 1972; Meinhardt and Gierer, 1974). Valuable experimental approaches showed the recurrence of this mechanism in various developmental behaviors, such as: spacing of leaves, tissue regeneration in Hydra, epidermis of insects, pigmentation in Zebra fish, embryogenesis in Drosophila, and also differentiation in some prokaryotes (for a review see Schweisguth and Corson, 2019). In all these developmental situations several aspects of the molecular interactions involved to generate patterning remain to be elucidated.

Among prokaryotes, several members of the Cyanobacteria phylum are capable of cell differentiation. The molecular basis of differentiation has been well documented for the cyanobacterium Anabaena/Nostoc PCC 7120 (referred herein as Nostoc). Nostoc is a diazotrophic strain which can differentiate into specific type of cells responsible for fixing atmospheric nitrogen. When combined nitrogen is abundant Nostoc forms long filaments consisting of a single cell type. When the filaments of Nostoc are deprived of combined nitrogen, around 10% of the vegetative cells differentiate into heterocysts. These micro-oxic cells, which provide a suitable environment for N₂-fixation, are non-dividing and are distributed semi-regularly along the filaments. Nostoc differentiation follows therefore a one-dimensional pattern of heterocysts separated by 10 vegetative cells. Heterocysts are unable to undergo cell division, but as vegetative cells continue dividing, the filaments grow and new heterocysts form in the middle of the intervals between pre-existing heterocysts. Consequently, the pattern is dynamic and persists throughout the growth (Kumar et al., 2010; Flores and Herrero, 2010). The molecular signal inducing heterocyst differentiation is 2-oxoglutarate (2-OG), which accumulates in response to combined nitrogen starvation (Laurent et al., 2005). Among the various genes involved in the regulation of heterocyst formation and patterning (Herrero et al., 2016), the global regulator NtcA and the specific master regulator HetR are key transcriptional factors in the cascade resulting in heterocyst development. Upon combined nitrogen starvation, NtcA interacts with 2-OG and induces heterocyst differentiation by controlling, directly or indirectly, the expression of several genes, including hetR (Herrero et al., 2004; Valladares et al., 2008). HetR is essential for cell differentiation: its deletion abolishes differentiation, and its overexpression induces differentiation of multiple contiguous heterocysts under combined nitrogen-starvation and allows differentiation even under non-permissive conditions (Buikema and Haselkorn, 1991). It exists in different oligomeric states among which dimer and tetramer have been proposed to interact with DNA (Huang et al., 2004; Valladares et al., 2016). The HetR regulon includes hundreds of genes, which are either activated in response to nitrogen starvation or repressed in nitrogen-replete conditions (Flaherty et al., 2014; Mitschke et al., 2011; Videau et al., 2014). The structure of HetR shows a unique fold comprising three domains: helix-turn-helix, flap and hood; with the latter encompassing the binding site of PatS (Kim et al., 2011; Hu et al., 2015). A key event in pattern formation in Nostoc is the positive autoregulation of hetR occurring specifically in the differentiating cell (Black et al., 1993; Rajagopalan and Callahan, 2010) and the inhibition of HetR in neighboring cells by the product of patS (Golden and Yoon, 2003). The deletion of the patS gene leads to the formation of multiple contiguous heterocysts, and its overexpression inhibits the differentiation process and hence induces a lethal phenotype under combined nitrogen starvation (Yoon and Golden, 1998). patS encodes a 13–17 amino acid peptide containing at its carboxy-terminal end a RGSGR pentapeptide (PatS-5) that interacts with HetR, which inhibits its DNA-binding activity (Huang et al., 2004; Feldmann et al., 2011) and blocks cell differentiation when added to culture medium (Yoon and Golden, 1998). The hexapeptide ERGSGR (PatS-6) derived from PatS is also able to interact with HetR and to inhibit its activity (Hu et al., 2015). A mutant strain producing a variant of HetR, R223W, that is no longer able to interact with PatS, forms multiple contiguous heterocysts (Khudyakov and Golden, 2004; Hu et al., 2015). PatS-dependent signals are considered to diffuse along the filament; inhibiting HetR in the cells adjacent to the heterocyst, so preventing them from differentiating (Risser and Callahan, 2009; Corrales-Guerrero et al., 2013). The RGSGR pentapeptide is also present in HetN; a protein required for the maintenance of the pattern (Black and Wolk, 1994; Higa et al., 2012; Corrales-Guerrero et al., 2014), and recently a third protein (PatX) containing a RGTGR motif, has been reported to inhibit heterocyst differentiation when overproduced in Nostoc (Elhai and Khudyakov, 2018). The transcription of patX is induced in the heterocyst and is under the control of NtcA (Elhai and Khudyakov, 2018).

The HetR/PatS regulatory loop fits the local activation/long range inhibition scheme that has been adapted from the Turing model explaining pattern formation (Brown and Rutenberg, 2014; Turing, 1952; Turing, 1990; Gierer and Meinhardt, 1972; Figure 1A). Furthermore, the action of HetN and PatX as inhibitory factors, the stochastically noisy expression of regulatory proteins (HetR and NtcA) among other features specific to cell differentiation of Nostoc, allow the emergence of more elaborated mathematical models that outline the principles governing pattern formation in cyanobacteria (Di Patti et al., 2018; Muñoz-García and Ares, 2016).

Figure 1 with 1 supplement see all

Download asset Open asset

Figure 1—source data 1

Source data to Figure 1B.

(upper) Bacterial two hybrid assay between HetL and HetR. BTH101 strain was transformed with pKT25-HetL and pUT18C-HetR, β-galactosidase activities were measured.

https://cdn.elifesciences.org/articles/59190/elife-59190-fig1-data1-v2.xlsx

Download elife-59190-fig1-data1-v2.xlsx

Figure 1—source data 2

Source data to Figure 1C.

BLi assay between HetL and HetR. 2.2 µM of biotinylated HetR was loaded onto streptavidin biosensors. A 30 s baseline in PBS was performed before a 120 s association step with various concentrations of HetL at 2.5, 5, 10 and 20 µM followed by a 120 s dissociation step.

https://cdn.elifesciences.org/articles/59190/elife-59190-fig1-data2-v2.xlsx

Download elife-59190-fig1-data2-v2.xlsx

Because Nostoc is the simplest model to address development in a one-dimensional self-organizing system, valuable genetics and biochemical studies have provided an accurate picture of heterocyst patterning. Nevertheless, several questions are still unanswered and deserve investigation. In particular, how the differentiating cell, where patS is expressed at its highest level, becomes immune to self-inhibition is not fully understood yet. A genetic analysis of patS suggested that the seven amino acids at the N-terminus of PatS protect the producing cell from inhibition likely concomitantly with the export of the active form of PatS (Corrales-Guerrero et al., 2013). Intriguingly, if patS or patS-5 are expressed specifically in the vegetative cells, or when PatS-5 is added to the culture medium, cell differentiation is abolished. However when produced in cells that have already initiated development (proheterocysts), PatS-5 is not able to inhibit differentiation (Yoon and Golden, 2001; Wu et al., 2004). These observations suggest that proheterocysts must acquire additional protection post-PatS export/processing.

The hetL gene (all3740) was unearthed in a genetic screen aiming to identify factors involved in PatS signaling (Liu and Golden, 2002). HetL is a single domain protein composed of 40 pentapeptides (A(D/N)LXX), adopting a right-handed quadrilateral beta helix typical of an Rfr-fold common to all pentapeptide repeats containing proteins (PRPs) (Ni et al., 2009). The ectopic expression of hetL in a background of patS overexpression restores the ability of the strain to differentiate heterocysts (Liu and Golden, 2002). hetL overexpression stimulates differentiation also when PatS-5 is added to the culture medium (Liu and Golden, 2002). Henceforth, HetL interferes with PatS inhibition but the molecular mechanism involved is unknown.

This study aims to further explore the function of HetL in PatS signaling. We show that HetL interacts with HetR at the same interface as PatS. This interaction is needed for HetR to escape PatS inhibition, and does not inhibit its DNA-binding activity. Analyzing hetL transcription, we found that this gene is induced shortly after initiation of cell differentiation and that HetR is required for its expression. Finally, we show that the expression of hetL in heterocysts, but not in vegetative cells, is necessary to counteract PatS inhibitory effect. We conclude that, by interacting with HetR, HetL interferes with PatS fixation and therefore provides immunity to the developing cell.

Eighteen years ago, the hetL gene was discovered on the basis of its ability, when overexpressed, to bypass the inhibitory effect of patS overexpression on cell differentiation (Liu and Golden, 2002). In their conclusion, the authors of this study speculated that PatS and HetL might act by modulating HetR activity. This speculation reveals to be an accurate prediction as demonstrated by the results of the experiments presented in this manuscript, added to all the knowledge accumulated on HetR and PatS functions from previous valuable studies that are fundamental to our investigation. In particular, as the unique structural fold of HetR with its two exposed domains (flap and hood) was proposed to favor protein-protein interactions, we questioned the possible interaction of HetR with proteins involved in patterning. In this context, HetL was found to interact strongly with HetR without abolishing its DNA-binding activity, which suggests that HetR-HetL complex may be active regarding gene regulation in vivo (Figure 1). A possible mechanism to explain the role of HetL in PatS signaling is the titration by HetL of the inhibiting peptide. This hypothesis was ruled out since ITC assay did not show any interaction between HetL and PatS (Figure 3). Alternatively, a site-exclusion mechanism can be proposed for HetL. The observation that HetL and PatS-5 (or PatS-6) interact with HetR at the same interface is in agreement with this suggestion (Figure 2B). In Bli assays, increasing concentrations of PatS-5 interfered with HetR-HetL interaction, and in EMSA experiments addition of PatS-5 abolished the formation of HetR-HetL complex in a concentration-dependent manner (Figure 2C–D). In addition to confirming the in silico docking model predicting a same HetR-interaction interface for PatS and HetL, these data imply that the concentration of HetL in the (pro)heterocyst must be higher than the PatS peptide, which is plausible because (i) PatS peptide is diffusible (Wu et al., 2004; Yoon and Golden, 2001), (ii) HetL features do not include any motif or domain that predicts a putative translocation/secretion or association with membranes that could decrease its amount in the producing cell. Moreover, as hetL transcription is activated by HetR (Figure 4), HetL protein is likely to accumulate in the cytoplasm of differentiating cells. The diffusion of the inhibiting peptides (derived from PatS and HetN) along the filament has been shown to occur through septal junctions (SJ) connecting adjacent cells (Mariscal et al., 2016). Electron cryotomography analysis showed that these SJ consist of tubes traversing the septal peptidoglycan (Weiss et al., 2019). Interestingly, the lumen of the tubes measures around 7 nm which is compatible with the diffusion of the inhibiting peptides but probably not with the transfer of HetL between cells. HetL protein is 3.5 nm wide and 6.5 nm length (Ni et al., 2009), which might suggest that a monomer could be transferred via the SJ. However, it is important to note that that HetL is capable of forming dimers (or multimers) (Figure 1B), the size of which is not consistent with transfer via the SJ. In addition, it is also possible that HetL surface charge might not be compatible with its transfer across the SJ tubes.

Alternatively, it can be assumed that in the (pro)heterocyst the affinity of HetR for HetL must be higher than that for PatS. Bli and ITC experiments showed that in vitro, the affinity of HetR for PatS is 10-fold higher than for HetL (compare data of Figure 2C and Figure 3). One might speculate that in vivo, and especially in the (pro)heterocyst, the affinity of HetR for HetL increases either due to a modification of HetR or to its interaction with another factor. HetR has been shown to be regulated by phosphorylation (Valladares et al., 2016; Roumezi et al., 2019), if this posttranslational modification occurs only in the developing cell it could explain a high affinity of HetR for HetL specifically in the heterocyst. In addition, the observation that the overexpression of hetL in vegetative cells, from petE or rbcL promoters, did not lead to their differentiation into heterocsyts is rather in favor of a mechanism promoting the association between HetR and HetL specifically in the heterocyst. Resolving the structure of HetR-HetL complex and following the behavior of HetL protein and PatS in vivo will provide more information about the dynamics and the nature of HetR complexes in the two cell types through the differentiation process.

A deletion mutant of hetL differentiates heterocysts as well as the wild-type strain (Liu and Golden, 2002). The possibility that hetL is not essential for heterocyst differentiation and that only its overexpression affects the differentiation process was one of the hypotheses proposed to explain the phenotype of the hetL mutant. The second hypothesis was functional redundancy based on a crosstalk between HetL and another PRP (Liu and Golden, 2002). The observation that at least one homolog of HetL (All4303) interacted with HetR, is rather in favor of functional redundancy. A global transcription study using RNA sequencing has shown that all4303 transcription is induced in response to nitrogen starvation (Flaherty et al., 2011). The hetL gene has also been reported in this study to be induced after nitrogen starvation, which is consistent with our data. The other PRPs coding genes whose transcription has also been reported to be regulated in this condition are reported in Supplementary file 1. At this stage of our investigation, we cannot rule out that homologs of HetL may also be part of the PatS signaling, but the fact that the genetic suppressor screen for suppression of PatS inhibition selected only hetL would rather point to it as the principal factor involved in the differentiation process (Liu and Golden, 2002). Because of the presence of multiple homologs, cross-complementation might occur when hetL happens to be mutated.

Genes encoding proteins containing tandem pentapeptide repeats are abundant in all cyanobacterial genomes, which render the study of their function by gene-deletion approaches unjustified. Interestingly, despite their high amino acid homology, their structures possess distinctive features that can help to investigate their function. Exploring these structural features and searching for potential binding-partners can be a successful strategy in their study. The gyrase-binding PRP is the first protein of this family whose partner was identified (Tran et al., 2005). PRPs from this family are largely conserved among bacteria, where number of them have been, based on their ability, proposed to provide resistance to quinolone-type antibiotics. Structural studies revealed the distribution of large contiguous patches of negative electrostatic potential resembling DNA which gave insights into their function as mimicking DNA structure for binding to gyrase (Xiong et al., 2011). The structure of HetL does not show such a large distribution of negative charges, but instead contiguous patches of positively and negatively charged surfaces are distributed at the surface of the structure. These patches have been suggested to mediate the binding to unknown potential partners, other than DNA (Ni et al., 2009). In docking simulation analyses reported in this study, these charged surfaces especially those localized in face 2 and face 3 mediate the interaction with the HetR-Hood domain. To the best of our knowledge, the interaction of HetL with HetR constitutes the second case of protein-protein complex identified for a PRP member. In Nostoc two other integral PRPs (PatL [Liu and Wolk, 2011] and FraF [Merino-Puerto et al., 2013]) and a pentapeptide repeat domain-containing protein (HglK, Black et al., 1995) are involved in cell differentiation, but their mechanism of action is not fully uncovered. Analyzing their interaction with their physiological partners will provide a better understanding of the role played by PRPs in the developmental program of Nostoc.

Genetic experiments showed that HetL interaction with HetR allows the differentiating cell to escape from PatS overproduction and from the addition to the culture medium of the (E)RGSGR, (H)RGTGR inhibiting peptides (Figures 4 and 6). These data suggest that HetL provides immunity for the developing cells all along the differentiation process. HetL might therefore be important for pattern initiation and establishment, exerted by PatX and PatS, and also for pattern maintenance mediated by HetN and PatS. Therefore, we propose that HetR can be engaged in two different networks depending on cell types: a HetR-HetL-positive network in the (pro)heterocyt as a consequence of hetL expression, and a negative HetR-inhibiting peptide complex taking place in the HetL-free vegetative cells. The HetR/HetL complex therefore enriches the ‘local-activation and long-range inhibition model’ with a third module that we propose to call ‘local protection’ since this complex allows the differentiating cell to become immune to self-inhibition (Figure 7C). An interesting particularity of patterning in Nostoc is that future heterocysts do not emerge as isolated cells. Rather, strings of 3–4 developing cells appear before resolving into a single proheterocyst. (Wilcox et al., 1973). These clusters of cells ‘competent’ for differentiation were suggested to be correlated to the stochastic fluctuation in hetR expression (Corrales-Guerrero et al., 2015). In addition, we propose that a stochastic variation of hetL expression among the string of competent cells might also be part of the resolving mechanism fixing the decision to differentiate in the cell that inherits enough HetL to be protected from self-inhibition. Microfluidic approaches probing multiple and integrated aspects of heterocyst formation along with mathematical models should shed light on how patterning emerges, resolves and persists in Nostoc. An interesting evolutionary aspect resulting from the conservation of PRPs coding-genes in cyanobacterial genomes is that the function of HetL described here might be accurate for other heterocyst-forming cyanobacteria. It is also likely that understanding all the parameters and factors governing heterocyst-pattern formation will be useful in uncovering the molecular mechanisms underlying patterning in more complex biological systems.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files are provided for bacterial two hybrid analysis, qRT-PCR and heterocyst intervals and percentages.

1. 1. Black TA
   2. Cai Y
   3. Wolk CP

   (1993) Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena

   Molecular Microbiology 9:77–84.

   https://doi.org/10.1111/j.1365-2958.1993.tb01670.x

   • PubMed
   • Google Scholar
2. 1. Black K
   2. Buikema WJ
   3. Haselkorn R

   (1995) The hglK gene is required for localization of heterocyst-specific glycolipids in the Cyanobacterium anabaena sp. strain PCC 7120

   Journal of Bacteriology 177:6440–6448.

   https://doi.org/10.1128/JB.177.22.6440-6448.1995

   • Google Scholar
3. 1. Black TA
   2. Wolk CP

   (1994) Analysis of a het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing

   Journal of Bacteriology 176:2282–2292.

   https://doi.org/10.1128/JB.176.8.2282-2292.1994

   • PubMed
   • Google Scholar
4. 1. Brown AI
   2. Rutenberg AD

   (2014) A storage-based model of heterocyst commitment and patterning in cyanobacteria

   Physical Biology 11:016001.

   https://doi.org/10.1088/1478-3975/11/1/016001

   • PubMed
   • Google Scholar
5. 1. Buikema WJ
   2. Haselkorn R

   (1991) Characterization of a gene controlling heterocyst differentiation in the Cyanobacterium anabaena 7120

   Genes & Development 5:321–330.

   https://doi.org/10.1101/gad.5.2.321

   • PubMed
   • Google Scholar
6. 1. Cai YP
   2. Wolk CP

   (1990) Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences

   Journal of Bacteriology 172:3138–3145.

   https://doi.org/10.1128/JB.172.6.3138-3145.1990

   • PubMed
   • Google Scholar
7. 1. Corrales-Guerrero L
   2. Mariscal V
   3. Flores E
   4. Herrero A

   (2013) Functional dissection and evidence for intercellular transfer of the heterocyst-differentiation PatS morphogen

   Molecular Microbiology 88:1093–1105.

   https://doi.org/10.1111/mmi.12244

   • PubMed
   • Google Scholar
8. 1. Corrales-Guerrero L
   2. Mariscal V
   3. Nürnberg DJ
   4. Elhai J
   5. Mullineaux CW
   6. Flores E
   7. Herrero A

   (2014) Subcellular localization and clues for the function of the HetN factor influencing heterocyst distribution in Anabaena sp. strain PCC 7120

   Journal of Bacteriology 196:3452–3460.

   https://doi.org/10.1128/JB.01922-14

   • PubMed
   • Google Scholar
9. 1. Corrales-Guerrero L
   2. Tal A
   3. Arbel-Goren R
   4. Mariscal V
   5. Flores E
   6. Herrero A
   7. Stavans J

   (2015) Spatial fluctuations in expression of the heterocyst differentiation regulatory gene hetR in Anabaena filaments

   PLOS Genetics 11:e1005031.

   https://doi.org/10.1371/journal.pgen.1005031

   • Google Scholar
10. 1. Di Patti F
    2. Lavacchi L
    3. Arbel-Goren R
    4. Schein-Lubomirsky L
    5. Fanelli D
    6. Stavans J

    (2018) Robust stochastic turing patterns in the development of a one-dimensional cyanobacterial organism

    PLOS Biology 16:e2004877.

    https://doi.org/10.1371/journal.pbio.2004877

    • PubMed
    • Google Scholar
11. 1. Elhai J
    2. Khudyakov I

    (2018) Ancient association of cyanobacterial multicellularity with the regulator HetR and an RGSGR pentapeptide-containing protein (PatX)

    Molecular Microbiology 110:931–954.

    https://doi.org/10.1111/mmi.14003

    • PubMed
    • Google Scholar
12. 1. Feldmann EA
    2. Ni S
    3. Sahu ID
    4. Mishler CH
    5. Risser DD
    6. Murakami JL
    7. Tom SK
    8. McCarrick RM
    9. Lorigan GA
    10. Tolbert BS
    11. Callahan SM
    12. Kennedy MA

    (2011) Evidence for direct binding between HetR from Anabaena sp. PCC 7120 and PatS-5

    Biochemistry 50:9212–9224.

    https://doi.org/10.1021/bi201226e

    • PubMed
    • Google Scholar
13. 1. Flaherty BL
    2. Van Nieuwerburgh F
    3. Head SR
    4. Golden JW

    (2011) Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation

    BMC Genomics 12:332.

    https://doi.org/10.1186/1471-2164-12-332

    • PubMed
    • Google Scholar
14. 1. Flaherty BL
    2. Johnson DB
    3. Golden JW

    (2014) Deep sequencing of HetR-bound DNA reveals novel HetR targets in Anabaena sp. strain PCC7120

    BMC Microbiology 14:255.

    https://doi.org/10.1186/s12866-014-0255-x

    • PubMed
    • Google Scholar
15. 1. Flores E
    2. Herrero A

    (2010) Compartmentalized function through cell differentiation in filamentous cyanobacteria

    Nature Reviews Microbiology 8:39–50.

    https://doi.org/10.1038/nrmicro2242

    • PubMed
    • Google Scholar
16. 1. Gierer A
    2. Meinhardt H

    (1972) A theory of biological pattern formation

    Kybernetik 12:30–39.

    https://doi.org/10.1007/BF00289234

    • PubMed
    • Google Scholar
17. 1. Golden JW
    2. Yoon HS

    (2003) Heterocyst development in anabaena

    Current Opinion in Microbiology 6:557–563.

    https://doi.org/10.1016/j.mib.2003.10.004

    • PubMed
    • Google Scholar
18. 1. Herrero A
    2. Muro-Pastor AM
    3. Valladares A
    4. Flores E

    (2004) Cellular differentiation and the NtcA transcription factor in filamentous cyanobacteria

    FEMS Microbiology Reviews 28:469–487.

    https://doi.org/10.1016/j.femsre.2004.04.003

    • PubMed
    • Google Scholar
19. 1. Herrero A
    2. Stavans J
    3. Flores E

    (2016) The multicellular nature of filamentous heterocyst-forming cyanobacteria

    FEMS Microbiology Reviews 40:831–854.

    https://doi.org/10.1093/femsre/fuw029

    • PubMed
    • Google Scholar
20. 1. Higa KC
    2. Rajagopalan R
    3. Risser DD
    4. Rivers OS
    5. Tom SK
    6. Videau P
    7. Callahan SM

    (2012) The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120

    Molecular Microbiology 83:682–693.

    https://doi.org/10.1111/j.1365-2958.2011.07949.x

    • PubMed
    • Google Scholar
21. 1. Hu HX
    2. Jiang YL
    3. Zhao MX
    4. Cai K
    5. Liu S
    6. Wen B
    7. Lv P
    8. Zhang Y
    9. Peng J
    10. Zhong H
    11. Yu HM
    12. Ren YM
    13. Zhang Z
    14. Tian C
    15. Wu Q
    16. Oliveberg M
    17. Zhang CC
    18. Chen Y
    19. Zhou CZ

    (2015) Structural insights into HetR−PatS interaction involved in cyanobacterial pattern formation

    Scientific Reports 5:16470.

    https://doi.org/10.1038/srep16470

    • PubMed
    • Google Scholar
22. 1. Huang X
    2. Dong Y
    3. Zhao J

    (2004) HetR Homodimer is a DNA-binding protein required for heterocyst differentiation, and the DNA-binding activity is inhibited by PatS

    PNAS 101:4848–4853.

    https://doi.org/10.1073/pnas.0400429101

    • PubMed
    • Google Scholar
23. 1. Karimova G
    2. Pidoux J
    3. Ullmann A
    4. Ladant D

    (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway

    PNAS 95:5752–5756.

    https://doi.org/10.1073/pnas.95.10.5752

    • PubMed
    • Google Scholar
24. 1. Khudyakov IY
    2. Golden JW

    (2004) Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp. PCC 7120, can be separated by mutation

    PNAS 101:16040–16045.

    https://doi.org/10.1073/pnas.0405572101

    • PubMed
    • Google Scholar
25. 1. Kim Y
    2. Joachimiak G
    3. Ye Z
    4. Binkowski TA
    5. Zhang R
    6. Gornicki P
    7. Callahan SM
    8. Hess WR
    9. Haselkorn R
    10. Joachimiak A

    (2011) Structure of transcription factor HetR required for heterocyst differentiation in cyanobacteria

    PNAS 108:10109–10114.

    https://doi.org/10.1073/pnas.1106840108

    • PubMed
    • Google Scholar
26. 1. Kumar K
    2. Mella-Herrera RA
    3. Golden JW

    (2010) Cyanobacterial heterocysts

    Cold Spring Harbor Perspectives in Biology 2:a000315.

    https://doi.org/10.1101/cshperspect.a000315

    • Google Scholar
27. 1. Laurent S
    2. Chen H
    3. Bédu S
    4. Ziarelli F
    5. Peng L
    6. Zhang CC

    (2005) Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120

    PNAS 102:9907–9912.

    https://doi.org/10.1073/pnas.0502337102

    • PubMed
    • Google Scholar
28. 1. Liu D
    2. Golden JW

    (2002) hetL overexpression stimulates heterocyst formation in Anabaena sp. strain PCC 7120

    Journal of Bacteriology 184:6873–6881.

    https://doi.org/10.1128/JB.184.24.6873-6881.2002

    • PubMed
    • Google Scholar
29. 1. Liu J
    2. Wolk CP

    (2011) Mutations in genes patA and patL of Anabaena sp. strain PCC 7120 result in similar phenotypes, and the proteins encoded by those genes may interact

    Journal of Bacteriology 193:6070–6074.

    https://doi.org/10.1128/JB.05523-11

    • PubMed
    • Google Scholar
30. 1. Mariscal V
    2. Nürnberg DJ
    3. Herrero A
    4. Mullineaux CW
    5. Flores E

    (2016) Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena

    Molecular Microbiology 101:968–981.

    https://doi.org/10.1111/mmi.13436

    • PubMed
    • Google Scholar
31. 1. Meinhardt H
    2. Gierer A

    (1974)

    Applications of a theory of biological pattern formation based on lateral inhibition

    Journal of Cell Science 15:321–346.

    • PubMed
    • Google Scholar
32. 1. Merino-Puerto V
    2. Herrero A
    3. Flores E

    (2013) Cluster of genes that encode positive and negative elements influencing filament length in a heterocyst-forming Cyanobacterium

    Journal of Bacteriology 195:3957–3966.

    https://doi.org/10.1128/JB.00181-13

    • PubMed
    • Google Scholar
33. 1. Mitschke J
    2. Vioque A
    3. Haas F
    4. Hess WR
    5. Muro-Pastor AM

    (2011) Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120

    PNAS 108:20130–20135.

    https://doi.org/10.1073/pnas.1112724108

    • PubMed
    • Google Scholar
34. 1. Muñoz-García J
    2. Ares S

    (2016) Formation and maintenance of nitrogen-fixing cell patterns in filamentous cyanobacteria

    PNAS 113:6218–6223.

    https://doi.org/10.1073/pnas.1524383113

    • PubMed
    • Google Scholar
35. 1. Ni S
    2. Sheldrick GM
    3. Benning MM
    4. Kennedy MA

    (2009) The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the Cyanobacterium nostoc sp. strain PCC 7120

    Journal of Structural Biology 165:47–52.

    https://doi.org/10.1016/j.jsb.2008.09.010

    • PubMed
    • Google Scholar
36. 1. Rajagopalan R
    2. Callahan SM

    (2010) Temporal and spatial regulation of the four transcription start sites of hetR from Anabaena sp. strain PCC 7120

    Journal of Bacteriology 192:1088–1096.

    https://doi.org/10.1128/JB.01297-09

    • PubMed
    • Google Scholar
37. 1. Rippka R D
    2. Wterbury JB J
    3. Herdman M
    4. Stanier RY

    (1979) Generic assignments, strain stories and properties of pure cultures of cyanobacteria

    J. Gent. Microbiol 111:1–61.

    https://doi.org/10.1099/00221287-111-1-1

    • Google Scholar
38. 1. Risser DD
    2. Callahan SM

    (2009) Genetic and cytological evidence that heterocyst patterning is regulated by inhibitor gradients that promote activator decay

    PNAS 106:19884–19888.

    https://doi.org/10.1073/pnas.0909152106

    • PubMed
    • Google Scholar
39. 1. Roumezi B
    2. Xu X
    3. Risoul V
    4. Fan Y
    5. Lebrun R
    6. Latifi A

    (2019) The Pkn22 kinase of Nostoc PCC 7120 is required for cell differentiation via the phosphorylation of HetR on a residue highly conserved in genomes of Heterocyst-Forming cyanobacteria

    Frontiers in Microbiology 10:3140.

    https://doi.org/10.3389/fmicb.2019.03140

    • Google Scholar
40. 1. Schweisguth F
    2. Corson F

    (2019) Self-Organization in pattern formation

    Developmental Cell 49:659–677.

    https://doi.org/10.1016/j.devcel.2019.05.019

    • PubMed
    • Google Scholar
41. 1. Tran JH
    2. Jacoby GA
    3. Hooper DC

    (2005) Interaction of the Plasmid-Encoded quinolone resistance protein qnr with Escherichia coli DNA gyrase

    Antimicrobial Agents and Chemotherapy 49:118–125.

    https://doi.org/10.1128/AAC.49.1.118-125.2005

    • Google Scholar
42. 1. Turing A

    (1952) The chemical basis of morphogenesis

    Philosophical Transactions of the Royal Society B 237:37–72.

    https://doi.org/10.1098/rstb.1952.0012

    • Google Scholar
43. 1. Turing AM

    (1990) The chemical basis of morphogenesis. 1953

    Bulletin of Mathematical Biology 52:153–197.

    https://doi.org/10.1016/S0092-8240(05)80008-4

    • PubMed
    • Google Scholar
44. 1. Valladares A
    2. Flores E
    3. Herrero A

    (2008) Transcription activation by NtcA and 2-oxoglutarate of three genes involved in heterocyst differentiation in the Cyanobacterium anabaena sp. strain PCC 7120

    Journal of Bacteriology 190:6126–6133.

    https://doi.org/10.1128/JB.00787-08

    • PubMed
    • Google Scholar
45. 1. Valladares A
    2. Flores E
    3. Herrero A

    (2016) The heterocyst differentiation transcriptional regulator HetR of the filamentous Cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation

    Molecular Microbiology 99:808–819.

    https://doi.org/10.1111/mmi.13268

    • PubMed
    • Google Scholar
46. 1. van Zundert GCP
    2. Rodrigues J
    3. Trellet M
    4. Schmitz C
    5. Kastritis PL
    6. Karaca E
    7. Melquiond ASJ
    8. van Dijk M
    9. de Vries SJ
    10. Bonvin A

    (2016) The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes

    Journal of Molecular Biology 428:720–725.

    https://doi.org/10.1016/j.jmb.2015.09.014

    • PubMed
    • Google Scholar
47. 1. Videau P
    2. Ni S
    3. Rivers OS
    4. Ushijima B
    5. Feldmann EA
    6. Cozy LM
    7. Kennedy MA
    8. Callahan SM

    (2014) Expanding the direct HetR regulon in Anabaena sp. strain PCC 7120

    Journal of Bacteriology 196:1113–1121.

    https://doi.org/10.1128/JB.01372-13

    • PubMed
    • Google Scholar
48. 1. Weiss GL
    2. Kieninger AK
    3. Maldener I
    4. Forchhammer K
    5. Pilhofer M

    (2019) Structure and function of a bacterial gap junction analog

    Cell 178:374–384.

    https://doi.org/10.1016/j.cell.2019.05.055

    • PubMed
    • Google Scholar
49. 1. Wilcox M
    2. Mitchison GJ
    3. Smith RJ

    (1973)

    Pattern formation in the blue-green alga, Anabaena. I. basic mechanisms

    Journal of Cell Science 12:707–723.

    • PubMed
    • Google Scholar
50. 1. Wu X
    2. Liu D
    3. Lee MH
    4. Golden JW

    (2004) patS minigenes inhibit heterocyst development of Anabaena sp. strain PCC 7120

    Journal of Bacteriology 186:6422–6429.

    https://doi.org/10.1128/JB.186.19.6422-6429.2004

    • PubMed
    • Google Scholar
51. 1. Xiong X
    2. Bromley EH
    3. Oelschlaeger P
    4. Woolfson DN
    5. Spencer J

    (2011) Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a qnr protein from a gram-negative bacterium

    Nucleic Acids Research 39:3917–3927.

    https://doi.org/10.1093/nar/gkq1296

    • PubMed
    • Google Scholar
52. 1. Yoon HS
    2. Golden JW

    (1998) Heterocyst pattern formation controlled by a diffusible peptide

    Science 282:935–938.

    https://doi.org/10.1126/science.282.5390.935

    • PubMed
    • Google Scholar
53. 1. Yoon HS
    2. Golden JW

    (2001) PatS and products of nitrogen fixation control heterocyst pattern

    Journal of Bacteriology 183:2605–2613.

    https://doi.org/10.1128/JB.183.8.2605-2613.2001

    • PubMed
    • Google Scholar
54. 1. Zubay G
    2. Morse DE
    3. Schrenk WJ
    4. Miller JH

    (1972) Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli

    PNAS 69:1100–1103.

    https://doi.org/10.1073/pnas.69.5.1100

    • PubMed
    • Google Scholar

Author details

1. Xiaomei Xu

   Aix Marseille Univ, CNRS, LCB, Laboratoire de Chimie Bactérienne, Marseille, France

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

2. Véronique Risoul

   Aix Marseille Univ, CNRS, LCB, Laboratoire de Chimie Bactérienne, Marseille, France

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

3. Deborah Byrne

   Aix Marseille Univ, CNRS, Protein Expression Facility, Institut de Microbiologie de la Méditerranée, Marseille, France

   Contribution

   Data curation, Software, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Stéphanie Champ

   Aix Marseille Univ, CNRS, LCB, Laboratoire de Chimie Bactérienne, Marseille, France

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

5. Badreddine Douzi

   Université de Lorraine, INRAE, DynAMic, Nancy, France

   Contribution

   Conceptualization, Data curation, Software, Formal analysis

   Competing interests

   No competing interests declared

6. Amel Latifi

   Aix Marseille Univ, CNRS, LCB, Laboratoire de Chimie Bactérienne, Marseille, France

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   latifi@imm.cnrs.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0776-7349

• 1,221

  views

• 178

  downloads

• 7

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.