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R S R S N Action Heterocysts HetR NtcA -N PatS HetN + + A B (early)
(late) A B Fig. 1: Model and proposed realization of a reaction-diffusion system. (A) Generic Turing/Meinhardt model. A molecule R regulates some action of interest. It also exerts positive feedback on its own synthesis or activity and increases the synthesis or activity of a second molecule, S, a suppressor of R. S is able to diffuse, while R remains at the site of synthesis. (B) Model of regulation of heterocyst differentiation in Anabaena PCC HetR plays the role of the R morphogen, here activating heterocyst differentiation. PatS and HetN collectively play the role of the S, suppressing HetR activity at different times during develeopment. NtcA monitors nitrogen status
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Fig. 2: Filaments of Anabaena PCC 7120 grown in the absence of fixed nitrogen. Green cells are vegetative cells, capable of photosynthesis. Cells stained with Alcian Blue are heterocysts, incapable of photosynthesis but sites of nitrogen fixation. Photo courtesy of AV Matveyev.
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Heterocyst Forming Unicellular Filamentous +HetN +PatS +HetR +PatX 1A
1B +HetR +PatX 2 1C 3 Filamentous 5 6 picocyanobacteria 7 4 8 9 Fig. 3. Overview of phylogenetic tree, rooted by Gloeobacter violaceus PCC 7421. See Methods section for details on construction and Figs. 4 for an expanded version of the tree. Strains highlighted in red are unicellular, blue heterocyst-forming, green other filamentous strains, and pink picocyanobacteria. The tree can be interpreted in multiple ways with regards to switches between unicellularity and filamentarity. What is shown is not the interpretation with the fewest switches but one that enforces the hypothesis that filamentarity arose only once. Group numbers correspond to those used by Howard-Azzeh et al (2014) (Ref. X). Arrows indicate proposed major evolutionary events.
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Nickname A R N S X +HetN ̶ PatS * * 1A
Nostoc piscinale cena Nos21 Nostoc PCC Nos7107 Nostoc PCC Nos7524 Nostoc NIES Nos3756 Anabaena variabilis ATCC Ana29413 Anabaena PCC Ana7120 Anabaena circinalis AWQC310F Ana310F Anabaena WA Ana102 Anabaena 90 Ana90 Anabaena PCC 7108 Ana7108 Anabaena cylindrica PCC 7122 Ana7122 Anabaena azollae AnaAzo Cylindrospermopsis raciborskii CS 505 CylR505 Raphidiopsis brookii D9 RapD9 Cylindrospermum stagnale PCC 7417 CylS7417 Nostoc punctiforme ATCC Nos29133 Nodularia spumigena CCY Nod9414 Tolypothrix PCC 7601 Tol7601 Calothrix PCC 7507 Cal7507 Microchaete PCC 7126 Mch7126 Hassallia byssoidea VB Has512170 Tolypothrix PCC 9009 Tol9009 Calothrix Cal336-3 Calothrix PCC Cal6303 Calothrix PCC 7103 Cal7103 Chlorogloeopsis fritschii PCC 6912 Chl6912 Chlorogloeopsis fritschii PCC 9212 Chl9212 Fischerella NIES 3754 Fis3754 Fischerella thermalis PCC Fis7521 Fischerella PCC Fis9339 Hapalosiphon MRB220 Hap220 Scytonema hofmanni PCC 7110 Scy7110 Tolypothrix bouteillei VB Tol521301 Scytonema tolypothrichoides VB Scy61278 Mastigocladopsis repens PCC Mas10914 Tolypothrix campylonemoides VB Tol511288 Richelia intracellularis HH01 RicHH01 Rivularia PCC 7116 Riv7116 Mastigocoleus testarum BC008 Mas008 +HetN ̶ PatS * * 1A Fig. 4. Phylogenetic tree of cyanobacterial genomes and presence of key regulatory proteins. See Fig. 3 for phylogenetic context and the Methods section for a description of how the tree came about. Colored circles on the right indicate the presence in the genome of A=NtcA, R=HetR, N=HetN, S=PatS, X=PatX. Small circles indicate protein assignments for which there is doubt owing to synteny concerns, and triangles indicate proteins with differences with respect to RGSGR of HetN or PatX or two of the conserved residues of HetR (see text). Asterisks indicate genomes that have a surprising presence of HetR or a surprising absence of PatX. Yellow circles and orange circles in the tree indicates bootstrap support of at least 90% and 67%, respectively. (A) Heterocyst-forming cyanobacteria. One strain (Raphiodopsis brookii D9) does not make functional heterocysts, but shows evidence by electron microscopy of differentiation of terminal cells [Ref 10] (B) Non-heterocyst-forming filamentous cyanobacteria and unicellular cyanobacteria.
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Nickname A R N S X ̶ HetR ̶ PatX * +HetR +PatX * ̶ HetR ̶ PatX 1B 2 1C
Fig 4: (B) Non--heterocyst-forming cyanobacteria, Nickname A R N S X Gloeocapsa PCC GloC Chroococcidiopsis thermalis PCC Chro7203 1B Microcystis aeruginosa NIES 843 MicA843 Synechocystis PCC 6803 Syn6803 Pleurocapsa PCC 7327 Ple7327 Cyanothece PCC 7424 Cyan7424 Cyanothece PCC Cyan7822 Cyanothece PCC 8802 Cyan8802 Cyanothece PCC Cyan8801 Epithemia turgida ETSB endosymbiont EpiTur Crocosphaera watsonii WH8501 CroC8501 Cyanothece CCY 0110 Cyan0110 Cyanothece ATCC Cyan51142 C. Atelocyanobacterium Thalassa UCYN-A Cyanobacterium stanieri PCC 7202 CyaS7202 Cyanobacterium aponinum PCC CyaA10605 Geminocystis herdmanii PCC 6308 Syn6308 Leptolyngbya PCC 7376 Lep7376 Synechococcus PCC 7002 Syn7002 ̶ HetR ̶ PatX 2 * Stanieria cyanosphaera PCC 7437 Sta7437 Spirulina subsalsa PCC 9445 Spi9445 Dactylococcopsis salina PCC 8305 Dac8305 Halothece PCC 7418 Halo7418 Microcoleus PCC 7113 Mic7113 Chamaesiphon minutus PCC Cha6605 Crinalium epipsammum PCC 9333 Cri9333 Oscillatoria PCC Osc10802 Oscillatoria acuminata PCC 6304 Osc6304 Geitlerinema PCC 7105 Gei7105 Phormidium OSCR PhoOSCR Microcoleus vaginatus FGP2 MicFGP2 Oscillatoria nigro-viridis PCC 7112 Osc7112 Trichodesmium erythraeum IMS101 Tri101 Planktothrix agardhii NIVA cya Pla126-8 Planktothrix prolifica NIVA cya 406 Pla406 Planktothrix rubescens NIVA cya 15 Pla15 Planktothrix rubescens NIVA cya 407 Pla407 Arthrospira platensis NIES 39 Arth39 Arthrospira maxima CS 328 Arth328 Arthrospira PCC 8005 Arth8005 Lyngbya PCC 8106 Lyn8106 Lyngbya aestuarii BL-J LynBLJ 1C +HetR +PatX 3 Geitlerinema PCC Gei7407 Oscillatoriales cyanobacterium JSC-12 Osc12 Leptolyngbya boryana PCC 6306 Lep6306 Leptolyngbya NIES 2104 Lep2104 Leptolyngbya NIES 3755 Lep3755 Leptolyngbya PCC 6406 Lep6406 Synechococcus PCC 7335 Syn7335 Leptolyngbya heron island J LepHIJ Prochlorothrix hollandica PCC ProH9006 Synechococcus elongatus PCC Syn6301 Synechococcus elongatus PCC 7942 Syn7942 PICOCYANOBACTERIA Acaryochloris marina MBIC Amar11017 Cyanothece PCC 7425 Cyan7425 Thermosynechococcus elongatus BP1 TherBP1 Thermosynechococcus NK55a Ther55a Pseudanabaena biceps PCC 7429 PseA7429 Pseudanabaena roaring creek PseARC Pseudanabaena PCC 6802 PseA6802 Pseudanabaena PCC 7367 PseA7367 synechococcus OS type A prime SynOSA synechococcus OS type B prime SynOSB 5 * // 6 ̶ HetR ̶ PatX 7 // 4 8 9 // Gloeobacter kilaueensis JS1 GloBJS1 Gloeobacter violaceus PCC 7421 GloB7421
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Figure 8: Conserved residues in PatS and PatX
4 Bits of information 2 B PxxxPPxxxPPxxx PatX (Het+) 4 11-36 aa Bits of information 2 C PatX (Het-) 4 0-47 aa Bits of information 2 Fig. 8. Conserved amino acid residues in PatS and PatX. Sequence logos representing the amount of information (associated with the degree of conservation) are shown for sequences of (A) PatS, (B) PatX (heterocyst-forming cyanobacteria), and (C) PatX (other cyanobacteria). Residues are colored blue (positively charged), red (negatively charged, green (polar), and black (non-polar). Variable spacing between clusters of aligned amino acids are shown. See Figs. 6 and 7 for amino acid sequences of each protein.
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DIF-containing regions in Ana7120
Figure 9: Regions upstream from patS and patX genes 17-18 nt DIF 2 nt patS A PatS Bits of information 1 PatX (Het+) 18 nt DIF 2 NtcA patX B Bits of information 1 PatX (Het-) DIF 2 patX C 17-18 nt 0-9 nt Bits of information 1 DIF 2 DIF-containing regions in Ana7120 D 17-18 nt Bits of information 1 Fig. 9. Conserved nucleotides upstream from PatS, PatX, and DIF-containing regions. Sequence logos representing the amount of information (associated with the degree of conservation) are shown for sequences upstream from (A) PatS, (B) PatX (heterocyst-forming cyanobacteria), (C) PatX (other cyanobacteria), and (D) HetR- and N-regulated genes of Anabaena PCC Variable spacing between clusters of aligned nucleotides are shown as are DIF and NtcA-binding motifs. The approximate position of transcriptional initiation for the appropriate gene from Anabaena PCC 7120 (ref X) is shown by an arrow. See Suppl. Figs. 5 – 7 for nucleotide acid sequences of each upstream sequence.
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