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The LysR
-
ype ansc ip ion ac o PacR is a global egula o
o pho osyn he ic ca bon assimila ion in Anabaena
Jou nal:
En i onmen al Mic obiology and En i onmen al Mic obiology Repo s
Manusc ip ID:
EMI-2014-1511.R1
Manusc ip Type:
EMI - Resea ch a icle
Jou nal:
En i onmen al Mic obiology
Da e Submi ed by he Au ho :
22-Jan-2015
Comple e Lis o Au ho s:
Picossi, Sil ia; Consejo Supe io de In es igaciones Cien í icas, Ins i u o de
Bioq´uímica Vege al y Fo osín esis
Flo es, En ique; Consejo Supe io de In es igaciones Cien í icas, Ins i u o
de Bioq´uímica Vege al y Fo osín esis
He e o, An onia; Consejo Supe io de In es igaciones Cien í icas, Ins i u o
de Bioquímica Vege al y Fo osín esis
Keywo ds:
bac e ia, en i onmen al signal/s ess esponses, gene
exp ession/ egula ion
Wiley-Blackwell and Socie y o Applied Mic obiology
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1
The LysR- ype ansc ip ion ac o PacR is a global egula o o pho osyn he ic 1
ca bon assimila ion in Anabaena 2
3
Sil ia Picossi, En ique Flo es and An onia He e o* 4
5
Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones 6
Cien í icas and Uni e sidad de Se illa, Amé ico Vespucio 49, E-41092, Se ille, Spain. 7
8
9
*Co esponding au ho . Tel.: +34 954489522. Fax: +34 954460165. E-mail add ess: 10
he e o@ib .csic.es 11
12
Keywo ds: ChIP; Cyanobac e ia; Oxygenic pho o ophy; Pho op o ec ion; RuBisCo 13
14
Running i le: Pho osyn he ic ca bon assimila ion egula o 15
16
Accession link o da a: 17
h p://www.ncbi.nlm.nih.go /geo/que y/acc.cgi? oken=a cxwkacxpyd ax&acc=GSE58861 18
19
20
21
22
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Summa y 23
Cyanobac e ia pe o m wa e -spli ing pho osyn hesis and a e impo an p ima y 24
p oduce s impac ing he ca bon and ni ogen cycles a global scale. They ix CO
2
25
h ough ibulose bisphospha e ca boxylase/oxygenase (RuBisCo) and ha e 26
e ol ed a dis inc CO
2
concen a ing mechanism (CCM) ha builds high CO
2
27
concen a ions in he icini y o RuBisCo a o ing i s ca boxylase ac i i y. 28
Filamen ous cyanobac e ia such as Anabaena ix CO
2
in pho osyn he ic 29
ege a i e cells, which dona e pho osyn ha e o he e ocys s ha ely on a 30
he e o ophic me abolism o ix N
2
. CCM elemen s a e induced in esponse o 31
ino ganic ca bon limi a ion, a cue ha exposes he pho osyn he ic appa a us o 32
pho odamage by o e - educ ion. An Anabaena mu an lacking he LysR- ype 33
ansc ip ion ac o All3953 g ows poo ly and dies unde high ligh . The bcL 34
ope on encoding RuBisCo is induced upon ca bon limi a ion in he wild ype bu 35
no in he mu an . ChIP-Seq analysis was used o globally iden i y All3953 a ge s 36
unde ca bon limi a ion. Ta ge s include, besides bcL, genes encoding CCM 37
elemen s, pho o espi a o y pa hway, pho osys em- and elec on anspo -38
ela ed componen s, and ac o s, including la odii on p o eins, wi h a 39
demons a ed o pu a i e unc ion in pho op o ec ion. qRT-PCR analysis o 40
selec ed All3953 a ge s showed egula ion in he wild ype bu no in he mu an . 41
All3953 (PacR) is a global egula o o ca bon assimila ion in an oxygenic 42
pho oau o oph. 43
44
45
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In oduc ion
46
As he o ganisms ha de eloped oxygenic pho osyn hesis, cyanobac e ia ha e played 47
a c ucial ole in Ea h's his o y and he e olu ion o li e in ou plane . Indeed, he 48
p oduc ion o O
2
as a esul o cyanobac e ial ac i i y was esponsible o he oxida ion 49
o he Ea h's a mosphe e abou 2.5-2.3 billion yea s ago (Lyons e al., 2014). 50
Fu he mo e, all he ex an plas ids o euka yo ic algae and plan s a e o cyanobac e ial 51
o igin. Cyanobac e ia we e he i s o ganisms o link he ac i i y o he wo ypes o 52
pho osys ems (PSI and PSII), which allowed he gene a ion o high elec ochemical 53
po en ial, and o combine hem wi h a H
2
O-spli ing complex. Nowadays, mos 54
cyanobac e ia a e pho o ophs elying on oxygenic pho osyn hesis o gene a e ATP 55
and educing equi alen s o he ixa ion o CO
2
and he assimila ion o ino ganic 56
ni ogen. Indeed, hey a e esponsible o an impo an ac ion o he p ima y 57
p oduc i i y in he Ea h's oceans, whe e hey a e impo an CO
2
and N
2
ixe s, hus 58
impac ing he C and N cycles a a global scale (Knoll 2008; P ice e al., 2008). 59
The enzyme esponsible o he bulk o CO
2
ixa ion in he biosphe e is ibulose-60
1,5-bisphospha e ca boxylase/oxygenase (RuBisCo), which has a ela i ely low a ini y 61
o CO
2
and, mo eo e , i can also accep O
2
as a subs a e. Compensa ing o his 62
ela i ely low pe o mance, RuBisCo is conside ed he mos abundan enzyme on 63
Ea h. As a ca boxylase, RuBisCo ca alyzes he i s s ep o he Cal in-Benson-64
Bassham (CBB) cycle, i.e., he inco po a ion o a mosphe ic CO
2
in o ibulose-1,5-65
bisphospha e o gi e wo molecules o 3-phosphoglyce a e. As an oxygenase, i 66
ca alyzes he inco po a ion o O
2
in o ibulose-bisphospha e, which p oduces 2-67
phosphoglycola e ha leads o pho o espi a ion wi h a subsequen loss o ixed C and 68
ene gy. To inc ease he e iciency o CO
2
ixa ion, cyanobac e ia ha e de eloped a 69
dis inc CO
2
concen a ing mechanism (CCM) cons i u ed by ino ganic ca bon (C
i
) 70
anspo e s ha inco po a e bica bona e and CO
2
in o he cell, and a p o einaceous 71
compa men , he ca boxysome, whe e RuBisCo, oge he wi h ca bonic anhyd ase, is 72
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con ined (P ice e al., 2008; Came on e al., 2014). Th ee high-a ini iy bica bona e 73
anspo e s ( he ABC- ype Cmp, also called BCT1, and he Na
+
-dependen BicA and 74
Sb A), and wo CO
2
anspo e s ( he high-a ini y NDH-I
3
and he low-a ini y NDH-I
4
) 75
ha e been iden i ied in di e en cyanobac e ia (see P ice, 2011). Th ough CCM, Ci in 76
he o m o CO
2
can concen a e in he icini y o cyanobac e ial RuBisCo, o allow high 77
speci ic ac i i y o p oduc ion o 3-phosphoglyce a e o le els much highe han in 78
plan s (Came on e al., 2014). Indeed, componen s o cyanobac e ial CCM ha e been 79
ans o med in obacco, wi h he esul o imp o ed speci ic ac i i y o CO
2
ixa ion, 80
which ep esen s a s ep owa ds imp o ed pho osyn hesis in plan s (Lin e al., 2014). 81
In unicellula cyanobac e ia, CCM elemen s a e egula ed by C
i
a ailabili y. 82
Especially genes encoding C
i
anspo e s a e induced, whe eas he s uc u al genes 83
o ca boxysome componen s and he bcL/S genes encoding RuBisCo a e only 84
mode a ely esponsi e (see P ice e al., 2008; Came on e al., 2014). In chemo ophic 85
bac e ia he p ocess o CO
2
ixa ion and he esponse o C
i
limi a ion a e usually 86
con olled by LysR- ype ansc ip ional egula o s (LTTRs). The genes encoding he 87
enzymes o he CBB cycle a e usually ound in clus e s egula ed by CbbR ac o s, 88
which cons i u e a sub- amily o LTTRs (Gibson and Tabi a, 1996). In unicellula 89
cyanobac e ia, a numbe o CbbR homologs ha e been cha ac e ized. CmpR is an 90
ac i a o o he cmp genes (Nishimu a e al., 2008), whe eas CcmR (aka NdhR) ac s as 91
a ansc ip ional ep esso o mul iple genes encoding o he C
i
anspo e s (e.g., Figge 92
e al., 2001; Wang e al., 2004). A hi d ype o CbbR-like p o ein, he ac i a o o he 93
RuBisCo genes, has no ye been iden i ied in cyanobac e ia. 94
Filamen ous he e ocys - o ming cyanobac e ia, o which Anabaena sp. PCC 95
7120 (he ea e Anabaena) is a model o ganism, addi ionally ha e he capaci y o cell 96
di e en ia ion o u n some O
2
-e ol ing pho osyn he ic cells o he ilamen in o 97
he e ocys s, which a e he e o ophic cells especialized in he ixa ion o a mosphe ic 98
N
2
. Thus, Anabaena is a uly plu icellula bac e ium wi h di e en cell ypes specialized 99
in di e en nu i ional asks ha exchange nu ien s and egula o s and con ibu e o 100
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he pe o mance o he ilamen as he o ganism uni (see Flo es and He e o, 2010). 101
Ni ogen assimila ion and he e ocys di e en ia ion in Anabaena a e egula ed by he 102
global ansc ip ion ac o N cA, which esponds o he cellula C- o-N a io, and he 103
he e ocys -speci ic ansc ip ion ac o He R (see He e o e al., 2013). The Anabaena 104
genomic sequence includes h ee genes anno a ed as CbbR-like LTTRs (Kaneko e 105
al., 2001). O hese, all0862 has been iden i ied as cmpR, and i s p oduc ac i a es he 106
exp ession o he cmp ope on (al 2877-al 2880) and he cmpR gene i sel in esponse 107
o C
i
limi a ion (López-Igual e al., 2012). No ably, his egula ion is e ec ed in 108
combina ion wi h N cA, hus e ealing a mode o co- egula ion by C and N a ailabili y 109
(López-Igual e al., 2012). Fu he mo e, in Anabaena he bcLXS ope on encoding 110
RuBisCo, which is mode a ely induced unde C
i
limi a ion (López-Igual e al., 2012), is 111
ep essed in he he e ocys s (Madan and Nie zwicki-Baue , 1993), a egula ion likely 112
exe ed by N cA (Ramasub amanian e al., 1994; Picossi e al., 2014). 113
He e we ha e iden i ied he CbbR-homolog All3953 as he ac i a o o he 114
RuBisCo-encoding ope on in Anabaena. We ha e de e mined he All3953 egulon by 115
ChIP-Seq, which has e ealed ha All3953 is a global egula o o C
i
assimila ion 116
genes and genes in ol ed in p o ec ion o he pho osyn he ic appa a us agains 117
oxida i e damage ha a e egula ed by Ci a ailabili y. 118
119
Resul s 120
All3953, an RbcR-like ac o in Anabaena sp. PCC 7120 121
To gain insigh in o he LTTR All3953 in Anabaena, he exp ession o he all3953 gene 122
unde di e en g ow h condi ions was analyzed by no he n blo . Th ee bands o 123
hyb idiza ion co esponding o ansc ip s o ca. 1.9, 1.6 and 1.3 kb, espec i ely, which 124
appea ed simila ly ep esen ed be o e and a e Ci limi a ion could be obse ed (Supp. 125
Fig. 1A). On he o he hand, all3953 exp ession did no signi ican ly espond o N 126
deple ion (Supp. Fig. 1B). The la e esul was consis en wi h p e ious global 127
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ansc ip ional s udies (Ehi a and Ohmo i, 2006; Flahe hy e al., 2011) and wi h he 128
lack o binding associa ed o all3953 o he N-con ol ansc ip ional egula o N cA 129
upon combined ni ogen deple ion (Picossi e al., 2014). 130
131
Isola ion and cha ac e iza ion o an all3953 mu an 132
To s udy he ole o All3953, a mu an s ain bea ing an inac i a ed e sion o all3953 133
was cons uc ed (see Expe imen al p ocedu es). In s ain CSS74 mos o he all3953 134
gene was dele ed and he C.S3 gene casse e (encoding Sm/Sp esis ance) was 135
in oduced o acili a e seg ega ion and main enance o he mu a ion in Anabaena 136
(Supp. Fig. 2A). As a con ol, s ain CSS77 bea ing he C.S3 gene casse e in he 137
Anabaena plasmid alpha was also cons uc ed. Fo cis complemen a ion, a wild- ype 138
e sion o all3953 was ans e ed o s ain CSS74 in an in eg a i e plasmid, 139
gene a ing s ain CSS74C (Supp. Fig. 2A). 140
S ain CSS74 exhibi ed poo g ow h unde s anda d g ow h condi ions wi h 141
ammonium as a ni ogen sou ce, and i o med sho ilamen s in liquid medium, 142
whe eas s ain CSS74C beha ed simila ly o CSS77 (no shown). To quan i y he 143
dele e ious e ec o he all3953 mu a ion, g ow h a es we e calcula ed in liquid 144
medium unde di e en illumina ion and C
i
-supply condi ions. G ow h a e o he con ol 145
s ain CSS77 was highes (0.892 days
-1
) unde high ligh (HL) and high ca bon (HC), 146
and was abou 30% lowe unde he o he es ed condi ions (Fig. 1A). In con as , 147
g ow h o s ain CSS74 was se e ely a ec ed unde HL HC condi ions (g ow h a e ca. 148
70% lowe han ha o he con ol) (Fig. 1A), unde which i ended up dying a e abou 149
5 days (Fig. 1B). Unde HL and low ca bon (LC) o low ligh (LL) HC, he de ec was 150
close o 30% wi h ega d o he con ol, al hough a e p olonged incuba ion he mu an 151
was mo e se e ely a ec ed unde HL LC. The de ec was he smalles (ca. 15%) unde 152
LL LC condi ions (Fig. 1A,B). In solid medium, g ow h o s ain CSS74 was simila , and 153
simila o ha o he con ol, in he p esence o ammonium, ni a e o no combined 154
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ni ogen unde LL LC condi ions, whe eas unde HL LC a se e e g ow h de ec was 155
obse ed wi h ega d o he con ol wi h any o he es ed ni ogen sou ces (no shown). 156
In summa y, he lack o All3953 had a dele e ious e ec on g ow h, especially unde 157
HL (and HC) condi ions. 158
To u he cha ac e ize he g ow h de ec o he all3953 mu an , he a e o 159
oxygen e olu ion using CO
2
as a inal elec on accep o was measu ed unde di e en 160
illumina ion condi ions in he CSS74 mu an in ela ion o he con ol s ain CSS77. 161
When exponen ially-g owing cells we e incuba ed o 24 h unde LL HC, he oxygen 162
e olu ion a e was sligh ly lowe in s ain CSS74 in compa ison o CSS77 (88 and 163
105 µmol O
2
·[mg Chl]
-1
·h
-1
, espec i ely). Unde HL he di e ence be ween he wo 164
s ains was la ge (167, o CSS77, and 110, o CSS74, µmol O
2
·[mg Chl]
-1
·h
-1
). 165
166
E ec o all3953 mu a ion on bcLXS exp ession 167
To es he e ec o he all3953 mu a ion on he exp ession o he bcLXS ope on, 168
no he n blo analysis was pe o med wi h RNA isola ed om cells o he con ol and 169
mu an s ains g own wi h HC and ans e ed o LC condi ions. A e 1 h incuba ion 170
wi h LC, a ca. 2- old inc ease in he amoun o he bcLXS ansc ip s could be 171
obse ed in he con ol s ain. No induc ion could be de ec ed in he mu an (Fig. 2). In 172
he complemen ed s ain CSS74C he exp ession o bcLXS inc eased in LC simila ly 173
o he con ol (Fig. 2). These esul s indica ed ha he induc ion o he bcLXS ope on 174
upon C
i
de iciency was dependen on All3953 and ha he de ec in s ain CSS74 was 175
exclusi ely due o he lack o All3953. 176
177
ChIP-Seq analysis o he All3953 a ge s 178
To de e mine he DNA a ge s o All3953 a a genomic le el, we used ch oma in 179
immunop ecipi a ion ollowed by high- h oughpu sequencing analysis. To his end, we 180
cons uc ed a s ain (CSS57) exp essing om he all3953 p omo e a e sion o 181
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All3953 C- e minally ussed o TAP- ag (Rigau e al., 1999), as well as a con ol s ain 182
(CSS68) exp essing he TAP- ag alone unde he con ol o he all3953 p omo e 183
(Supp. Fig. 2B, see Expe imen al p ocedu es). Immunop ecipi a ion was ca ied ou 184
using cells o s ains CSS57 and CSS68 g own wi h ammonium as he ni ogen sou ce 185
unde HC condi ions and incuba ed o 3 h wi h ammonium unde LC condi ions. 186
The analysis o he sequences esul ed in a o al o 142 All3953 binding 187
egions, o which 127 we e loca ed in he ch omosome, 10 in plasmid alpha, h ee in 188
plasmid be a and wo in plasmid gamma. Each binding egion was asc ibed o one o 189
wo genes acco ding o he loca ion (midpoin ) o he egion, and he ela i e loca ion 190
wi h espec o he assigned gene was also indica ed (Table 1 and Supp. Table 1). A 191
o al o 175 genes we e asc ibed o he 142 binding egions. The binding egions we e 192
mos ly loca ed ups eam o he asc ibed genes (72%), whe eas 21% we e in agenic 193
and 7% we e loca ed downs eam o genes. The esul s o he ChIP-Seq analysis a e 194
a ailable a GEO accession numbe GSE58861 195
(h p://www.ncbi.nlm.nih.go /geo/que y/acc.cgi?acc= GSE58861). 196
The 175 asc ibed genes we e classi ied acco ding o hei unc ional ca ego y 197
(Table 2). Rema kably, he e we e 19 genes encoding p o eins ela ed o 198
pho osyn hesis and espi a ion, and 21 genes encoding egula o y p o eins, including 199
All7179, a SigB homolog. The es we e mos ly genes encoding hypo he ical o 200
unknown p o eins (42%), bu also genes encoding p o eins in ol ed in ansla ion, in 201
biosyn hesis o amino acids and co ac o s, p os he ic g oups and ca ie s, in anspo , 202
and in o he cellula p ocesses. Table 3 highligh s All3953-binding egions ela ed o 203
pho osyn hesis and espi a ion among which, con i ming ou esul s o gene 204
inac i a ion, he gene encoding he la ge subuni o he RuBisCo ( bcL; binding egion 205
#37) is included. The ac ha a high numbe o genes in ol ed in pho osyn hesis and 206
C ixa ion, including bcL, we e iden i ied as a ge s o RbcR sugges s ha his p o ein 207
is a global ansc ip ion ac o o pho osyn he ic C assimila ion. 208
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pCSE120 [S.K3/L.HEH2 (BamHI)/C.S3 (BamHI); nomencla u e as in (Elhai and Wolk, 374
1988)], was inse ed ob aining plasmid pCSS162. Plasmid pCSS162 was ans e ed o 375
s ain PCC 7120 by conjuga ion (Elhai e al., 1997). Exconjugan s esis an o Sm and 376
Sp, which had he ∆all3953::C.S3 cons uc in eg a ed by double ecombina ion we e 377
selec ed, ob aining s ain CSS74. The seg ega ion o he mu a ion was es ed by PCR 378
(Supp. Fig. 2) wi h p ime s all3953-14, all3953-15, all3953-17 and all3953-20. 379
To cons uc a con ol s ain exp essing Sm
and Sp
plasmid pCSS163, a 380
de i a i e o plasmid pCSEL24 (Olmedo-Ve d e al., 2006) con aining he C.S3 gene 381
casse e, was ans e ed o Anabaena by conjuga ion. Exconjugan s ha had he 382
pCSS163 in eg a ed in he alpha plasmid o Anabaena we e selec ed, ob aining he 383
s ain CSS77 (Supp. Fig. 2). 384
To complemen he all3953 mu a ion o s ain CSS74, a DNA agmen 385
encompassing he whole all3953 gene and sequences ups eam om i was amplified 386
by PCR using he p ime pai all3953-24/all3953-25, bo h including EcoRI si es, and 387
s ain PCC 7120 DNA as he empla e. This agmen was cloned in he EcoRI si e o 388
he mobilizable Nm
encoding ec o pRL424 (Elhai and Wolk, 1988) p oducing plasmid 389
pCSS164, which was ans e ed o s ain CSS74 by conjuga ion ollowed by selec ion 390
o Nm
. The genomic s uc u e o he exconjugan s in he all3953 egion (Supp. Fig. 2) 391
was co obo a ed by PCR. 392
To cons uc a s ain exp essing All3953-C-TAP, he all3953 gene (including he 393
ups eam egion) was PCR-ampli ied wi h p ime s all3953-11 and all3953-12 and DNA 394
o PCC 7120 as he empla e. The TAP- ag was PCR-ampli ied wi h p ime s TAP ag-1 395
and TAP ag-2 using DNA o plasmid pBS1479 as he empla e (Puig e al., 2001). The 396
wo PCR p oduc s we e diges ed wi h SalI and liga ed, a e which he liga ion p oduc 397
was used as he empla e o an o e lapping PCR using p ime s all3953-11 and 398
TAP ag-2. The PCR p oduc was diges ed wi h Ps I and liga ed o he mobilizable 399
ec o pCSV3 (Vallada es e al., 2011) diges ed wi h Ps I, ende ing plasmid pCSS107. 400
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To cons uc a con ol s ain wi h he TAP- ag unde he con ol o he all3953 401
p omo e , a 0.4-kb egion ups eam o all3953 was PCR-ampli ied using p ime s 402
all3953-11 and all3953-18 and DNA o pCSS107 as he empla e. The PCR p oduc 403
was diges ed wi h SalI and liga ed o he PCR-ampli ied TAP- ag diges ed wi h SalI, 404
a e which he liga ion p oduc was used as he empla e o an o e lapping PCR using 405
p ime s all3953-11 and TAP ag-2. The PCR p oduc was diges ed wi h Ps I and liga ed 406
o Ps I-diges ed pCSV3 o gi e plasmid pCSS157. Plasmids pCSS107 and pCSS157 407
we e ans e ed by conjuga ion o s ain PCC 7120 and single Sm
Sp
ecombinan s 408
we e selec ed, ob aining s ain CSS57 and CSS68, espec i ely. Wes e n blo s using 409
Pe oxidase-An i-Pe oxidase Soluble Complex (PAP, Sigma-Ald ich) we e pe o med o 410
ensu e ha he wo s ains exp essed he TAP- ag (Supp. Fig. 2B). 411
412
Ch oma in immunop ecipi a ion 413
Cells o s ains CSS57 g owing exponen ially (3-5 µg Chl·ml
-1
) in he ligh (80 414
µE·m
-2
·s
-1
) in medium supplemen ed wi h 2 µg·ml
-1
Sm and Sp, in HC condi ions we e 415
incuba ed wi h LC o 3 h. Fo maldehyde was hen added o he cul u es o a inal 416
concen a ion o 1%, and he cul u es we e incuba ed o 15 min. Glycine was added a 417
125 mM inal concen a ion and he incuba ion was con inued o 5 min o s op he 418
ixing eac ion. The cells we e hen il e ed, washed wi h cold TBS (20 mM T is-HCl, pH 419
7.4, 140 mM NaCl) and collec ed in ubes (25 ml o cul u e pe ube). The pelle s we e 420
ozen in liquid ni ogen and s o ed a -20°C un il used. Pelle s co esponding o abou 421
25 ml o cul u e we e esuspended in 500 µl o lysis bu e (50 mM HEPES-KOH, pH 422
7.5, 140 mM NaCl, 1 mM EDTA, 1% T i on X-100, 0.1% sodium deoxychola e, 423
supplemen ed wi h Mini EDTA- ee p o ease inhibi o cock ail [Roche]) and, a e 424
addi ion o 150 µl o glass beads (acid-washed, 425-600 µm [Sigma]), he cells we e 425
b oken in a mul i o exe a 2,000 pm o 1 h a 4°C. The cell lysa es we e collec ed by 426
cen i uga ion and he ex ac s we e subjec ed o sonica ion o shea he DNA o abou 427
300-bp agmen s (60 cycles o 10 s, 20 s ice, 15% ampli ude, in a B anson Digi al 428
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Soni ie ). A e cen i uga ion o elimina e cell deb is, he whole-cell ex ac s we e 429
s o ed a −20°C o immedia ely used o immunop ecipi a ion. 430
Immunop ecipi a ion o DNA was ca ied ou as desc ibed (Picossi e al., 2014), 431
wi h some modi ica ions. Whole-cell ex ac s we e p epa ed a 4 mg·ml
-1
o o al p o ein 432
wi h lysis bu e (in 500 µl o al olume). A 50-µl sample was aken as he inpu sample, 433
and he ex ac s we e incuba ed wi h 15 µl IgG-conjuga ed Dynabeads (abou 6 µg 434
IgG) a 4ºC wi h o a ion o 12-14h. The washes o he Dynabeads, as well as he 435
elu ion o he immunop ecipi a ed ma e ial, he c osslinking e e sion and he isola ion 436
o he DNA we e pe o med as in (Picossi e al., 2014). 437
438
Massi e sequencing o he immunop ecipi a ed DNA 439
Inpu and ChIP DNA samples we e sen o sequencing o he Func ional Genomics 440
Co e Facili y o he Ins i u e o Resea ch in Biomedicine, Ba celona (Spain). Nex 441
gene a ion sequencing was ca ied ou using Illumina’s sequencing echnology. ChIP 442
DNA Sample P ep Ki (Illumina) was used o lib a y p epa a ion. Lib a ies we e loaded 443
a 8 pM concen a ion in o he low cell using he Clus e S a ion unning ecipe V7 wi h 444
he Single-Read Clus e Gene a ion Ki 4 (all Illumina). The low cell was loaded in o 445
he Genome Analyze II and samples we e sequenced o 120 nucleo ides om a 446
single end using he Sequencing Ki 5 and ecipe 8 (all Illumina). Manu ac u e ’s 447
ecommenda ions we e s ic ly ollowed. Illumina sequencing da a we e p e-p ocessed 448
wi h he s anda d Illumina pipeline e sion 1.5 and sequences we e aligned o he PCC 449
7120 genome (h p://genome.mic obedb.jp/cyanobase/Anabaena) wi h he Bow ie 450
so wa e 0.12.5 (Langmead e al., 2009). The analysis o he esul s we e ca ied ou 451
using he T i o m algo i hm (Ko nacke e al., 2012) as in (Picossi e al., 2014). The 452
sequences in he ChIP samples o s ain CSS68 (TAP con ol) we e used as he 453
backg ound o he sequences ound o s ain CSS57 (All3953-TAP), and hus o 454
de e mine he speci ic binding egions o All3953-TAP in he genome o Anabaena. 455
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The binding egions we e isualized and analyzed using he UCSC Mic obial Genome 456
B owse (Scheneide e al., 2006). They we e asc ibed o one o wo genes, in case i 457
was no possible o asc ibe hem o only one, and classi ied as ups eam o he gene, i 458
he midpoin o he binding egion was loca ed ups eam o he s a o he gene, 459
in e nal, i he midpoin o he binding egion was inside he gene, o downs eam, i he 460
midpoin o he binding egion was loca ed downs eam o he end o he gene o which 461
i had been asc ibed. 462
463
No he n and qRT-PCR analyses 464
Isola ion o o al RNA om Anabaena was done as desc ibed p e iously (Mohamed 465
and Jansson, 1989). No he n analysis was pe o med as desc ibed p e iously (López-466
Igual e al., 2012). 467
Fo qRT-PCR, 750 ng o DNA- ee RNA samples we e used o all he PCR 468
p ime pai s. Fo he RT eac ion, he Quan i ech Re e se ansc ip ion ki (Qiagen), 469
wi h he Random Hexame P ime mix (100 ng pe sample) (Bioline) was used. The 470
cDNA p oduced was dilu ed 7.5 imes o use 2 µl o cDNA pe PCR eac ion. PCR was 471
done using he Quan imix Easy SYG Ki (Bio ools) (SYBR g een I) in a iCycle iQ Mul i-472
Colo Real Time PCR De ec ion Sys em (Bio-Rad). The abundance o a ansc ip in 473
he RNA sample was calcula ed as: abundance= 2^[C (sample)-C (con ol)], whe e he 474
RNA sample o he con ol s ain CSS77 in HC condi ion (0) was used as he con ol. 475
476
Oxygen e olu ion 477
2-ml samples o exponen ially g own cul u es in HC LL o HC HL condi ions we e used 478
o measu e O
2
e olu ion wi h an O
2
elec ode calib a ed wi h cul u e medium and 479
Na
2
S
2
O
4
as he educing agen . O
2
p oduc ion was measu ed in he ligh (400 480
µE·m
-2
·s
-1
) a e a se en-minu e incuba ion in he da k. 481
482
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Acknowledgmen s 483
This wo k was suppo ed by g an s BFU2010-17980 and BFU2013-44686-P om he 484
Spanish go e nmen , co- inanced by FEDER. The au ho s a e g a e ul o K. Ko nacke 485
o ca ying ou he T i o m and CisFinde analyses, and o H. Aue and I. Pons, om 486
he Func ional Genomics Co e Facili y o he IRB, Ba celona (Spain); o F. Monje-487
Casas and Yagu Allah e diye a-Rinne o c i ical eading o he manusc ip and o M. 488
J. Hue as and A. To ado o help wi h O
2
p oduc ion measu emen s. 489
490
REFERENCES 491
492
Black, T.A., Cai, Y., and Wolk C.P. (1993) Spa ial exp ession and au o egula ion o 493
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Came on, C.J., Su e , M., and Ke eld, C.A. (2014) The ca boxysome: Func ion, 496
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He e o A (Cais e Academic P ess, No olk), pp. 171-188. 498
Ehi a, S., and Ohmo i, M, (2006) N A, a ni ogen- esponsi e esponse egula o 499
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Kaneko, T., Nakamu a, Y., Wolk, C.P., Ku i z, T., Sasamo o, S., and Wa anabe, A. 529
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Knoll, A.H. (2008) Cyanobac e ia and Ea h his o y. The Cyanobac e ia: Molecula 532
Biology, Genomics and E olu ion, eds. He e o A, Flo es E (Cais e Academic 533
P ess, No olk), pp. 1-19. 534
Ko nacke , K., Rye, M.B., Hands ad, T., and D ablos, F. (2012) The T i o m algo i hm: 535
imp o ed sensi i i y and speci ici y in ChIP-Seq peak inding. BMC Bioin o ma ics 536
13: 176. 537
Langmead, B., T apnell, C., Pop, M., and Salzbe g, S.L. (2009) Ul a as and memo y-538
e icien alignmen o sho DNA sequences o he human genome. Genome Biol 10: 539
R25. 540
Lin, M.T., Occhialini, A., And alojc, P.J., Pa y, M.A.J., and Hanson, M.R. (2014) A 541
as e Rubisco wi h po en ial o inc ease pho osyn hesis in c ops. Na u e 513: 547-542
550. 543
López-Igual, R., Picossi, S., López-Ga ido, J., Flo es, E., and He e o, A. (2012) N 544
and C con ol o ABC- ype bica bona e anspo e Cmp and i s LysR- ype 545
ansc ip ional egula o CmpR in a he e ocys - o ming cyanobac e ium, Anabaena 546
sp. En i on Mic obiol 14: 1035-1048. 547
Lyons, T.W., Reinha d, C.T., and Plana sky, N.J. (2014) The ise o oxygen in Ea h's 548
ea ly ocean and a mosphe e. Na u e 506: 307-315. 549
Madan, A.P., and Nie zwicki-Baue , S.A. (1993) In si u de ec ion o ansc ip s o 550
ibulose-1,5-bisphospha e ca boxylase in cyanobac e ial he e ocys s. J Bac e iol 551
175: 7301-7306. 552
Maddocks, S.E., and Oys on, P.C. (2008) S uc u e and unc ion o he LysR- ype 553
ansc ip ional egula o (LTTR) amily p o eins. Mic obiology 154: 3609-3623. 554
Mohamed, A., and Jansson, C. (1989) In luence o ligh on accumula ion o 555
pho osyn hesis-speci ic ansc ip s in he cyanobac e ium Synechocys is 6803. Plan 556
Mol Biol 13: 693-700. 557
Nie zwicki-Baue , S.A., Cu is, S.E., and Haselko n, R. (1984) Co ansc ip ion o genes 558
encoding he small and la ge subuni s o ibulose-1,5-bisphospha e ca boxylase in 559
he cyanobac e ium Anabaena 7120. P oc Na l Acad Sci USA 81: 5961-5965. 560
Nishimu a, T., Takahashi, Y., Yamaguchi, O., Suzuki, H., Maeda, S., and Oma a, T. 561
(2008) Mechanism o low CO
2
-induced ac i a ion o he cmp bica bona e anspo e 562
ope on by a LysR amily p o ein in he cyanobac e ium Synechococcus elonga us 563
s ain PCC 7942. Mol Mic obiol 68: 98-109. 564
Olmedo-Ve d, E., Mu o-Pas o , A.M., Flo es, E., and He e o, A. (2006) Localized 565
induc ion o he n cA egula o y gene in de eloping he e ocys s o Anabaena sp. 566
s ain PCC 7120. J Bac e iol 188: 6694-6649. 567
Picossi, S., Flo es, E. and He e o, A. (2014) ChIP analysis un a els an excep ionally 568
wide dis ibu ion o DNA binding si es o he N cA ansc ip ion ac o in a 569
he e ocys - o ming cyanobac e ium. BMC Genomics 15: 22. 570
Polla i, M., Ruo salainen, V., Ran amaki, S., Tyys ja i, E., and Tyys ja i, T. (2009). 571
Simul aneous inac i a ion o sigma ac o s B and D in e e es wi h ligh acclima ion 572
o he cyanobac e ium Synechocys is sp. s ain PCC 6803. J Bac e iol 191: 3992-573
4001. 574
Puig, O., Caspa y, F., Rigau , G., Ru z, B., Bou e e , E., B agado-Nilsson, E., Wilm, 575
M., and Se aphin, B. (2001) The andem a ini y pu i ica ion (TAP) me hod: a 576
gene al p ocedu e o p o ein complex pu i ica ion. Me hods 24: 218-229. 577
P ice, G.D., Badge , M.R., Woodge , F.J., and Long, B.M. (2008). Ad ances in 578
unde s anding he cyanobac e ial CO
2
-concen a ing-mechanism (CCM): unc ional 579
componen s, Ci anspo e s, di e si y, gene ic egula ion and p ospec s o 580
enginee ing in o plan s. J Exp Bo. 59: 1441-1461. 581
P ice, G.D. (2011) Ino ganic ca bon anspo e s o he cyanobac e ial CO
2
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concen a ing mechanism. Pho osyn h Res 109: 47-57. 583
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Ramasub amanian, T.S., Wei, T.-F. and Golden, J.W. (1994) Two Anabaena sp. s ain 584
PCC 7120 DNA-binding ac o s in e ac wi h ege a i e cell- and he e ocys -speci ic 585
genes. J Bac e iol 176: 1214-1223. 586
Rigau , G., She chenko, A., Ru z, B., Wilm, M., Mann, M., and Se aphin, B. (1999) A 587
gene ic p o ein pu i ica ion me hod o p o ein complex cha ac e iza ion and 588
p o eome explo a ion. Na. Bio echnol 17: 1030-1032. 589
Rippka, R., De uelles, J., Wa e bu y, J.B., He dman, M., and S anie , R.Y. (1979) 590
Gene ic assignmen s, s ain s o ies and p ope ies o pu e cul u es o 591
cyanobac e ia. J Gen Mic obiol 111: 1-61. 592
Schae e , M.R., and Golden, S.S. (1989) Di e en ial exp ession o membe s o a 593
cyanobac e ial psbA gene amily in esponse o ligh . J Bac e iol 171: 3973-3781. 594
Scheneide , K.L., Polla d, K.S., Bae sch, R., Pohl, A., and Lowe, T.M. (2006) The 595
UCSC a chaeal genome b owse . Nucleic Ac Res 34: D407–D410. 596
Vallada es, A., Rod íguez, V., Cama go, S., Ma ínez-Nöel, G.M., He e o, A., and 597
Luque, I. (2011) Speci ic ole o he cyanobac e ial PipX ac o in he he e ocys s o 598
Anabaena sp. s ain PCC 7120. J Bac e iol 193(5): 1172-1182. 599
Wang, H.L., Pos ie , B.L., and Bu nap, R.L. (2004) Al e a ions in global pa e ns o 600
gene exp ession in Synechocys is sp. PCC 6803 in esponse o ino ganic ca bon 601
limi a ion and he inac i a ion o ndhR, a LysR amily egula o . J Biol Chem 279: 602
5739-5751. 603
604
605
606
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Figu e legends: 607
Fig. 1. G ow h o he all3953 mu an . A, The g ow h a e cons an (µ = ln2/
d
, whe e
d
is 608
he doubling ime)
was calcula ed om he inc ease o p o ein con en de e mined in 609
0.2 ml samples o cul u es. The able shows he mean and s anda d de ia ion om 3 610
independen cul u es o each s ain and condi ion. ∆all3953 is s ain CSS74; CSS77 is 611
a con ol s ain ha ca ies he Sm/Sp- esis an de e minan in a wild- ype backg ound. 612
B, Samples o cul u es we e pho og aphed a e 5 days o incuba ion unde he 613
indica ed condi ions. HL, high ligh ; LL, low ligh ; HC, high ca bon; LC, low ca bon. 614
615
Fig. 2. Exp ession o bcLXS in he ∆all3953 mu an and complemen ed s ain. 616
No he n analysis ca ied ou wi h RNA om s ains CSS77 (con ol) CSS74 (∆all3953) 617
and CSS74C (CSS74 complemen ed) was isola ed om cells g own wi h HC (0) and 618
incuba ed o 1h (1) wi h LC. The memb anes we e hyb idized wi h an in e nal 619
agmen o he bcL gene (uppe panels) and, as a loading and ans e con ol, o he 620
npB gene (lowe panels). A owheads poin o he main ansc ip s de ec ed wi h he 621
bcL gene p obe (app oxima e sizes a e indica ed). 622
623
Fig. 3. Consensus All3953 binding sequence and bcL p omo e . A, The p ima y 624
consensus mo i based on 142 high con idence CSS57 ChIP-Seq peak sequences is 625
shown wi h indica ion o he p obabili y o occu ence o each base along he 22-n 626
sequence. W is A o T; Y is C o T; B is C, G o T. B, S uc u e o he bcLXS p omo e 627
egion. The ansc ip ion ini ia ion poin o he ope on (+1) and he -10 and -35 boxes 628
( om Nie zwicki-Baue e al., 1984) a e indica ed in ed. The N cA-binding si e 629
(GTAN
8
TAC) is indica ed in g een, and he h ee pu a i e binding si es o All3953 (Box 630
I, Box II and Box III) a e indica ed in blue. 631
632
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Fig. 4. qRT-PCR analysis o he exp ession o selec ed pho osyn hesis and espi a ion-633
ela ed All3953 gene a ge s. T ansc ip ional esponse o he indica ed genes o C
i
634
limi a ion in he con ol (CSS77) and ∆all3953 mu an (CSS74) s ains was 635
in es iga ed. RNA was isola ed om cells g own wi h 10 mM NaHCO
3
-supplemen ed 636
medium bubbled wi h 1%CO
2
in ai (0) incuba ed o 1 h (1) o 3 h (3) in NaHCO
3
- ee 637
medium bubbled wi h ai . Ba s ep esen he mean ansc ip le els (± s anda d 638
de ia ion) in h ee independen expe imen s. 639
640
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1
The LysR- ype ansc ip ion ac o PacR is a global egula o o pho osyn he ic 1
ca bon assimila ion in Anabaena 2
3
Sil ia Picossi, En ique Flo es and An onia He e o* 4
5
Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones 6
Cien í icas and Uni e sidad de Se illa, Amé ico Vespucio 49, E-41092, Se ille, Spain. 7
8
9
*Co esponding au ho . Tel.: +34 954489522. Fax: +34 954460165. E-mail add ess: 10
he [email p o ec ed] 11
12
Keywo ds: ChIP; Cyanobac e ia; Oxygenic pho o ophy; Pho op o ec ion; RuBisCo 13
14
Running i le: Pho osyn he ic ca bon assimila ion egula o 15
16
Accession link o da a: 17
h p://www.ncbi.nlm.nih.go /geo/que y/acc.cgi? oken=a cxwkacxpyd ax&acc=GSE58861 18
19
20
21
22
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upon C
i
de iciency was dependen on All3953 and ha he de ec in s ain CSS74 was 182
exclusi ely due o he lack o All3953. 183
184
ChIP-Seq analysis o he All3953 a ge s 185
To de e mine he DNA a ge s o All3953 a a genomic le el, we used ch oma in 186
immunop ecipi a ion ollowed by high- h oughpu sequencing analysis. To his end, we 187
cons uc ed a s ain (CSS57) exp essing om he all3953 p omo e a e sion o 188
All3953 C- e minally ussed o TAP- ag (Rigau e al., 1999), as well as a con ol s ain 189
(CSS68) exp essing he TAP- ag alone unde he con ol o he all3953 p omo e 190
(Supp. Fig. 2B, see Expe imen al p ocedu es). Immunop ecipi a ion was ca ied ou 191
using cells o s ains CSS57 and CSS68 g own wi h ammonium as he ni ogen sou ce 192
unde HC condi ions and incuba ed o 3 h wi h ammonium unde LC condi ions. 193
The analysis o he sequences esul ed in a o al o 142 All3953 binding 194
egions, o which 127 we e loca ed in he ch omosome, 10 in plasmid alpha, h ee in 195
plasmid be a and wo in plasmid gamma. Each binding egion was asc ibed o one o 196
wo genes acco ding o he loca ion (midpoin ) o he egion, and he ela i e loca ion 197
wi h espec o he assigned gene was also indica ed (Table 1 and Supp. Table 1). A 198
o al o 175 genes we e asc ibed o he 142 binding egions. The binding egions we e 199
mos ly loca ed ups eam o he asc ibed genes (72%), whe eas 21% we e in agenic 200
and 7% we e loca ed downs eam o genes. The esul s o he ChIP-Seq analysis a e 201
a ailable a GEO accession numbe GSE58861 202
(h p://www.ncbi.nlm.nih.go /geo/que y/acc.cgi?acc= GSE58861). 203
The 175 asc ibed genes we e classi ied acco ding o hei unc ional ca ego y 204
(Table 2). Rema kably, he e we e 19 genes encoding p o eins ela ed o 205
pho osyn hesis and espi a ion, and 21 genes encoding egula o y p o eins, including 206
All7179, a SigB homolog. The es we e mos ly genes encoding hypo he ical o 207
unknown p o eins (42%), bu also genes encoding p o eins in ol ed in ansla ion, in 208
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biosyn hesis o amino acids and co ac o s, p os he ic g oups and ca ie s, in anspo , 209
and in o he cellula p ocesses. Table 3 highligh s All3953-binding egions ela ed o 210
pho osyn hesis and espi a ion among which, con i ming ou esul s o gene 211
inac i a ion, he gene encoding he la ge subuni o he RuBisCo ( bcL; binding egion 212
#37) is included. The ac ha a high numbe o genes in ol ed in pho osyn hesis and 213
C ixa ion, including bcL, we e iden i ied as a ge s o RbcR sugges s ha his p o ein 214
is a global ansc ip ion ac o o pho osyn he ic C assimila ion. 215
A CisFinde analysis o he p ima y consensus mo i was ca ied ou based on 216
142 high-con idence ChIP-Seq peak sequences (Fig. 3A). The consensus mo i ound 217
has a dyad-symme y a chi ec u e and ma ches he consensus o LysR- ecogni ion 218
binding si es (RBS) (T-N
11
-A) (Maddocks and Oys on, 2008), as well as he consensus 219
binding si es p oposed o CbbR ac o s (TNA-N
7/8
-TNA). The p ima y mo i s iden i ied 220
in he cen al 100 n o he binding egions a e indica ed in Supp. Table 1 ( o some 221
binding egions mo e han one mo i ha e been iden i ied). 222
223
Exp ession analysis o some All3953 a ge s in Anabaena 224
To co obo a e ou ChIP-Seq analysis and o suppo he no ion ha All3953 is indeed 225
a global egula o o C ixa ion genes, he exp ession le els o some o he 226
pho osyn hesis- and C- ixa ion- ela ed a ge genes, i s esponse o C
i
limi a ion and i s 227
dependence on All3953 was u he analyzed by qRT-PCR (Fig. 4). S ains CSS77 and 228
CSS74 we e g own in ammonium and HC unde s anda d ligh condi ions (80 229
µE·m
-2
·s
-1
) a 30ºC up o he exponen ial phase. They we e hen ans e ed o medium 230
wi h ammonium unde LC condi ions. As p e iously shown by no he n analysis, 231
ansc ip ion le els o he all3953 gene did no signi ican ly change a e C
i
dep i a ion 232
in he con ol s ain CSS77. As expec ed, all3953 ansc ip le els we e no de ec able 233
in CSS74, co obo a ing ha he all3953 mu a ion was seg ega ed in his s ain. The 234
bcL gene (al 1524) was 4.6- old induced a 3 h a e ans e o LC in s ain CSS77, 235
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whe eas no induc ion was obse ed in CSS74, hus co obo a ing he dependence on 236
All3953. In e es ingly, unde HC he exp ession o bcL in he mu an was highe han 237
in he con ol s ain, sugges ing ha besides as an ac i a o unde LC, All3953 could 238
be ac ing as a ep esso o bcL unde HC condi ions. 239
ORF all4446 ( l 4) was highly induced (abou 100- old), and i s le el was 240
maximum 1 h a e he shi o LC (Fig. 4). In he all3953 mu an , he basal ansc ip ion 241
o all4446 in HC was abou 8- old lowe han in he con ol s ain, and i was only 242
sligh ly induced (2- old) upon he shi o LC. all3891 ( l 1A) was induced abou 6- old 3 243
h a e he shi o LC in he con ol s ain, whe eas only a 2- old induc ion was 244
obse ed in he all3953 mu an . all1304 (bica bona e anspo e homolog) and al 4156 245
(NdhF homolog) we e bo h highly induced (up o 30- and 20- old, espec i ely) upon 246
ans e o s ain CSS77 o LC. In con as , no induc ion o al 4156 and only a small 247
induc ion o all1304 ook place in s ain CSS74. Exp ession o al 4592 (psbAIII) 248
inc eased abou 5- old upon C
i
dep i a ion in CSS77, bu did no app eciably change in 249
CSS74. The al 0223 (NdhA homolog) gene was abou 2- old induced unde C
i
250
dep i a ion in CSS77 bu no in CSS74. Finally, he exp ession o al 1004 (alanine-251
glyoxyla e amino ans e ase) was ep essed by 5- old unde LC condi ions in CSSL77 252
bu no in CSS74. These esul s con i m ha exp ession o he abo e s udied genes is 253
egula ed, ei he posi i ely o nega i ely, by All3953. 254
255
Discussion 256
We ha e iden i ied he LTTR All3953 as he ac i a o o he RuBisCo-encoding genes 257
in he cyanobac e ium Anabaena sp. PCC 7120. All3953 appea s o ac i a e he bcL 258
ope on unde C
i
limi a ion and o ep ess i when C
i
is abundan . An LTTR ac o 259
egula ing he exp ession o he bcL ope on has no , o ou knowledge, been 260
desc ibed in any cyanobac e ium. The exp ession o all3953 does no espond o C
i
261
limi a ion (Supp. Fig. 1) and, indeed, no binding egion o All3953 was ound adsc ibed 262
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o all3953. Thus, he all3953 gene seems o belong o he non-au o egula ed LTTRs. 263
All3953 sha es 28 and 26% iden ical esidues wi h NdhR om Synechocys is sp. PCC 264
6803 and Synechococcus sp. PCC 7002 (CcmR), espec i ely. 265
By ChIP-Seq analysis o a s ain bea ing a TAP- agged e sion o All3953, we 266
ha e de e mined 142 All3953-bound egions a 3 h a e ans e om high o low C
i
267
condi ions, which ha e been assigned o 177 genes. Apa om genes encoding 268
unknown o hypo he ical p o eins, he la ge ca ego y is o genes encoding egula o y 269
p o eins, including he ansc ip ional egula o s Al 0353 (a LTTR) and All4500 (CRP-270
like), he wo-componen esponse egula o All3348, he wo-componen senso 271
his idine-kinase All1145 and he g oup 2-sigma 70- ype sigma ac o All7179 (Supp. 272
Table 1). In e es ingly, when compa ing o Synechocys is sigma ac o s, All7179 273
(SigB4) is mo e simila oa homolog o Synechocys is SigB, which, along wi h SigD has 274
been shown o be impo an o PSII eco e y in his unicellula cyanobac e ium (Polla i 275
e al., 2009). All3953 also binds ups eam o genes pa S and he N, whose p oduc s 276
egula e he e ocys di e en ia ion (Supp. Table 1). The ac ha All3953 binds o he 277
p omo e egion o genes encoding o he egula o y p o eins sugges s a wide ole o 278
his p o ein in he physiology o he o ganism. 279
In he p omo e egion o he bcL ope on we ha e ound h ee pu a i e binding 280
si es o All3953, Box I, Box II, and Box III (Fig. 3B) ha esemble he consensus 281
ecogni ion sequence ound by Cis inde analysis (Fig. 3A). I is concei able ha , like in 282
some o he LTTRs hese boxes combine ep ession and ac i a ion si es. In his ega d, 283
binding o All3953 o Box III, o e lapping gene p omo e elemen s, could be ela ed o 284
he ep ession o bcL obse ed unde high C
i
(Fig. 4). On he o he hand, as 285
men ioned abo e, he bcL ope on is ep essed in he e ocys s by he global 286
ansc ip ional egula o N cA, o which a binding si e is ound o e lapping he ope on 287
TSP (Ramasub amanian e al., 1994; Picossi e al., 2014) (Figu e 3B). I is concei able 288
ha N cA binding in hese di e en ia ed cells in e e e wi h All3952-media ed ac i a ion. 289
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Besides being he ac i a o o he RuBisCo genes, All3953 a ge s include 290
genes in ol ed in o he p ocesses ela ed o ca bon assimila ion, such as C
i
anspo 291
(all1304, encoding an homolog o he BicA bica bona e anspo e ; al 4156 encoding a 292
homolog o he NdhF3 subuni o he NDH-1
3
CO
2
up ake sys em; and al 0869 293
encoding a homolog o he NdhF4 subuni o he NDH-1
4
CO
2
up ake sys em); 294
componen s o he ca boxysome shell (all0868, pu a i e ccmK) and 2-295
phosphoglycola e me abolism (al 1004 and al 2873, possibly ela ed o 296
pho o espi a ion [Eisenhu e al., 2008]). No ably, All3953 a ge s include also a 297
numbe o genes encoding pho osys em componen s, such as al 5154 (psaA, 298
encoding he PSI co e p o ein PsaA), al 3727 (psbAII, encoding a componen o o m II 299
o he PSII co e p o ein PsbA [D1]), al 4592 (psbAIII, encoding ano he componen o 300
o m II o PsbA) and al 1216 (PSII 12 kD ex insic p o ein PsbU), and genes ela ed o 301
PS ac i i y. In he la e g oup a e al 4149 (bili e din educ ase, pu a i ely in ol ed in 302
phycobilisome -PSII an enna- syn hesis), and genes ha can pa icipa e in 303
pho osyn he ic elec on ans e , such as al 0223 and al 0348 (ndhA and ndhD, 304
subuni s o o he pu a i e NADH dehyd ogenases), al 1576 (dehyd ogenase subuni ), 305
all0737 ( hio edoxin educ ase), all1365 (Cy M cy och ome), all4148 ( e edoxin I), 306
all3891 and all4446 ( la odii on p o eins Fl 1A and Fl 4, espec i ely). (Besides in CO
2
307
up ake, NdhF3 and NdhF4 can also pa icipa e in elec on ans e .) 308
Repo s on gene exp ession egula ion by C
i
a e sca ce o Anabaena. 309
Howe e , in he unicellula cyanobac e ium Synechocys is sp. PCC 6803 310
ansc ip omic analysis has al eady shown down- egula ion o some genes encoding 311
polypep ides o PSI and PSII complexes as well as o phycobilisome componen s, 312
upon ans e o C
i
limi a ion condi ions, likely as an adap a ion o lowe assimila o y 313
powe demand, and up- egula ion o some PSII co e polypep ides, in e p e ed as 314
adap a ion o condi ions o sho age o elec on accep o s ha could lead o 315
pho odamage and inc eased u no e o PS co e componen s (Wang e al., 2004). Ou 316
analysis ex ends he a ay o pho osyn he ic genes esponding o C
i
limi a ion, 317
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ema kably o include he co e PSI eac ion cen e psaA gene. Mo eo e , excep o 318
al 1004 ha esponds nega i ely, all he Anabaena pho osyn he ic genes men ioned 319
abo e inc ease exp ession upon he shi o C
i
limi a ion. 320
No ewo hy, o he majo i y o pho osyn he ic gene a ge s o All3953 in 321
Anabaena, a unc ion in p o ec ion agains eac i e oxygen species, which can be 322
gene a ed by C
i
limi a ion o exposu e o HL, has ei he been desc ibed o could be 323
p edic ed. Thus, he psbAII and psbAIII genes a e induced unde HL in he unicellula 324
Synechococcus sp. PCC 7942, and cells cul u ed unde HL showed mo e o m II, and 325
less o m I (encoded by psbAI), o D1 compa ed o cells unde LL (Schae e and 326
Golden, 1989). Rega ding la odii on p o eins, genes, l 1A, l 3A, and specially l 2 327
and l 4 o Anabaena ha e been shown up- egula ed in ege a i e cells in low Ci, and 328
l 1A and l 3A also in high ligh , whe eas l 1B and l 3B a e exp essed exclusi ely in 329
he e ocys s (E mako a e al., 2013). Whe eas Fl 1A and Fl 3A appea in ol ed in 330
pho o educ ion o oxygen o wa e by emo ing excess elec ons om PSI h ough 331
NAD(P)H dehyd ogenases, Fl 2 and Fl 4 could ha e a ole in pho op o ec ion o PSII 332
unde low Ci (E mako a e al., 2013). Rega ding pho o espi a ion, i has also been 333
conside ed o ha e a ole in emo al o excess O
2
(Eisenhu e al., 2008). To he bes 334
o ou knowledge, he egula o esponsible o he esponse o C
i
a ailabili y o any 335
pho osyn he ic gene has no been iden i ied in cyanobac e ia (oxygenic pho o ophs). 336
Ou ChIP-Seq and exp ession analysis indica e ha All3953 is a egula o o 337
pho osyn he ic genes in Anabaena. 338
The g ow h a e o Anabaena is highes unde HL HC condi ions (Fig. 1), 339
implying ha his cyanobac e ium has mechanisms o ge p o i o HC while 340
coun e ac ing HL s ess. The all3953 mu an s ain CSS74 exhibi ed a g ow h de ec in 341
all he condi ions es ed, bu especially unde HL, whe e i ends-up dying (Fig. 1). This 342
sha es he idea ha in Anabaena All3953 is equi ed o cope wi h HL s ess. The e ec 343
o he lack o All3953 seems mo e de imen al in ela ion o impai ed pho op o ec ion 344
han o impai ed C
i
sca enging (p e e ence o he mu an o LL LC o e HL HC 345
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condi ions). Indeed, e en in LL, he mu an seems o pe o m sligh ly be e wi h LC 346
han wi h HC (Fig. 1). Al hough LC could suppose a limi a ion o elec on accep o s, an 347
inc eased a e o pho o espi a ion unde LC oge he wi h he ac ha some o he 348
All3953 a ge s ha could cope wi h excess oxygen a e esidually induced upon he 349
ans e om HC o LC in he CSS74 mu an (Fig. 4) could con ibu e o he p e e ence 350
o his s ain o LC o e HC, especially unde HL. 351
Ou esul s show ha in Anabaena he esponses o C
i
a ailabili y include 352
egula ion o genes encoding elemen s o CCM and RuBisCo, bu also o 353
pho osyn he ic genes o adjus gene a ion o assimila o y powe while p ese ing he 354
pho osyn he ic appa a us om oxida i e damage, which is specially ele an in 355
oxygenic pho o ophs. Because All3953 is a ansc ip ional egula o globally 356
coo dina ing hese esponses, we ha e named i PacR (Pho osyn he ic assimila ion o 357
ca bon Regula o ). 358
359
Expe imen al p ocedu es 360
S ains 361
Anabaena sp. s ain PCC 7120 was g own pho oau ophically a 30°C wi h illumina ion 362
(80 µE·m
-2
·s
-1
) in liquid BG11
0
medium (Rippka e al., 1979) supplemen ed wi h 3 mM 363
NH
4
Cl, 6 mM TES bu e and 10 mM NaHCO
3
and bubbled wi h a mix u e o CO
2
and 364
ai (1% / ) (HC). O he condi ions used we e no NaHCO
3
supplemen and bubbling 365
wi h ai (LC); 12 µE·m
-2
·s
-1
(LL); 175 µE·m
-2
·s
-1
(HL). Fo g ow h on pla es illumina ion 366
was 9 µE·m
-2
·s
-1
(LL) o 34 µE·m
-2
·s
-1
(HL). Fo he mu an s gene a ed in his wo k, 367
an ibio ics we e used a he ollowing concen a ions: Sm, 2 µg ml
−1
; Sp, 2 µg ml
−1
; and 368
Nm, 25 µg ml
−1
o bubbled cul u es; and Sm, 5 µg ml
−1
; Sp, 5 µg ml
−1
; and Nm, 369
40 µg ml
−1
o cul u es in solid medium. 370
371
S ain cons uc ion 372
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To cons uc a mu an o he all3953 gene, he 5' and 3' end o he gene, along wi h he 373
lanking egions, we e PCR ampli ied using ch omosomal DNA o PCC 7120 as he 374
empla e and p ime s all3953-14 (BglII) and all3953-15 (SalI), and p ime s all3953-16 375
(SalI) and all3953-17 (Ps I), espec i ely (all p ime s a e speci ied in Supp. Table 2). 376
The PCR p oduc s we e diges ed wi h SalI and liga ed. The esul ing mix u e was used 377
as a empla e o o e lapping PCR wi h p ime s all3953-14 and all3953-17. The new 378
PCR p oduc was diges ed wi h BglII and Ps I and liga ed o he BglII-Ps I-diges ed 379
pRL271 (Black e al., 1993), ob aining plasmid pCSS161. Plasmid pCSS161 was 380
diges ed wi h SpeI and he 2-kb Sm
Sp
gene casse e C.S3, excised wi h XbaI om 381
pCSE120 [S.K3/L.HEH2 (BamHI)/C.S3 (BamHI); nomencla u e as in (Elhai and Wolk, 382
1988)], was inse ed ob aining plasmid pCSS162. Plasmid pCSS162 was ans e ed o 383
s ain PCC 7120 by conjuga ion (Elhai e al., 1997). Exconjugan s esis an o Sm and 384
Sp, which had he ∆all3953::C.S3 cons uc in eg a ed by double ecombina ion we e 385
selec ed, ob aining s ain CSS74. The seg ega ion o he mu a ion was es ed by PCR 386
(Supp. Fig. 2) wi h p ime s all3953-14, all3953-15, all3953-17 and all3953-20. 387
To cons uc a con ol s ain exp essing Sm
and Sp
plasmid pCSS163, a 388
de i a i e o plasmid pCSEL24 (Olmedo-Ve d e al., 2006) con aining he C.S3 gene 389
casse e, was ans e ed o Anabaena by conjuga ion. Exconjugan s ha had he 390
pCSS163 in eg a ed in he alpha plasmid o Anabaena we e selec ed, ob aining he 391
s ain CSS77 (Supp. Fig. 2). 392
To complemen he all3953 mu a ion o s ain CSS74, a DNA agmen 393
encompassing he whole all3953 gene and sequences ups eam om i was amplified 394
by PCR using he p ime pai all3953-24/all3953-25, bo h including EcoRI si es, and 395
s ain PCC 7120 DNA as he empla e. This agmen was cloned in he EcoRI si e o 396
he mobilizable Nm
encoding ec o pRL424 (Elhai and Wolk, 1988) p oducing plasmid 397
pCSS164, which was ans e ed o s ain CSS74 by conjuga ion ollowed by selec ion 398
o Nm
. The genomic s uc u e o he exconjugan s in he all3953 egion (Supp. Fig. 2) 399
was co obo a ed by PCR. 400
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To cons uc a s ain exp essing All3953-C-TAP, he all3953 gene (including he 401
ups eam egion) was PCR-ampli ied wi h p ime s all3953-11 and all3953-12 and DNA 402
o PCC 7120 as he empla e. The TAP- ag was PCR-ampli ied wi h p ime s TAP ag-1 403
and TAP ag-2 using DNA o plasmid pBS1479 as he empla e (Puig e al., 2001). The 404
wo PCR p oduc s we e diges ed wi h SalI and liga ed, a e which he liga ion p oduc 405
was used as he empla e o an o e lapping PCR using p ime s all3953-11 and 406
TAP ag-2. The PCR p oduc was diges ed wi h Ps I and liga ed o he mobilizable 407
ec o pCSV3 (Vallada es e al., 2011) diges ed wi h Ps I, ende ing plasmid pCSS107. 408
To cons uc a con ol s ain wi h he TAP- ag unde he con ol o he all3953 409
p omo e , a 0.4-kb egion ups eam o all3953 was PCR-ampli ied using p ime s 410
all3953-11 and all3953-18 and DNA o pCSS107 as he empla e. The PCR p oduc 411
was diges ed wi h SalI and liga ed o he PCR-ampli ied TAP- ag diges ed wi h SalI, 412
a e which he liga ion p oduc was used as he empla e o an o e lapping PCR using 413
p ime s all3953-11 and TAP ag-2. The PCR p oduc was diges ed wi h Ps I and liga ed 414
o Ps I-diges ed pCSV3 o gi e plasmid pCSS157. Plasmids pCSS107 and pCSS157 415
we e ans e ed by conjuga ion o s ain PCC 7120 and single Sm
Sp
ecombinan s 416
we e selec ed, ob aining s ain CSS57 and CSS68, espec i ely. Wes e n blo s using 417
Pe oxidase-An i-Pe oxidase Soluble Complex (PAP, Sigma-Ald ich) we e pe o med o 418
ensu e ha he wo s ains exp essed he TAP- ag (Supp. Fig. 2B). 419
420
Ch oma in immunop ecipi a ion 421
Cells o s ains CSS57 g owing exponen ially (3-5 µg Chl·ml
-1
) in he ligh (80 422
µE·m
-2
·s
-1
) in medium supplemen ed wi h 2 µg·ml
-1
Sm and Sp, in HC condi ions we e 423
incuba ed wi h LC o 3 h. Fo maldehyde was hen added o he cul u es o a inal 424
concen a ion o 1%, and he cul u es we e incuba ed o 15 min. Glycine was added a 425
125 mM inal concen a ion and he incuba ion was con inued o 5 min o s op he 426
ixing eac ion. The cells we e hen il e ed, washed wi h cold TBS (20 mM T is-HCl, pH 427
7.4, 140 mM NaCl) and collec ed in ubes (25 ml o cul u e pe ube). The pelle s we e 428
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ozen in liquid ni ogen and s o ed a -20°C un il used. Pelle s co esponding o abou 429
25 ml o cul u e we e esuspended in 500 µl o lysis bu e (50 mM HEPES-KOH, pH 430
7.5, 140 mM NaCl, 1 mM EDTA, 1% T i on X-100, 0.1% sodium deoxychola e, 431
supplemen ed wi h Mini EDTA- ee p o ease inhibi o cock ail [Roche]) and, a e 432
addi ion o 150 µl o glass beads (acid-washed, 425-600 µm [Sigma]), he cells we e 433
b oken in a mul i o exe a 2,000 pm o 1 h a 4°C. The cell lysa es we e collec ed by 434
cen i uga ion and he ex ac s we e subjec ed o sonica ion o shea he DNA o abou 435
300-bp agmen s (60 cycles o 10 s, 20 s ice, 15% ampli ude, in a B anson Digi al 436
Soni ie ). A e cen i uga ion o elimina e cell deb is, he whole-cell ex ac s we e 437
s o ed a −20°C o immedia ely used o immunop ecipi a ion. 438
Immunop ecipi a ion o DNA was ca ied ou as desc ibed (Picossi e al., 2014), 439
wi h some modi ica ions. Whole-cell ex ac s we e p epa ed a 4 mg·ml
-1
o o al p o ein 440
wi h lysis bu e (in 500 µl o al olume). A 50-µl sample was aken as he inpu sample, 441
and he ex ac s we e incuba ed wi h 15 µl IgG-conjuga ed Dynabeads (abou 6 µg 442
IgG) a 4ºC wi h o a ion o 12-14h. The washes o he Dynabeads, as well as he 443
elu ion o he immunop ecipi a ed ma e ial, he c osslinking e e sion and he isola ion 444
o he DNA we e pe o med as in (Picossi e al., 2014). 445
446
Massi e sequencing o he immunop ecipi a ed DNA 447
Inpu and ChIP DNA samples we e sen o sequencing o he Func ional Genomics 448
Co e Facili y o he Ins i u e o Resea ch in Biomedicine, Ba celona (Spain). Nex 449
gene a ion sequencing was ca ied ou using Illumina’s sequencing echnology. ChIP 450
DNA Sample P ep Ki (Illumina) was used o lib a y p epa a ion. Lib a ies we e loaded 451
a 8 pM concen a ion in o he low cell using he Clus e S a ion unning ecipe V7 wi h 452
he Single-Read Clus e Gene a ion Ki 4 (all Illumina). The low cell was loaded in o 453
he Genome Analyze II and samples we e sequenced o 120 nucleo ides om a 454
single end using he Sequencing Ki 5 and ecipe 8 (all Illumina). Manu ac u e ’s 455
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(GTAN
8
TAC) is indica ed in g een, and he h ee pu a i e binding si es o All3953 (Box 641
I, Box II and Box III) a e indica ed in blue. 642
643
Fig. 4. qRT-PCR analysis o he exp ession o selec ed pho osyn hesis and espi a ion-644
ela ed All3953 gene a ge s. T ansc ip ional esponse o he indica ed genes o C
i
645
limi a ion in he con ol (CSS77) and ∆all3953 mu an (CSS74) s ains was 646
in es iga ed. RNA was isola ed om cells g own wi h 10 mM NaHCO
3
-supplemen ed 647
medium bubbled wi h 1%CO
2
in ai (0) incuba ed o 1 h (1) o 3 h (3) in NaHCO
3
- ee 648
medium bubbled wi h ai . Ba s ep esen he mean ansc ip le els (± s anda d 649
de ia ion) in h ee independen expe imen s. 650
651
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Table 1. Resul s o he ChIP-Seq analysis o All3953 binding o DNA a 3 h a e C
i
limi a ion
*Some o he binding egions we e asc ibed o mo e han one gene (see Supp. Table 1).
Binding egions ound
Genes asc ibed
Posi ion o he binding egion wi h espec o he gene
ups eam
in e nal
downs eam
Ch omosome
127
157
118
29
10
Alp
ha
10
13
8
4
2
Be a
3
3
0
3
0
Gamma
2
2
0
1
1
To al
142
175
126
37
13
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Table 2. Func ional ca ego ies o he genes asc ibed o he All3953 binding egions.
Func ional ca ego y Numbe
Amino acid biosyn hesis 5
Biosyn hesis o co ac o s, p os he ic g oups, and ca ie s 4
Cell en elope 2
Cellula p ocesses 5
Cen al in e media y me abolism 3
DNA eplica ion, ecombina ion and epai 3
Ene gy me abolism 4
O he ca ego ies 19
Pho osyn hesis and espi a ion 19
Pu ines, py imidines, nucleosides and nucleo ides 2
Regula o y p o eins 21
T ansla ion 9
T anspo and binding p o eins 4
Unknown and hypo he ical p o eins 75
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Table 3. All3953-binding egions assigned o pho osyn hesis and espi a ion- ela ed genes.
BR NLQ LOC GENE POSITION FUNCTION
¶
ST
START SEQ
7
172,84
239694
al 0223*
ups eam NADH dehyd ogenase subuni 1; NdhA + 239712
AGGTATTAGTTTAACTAATGTT
12
38,58
398948
al 0348 ups eam close
NADH dehyd ogenase subuni 4; NdhD + 398912
AACAATTCCTTAATATAATGTA
19
62,81
859596
all0737 ups eam hio edoxin educ ase - 859596
ATTCATAAAAGCGTTTTATATC
25
238,45
997693
all0868 ups eam CO
2
concen a ing mechanism p o ein CcmK
+ 997698
ATGTATAAGTTTTATTAATATG
al 0869 ups eam NADH dehyd ogenase subuni 5
27
66,90
1175262
al 1004*
ups eam alanine--glyoxyla e amino ans e ase + 1175259
GTATATAGGCGATCATTATGGC
31
36,15
1433154
al 1216 ups eam pho osys em II 12 kD ex insic p o ein PsbU + 1433147
AAATATTGTGAGCATTAATAAG
34
385,11
1547013
all1304*
ups eam bica bona e anspo e - 1546894
GTGCATTTGCAATAGTTATTAT
35
31,96
1620674
all1365 downs eam cy och ome Cy M + 1620645
CGTAATAAATTTTAATCATCAT
37
63,26
1785455
al 1524*
ups eam RbcL + 1785446
ACTTATGCCATTTCTTGATATA
38
204,06
1843221
al 1576 ups eam a dehyd ogenase subuni - 1843218
AGTAATAACTGCTACTTATTAC
61
29,78
3499605
al 2873 in e nal 3' end possible glyce a e kinase - 1843218
CAAAATTAAACTGTCTAATTTC
86
170,25
4499861
al 3727 ups eam pho osys em II p o ein D1 (psbAII) + 4499883
GTATATATATTTTAGTAATATT
91
155,06
4693128
all3891*
ups eam la op o ein ( l 1A) + 4693099
ATTTATAAGTTTTACTTAAGCT
98
166,48
4993006
all4148 ups eam e edoxin I - 4993375
AACCATAAATTTTTCTAATAAC
99
61,47
4999503
al 4156*
ups eam NADH dehyd ogenase subuni 5; NdhF + 4999503
AAAGATAAATTTGCCTTATTTA
108
163,25
5332512
all4446*
ups eam la op o ein ( l 4) - 5332500
AATAATAAATTTTACTAATAAA
112
233,71
5489698
al 4592*
ups eam pho osys em II p o ein D1 (psbAIII) + 5489979
CTATATAGTTTTTACTCATATT
121
51,04
6151146
al 5154 ups eam pho osys em I co e p o ein A1 + 6151133
GGACATAAGTTTTACGAATTGT
*Genes whose exp ession has been s udied by qRT-PCR
¶
Func ions a e as speci ied in cyanobase (h p://genome.mic obedb.jp/cyanobase/Anabaena).
BR: binding egion, NLQ: -logQ alue , LOC: ch omosome loca ion o he midpoin o he binding egion, ST: DNA s and, START: ch omosome loca ion o he 5’ end o he
pu a i e binding sequence o All3953 (SEQ).
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