Microbiology (2014), 160, 844–850
DOI 10.1099/mic.0.076703-0
NrrA directly regulates expression of the fraF gene
and antisense RNAs for fraE in the heterocystforming cyanobacterium Anabaena sp. strain PCC
7120
Shigeki Ehira1,2,3 and Masayuki Ohmori2,4
Correspondence
Shigeki Ehira
1
[email protected]
2
Department of Biological Sciences, Graduate school of Science and Engineering,
Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan
Department of Biological Science, Faculty of Science and Engineering, Chuo University,
Bunkyo-ku, Tokyo 112-8551, Japan
3
PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
4
Consolidated Research Institute for Advanced Science and Medical Care, Waseda University,
Shinjuku-ku, Tokyo 162-8480, Japan
Received 3 January 2014
Accepted 14 February 2014
The heterocystous cyanobacterium Anabaena sp. strain PCC 7120 grows as linear multicellular
filaments that can contain hundreds of cells. Heterocysts, which are specialized cells for nitrogen
fixation, are regularly intercalated among photosynthetic vegetative cells, and these cells are
metabolically dependent on each other. Thus, multicellularity is essential for diazotrophic growth
of heterocystous cyanobacteria. In Anabaena sp. strain PCC 7120, the fraF gene, which is
required to limit filament length, is induced by nitrogen deprivation. The fraF transcripts extend to
the fraE gene, which lies on the opposite DNA strand and could possess dual functionality,
mRNAs for fraF and antisense RNAs for fraE. In the present study, we found that NrrA, a nitrogenregulated response regulator, directly regulated expression of fraF. Induction of fraF by nitrogen
deprivation was abolished by the nrrA disruption. NrrA specifically bound to the promoter region
of fraF, and recognized an inverted repeat sequence. Thus, it is concluded that NrrA controls
expression of mRNAs for fraF and antisense RNAs for fraE in response to nitrogen deprivation.
INTRODUCTION
Cyanobacteria are a large group of bacteria characterized
by O2-evolving photosynthesis. Anabaena sp. strain PCC
7120 (hereafter Anabaena) is a filamentous cyanobacterium
in which certain vegetative cells differentiate into heterocysts
with a semi-regular spacing of 10–15 cells upon limitation of
combined nitrogen in the medium (Flores & Herrero, 2010;
Kumar et al., 2010). Heterocysts are terminally differentiated
cells specialized for nitrogen fixation. The intracellular
environment of heterocysts is micro-oxic for protection
of the oxygen-labile nitrogenase as a result of increased
respiration, inactivation of O2-evolving photosystem II
and formation of a thick envelope outside of the cell wall
(Wolk et al., 1994). Since heterocysts are unable to carry
out photosynthesis, vegetative cells supply heterocysts with
carbohydrate required for nitrogen fixation. In return,
vegetative cells receive nitrogen fixation products from
Abbreviations: asRNA, antisense RNA; IR, inverted repeat; qRT-PCR,
quantitative reverse transcription PCR; TIS, transcription initiation site.
Microarray data have been deposited in the KEGG Expression Database
(accession numbers ex0001949 to ex0001954).
844
heterocysts. Thus, multicellularity and division of labour
between two cell types are essential for heterocyst-forming
cyanobacteria to grow.
In Anabaena, genes influencing filament integrity have
been identified (Ehira & Ohmori, 2012; Flores et al., 2007;
Merino-Puerto et al., 2010, 2013; Nayar et al., 2007).
Inactivation of genes in the fraC operon, which is composed
of three genes, fraC, fraD and fraE, produces filament
fragmentation under conditions of nitrogen deprivation and
impairs diazotrophic growth (Merino-Puerto et al., 2010).
Deletion of the fraF gene, which is located downstream of
fraE in the opposite orientation, results in a large increase
of filament length and impairs diazotrophic growth on
solid medium (Merino-Puerto et al., 2013). Thus, FraF and
products of the fraC operon have an opposite effect
on filament length. Expression of fraF is induced by
nitrogen deprivation, while the fraC operon is constitutively
expressed. Induction of fraF is dependent on NtcA, the
global nitrogen regulator of cyanobacteria (Herrero et al.,
2004), but no putative NtcA-binding site is found within its
promoter region. The fraF transcripts extend as antisense
RNAs (asRNAs) to the fraE gene, and prevention of asRNA
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Printed in Great Britain
Regulation of the excludon in Anabaena
production results in an increased length of filaments,
suggesting that the asRNAs could affect the expression of the
fraC operon. Thus, the fraF transcripts are likely to possess
dual functionality, mRNAs for fraF and asRNAs for fraE
(Merino-Puerto et al., 2013).
NrrA is a nitrogen-regulated response regulator of cyanobacteria belonging to the OmpR family. In Anabaena,
expression of nrrA is induced by nitrogen deprivation and is
directly regulated by NtcA (Ehira & Ohmori, 2006a; MuroPastor et al., 2006). NrrA is involved in regulation of
heterocyst differentiation. NrrA binds specifically to the
region upstream of the nitrogen-responsive transcription
initiation sites of the hetR gene, the master regulator of
heterocyst differentiation (Buikema & Haselkorn, 1991,
2001). Inactivation of nrrA diminishes the induction of hetR
and delays heterocyst differentiation, and overexpression of
nrrA results in enhanced hetR expression and heterocyst
formation (Ehira & Ohmori, 2006a, b). NrrA also controls
glycogen catabolism under nitrogen-deprived conditions.
NrrA directly regulates expression of the glgP1 gene,
encoding glycogen phosphorylase. Glycogen is utilized to
provide reductant and ATP required for nitrogen fixation
(Ernst et al., 1990). In the nrrA mutant, the reduced
expression of glgP1 is accompanied by decreased degradation of glycogen, resulting in depressed nitrogen fixation
activity (Ehira & Ohmori, 2011). In the present study, new
members of the NrrA regulon were sought using DNA
microarray. We identified 46 genes downregulated by
inactivation of nrrA. The fraF gene was included in genes
under the control of NrrA. Induction of fraF by nitrogen
deprivation was abolished by nrrA inactivation and an
NrrA-binding site within the fraF promoter region was
identified. Thus, NrrA directly regulates expression of fraF
and asRNAs for fraE in response to nitrogen deprivation.
significantly different transcript levels (P,0.05; Student’s t-test) by at
least a factor of two were determined.
Quantitative reverse transcription PCR (qRT-PCR). All primers
used in this study are listed in Table 1. cDNA was synthesized from
1 mg total RNA with random hexamer primers using a PrimeScript
1st strand cDNA synthesis kit (Takara Bio). qRT-PCR was performed
with a Thermal Cycler Dice Real-time System (TP900; Takara Bio) in
a 20 ml reaction mixture containing 10 ml SYBR Premix Ex Taq II
(Takara Bio), 0.2 mM each gene-specific forward and reverse primer
and cDNA. Relative ratios were normalized with the value for 16S
rRNA and are represented as means of triplicate experiments.
Gel mobility shift assay. His–NrrA protein was overproduced
and purified from Escherichia coli as described previously (Ehira &
Ohmori, 2006b). The promoter region of fraF (P2395), corresponding to the region from 2307 to +3 with respect to the translation
start site of the fraF gene, was amplified by PCR using the primer pair
P2395-F and P2395-R and cloned into the HincII site of pBluescript II
SK+ (Agilent Technologies) to construct pBP2395. A digoxigeninlabelled probe was prepared by PCR using a digoxigenin-labelled T7promoter primer in combination with P2395-F and pBP2395 as a
template. Gel mobility shift assay was carried out using 1 fmol of the
digoxigenin-labelled probe as described previously (Ehira & Ohmori,
2006b). Deletion probes D1, D2, D3 and D4 were prepared by PCR
using the forward primers P2395-F4, P2395-F3, P2395-F5 and P2395F2 in combination with the reverse primer P2395-R, respectively.
Deletion probes of each side of the inverted repeat sequence, DL and
DR, were prepared with the PrimeSTAR mutagenesis basal kit
(Takara Bio) using the primer pairs P2395D3-F and P2395D3-R, and
P2395D4-F and P2395D4-R, respectively.
RESULTS
NrrA positively regulates expression of the fraF
gene
We carried out DNA microarray analysis to identify genes
regulated by NrrA. Since the expression of nrrA is induced
within 3 h of nitrogen deprivation (Ehira & Ohmori,
METHODS
Bacterial strains and culture conditions. Anabaena sp. strain PCC
7120 and its derivative were grown in BG-11 medium (containing
NaNO3 as a nitrogen source) as described previously (Ehira & Ohmori,
2006a). The mutant strain DR4312S (Ehira & Ohmori, 2006a) was used
as the nrrA disruptant in this study. Liquid cultures were bubbled with
air containing 1.0 % (v/v) CO2. For nitrogen deprivation experiments,
cells grown in BG-11 medium until they reached an OD750 of 0.4–0.5
were washed twice with nitrogen-free BG-11 medium (BG-110) and
then resuspended in BG-110 medium. Spectinomycin was added to the
medium at a final concentration of 10 mg ml21 when required. Genome
sequence and annotation were based on CyanoBase (Fujisawa et al.,
2014).
DNA microarray analysis. Total RNA was extracted from whole
filaments according to Pinto et al. (2009) and was treated with DNase I
(Takara Bio). Global gene expression was analysed using the Anabaena
oligonucleotide microarray as described previously (Ehira & Ohmori,
2006a). Microarray analyses were carried out with three sets of RNA
samples isolated from independently grown cultures. Two hybridization reactions were performed with different combination of Cy-dyes
for each set of RNA samples. Thus, a total of six replicates per gene
were available to determine changes in gene expression. Genes with
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Table 1. Primers used in this study
Primer
RTrrn16SF2
RTrrn16SR2
RT2395-F
RT2395-R
P2395-F
P2395-R
T7promoter
P2395-F2
P2395-F3
P2395-F4
P2395-F5
P2395D3-F
P2395D3-R
P2395D4-F
P2395D4-R
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Sequence (5§A3§)
GCAAGTCGAACGGTCTCTTC
GGTATTAGCCACCGTTTCCA
TGCCAATTTGTTTGGCTGTA
TCGGCATCTGTCAAATTAGC
TTACCTGAATATGCCACAGC
CACCTGCGAGTGTCAATGAG
TAATACGACTCACTATAGGG
GTGTTTCATGACTTTGATCAGC
TGAGTGCTGATTTTCTCGTCA
GAAAAAGTGAAACCTAAACTTAGCA
CCCATCACCTTGCTCATTATT
CTTGCTCGTGTTTCATGACTTTGAT
TGAAACACGAGCAAGGTGATGGGGAA
GTGTTTCAGATCAGCTATCTATGTTG
GCTGATCTGAAACACTAATAATGAG
845
S. Ehira and M. Ohmori
2006a), gene expression patterns of the WT strain and the
nrrA disruptant DR4312S (Ehira & Ohmori, 2006a) after
3 h of nitrogen deprivation were compared to minimize
indirect effects of nrrA disruption. The transcript levels of
46 genes in the nrrA disruptant were lower than those in
the WT (Table 2), most of which were upregulated by
nitrogen deprivation (data not shown), suggesting reduced
induction in the nrrA disruptant. NrrA is required for
the full induction of hetR and facilitates heterocyst
differentiation (Ehira & Ohmori, 2006a). In addition to
Table 2. Genes downregulated by nrrA disruption
Gene
asr0064
asr0098
alr0099
asr0100
alr0101
all0313
alr0444
alr0445
alr0446
alr0447
all0565
all0955
all0968
all1272
all1454
all1455
all1692
asl2301
asl2332
alr2339
all2395
asl2397
alr2463
all2705
all2747
alr2817
alr2830
alr2831
alr2833
alr2837
alr2838
alr2839
alr2840
alr3240
alr3241
alr3676
all3696
alr3732
alr3808
all4000
asr4638
alr4685
alr4686
asr4987
all4991
all5369
Product
hetZ
patU5
patU3
fabG
glgP1
nifD
nifH
sigC
patS
hetR
fraF
hetC
rfbC
pknE
desC
Hypothetical protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Hypothetical protein
Hypothetical protein
ATP-binding protein of ABC transporter
Unknown protein
Hypothetical protein
3-Ketoacyl-acyl carrier protein reductase
Glycogen phosphorylase
Nitrogenase MoFe protein subunit alpha
Nitrogenase iron protein
RNA polymerase group 2 sigma subunit
Heterocyst-inhibiting signalling peptide
Unknown protein
Heterocyst differentiation protein
Protein restricting filament length
Unknown protein
Unknown protein
Hypothetical protein
Similar to precorrin methylase
Heterocyst differentiation protein
dTDP-4-dehydrorhamnose 3,5-epimerase
Probable NAD(P)-dependent oxidoreductase
Hypothetical protein
Glycosyltransferase
Unknown protein
Glycosyltransferase
Glycosyltransferase
Permease protein of ferrichrome ABC transporter
ATP-binding protein of ferrichrome ABC transporter
Unknown protein
Unknown protein
Protein serine-threonine kinase
Nutrient-stress-induced DNA-binding protein
Photosystem II CP43 protein PsbC homologue
Unknown protein
Unknown protein
Cytochrome P450
Unknown protein
Delta-9 desaturase
Unknown protein
DnrrA/WT*
PD
21.61
21.05
21.08
21.03
21.01
21.29
22.22
21.65
21.30
21.11
21.24
21.75
21.49
21.87
21.58
21.11
21.57
21.70
21.86
21.44
21.09
21.15
21.36
21.67
21.60
21.31
21.28
21.06
21.99
22.80
21.24
21.76
22.49
21.06
21.37
21.13
21.04
21.33
21.16
21.23
22.14
23.86
21.81
21.08
21.04
22.72
9.3e205
2.8e202
1.4e202
1.0e203
5.1e205
3.9e202
2.0e202
3.1e203
7.2e203
4.7e203
1.4e202
1.6e202
9.3e204
1.4e204
3.7e203
6.5e204
1.1e203
2.1e203
1.7e203
9.3e205
2.0e203
2.1e204
5.1e204
5.0e205
1.4e204
2.5e202
3.1e203
1.5e203
4.7e202
4.3e204
2.2e202
1.6e202
6.5e203
3.0e204
4.3e204
1.5e202
1.1e202
4.7e203
6.7e203
5.8e204
2.2e202
1.7e203
7.9e205
2.8e202
2.3e202
9.0e204
*Relative ratios of transcript level in the nrrA disruptant to those in the WT 3 h after nitrogen deprivation are shown in the base 2 logarithm.
DP values for the relative ratios were determined by Student’s t-test.
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Microbiology 160
Regulation of the excludon in Anabaena
4.0
P2395
Relative transcript level
3.5
NrrA
PhetR2
0
3
10
30
30
30
30
1
2
3
4
5
6
7
Competitor
30 (nM)
3.0
2.5
2.0
1.5
1.0
0.5
8
0
0
5
10 15 20 25
Time after nitrogen deprivation (h)
Fig. 1. Changes in the transcript level of all2395 after nitrogen
deprivation. Relative transcript levels of fraF at the indicated times
were determined by qRT-PCR in the WT strain ($) and the nrrA
disruptant DR4312S (#). Transcript levels were determined in
duplicate using three independently grown cultures. The transcript
level of the WT strain at 0 h was taken as 1.
hetR, genes involved in heterocyst differentiation, such as
hetZ-patU5-patU3, sigC and patS (Khudyakov & Golden,
2001; Yoon & Golden, 1998; Zhang et al., 2007), were
downregulated by nrrA disruption (Table 2). However, no
interaction between NrrA and promoter regions of hetZ,
sigC and patS was observed (data not shown), indicating
indirect regulation of these genes by NrrA.
The fraF gene (all2395) was recently shown to negatively
influence the length of filaments in Anabaena (MerinoPuerto et al., 2013). Expression of fraF is increased by
nitrogen deprivation in a NtcA-dependent manner, but no
putative NtcA-binding site could be identified within its
promoter region, suggesting regulation by NtcA is indirect
(Merino-Puerto et al., 2013). The fraF transcript level was
decreased in the nrrA disruptant (Table 2). Changes in
expression of fraF after nitrogen deprivation were analysed
by qRT-PCR in the WT and nrrA disruptant (Fig. 1). In the
WT, the transcript levels of fraF were increased within 3 h
of nitrogen deprivation, with increases about 3-fold after
8 h. In the nrrA disruptant, the fraF transcript level was
decreased to half of the WT level in the presence of nitrate
and slightly increased following nitrogen deprivation, but
its level was lower than that of the WT before induction.
Thus, NrrA is a positive regulator of fraF.
NrrA binds to the promoter region of fraF
Gel mobility shift assays were carried out with purified
His–NrrA and DNA probe P2395, corresponding to the
region from 2307 to +3 with respect to the translational
start site of fraF (Fig. 2). His–NrrA reduced the electrophoretic mobility of probe P2395, and the amount of
the NrrA-P2395 complex increased in proportion to the
concentration of His–NrrA (Fig. 2, lanes 1–4). The band
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Fig. 2. Gel mobility shift assays with His–NrrA and the promoter
region of fraF. Digoxigenin-labelled probe P2395 (50 pM)
including the promoter region of fraF was mixed with His–NrrA
in the amounts indicated above each lane, and the mixtures were
subjected to electrophoresis. Non-labelled fragments of P2395
(lanes 5 and 6) and PhetR2 (lanes 7 and 8) were added at a final
concentration of 1.5 nM (lanes 5 and 7) or 5 nM (lanes 6 and 8).
Filled and open arrowheads indicate the NrrA-P2395 complex and
free probe, respectively.
intensity of the NrrA-P2395 complex was decreased upon
addition of a non-labelled P2395 fragment to the assay
mixture (Fig. 2, lanes 5 and 6). The addition of a fragment
PhetR2, which does not interact with His–NrrA (Ehira &
Ohmori, 2006b), did not affect the amount of the NrrAP2395 complex (Fig. 2, lanes 7 and 8). These observations
led to the conclusion that NrrA binds to the region
upstream of fraF in a sequence-specific manner.
The 59 ends of fraF transcripts have been identified by
primer extension analysis (Merino-Puerto et al., 2013) and
RNA sequencing (Mitschke et al., 2011). The transcription
initiation site (TIS) positioned at 137 bp upstream of the
translational start site of fraF was detected in both analyses
and is likely to be a major nitrogen-regulated TIS. To
determine the NrrA-binding site within the fraF promoter
region, a deletion series of four probes was used for gel
mobility shift assays (Fig. 3a). NrrA bound to probes D1,
D2 and D3 in a similar way to P2395, but no interaction
between D4 and NrrA was observed (Fig. 3b), indicating
that a sequence required for NrrA binding to the fraF
promoter was present in the region from 2205 to 2184.
We have previously indicated that the sequence AA
(A/T)GTCA is required for NrrA binding to the glgP1
and hetR promoters (Ehira & Ohmori, 2006b, 2011).
Eight nucleotides downstream of these sequences, we
found incomplete inverted sequences (Fig. 3a). The inverted
repeat (IR) sequence (aTtAN8TGAC) was also found
within the fraF promoter region, which was located from
–187 to 2172 (Fig. 3a). Removal of each side of the IR
sequence from P2395 decreased the DNA-binding affinity
of NrrA, although weak interactions between NrrA and
probes DL and DR were still observed (Fig. 3c). These
results indicate that NrrA binds to the IR sequence in
dimeric form.
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847
S. Ehira and M. Ohmori
(a) P2395(–307)
D1(–275)
D3(–205)
D2(–232)
DL
D4(–183)
DR
–10 Promoter
–137
glgP1
hetR
Consensus
(c)
(b)
NrrA
P2395
D1
– + – +
D2
– +
D3
– +
D4
– +
NrrA
P2395
+
–
DL
–
+
–
DR
+
Fig. 3. Identification of the NrrA-recognition sequence. (a) Nucleotide sequence of the fraF promoter region. The transcription
initiation site and the ”10 promoter region are indicated by a bent arrow and an open box, respectively. An incomplete inverted
repeat sequence is indicated with arrows. Sequences of the NrrA-binding region within glgP1 and hetR promoters and the
consensus sequence are shown. (b) Gel mobility shift assays with a deletion series of probes. Each probe contains the fraF
promoter region indicated in (a). Each probe was incubated with (+) or without (”) His–NrrA (0.1 mM) and then subjected to
electrophoresis. DNA was visualized by staining with ethidium bromide. (c) Effects of removal of the inverted-repeat sequence
on interaction between NrrA and the promoter region of fraF. The left and right sides of the inverted-repeat sequence indicated
with arrows in (a) were removed from probe P2395 to generate probe DL and DR, respectively. Each probe was incubated with
(+) or without (”) His–NrrA (0.3 mM) and then subjected to electrophoresis.
DISCUSSION
In the present study, we indicated that nitrogen-dependent
expression of the fraF gene was regulated by NrrA. NrrA
bound to the promoter region of fraF (Fig. 2) and
upregulated expression of fraF in response to nitrogen
deprivation (Fig. 1). It has been shown that expression of
fraF is under the control of NtcA (Merino-Puerto et al.,
2013). Since NrrA is directly regulated by NtcA (MuroPastor et al., 2006), the nitrogen limitation signal induces
the expression of fraF via two transcriptional regulators,
NtcA and NrrA. Expression of the fraF is higher in
heterocysts than in vegetative cells (Merino-Puerto et al.,
2013), which is consistent with the higher expression of
nrrA in heterocysts (Ehira & Ohmori, 2006a). The fraF
transcripts possess dual functionality, mRNAs for fraF and
asRNAs for fraE (Merino-Puerto et al., 2013). Recently,
the excludon, a new paradigm in RNA-mediated gene
regulation, has been defined to describe a genetic locus for
which a transcript activates expression of one gene or
operon while excluding or inhibiting expression of another
(Wurtzel et al., 2012). A genetic locus containing the fraF
gene and the fraC operon is an example of the excludon
in Anabaena. NrrA could influence filament length by
regulating expression of fraF and asRNAs for fraE.
848
The NrrA recognition sequence was determined by analysis
of the promoter region of fraF (Fig. 3). In a previous study
(Ehira & Ohmori, 2011), we have shown that the sequence
AAAGTCA within the glgP1 promoter and the sequence
AATGTCA within the hetR promoter, which include the
upstream side of the IR sequence, are indispensable for the
binding of NrrA (Fig. 3a). Deletion up to the upstream side
of the IR sequence within the fraF promoter (probe D4)
also abolished the interaction between NrrA and the fraF
promoter (Fig. 3b). Moreover, removal of each side of the
IR sequence from probe P2395 impaired the binding of
NrrA (Fig. 3c), indicating that NrrA recognized the IR
sequence. The IR sequence plays a pivotal role in NrrA
binding; only a faint shift band was observed using probes
DL and DR (Fig. 3c). As the shift bands observed in DL and
DR migrated faster than that of P2395, NrrA might bind to
half of the IR sequence in monomeric form or be detached
from DNA during electrophoresis because of the weak
interaction. The NrrA-binding site is centred 42.5 nt
upstream from the TIS positioned at 2137, indicating that
NrrA activates this promoter by the class II mechanism
(Browning & Busby, 2004).
Although expression of genes involved in heterocyst
differentiation, such as hetZ-patU5-patU3, sigC and patS,
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Microbiology 160
Regulation of the excludon in Anabaena
was significantly decreased by the nrrA disruption (Table
2), no interaction between NrrA and the promoter regions
of these genes was observed. Since HetR binds to the
promoter regions of hetZ and patS (Du et al., 2012; Huang
et al., 2004), downregulation of hetR could affect the
transcript levels of hetZ and patS in the nrrA mutant.
We were unable to detect interaction between the sigC
promoter and HetR (data not shown). Thus, mechanisms
of sigC regulation by NrrA remain unknown.
Orthologues of NrrA are present in most cyanobacteria
except marine picocyanobacteria. NrrA orthologues are
also induced by nitrogen deprivation under the control of
NtcA, and regulate glycogen catabolism in the unicellular
cyanobacteria Synechocystis PCC 6803 (Azuma et al., 2011)
and Synechococcus PCC 7002 (unpublished data). Thus,
regulation of nrrA expression by NtcA and glycogen
catabolism by NrrA are likely to be common features in
cyanobacteria. In Anabaena, NrrA regulates hetR and fraF
in addition to glgP1. The hetR gene is conserved among
filamentous cyanobacteria (Zhang et al., 2009), and the
fraF gene is frequently found in filamentous heterocystforming cyanobacteria (Merino-Puerto et al., 2013). NrrA
of Anabaena seems to have acquired new members of the
regulon to regulate filament length and cellular differentiation in response to nitrogen deprivation.
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ACKNOWLEDGEMENTS
This work was supported by PRESTO of the Japan Science and
Technology Agency and Grant-in-aid for Scientific Research (C) from
the Japan Society for the Promotion of Science (23603005) to S. E.
tion of three group 2 sigma factor genes in Anabaena sp. strain PCC
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