ChlH, the H subunit of the Mg-chelatase, is an
- FileName: 6860.full.pdf
Abstract: ChlH, the H subunit of the Mg-chelatase, is ananti-sigma factor for SigE in Synechocystis sp.PCC 6803Takashi Osanaia,d,1, Masahiko Imashimizua, Asako Sekia, Shusei Satob, Satoshi Tabatab, Sousuke Imamurac,
ChlH, the H subunit of the Mg-chelatase, is an
anti-sigma factor for SigE in Synechocystis sp.
Takashi Osanaia,d,1, Masahiko Imashimizua, Asako Sekia, Shusei Satob, Satoshi Tabatab, Sousuke Imamurac,
Munehiko Asayamac, Masahiko Ikeuchid, and Kan Tanakaa,e
aInstitute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; bKazusa DNA Research Institute, 2-6-7
Kazusa-Kamatari, Chiba 292-0818, Japan; cCollege of Agriculture, Ibaraki University, Ami, Inashiki, Ibaraki 300-0393, Japan; dDepartment of Life Sciences
(Biology), University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan; and eGraduate School of Horticulture, Chiba University, 648 Matsudo,
Matsudo, Chiba 271-8510, Japan
Edited by Carl E. Bauer, Indiana University, Bloomington, Indiana, and accepted by the Editorial Board February 26, 2009 (received for review
October 7, 2008)
Although regulation of sigma factors has been intensively inves- oxygenic photosynthetic organisms, yeast two-hybrid analyses
tigated, anti-sigma factors have not been identiﬁed in oxygenic have shown that the proteins SapG of the unicellular cyanobac-
photosynthetic organisms. A previous study suggested that the terium Synechococcus sp. PCC 7002 and SibI of A. thaliana
sigma factor, SigE, of the cyanobacterium Synechocystis sp. PCC interact with the sigma factor SigG of Synechococcus sp. PCC
6803, a positive regulator of sugar catabolism, is posttranslation- 7002 and the plastid sigma factor SIG1 of A. thaliana, respec-
ally activated by light-to-dark transition. In the present study, we tively, although the biochemical functions of these interactions
found that the H subunit of Mg-chelatase ChlH interacts with sigma remain unknown (10, 11). To date, no anti-sigma factor has been
factor SigE by yeast two-hybrid screening, and immunoprecipita- identified in oxygenic photosynthetic organisms.
tion analysis revealed that ChlH associates with SigE in a light- Group-2 sigma factors are known as ‘‘primary-like’’ sigma
dependent manner in vivo. We also found that Mg2 promotes the factors, sharing similar promoter recognition with group 1 (or
interaction of SigE and ChlH and determines their localization in primary) sigma factors, but are not essential for cellular viability
vitro. In vitro transcription analysis demonstrated that ChlH inhib- (12, 13). A non–nitrogen-fixing, unicellular cyanobacterium,
its the transcription activity of SigE. Based on these results, we Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803), con-
propose a model in which ChlH functions as an anti-sigma factor, tains 4 group 2 sigma factors (sigB–E) (14). A previous study
transducing light signals to SigE in a process mediated by Mg2 . revealed that SigE is a positive regulator of sugar catabolism,
such as glycolysis, the oxidative pentose phosphate (OPP) path-
cyanobacteria transcription way, and glycogen catabolism (15). Sugar catabolism in photo-
synthetic organisms, including cyanobacteria, is essential under
heterotrophic and dark conditions (16). Metabolomic analysis
R ecent investigations have revealed that metabolic enzymes
play multiple roles as well as catalyzing metabolic reactions,
particularly in transcriptional regulation (1). For example,
with Synechocystis 6803 cells has shown that glucose is degraded
mainly through the OPP pathway under heterotrophic condi-
GAPDH in mammalian cells acts as a coactivator of transcrip- tions (17); consistently, the transcript levels of genes in the OPP
tion from the histone H2B promoter during the S phase (2). In pathway [zwf (slr1843), opcA (slr1734), gnd (sll0329), and tal
yeast mitochondria, Arg 5,6, an enzyme involved in arginine (slr1793)] are increased 1 h after transition from light to dark
biosynthesis, directly binds to the promoter region of COX1 conditions in a SigE-dependent manner (15). SigE levels are not
(encoding a subunit of cytochrome c oxidase) and activates its increased by light-to-dark transition (18), however, suggesting
transcription (3). In Bacillus subtilis, glutamine synthetase inter- that SigE activity is up-regulated at the posttranslational level
acts with TnrA, a global nitrogen transcription factor conserved under dark conditions. In this study, we show that the H subunit
among Bacillus species, inhibiting the access of TnrA to the of the Mg-chelatase ChlH interacts with SigE and represses
promoters of its regulatory genes (4). In higher plants, Arabi- transcription by SigE, indicating that this metabolic enzyme
dopsis thaliana hexokinase1 (Hxk1) forms a complex in the functions as an anti-sigma factor, possibly transducing light/dark
nucleus with VHA-B1 (1 of the 3 expressed isoforms of signals to the SigE.
the B-subunit of the V1 complex in V-ATPase) and RPT5B (1 Results
of the 2 expressed isoforms of the 19S regulatory particle triple-A
Determination of ChlH as a SigE-Binding Protein and the SigE–ChlH
ATPase) and is essential for glucose-dependent transcriptional
Interaction In Vitro and In Vivo. Data from a large-scale protein–
repression (5). Metabolic enzymes regulating transcription have
been discovered in species from several different kingdoms. protein interaction analysis showed that SigE interacted with 4
Transcription in bacteria is initiated by recognition of pro- proteins: ORFs sll1965, slr1055, slr1702, and ssl1707 (19). We
moter sequences by a sigma factor of an RNA polymerase. Sigma performed yeast two-hybrid screening of 3.8 106 independent
clones of a Synechocystis 6803 genomic library with Synechocystis
factors are regulated at transcriptional, translational, and post-
6803 SigE as bait, yielding positive clones including 27 ORFs
translational levels. The role of anti-sigma factors in posttrans-
lational regulation has been intensively investigated (6). The first
identification of a protein inhibiting a sigma factor was in the T4 Author contributions: T.O. and K.T. designed research; T.O., M. Imashimizu, A.S., S.S., S.T.,
bacteriophage (7). Subsequently, it has been found that unin- S.I., M.A., M. Ikeuchi, and K.T. performed research; T.O. analyzed data; and T.O., M. Ikeuchi,
fected Escherichia coli also contains similar mechanisms, such as and K.T. wrote the paper.
FlgM, which inactivates F to transcribe the structural genes The authors declare no conﬂicts of interest.
necessary for the late stage of flagella biogenesis (8). It also is This article is a PNAS Direct Submission. C.E.B. is a guest editor invited by the Editorial Board.
known that sigma factors of Gram-positive bacteria, such as B. 1To whom correspondence should be addressed. E-mail: email@example.com.
subtilis, are regulated by anti-sigma factors necessary for the This article contains supporting information online at www.pnas.org/cgi/content/full/
control of sporulation and stress responses (9). In the case of 0810040106/DCSupplemental.
6860 – 6865 PNAS April 21, 2009 vol. 106 no. 16 www.pnas.org cgi doi 10.1073 pnas.0810040106
Fig. 2. Immunoprecipitation analysis. Proteins from GT Synechocystis 6803
cells grown in modiﬁed BG-11 (A750 2.0) under light or dark conditions (1
h) were mixed with 50 L of rabbit preimmune antiserum, rabbit antiserum
against ChlH, or rat antiserum against SigE. Proteins from the collected cells
were extracted in PBS containing 1 mM MgCl2. Precipitated SigE or ChlH
proteins were detected by immunoblotting with rabbit or rat SigE or rabbit
light conditions (Fig. 3A). To confirm that the decreased protein
levels of ChlH in G50 actually resulted from sigE deficiency, we
performed a complementation experiment with a plasmid car-
Fig. 1. Interaction between SigE and ChlH in vitro: GST-pulldown analysis. rying the wild-type sigE gene, pVZ322:sigE (22). Immunoblot-
Various amounts of GST or GST-ChlH were bound to glutathione-sepharose 4B ting revealed that wild-type sigE gene restored protein levels of
beads and mixed with His-SigE (1.0 g), after which bead-bound proteins were
ChlH (Fig. 3B).
subjected to immunoblotting with antiserum to SigE. Data represent mean
SD of values from 3 independent experiments. The level is calibrated relative
to the pull-down value at 6 g GST-ChlH, which is set at 100%. Mg2 -Dependent Interaction Between SigE and ChlH. Because it has
been suggested that Mg2 and ATP associate with Synechocystis
6803 ChlH (23), we tested the effects of these cofactors on the
[supporting information (SI) Table S1]. Combining the 2 results,
only 1 ORF (slr1055) was included as a positive clone in both
experiments. The ORF slr1055 encodes ChlH, the H subunit of
Mg-chelatase. Mg-chelatase is an enzyme in the chlorophyll
biosynthesis pathway that catalyzes the reaction from protopor-
phyrin IX (Proto) to Mg-protoporphyrin IX (Mg-Proto) by
integrating an Mg2 ion (20, 21). In plants, ChlH is a multifunc-
tional protein with roles in plastid-to-nucleus and plant hormone
signal transduction pathways (see below).
Subsequently, we purified histidine-tagged SigE (His-SigE)
and GST-tagged ChlH (GST-ChlH) from E. coli (Fig. S1 A).
Using GST-ChlH or GST as bait proteins, the amounts of
coprecipitated His-SigE were found to increase in a concentra-
tion-dependent manner with GST-ChlH, but not with GST (Fig.
1), indicating that ChlH interacts with His-SigE in vitro. Because
yeast two-hybrid screening suggested that other proteins also
might interact with SigE, we tested another protein, glutamine
synthetase (GS), for an interaction with His-SigE. Using purified
GST-tagged GS (GST-GS), we performed a GST-pulldown
analysis; the results show that GST-GS interacts with His-SigE
at background levels (Fig. S1B). In addition, we tested whether
another sigma factor interacts with ChlH. GST-pulldown anal-
ysis revealed that purified histidine-tagged SigA (His-SigA) did
not bind GST-ChlH (Fig. S1C).
We then investigated the interaction between SigE and ChlH
in vivo. To examine the effect of light conditions, we used
proteins from cells grown under light or dark conditions for 1 h.
SigE and ChlH proteins from light-grown cells were coimmu-
noprecipitated by rat anti-SigE antiserum or rabbit anti-ChlH
antiserum, but neither was precipitated by rabbit preimmune
antiserum (Fig. 2). Immunoprecipitation with anti-ChlH or
SigE antiserum also revealed that SigE or ChlH proteins from
dark-grown cells were not coimmunoprecipitated (Fig. 2). Fig. 3. (A) Amounts of ChlH proteins in the GT strain of Synechocystis 6803
Next, we examined the protein levels of ChlH during light- and the sigE mutant (G50) under light and dark conditions. Total protein (5 g)
was subjected to immunoblotting. (Lower Panel) GS protein levels as a control.
to-dark transition. Immunoblotting with antiserum to ChlH
Data represent mean SD of values from 3 independent experiments. The
revealed that ChlH protein levels decreased to about 80% and level is calibrated relative to the value in the GT strain of Synechocystis 6803
40% of those under light conditions at 1 h and 4 h after under light conditions, which is set at 100%. (B) Amounts of ChlH proteins
light-to-dark transition, respectively (Fig. 3A). In addition, dis- in GT, G50, and G50 carrying pVZ322:sigE. Cells were grown in modiﬁed
ruption of sigE, such as that in the sigE insertion mutant G50 BG-11 under light conditions, and total protein (5 g) was subjected to
(15), resulted in a 40% decrease in ChlH protein levels under immunoblotting.
Osanai et al. PNAS April 21, 2009 vol. 106 no. 16 6861
Fig. 5. ChlH repressed the transcriptional activity of SigE in vitro. An in vitro
transcription analysis was performed by mixing puriﬁed His-SigA or His-SigE
(1.5 pmol) with GST-ChlH (0, 1.5, 3, or 9 pmol) or GST (9 pmol), after which the
native core of RNA polymerase (1 pmol) and 0.3 pmol of template DNA
pKK223–3 was added. The resultant mRNAs were resolved by 5% urea-PAGE,
and radioactivity was detected using a BAS-1000 image analyzer.
Previous experiments have found that ChlH fractionates within
the membrane fraction at Mg2 concentrations exceeding 5 mM
and within the soluble fraction at concentrations below 1 mM
(24, 25). We carried out a similar experiment using glucose-
tolerant (GT) cells of Synechocystis 6803 (the GT strain of
Synechocystis 6803). GT Synechocystis 6803 cells were disrupted
in lysis buffer with or without 10 mM MgCl2, and soluble and
membrane fractions were separated by ultracentrifugation (see
Materials and Methods). As is the case in higher plants, Synecho-
cystis 6803 ChlH proteins were detected mostly within the
membrane fraction in the presence of Mg2 , whereas they were
Fig. 4. Mg2 -dependent interaction between SigE and ChlH. (A) Effect of detected in the soluble fraction in the absence of Mg2 (Fig. 4C).
Mg2 and ATP on the SigE–ChlH interaction. GST or GST-ChlH (2.0 g) bound Like the ChlH proteins, most SigE proteins were detected within
to glutathione-sepharose 4B beads and mixed with His-SigE (1.0 g) in the
the membrane fraction in the presence of Mg2 or in the soluble
absence or presence of 2 or 20 mM MgCl2 and with or without 1 mM ATP.
Precipitated proteins were detected by immunoblotting. Data represent fraction in the absence of Mg2 (Fig. 4C). In contrast, 3 subunits
mean SD of values from 4 independent experiments. The level is calibrated of Mg-chelatase or RNA polymerase—ChlI, RpoB (beta sub-
relative to the value obtained in the absence of both MgCl2 and ATP, which is unit), and RpoC2 (beta prime subunit)—were detected within
set at 100%. (B) Speciﬁc effect of Mg2 on the SigE–ChlH interaction. GST-ChlH the soluble fraction irrespective of the presence or absence of
(2.0 g) bound to glutathione-sepharose 4B beads and mixed with His-SigE Mg2 (Fig. 4C). Disruption of sigE did not affect the localization
(1.0 g) in the absence or presence of 10 mM MgCl2 or MgSO4 or 20 mM NaCl.
of ChlH (Fig. S3).
Precipitated proteins were detected by immunoblotting. (C) Colocalization of
ChlH and SigE depending on Mg2 concentration. Cells were disrupted in the
absence or presence of 10 mM MgCl2 (whole cell extract), and divided into ChlH Represses Transcription by RNA Polymerase Containing SigE In
soluble and membrane fractions (Sup and Ppt, respectively) by ultracentrifu- Vitro. We performed in vitro transcription analysis to examine
gation. Proteins (5 g/lane) were detected by immunoblotting. the posttranslational regulation of SigE by ChlH. Fig. 5 shows
that transcription by RNA polymerase containing the group-1
SigE–ChlH interaction. GST-pulldown analysis revealed that the sigma factor SigA was not affected by the addition of GST-ChlH;
addition of MgCl2 strengthened the interaction between GST- however, transcription by RNA polymerase containing SigE was
ChlH and His-SigE (Fig. 4A). The amounts of coprecipitated repressed in the presence of GST-ChlH in a ChlH concentra-
His-SigE proteins in the presence of 2 or 20 mM MgCl2 were 2.1 tion–dependent manner. To exclude a possible effect of GST, we
or 3.2 times those in the absence of MgCl2, respectively (Fig. 4A). purified histidine-tagged ChlH (His-ChlH) from E. coli (Fig.
MgSO4 strengthened the interaction, but NaCl did not, indicat- S4A). In vitro transcription analysis revealed that His-ChlH also
ing that Mg2 specifically promoted the interaction (Fig. 4B). In inhibited transcription by RNA polymerase containing SigE, but
contrast, the addition of ATP inhibited the interaction (Fig. 4A). not transcription by RNA polymerase containing SigA (Fig.
The amount of His-SigE in the presence of 1 mM ATP was 54% S4B). Although we tested the effects of increased concentrations
of that in the absence of ATP. The inhibitory effect of ATP was of Mg2 (from 3 mM to 20 mM), transcription itself was
eliminated by increasing the Mg2 concentration (Fig. 4A). abolished in the presence of 20 mM MgCl2; thus, we could not
EDTA did not inhibit the interaction, suggesting that the inhib- examine the effect of Mg2 on in vitro transcription (Fig. S4C).
itory effect of ATP was not due to chelating Mg2 (Fig. S2). We also found that an excess amount of GST-ChlH completely
ChlH proteins of higher plants fractionate differently, depend- abolished the transcription by RNA polymerase containing SigE
ing on the concentration of Mg2 in the lysis buffer (24, 25). (Fig. S4D).
6862 www.pnas.org cgi doi 10.1073 pnas.0810040106 Osanai et al.
Proto or Mg-Proto, and regulates SigE at the posttranslational
level. This model is consistent with the data showing that ChlH
is a negative regulator of SigE transcription (Fig. 5). This model
also is consistent with a previous study demonstrating increased
expression of OPP pathway genes by light-to-dark transition in
a SigE-dependent manner (15).
ChlH is known as a multifunctional protein. Shepherd et al.
(27) reported that ChlH of Synechocystis 6803 accelerates the
catalytic activity of ChlM, which catalyzes the conversion of
Mg-Proto to Mg-protoporphyrin IX monomethyl ester. In higher
plants, the chlH mutant gun5 cannot transduce signals from
plastid to nucleus in A. thaliana (28). In A. thaliana, ChlH also
functions as a receptor of the plant hormone abscisic acid (ABA)
and as a positive regulator of ABA signal transduction (29). We
propose a novel function of ChlH as an anti-sigma factor,
repressing the transcription of SigE via a protein–protein inter-
action. In higher plants, ChlH and several sigma factors are
localized within the plastid; however, phylogenetic analysis
suggests that sigma factors in eukaryotes are derived from
group-1 sigma factors of ancient cyanobacteria (30); therefore,
regulation of a sigma factor by ChlH may be restricted to several
cyanobacterial strains. Nevertheless, a recent study revealed that
transcription within chloroplasts was regulated by Mg-Proto
level, indicating the significance of tetrapyrrole metabolism for
transcription within plastids (31). Thus, future studies may reveal
that plastids also have regulatory mechanisms regulating sigma
factors via protein–protein interactions with other proteins,
including metabolic enzymes, similar to the repression of SigE by
ChlH in cyanobacteria.
Materials and Methods
Bacterial Strains and Culture Conditions. The GT strain of Synechocystis sp. PCC
6803, isolated by Williams (32), was grown in BG-110 liquid medium with 5 mM
NH4Cl (buffered with 20 mM Hepes-KOH; pH 8.0), designated modiﬁed BG-11
medium. Liquid cultures were bubbled with 2% (vol/vol) CO2 in air at 30 °C
Fig. 6. Probable model for the regulation of SigE by ChlH. Under light
under continuous white light (ca. 70 mol photons m 2 s 1) (33). For plate
conditions, ChlH is membrane-associated and interacts with SigE, leading to
cultures, BG-11 medium (17.5 mM NaNO3 and 20 mM Hepes-KOH; pH 8.0) was
repression of transcription by RNA polymerase interacting with SigE. When
solidiﬁed using 1.5% (wt/vol) agar (Nissui) and incubated in air containing 2%
Proto binds to ChlH, ChlH is released from SigE and functions as a Mg-
chelatase. During the transition from light to dark, ChlH dissociates from the (vol/vol) CO2 at 30 °C under continuous white light (ca. 70 mol photons m 2
membrane, and concomitantly, SigE dissociates from ChlH due to the decreas- s 1). Growth and cell densities were measured at A750 with a Beckman DU640
ing Mg2 concentration. As a result, free SigE can complex with the RNA spectrophotometer.
polymerase core, resulting in activation of the transcription of SigE regulons.
Yeast Two-Hybrid Analysis. The full-length sigE gene (sll1689) of Synechocystis
6803 was ampliﬁed by PCR using Pfu polymerase (Clontech) and the speciﬁc
Discussion primers 5 -GAGGGCGCGCCATGAGCGATATGTCTTCC-3 and 5 -CGGTATC-
TATAACCAACCTTTGAG-3 , and then subcloned into the pAS2–1-AscI bait
Our findings demonstrate that the H subunit of Mg-chelatase, vector constructed by Sato et al. (19). The details of the yeast two-hybrid
ChlH, interacts with the sigma factor SigE and represses tran- screening procedure were as described by Sato et al. (19).
scriptional activity in vitro, and that this interaction is controlled
by light conditions in vivo. It has been shown that Mg2 Affinity Purification of GST-ChlH and His-SigE. A region of the Synechocystis
concentrations in the stroma of spinach chloroplasts are altered 6803 genome encoding ChlH was ampliﬁed by PCR using KOD polymerase
by light-dark transition (26). The free Mg2 concentration is (Toyobo) and the speciﬁc primers 5 -CGGGATCCGGATGTTTACTAACGT-
about 0.5 mM under dark conditions and increases to about 2 CAAGTC-3 and 5 -ACTCGAGTTTATTCAACCCCTTCAATG-3 , digested with
mM during illumination. Although changes in the in vivo con- BamHI and XhoI (Takara), and inserted into the BamHI-XhoI sites of pGEX5X-2
(Amersham Pharmacia Bioscience). The resultant plasmid was designated
centrations of free Mg2 were not investigated in Synechocystis
pGEXChlH. Full-length sigE was ampliﬁed using KOD polymerase (Toyobo)
6803, our preliminary analysis with a fluorescence probe mag-
and the speciﬁc primers 5 -CCGCATGCATGAGCGATATGTCTTCC-3 and 5 -
fura 2 (Invitrogen) revealed that free Mg2 concentration in a CCGATATCCTATAACCAACCTTTGAG-3 , digested with SphI and EcoRV
Synechocystis cell is about 1 mM under light conditions (data not (Takara), and inserted into the SphI-SmaI sites of pQE80L (Qiagen). The
shown). Further analysis is needed to determine the changes of constructed plasmids encoding GST-ChlH or His-SigE were introduced sepa-
Mg2 concentration by light-to-dark transition. rately into E. coli BL21 Codon Plus cells (Stratagene) by transformation.
Based on these data, we propose a probable model for SigE Expression was induced by the addition of 1 mM isopropyl- -D-thiogalacto-
regulation by ChlH (Fig. 6). Under light conditions, SigE pyranoside (Wako) to 1 L of LB medium, and the cells were cultured overnight
interacts with ChlH, which is anchored to plasma or thylakoid at 27 °C. The cells were then collected by centrifugation and lysed in 30 mL of
lysis buffer [40 mM Tris-HCl (pH 8.0), 5% glycerol, 5 mM EDTA, and 4.5% Triton
membranes (Fig. 4C). During transition from light to dark, the
X-100] by sonication (Branson Soniﬁer 450). The soluble fraction and the
decreased Mg2 concentration causes ChlH to dissociate from insoluble fraction that contained recombinant protein were divided by cen-
the membrane, and the interaction between SigE and ChlH is trifugation of the cell lysate at 17,400 g for 20 min at 4 °C. For puriﬁcation
abolished. Then free SigE is able to associate with the RNA of GST-ChlH, 800 L of glutathione-sepharose 4B (GE Healthcare) was added
polymerase core and activate transcription of SigE regulons (Fig. to soluble fraction and gently mixed for 1 h at 4 °C. After centrifugation at
6). In this model, ChlH transduces light signals by Mg2 , not by 300 g, resin incubated with GST-ChlH was washed 5 times with PBS con-
Osanai et al. PNAS April 21, 2009 vol. 106 no. 16 6863
taining 0.1% Triton X-100 and eluted 3 times with 400 L of GST elution buffer g for 4 min and then resuspended in 15 mL of Hepes-KOH (pH 8.0) with or
[50 mM Tris-HCl (pH 8.0) and 10 mM reduced glutathione]. For puriﬁcation of without 10 mM MgCl2. After disruption of cells by sonication, undisrupted
His-SigE, the insoluble fraction was washed with cell lysis buffer, suspended in cells were removed by centrifugation at 17,400 g for 20 min; the resulting
sterilized water, and solubilized by the addition of half a volume of a solution supernatant was designated the whole cell extract. Then soluble and mem-
containing 8 M urea, 50 mM Tris-HCl (pH 8.0), and 10 mM DTT and incubated brane fractions were divided by ultracentrifugation at 100,000 g for 1 h. The
for 1 h at 37 °C. The solubilized His-SigE proteins were dialyzed against protein concentrations of each fraction were estimated using a BCA protein
His-binding buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 0.1% Triton assay kit (Pierce).
X-100] and puriﬁed with HIS-Select resin (Sigma). Protein concentration was
determined using a Bio-Rad protein assay. Affinity Purification of His-ChlH, GST-GS, and His-ChlI. pGEXChlH was digested
by BamHI and XhoI, and the resultant fragment was cloned into pQE82L
GST-Pulldown Analysis. Puriﬁed GST or GST-ChlH was bound to 20 L of (Qiagen) digested by BamHI and SalI (Toyobo). The constructed plasmid
glutathione-sepharose 4B for 30 min at room temperature, mixed with His- encoding His-ChlH was introduced into E. coli BL21 Codon Plus (Stratagene) by
SigE in 500 L of Hepes-binding buffer [50 mM Hepes-KOH (pH 8.0), 5% transformation. The expression of His-ChlH was induced by the addition of 1
glycerol, and 0.1% Triton X-100] with MgCl2 at the concentrations indicated mM isopropyl- -D-thiogalactopyranoside (Wako) to 1 L of LB medium, after
in the ﬁgure legends, and incubated for 1 h at room temperature. The resin which the cells were cultured overnight at 27 °C. The cells were then collected
was washed twice with 150 L of Hepes-binding buffer, suspended in 50 L of by centrifugation, lysed in 30 mL of His-binding buffer by sonication (Branson
SDS sample buffer [250 mM Tris-HCl (pH 6.8), 20% sucrose, 20% 2-mercapto- Soniﬁer 450), and centrifuged at 17,400 g for 20 min at 4 °C. The soluble
ethanol, 8% SDS, and 0.04% bromophenol blue], and then heated for 5 min fraction was gently mixed with 800 L of HIS-Select for 1 h at 4 °C. After
at 96 °C. The released proteins were then subjected to SDS-PAGE and detected centrifugation at 300 g, resin incubated with His-ChlH was washed 5 times
by immunoblot analysis with antiserum to SigE (15). with His-binding buffer containing 5 mM imidazol and eluted with 400 L of
His-binding buffer containing 500 mM imidazol.
A region of glnA encoding GS was ampliﬁed by PCR using KOD polymerase
Rabbit ChlH Antiserum and Rat SigE Antiserum Production and Immunoblot
(Toyobo) and the primers 5 -CATAGATCTTAATGGCCAGAACCCCCCA-3 and
Analysis. Antisera to ChlH or SigE were produced commercially by Tanpaku
5 -GGCTCGAGCCTTAGCAGTCGTAGTACAAG-3 , digested by BglII and XhoI,
Seisei Kogyo. Antiserum was raised in a rabbit using 2.0 mg of puriﬁed
and subcloned into pGEX5X-2 (Amersham Pharmacia Bioscience) digested by
GST-ChlH and in rats using 1.5 mg of puriﬁed His-SigE. Immunoblotting with
BamHI and XhoI. Expression and puriﬁcation of GST-GS were performed as
antiserum to ChlH or SigE was performed as described previously (12).
GST-ChlH. A region of chlI encoding the I subunit of Mg-chelatase was similarly
ampliﬁed and cloned into a pQE82L vector with the primers 5 -CATAGATC-
Immunoprecipitation Analysis. The GT strain of Synechocystis 6803 cells was TATGACTGCCACCCTTGCC-3 and 5 -AAGATATCTTAAGCTTCATCGACAAC-3 .
grown in modiﬁed BG-11 (A750 2.0) and collected by centrifugation (8,000 Expression and puriﬁcation of His-ChlI were performed as His-ChlH. Rabbit
g for 5 min). Cells from 0.5-L cultures were resuspended in 1.5 mL of PBS with 1 antisera to GS and ChlI was produced commercially by Tanpaku Seisei Kogyo.
mM MgCl2, 2 mM PMSF, and 1 tablet of Complete Mini, EDTA-free (Roche), and
then disrupted by sonication (Branson). After centrifugation at 17,400 g for 10 In Vitro Transcription Analysis. His-SigA was expressed from an expression
min at 4 °C, the protein concentration of the supernatant was estimated using a vector constructed previously (34) and puriﬁed from insoluble fractions as
BCA protein assay kit (Pierce), and the supernatant was used as a cell extract. described for His-SigE (see above). In vitro transcription analysis was per-
Anti-SigE or preimmune antiserum from rat (50 lL) was mixed with 400 L of formed as described by Imashimizu et al. (35) with some modiﬁcations.
protein G sepharose (GE Healthcare) and 500 L of PBS. After 30 min of shaking Puriﬁed His-SigA or His-SigE (1.5 pmol) was mixed with GST-ChlH or His-ChlH
at room temperature, the resins incubated with antibodies were washed 3 times in 10 L of transcription buffer [50 mM Hepes-KOH (pH 8.0), 3 mM MgCl2, 1
with 500 L of PBS. Then resins were suspended in 500 L of PBS, and 0.5 mg/mL mM DTT, 50 mM potassium glutamate, and 25 g/mL BSA], and the mixture
of disuccinimidyl suberate (DSS; Pierce) was added to cross-link antibodies to the was incubated for 10 min at 30 °C. Then the native core of the RNA polymerase
resins. After 40 –50 min of mixing at room temperature, the resins were washed (1 pmol) of the thermophilic cyanobacteria Thermosynechococcus elongatus
5 times with 500 L of glycine elution buffer (0.1 M glycine-HCl; pH 2.7) and 4 BP-1 was added to the mixture and incubated for 20 min at 30 °C. Subse-
times with PBS. Then aliquots of cell extracts conta
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