Appendix S1
Affinity purification of His-Rre12
A region of the Synechocystis 6803 genome encoding Rre12 (sll1291) was amplified by
PCR using KOD polymerase (Toyobo, Osaka, Japan) and the specific primers
5′-ATGGATCCATGCAATCTCCCCTG-3′ and 5′-TTAAACAGCTGCGGAGAT-3′.
The products were digested with BamHI (Takara Bio, Shiga, Japan), phosphorylated by
T4 polynucleotide kinase (Takara), and inserted into the BamHI–SmaI site of the
pQE80L vector (Qiagen Japan, Tokyo, Japan). The constructed plasmids encoding
His-Rre12 were introduced separately into Escherichia coli BL21 Codon Plus cells
(Agilent Technologies, Palo Alto, CA, USA) by transformation. Expression was induced
by addition of 10 M isopropyl--D-thiogalactopyranoside (Wako Chemicals, Osaka,
Japan) to 1 liter of LB medium, and the cells were cultured for 4 h at 30C. The cells
were collected by centrifugation and lysed in 30 ml cell lysis buffer [40 mM Tris-HCl
(pH 8.0), 5% glycerol, 5 mM EDTA, and 4.5% Triton X-100] by sonication (Sonifier
450, Branson, Danbury, CT, USA). The soluble and insoluble fractions that contained
His-Rre12 were divided by centrifugation of the cell lysate (at 17,400 g for 20 min at
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4C). The insoluble fraction was washed with 30 ml cell lysis buffer, suspended in
sterilized water, and solubilized by addition of half a volume of a solution containing 8
M urea, 50 mM Tris-HCl (pH 8.0), and 10 mM DTT, and incubated for 1 h at 37C. The
solubilized His-Rre12 proteins were dialyzed against His-binding buffer [50 mM
Tris-HCl (pH 8.0), 100 mM NaCl, and 0.1% Triton X-100] and bound with HIS-Select
resin (Sigma Aldrich Japan, Tokyo, Japan). After centrifugation, and several washes
with His-binding buffer containing 5 mM or 20 mM imidazole, recombinant proteins
were eluted by His-binding buffer with 200 mM imidazole. The protein concentration
was determined using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA).
Production of antisera and immunoblotting
Antisera against Rre37, PII, ChlH, ChlI and SigA were generated previously (Osanai et
al., 2005a; Seki et al., 2007; Osanai et al., 2009; Azuma et al., 2011; Osanai et al.,
2011). Antiserum against Rre12 was commercially produced by Tampaku Seisei Kogyo
Co. Ltd (Gunma, Japan). Immunoblotting was performed as described previously
(Osanai et al., 2013).
2
Results and discussion
First, three proteins induced by nitrogen depletion, namely Rre37, Rre12 and PII, were
quantified. Rre37 is increased during nitrogen starvation and is involved in transcription
of sugar catabolic genes (Azuma et al., 2011). Another response regulator, Rre12
(sll1291), which is an unknown protein belonging to the PatA-family, is also induced by
nitrogen depletion (Osanai et al., 2006; supplemental data, Table S3). Histidine-tagged
Rre12 proteins were expressed in E. coli and purified by affinity chromatography for
antibody production (Figure S7a). All three proteins increased during nitrogen
starvation in both strains, but were reduced by sigE overexpression (Figure S7b). The
knockout mutant of sigE showed an increased level of Rre37 protein (Azuma et al.
2011), indicating that SigE and Rre37 repress the expression of each other. The sigE
gene is regulated by protein–protein interaction with a H-subunit of Mg-chelatase ChlH
(Osanai et al., 2009). The protein levels of ChlH decreased under nitrogen depletion in
both strains, whereas the level in the sigE-overexpressing strain was lower than that of
GT after 6 h of nitrogen depletion (Figure S8). The protein levels of ChlI, which is
3
another subunit of Mg-chelatase, were unaffected by either nitrogen depletion or sigE
overexpression (Figure S9). The protein level of SigA, a primary sigma factor of
Synechocystis 6803, was increased by sigE overexpression (Figure S9). The SigA
protein level was transiently reduced by nitrogen depletion, and the level in the
sigE-overexpressing strain remained higher than that of the GT strain during nitrogen
starvation (Figure S8).
The level of sigA transcripts was strongly reduced by sigE
disruption (Lemeille et al. 2005); these results are consistent with the increased SigA
protein levels by sigE overexpression.
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