1 Supplementary material Supplemental Methods Plasmids and mutants generation Synechocystis mutant strains were generated by transformation with the plasmids described below and selection with the appropriate antibiotic. Segregation of the mutant strain was checked by PCR using the appropriate primers. The ΔAGP mutant strain was generated by a complete deletion of the slr1176 ORF. A two steps PCR was performed with primers glgC_UP_F/ ΔglgC_UP_R and ΔglgC_DO_F / glgC_DO_R which introduces a BamHI site and the product was ligated into pGEM-T. A chloramphenicol resistance cassette was introduced in BamHI in sense and antisense orientations generating pΔglgC-Cm(+) and pΔglgC- Cm(-), which were used to transform the WT strain. The ΔglgA1 and ΔglgA2 mutant strains were also generated by complete deletion of the sll0945 and sll1393 ORFs, respectively, with a similar strategy as for ΔAGP. The primers used were glgA1_UP_F/ ΔglgA1_UP_R and ΔglgA1_DO_F / glgA1_DO_R for ΔglgA1 and glgA2_UP_F/ ΔglgA2_UP_R and ΔglgA2_DO_F / glgA2_DO_R for ΔglgA2. A kanamycin and spectinomycin resistance cassettes were introduced in BamHI generating pΔglgA1-Km and pΔglgA2-Sp, respectively. For the strain complemented with the wild type AGP, a 2290 pb fragment including the complete glgC ORF was amplified by two steps PCR with primers glgC_UP_F/ glgC_UP_R and glgC_DO_F / glgC_DO_R, introducing a BamHI site after the glgC stop codon. This fragment was cloned into pGEM-T generating pGLGC plasmid. To obtain pGLGC-Km, a C.K.1 cassette was inserted into BamHI. Site-directed mutagenesis of glgC was performed by a two-step PCR. pET45b- GlgC was used as template and overlapping fragments were amplified incorporating the corresponding cysteine to serine substitution with the following
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1
Supplementary material
Supplemental Methods
Plasmids and mutants generation
Synechocystis mutant strains were generated by transformation with the plasmids
described below and selection with the appropriate antibiotic. Segregation of the
mutant strain was checked by PCR using the appropriate primers.
The ΔAGP mutant strain was generated by a complete deletion of the slr1176
ORF. A two steps PCR was performed with primers glgC_UP_F/ ΔglgC_UP_R and
ΔglgC_DO_F / glgC_DO_R which introduces a BamHI site and the product was
ligated into pGEM-T. A chloramphenicol resistance cassette was introduced in
BamHI in sense and antisense orientations generating pΔglgC-Cm(+) and pΔglgC-
Cm(-), which were used to transform the WT strain.
The ΔglgA1 and ΔglgA2 mutant strains were also generated by complete deletion
of the sll0945 and sll1393 ORFs, respectively, with a similar strategy as for ΔAGP.
The primers used were glgA1_UP_F/ ΔglgA1_UP_R and ΔglgA1_DO_F /
glgA1_DO_R for ΔglgA1 and glgA2_UP_F/ ΔglgA2_UP_R and ΔglgA2_DO_F /
glgA2_DO_R for ΔglgA2. A kanamycin and spectinomycin resistance cassettes
were introduced in BamHI generating pΔglgA1-Km and pΔglgA2-Sp, respectively.
For the strain complemented with the wild type AGP, a 2290 pb fragment including
the complete glgC ORF was amplified by two steps PCR with primers glgC_UP_F/
glgC_UP_R and glgC_DO_F / glgC_DO_R, introducing a BamHI site after the glgC
stop codon. This fragment was cloned into pGEM-T generating pGLGC plasmid.
To obtain pGLGC-Km, a C.K.1 cassette was inserted into BamHI.
Site-directed mutagenesis of glgC was performed by a two-step PCR. pET45b-
GlgC was used as template and overlapping fragments were amplified
incorporating the corresponding cysteine to serine substitution with the following
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primers: C45S_DO_F and C45S_UP_R for pET45b-GlgC45S, C315S_DO_F and
C315S_UP_R for pET45b-GlgC315S, C320S_DO_F and C320S_UP_R for
pET45b-GlgC320S, and C337S_DO_F and C337S_UP_R for pET45b-GlgC337S.
A 970 pb ClaI/ SmaI fragment comprising the four cysteine residues was
subcloned into pGLGC from the corresponding pET45b-GlgC plasmids, generating
pGLGC45S, pGLGC315S, pGLGC320S and pGLGC337S, respectively. A
kanamycin resistance cassette was introduced into the BamHI site of these
plasmids, which, as well as pGLGC-Km, were used to transform the ΔAGP strain,
generating C45S, C315S, C320S, C337S and WTc strains.
The STXC strain was generated by transforming the WT strain with pTRXC2
plasmid which contains the sll1057 gene interrupted with a CK1 cassette in the
HincII sites which deletes most of the ORF.
STXAc strain was constructed by insertion of a DNA fragment containing the trxA
ORF under the control of the gifB promoter in the nrsD gene. After complete
segregation of this mutant strain it was transformed with plasmid pTrxASp that
deletes the complete trxA gene. The PgifB::trxA DNA fragment was generated by
overlapping PCR using the oligonucleotides gifB1_SmaI/gifB2_NdeI and
trxA_Nde1/trxA_SmaI. The fragment was digested with SmaI and inserted in StuI
from pNIQ2 which contained the nrsD gene and a kanamycin resistance cassette.
For the trxA deletion an overlapping PCR was performed using oligonucleotides
Sll0586_F1/TrxA1_R1 and sll0585_F1/sll0585_R1 that generate a DNA fragment
with the flanking regions of the trxA gene and BamHI site. After cloning of this
fragment in pGEMT it was sequenced to confirm that no mutation was introduced
and a spectinomycin resistance cassette was inserted in the BamHI site generating
pTrxASp.
Table S1. Oligonucleotides used in this work.
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Primer Sequence
glgC-BamHI_F ccggggatccgGTGAAACGTGTCTTAGC
glgC-SalI_R ccgggtcgacCTAGATTACCGTGCCGTCG
glgA1-BamHI_F ggccggatccgATGAAGATTTTATTTG
glgA1-SalI_R ccgggtcgacTTAGCGATAGGAAGCAGTTAAC
glgA2-BamH_ F ggccggatccgATGTACATCGTTCAA
glgA2-SalI_R ggccgtcgacTTAAGCCCGGATGTATTC
glgC_UP_F CCGTTTACTAATGGCATCAACGGCG
ΔglgC_UP_R CTGGCCggatccAAGCAGACCTCTCGATTG
ΔglgC_DO_F CTGCTTggatccGGCCAGTTTCTTTCCTCG
glgC_DO_R GCGATCCTCCGGCTTAATCTGGGAG
glgA1_UP_F GCCTTATTCTGTTGCTACGTCAATG
ΔglgA1_UP_R AGATTGggatccACCGTCGTTATTCCAC
ΔglgA1_DO_F GACGGTggatccCAATCTCCCGGCAG
glgA1_DO_R TAGAATGAAGCTGGAAATCGGCTC
glgA2_UP_F GCGGCGCTTGGTATTTGTGGAAG
ΔglgA2_UP_R GAGGTGggatccAGAGGCTCCTGATAGCGG
ΔglgA2_DO_F GCCTCTggatccCACCTCGGGTTTGTAAC
glgA2_DO_R CCTGGGTAGGGTTATGGCTTTC
glgC_UP_R GGTGCGAGGAAAggatccGGCC
glgC_DO_F GGCCggatccTTTCCTCGCACC
C45S_DO_F ATTCCCGTCAGTAATtccATCAACTCAGAAATC
C45S_UP_R GATTTCTGAGTTGATggaATTACTGACGGGAAT
C315S_DO_F ATGATCGGGGAAGGTtccATGATTAAGCAA
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C315S_UP_R TTGCTTAATCATggaACCTTCCCCGATCAT
C320S_DO_F ATGATTAAGCAAtctCGCATCCACCACTCA
C320S_UP_R TGAGTGGTGGATGCGagaTTGCTTAATCAT
C337S_DO_F CGCATTGAATCTGATtccACCATTGAGGATACT
C337S_UP_R AGTATCCTCAATGGTggaATCAGATTCAATGCG
Sll0586_F1 CAAAGCAATGGTGGGTCACA
TrxA1_R1 GTCGGTCCTTGAGGGGTAGCACTCATACTG
sll0585_F1 GCTACCCCTCAAGGATCCACCCCCACC
sll0585_R1 CCATGGAACGCTATGGTCTTACCGAC
gifB1_SmaI GATCCCGGGCATCCAGCCCCAGTTCC
gifB2_NdeI CACTCATATGGCGCTCCTAATTGTTATTGAAG
trxA_NdeI TAGGAGCGCCATATGAGTGCTACCCCTCAAGTTTCC
trxA_SmaI AAACCCCGGGAAGTGATTGGCGAATTAG
Supplemental Figure legends
Figure S1. Protein purification and antibody production.
A. Purification of recombinant glycogen synthases and AGP. Recombinant proteins
were purified as described in Materials and methods, resolved in 12% SDS-PAGE
(2 µg) and Coomassie Blue stained
B. Antibody generation against glycogen synthases and AGP. 2.5 µg of
Synechocystis WT, ΔglgA1, ΔglgA2 and ΔAGP strains soluble extracts was
resolved by SDS-PAGE and analyzed by western blot with polyclonal antibodies
raised against purified recombinant GlgA1, GlgA2 and AGP.
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Figure S2. Glycogen synthases are not able to interact with TrxA.
A. Glycogen synthases do not interact with TrxA in Synechocystis extracts. 2.5 µg
of soluble extract were oxidized with 25 µM CuCl2 for 30 min and subsequently
incubated with or without 50 µM TrxAC35S for 1h. Proteins were resolved in 7%
reducing and non-reducing polyacrylamide gels and GlgA1 and GlgA2 were
detected by western blot.
B. Synechocystis glycogen synthases do not interact with TrxA in vitro. 1 µM
recombinant purified GlgA1 or GlgA2 was incubated for 1 h with 4 µM TrxAC35S,
resolved in 12% non-reducing polyacrylamide gels and probed with TrxA
antibodies.
Figure S3. Alignment of selected cyanobacterial and plant AGP proteins. Sequences were aligned using Clustal X program. The cysteines present in
Synechocystis AGP were highlighted in red, the ones involved in APS1
dimerization in green and other conserved cysteines in magenta. Transit peptides
to the chloroplast were removed from plant sequences. Conserved residues are
labeled as defined by Clustal X. Protein accession numbers are Solanum