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Supplementary tables Berendt et al. 2012
Supplementary Table 1:
Anabaena and its derivative strains used in this study
Strain Relevant characteristics Reference Anabaena sp. PCC 7120 heterocyst forming wild type Anabaena sp. PCC 7120 (pIM471)
alr0092-gfp fusion; Smr/Spr This study
Anabaena sp. PCC 7120 (pIM472)
alr0093-gfp fusion; Smr/Spr This study
Anabaena sp. PCC 7120 SR477
amiC1::pIM477, Smr/Spr This study
Anabaena sp. PCC 7120 SR478
amiC2::pIM478, Smr/Spr This study
Anabaena sp. PCC 7120 OE484
PpetE+alr0092;Smr, Spr; This study
Anabaena sp. PCC 7120 OE485
PpetE+alr0093;Smr, Spr This study
Anabaena sp. PCC 7120 SR477 (pIM496)
alr0092-gfp fusion; Smr/Spr, Nmr This study
Supplementary Table 2:
Plasmids used in this study
Plasmid Relevant characteristics Reference pCSEL19 Vector containing promotorless
gfp gene; Amr (1)
pCSEL24 Mobilizabel plasmid to integrate constructs into a neutral site (nucA-nuiA region) located in the α-megaplasmid of Anabaena sp. PCC 7120.
(2)
pGEM-T AT overhang, cloning Vektor; Amr
Promega
pIM451 Tat-amiC1(alr0092) inserted in pGEM-T; Amr
This study
pIM452 Tat-amiC1-gfp inserted in pEGM-T; Amr
This study
pIM453 Tat-amiC1-gfp inserted in pRL1049; Smr/Spr
This study
pIM454 Tat-amiC2-gfp inserted in pJET; Amr
This study
pIM455 Tat-amiC2-gfp inserted in pRL1049; Smr/Spr
This study
pIM471 promotor region of amiC1,2 inserted in EcoRI site of pIM453; Smr/Spr
This study
pIM472 promotor region of amiC1,2 inserted in EcoRI site of pIM455; Smr/Spr
This study
pIM475 Internal fragment of amiC1 in pGEM-T, Ampr
This study
pIM476 Internal fragment of amiC2 in pGEM-T, Ampr
This study
pIM477 Internal fragment of amiC1 in pRL277, Smr, Spr
This study
pIM478 Internal fragment of amiC2 in pRL277, Smr, Spr
This study
pIM480 alr0092 in pJET, Ampr This study pIM481 alr0093 in pJET, Ampr This study pIM482 PpetE+alr0092 in pJET, Ampr This study pIM483 PpetE+alr0093 in pJET, Ampr This study pIM484 PpetE+alr0092 in pCSEL24, Smr,
Spr This study
pIM485 PpetE+alr0093 in pCSEL24, Smr, Spr
This study
pIM496 PamiC1,2 -Tat-amiC1-gfp in pRL1049+C.K3; Sm/Sp, Km
This study
pJET Cloning vector; Amr Fermentas pRL277 Non-replicating, mobilizable
vector containing sacB; Smr, Spr
(3)
pRL442 Vector containing PsbA nptII cassette; Kmr/Nmr
(4)
pRL1049 multicopy replicating plasmid for use in Anabaena sp. PCC 7120
(5)
Supplementary Table 3:
Oligonucleotides used in this study for PCR and RT-PCR
Oligo Sequence F1 CTCGAGCAGTGGTGGCAATCCTCAAG XhoI R1 CTCGAGGGCTGGACGAGAACAACTAC XhoI F2 CTCGAGTTACGAGTTACAGGCGATG XhoI R2 CTCGAGGCGGTAAAGTCCAGAGGTTC XhoI F3 AGATCTGTGAAATTACACTGGTTACTATCTGG BglII R3 CTGCAGCTATCGTTTGAGGTACTGAAG PstI F4 AGATCTGTGAAATTACACTGGTTACTATCCG BglII R4 CTGCAGTTACTGTCTCTTTAAATATTGCAGGATACC PstI R5 ACCTATCTCAGGGATCTGTC Oligo698 GAATTCCCCCCAGAGTTAATATGCCAAAG EcoRV Oligo699 GGCGACGATAGTAATAAGACAGTTCCAACTTTGGCAATAGTGGCAT
GAGTCAG
Oligo700 CTCATGCCACTATTGCCAAAGTTGGAACTGTCTTATTACTATCGTCG Oligo706 GGATCCCAGGTCTGGACATTTATTTGTATAG BamHI Oligo703 GATGGTTCCAACCGTACCGGATAGTTTGGCAATAGTGGCATGAGTC Oligo704 CACTGACTCATGCCACTATTGCCAAACTATCCGGTACGGTTGGAAC Oligo727 CCCGGGTCGTTTGAGGTACTGAAGAATACCG SmaI Oligo730 CAATATTTAAAGAGACAGCCCGGGGATATCCATATGAGTAAAGGAG
AAG
Oligo731 CTTCTCCTTTACTCATATGGATATCCCCGGGCTGTCTCTTTAAATATTG
Oligo767 GAATTCGCTGAGGTACTGAGTACAC EcoRI Oligo768 AGATCTGTTCTCCTAACCTGTAGTTTTATTTTTC BglII Oligo807 GAATTCCAGAGTGCTGTAGGCAGATG EcoRI Oligo808 GAATTCCAATCCATTCTCCTGATTTGC EcoRI rnpB-for GGGGAGGAAAGTCCGGGCTCC rnpB-rev TAAGCCGGGTTCTGTTCTCTG amiC1-for CCCAGGAGCCATTGGTATTG amiC1-rev GCGTTAGCGTGGATACTGAC amiC2-for TAAAGATTCTGGCGCTCCTG amiC2-rev AGACGTAGAACCGAGCTTTG hepA-for ACATGATTTTGGCTGCTGATGC
hepA-rev GCTAAAACAACTTTGATAGCTGC ntcA-for CAGGAAACAAGTCGGATAGG ntcA-rev AACAGTAACGCGAGTAGAGC hetR-for CCGGATGACCGGACATTTGC hetR-rev CTGAGCGTCGGGAAGATTGC all4999-for CTCGAGTTTGGCTGCGCTCACTGGAC all4999-rev CTCGAGACGGCGAGGTTATGGGCTTG nifD-for CCTCGCTTGGAAGGTAACAC nifD-rev TGCATTTGACGGAAGGGAAG Oligo919 GAAGATACTGCGGAGCAAGGC
1. Muro-Pastor AM, Olmedo-Verd E, Flores E. All4312, an NtcA-regulated two-component response regulator in Anabaena sp. strain PCC 7120. FEMS Microbiol Lett. 2006;256(1):171-7. PubMed PMID: 16487336. 2. Olmedo-Verd E, Muro-Pastor AM, Flores E, Herrero A. Localized induction of the ntcA regulatory gene in developing heterocysts of Anabaena sp. strain PCC 7120. J Bacteriol. 2006;188(18):6694-9. PubMed PMID: 16952962. 3. Black TA, Cai Y, Wolk CP. Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol Microbiol. 1993;9(1):77-84. PubMed PMID: 8412673. 4. Elhai J, Wolk CP. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene. 1988;68(1):119-38. PubMed PMID: 2851487. 5. Black TA, Wolk CP. Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing. J Bacteriol. 1994;176(8):2282-92. PubMed PMID: 8157596.
Berendt et al. Fig.: S1
B kefB amiC1 amiC2 murI
Anabaena sp. PCC 7120
Anabaena variablilis
Nostoc punctiforme
Nostoc azollae
Nodularia spumigena
Synecchococcus PCC 7002
Cyanothece sp. PCC 8801
Arthrospira platensis
Trichodesmium erythraeum
alr0093 alr0092 alr0091 alr0094
Ava1466 Ava1465 Ava1464
Ava1467 AA
NpF1846 NpF1845 NpF1844 NpF1847
0861 0862 0863 0860
08355 08350 08360
0578 0577 0579
0589 0579
6750
0339
Alr0092 (Anabaena sp. PCC 7120)
Ava1465 (Anabaena variablilis)
NpF1845 (Noctoc punctiforme)
0862 (Nostoc azollae)
08355 (Nodularia spumigena)
6750 (Arthrospira platensis)
0339 (Trichodesmium erythraeum)
Amidase
Amidase
Amidase
Amidase
Amidase
Amidase
AMIN
AMIN
AMIN
AMIN
AMIN
AMIN
AMIN
AMIN
AMIN
AMIN
AMIN
AMIN
Amidase
Fila
men
tous
,
hete
rocy
st fo
rmer
s Fi
lam
ento
us
SS
SS
SS
SS
SS
SS
SS
A
Fila
men
tous
,
hete
rocy
st fo
rmer
s Fi
lam
ento
us
Uni
cellu
lar
Fig. S1: Orthologous of AmiC in several cyanobacteria. (A) Scheme of the structure of AmiC like proteins in filamentous cyanobacteria with and without differentiation potential. SS, Sec signal sequence; AMIN, Amidase N-termial domain. (B) Scheme of the genomic organization of the amiC region in different cyanobacteria.
Berendt et al. Fig.: S2
WT SR478
1301 bp
WT SR477 SR478
amiC2 amiC1 alr0091
pIM478
pIM477
F3 F1
F4
F2
R5
F1
F3
R5
R3
R1
R4
R2
amiC1 amiC1 amiC2 amiC2
rnpB rnpB
WT SR477 WT SR478
A
1884 bp
576 bp 461 bp
B
D C
WT SR477
F1/R5
1415 bp
F3/R5 F3/R5 F1/R5 F2/R5 F2/R5 F4/R5 F4/R5
F1/R1 F3/R3 F2/R2 F4/R4
Fig. S2: (A) Genomic organization of the amiC cluster in Anabaena PCC 7120 wild type (WT), amiC1-mutant strain SR477 and amiC2-mutant strains SR478 and the approximate positions of primers used for PCR analysis. (B, C) PCR analysis of Anabaena PCC 7120 WT, amiC1-mutant and amiC2-mutant. Sites of used primers are indicated in (B) R5 binds inside the integrated vector pRL277. Primer sequences are listed in supplementary table 3. (D) RT-PCR analysis of amiC1 and amiC2 in the amiC1 mutant SR477 and the amiC2-mutant SR478. The rnpB gene was used as a loading control. Oligonucleotides amiC1-for , amiC1-rev, amiC2-for, amiC2-rev, rnpB-for and rnpB-rev were used (Table S3)
R1
amiC2 alr0091
amiC1 alr0091
R3
R4 R2
F4
F2
Berendt et al. Fig.: S3
WT OE484
WT OE485
-‐
+
amiC1
-‐
+
amiC2
WT OE484 OE485
amiC1 amiC2
A B
Fig. S3: Expression of amiC1 and amiC2 was analyzed by RT-PCR in Anabaena PCC 7120 and in the amiC1- and amiC2-overexpression strains OE484 and OE485, respectively. (A)With (+) and without (-) CuSO4 treatment. (B) Comparison of the transcripts in the WT and the overexpressing strains. Overexpression of amiC1 had no effect on amiC2 and vice versa. Primers used for RT-PCR (sequence in Table S3): Transcript of amiC1: amiC1-for and amiC1-rev, binding inside the gene Transcript of amiC2: amiC2-for and amiC2-rev, binding inside the gene
Fig. S4: Two different filaments Anabaena PCC 7120 bearing plasmid pIM471 showing
subcellular localization of AmiC1-GFP 12 h after nitrogen step-down. Arrows show region
of the polar neck. BF, bright field; GFP, GFP-fluorescence; AF, auto-fluorescence GFP
+AF, overlay of GFP-fluorescence and autofluorescence. Bar = 5 µm.
AmiC1-‐GFP
AmiC1-‐GFP
BF AF GFP GFP + AF
Berendt et al. Fig.: S4
