SUPPLEMENTAL MATERIAL
Supplemental Figure S1
Disruption of ctaC1D1E1, ctaCII and cydAB in Synechocystis sp. PCC 6803.
Schematic representations of locus location in the Synechocystis genome (top) and the wild-type
(middle) and unmarked knockouts (bottom) profiles expected in (A) ∆ctaC1D1E1, (B) ∆ctaC2 and (C)
∆cydAB strains following amplification with primers flanking the deleted sequence. Regions deleted
in the mutant strains are shaded in black. (D) Amplification of genomic DNA in WT (Lane 1) and
∆ctaC1D1E1 (Lane 2) using COXf and COXr primers; in WT (Lane 3) and ∆ctaC2 (Lane 4) using Ctaf
and Ctar primers; in WT (Lane 5) and ∆cydAB (Lane 6) using Cydf and Cydr primers.
Supplemental Figure S2
Analysis of transcripts in regions downstream of ctaE1 and ctaCII.
Schematic representations of locus location in the Synechocystis genome showing the primers used
to detect transcripts (A) including both the ctaEI and trxA genes (1) and within trxA (2) and (B) within
Sll0814 (3) and including both the ctaCII and Sll0814 genes. (C) RT-PCR analysis of gDNA (lanes 1-4),
RT- (lanes 5-8) and RT+ (lanes 9-12) samples. No transcript was detected between the ctaEI/ trxA
and ctaCII/ Sll0814 genes indicating that these genes are not co-transcribed. Total RNA was
extracted from wild-type cells cultured under moderate light conditions.Supplemental Figure S3
Supplemental Figure S3
Correlation between the Abs(680nm)-Abs(750nm) value and amounts of chlorophyll measured
following methanol extraction.
Fifteen samples were measured at absorbance of 750nm and 680nm, followed by extraction with
methanol to measure chlorophyll concentration. Amount of chlorophyll was correlated with
absorbance (A680-A750). The regression line is shown. The slope of the regression line (R2=0.9854) is
equivalent to 10.854. was calculated.
Supplemental Figure S4
Growth of terminal oxidase mutants under different light conditions.
Growth of triplicate cultures was measured at optical densities of 750nm and 680nm under (A)
continuous moderate light; (B) continuous high light; (C) 12 hour moderate light/ dark cycles; (D) 12
hour high light/ dark cycles; (E) diurnal light conditions. Dark periods are indicated by black bars. The
750nm and 680nm values were used to determine chlorophyll levels per cell using the equation:
((Abs(680nm)-Abs(750nm)x10.854)/ Abs(750nm). The amount of chlorophyll per cell is an indication
of photobleaching.(F) Diurnal light input simulating a 12-hour day with sunrise at 8 am and a noon
peak intensity of 180 μmol/m2/s. (G) Legend.
Supplemental Figure S5
Validation of DCFH-DA as a tool to measure of oxidative damage in Synechocystis.
DCF fluorescence of wild-type samples exposed to 30 minutes of moderate light (L) or maintained in
the dark (D). Methyl viologen (MV) was added to one set of samples at a final concentration of
100µM. Results are from 3 biological replicates.
Supplemental Table S1
Number of COX (ctacC1), ARTO (ctacII), Cyd (cydA) and PTOX terminal oxidases present in the 60
cyanobacteria with sequenced genomes.
Strains with ctacC1, ctacII, or cydA always contained genes encoding for the other subunits of the
terminal oxidase complex. The presence of a nifH and nhaP gene respectively indicates that the cell
is capable of either nitrogen fixation (*except in the case of Microcoleus chthonoplastes PCC 7420
and Synechococcus sp. JA-3-3Ab) and possibly salt tolerance. A blank space indicates the absence of
a homologue in this species.
Strain
CtaCI
(COX)
ctaCII
(ARTO)
cydA
(Cyd)
PTOX
1
3
4
1
nifH
nhaP
Acaryochloris marina MBIC11017
1
Anabaena variabilis ATCC 29413
2
Arthrospira maxima CS-328
1
1
1
Arthrospira platensis str. Paraca
1
1
1
Crocosphaera watsonii WH 8501
1
1
Cyanobium sp. PCC 7001
1
1
Cyanothece sp. ATCC 51142
1
2
1
1
3
Cyanothece sp. CCY0110
1
1
1
1
2
Cyanothece sp. PCC 7424
1
1
1
1
2
Cyanothece sp. PCC 7425
1
1
1
2
Cyanothece sp. PCC 7822
1
1
1
3
Cyanothece sp. PCC 8801
1
1
2
Cyanothece sp. PCC 8802
1
1
2
Cylindrospermopsis raciborskii CS-505
2
Gloeobacter violaceus PCC 7421
1
Lyngbya sp. PCC 8106
2
Microcoleus chthonoplastes PCC 7420
1
Microcystis aeruginosa NIES-843
1
1
Nodularia spumigena CCY9414
2
1
1
Nostoc azollae' 0708
2
2
1
1
Nostoc punctiforme PCC 73102
2
2
Nostoc sp. PCC 7120
2
1
1
1
Oscillatoria sp. PCC 6506
1
1
1
Prochlorococcus marinus str. AS9601
1
Prochlorococcus marinus str. MIT 9202
1
Prochlorococcus marinus str. MIT 9211
1
Prochlorococcus marinus str. MIT 9215
1
Prochlorococcus marinus str. MIT 9301
1
Prochlorococcus marinus str. MIT 9303
1
Prochlorococcus marinus str. MIT 9312
1
Prochlorococcus marinus str. MIT 9313
1
Prochlorococcus marinus str. MIT 9515
1
Prochlorococcus marinus str. NATL1A
1
1
Prochlorococcus marinus str. NATL2A
1
1
Prochlorococcus marinus str. CCMP1375
1
Prochlorococcus marinus str. CCMP1986
1
Raphidiopsis brookii D9
1
1
Synechococcus elongatus
1
1
Synechococcus sp. BL107
1
Synechococcus sp. CB0101
1
1
Synechococcus sp. CB0205
1
1
Synechococcus sp. CC9311
1
1
3
2
1
1
1
2
2
1
1
1
1
2
1
1
1
1
1
1
1*
4
2
1
2
1
1
1
4
1
2
1
2
1
1
1
1
1
1
1
1
1
1
1
Synechococcus sp. CC9605
1
1
1
Synechococcus sp. CC9902
1
Synechococcus sp. JA-2-3B'a(2-13)
1
1
Synechococcus sp. JA-3-3Ab
1
1
Synechococcus sp. PCC 7002
1
1
Synechococcus sp. PCC 7335
1
1
Synechococcus sp. RCC307
1
1
Synechococcus sp. RS9916
1
1
Synechococcus sp. RS9917
1
1
Synechococcus sp. WH 5701
1
1
Synechococcus sp. WH 7803
1
1
Synechococcus sp. WH 7805
1
1
Synechococcus sp. WH 8102
1
1
Synechococcus sp. WH 8109
1
Synechocystis sp. PCC 6803
1
Thermosynechococcus elongatus BP-1
1
Trichodesmium erythraeum IMS101
1
cyanobacterium UCYN-A
1
1
1*
2
1
1
1
2
1
1
1
1
1
2
1
1
1
1
Supplemental Table S2
Respiratory rates are greatly reduced in strains deficient in thylakoid localized terminal oxidases.
Oxygen consumption was measured from three separate biological replicates and carried out in BG11 medium at 30 °C using DW1 liquid-phase oxygen electrode chambers with Oxygraph meters
(Hansatech Instruments, Norfolk).
Strain
Genotype
WT
Synechocystis sp. PCC 6803
Wild-type
∆CtaCIDIEI
∆CtaCII
∆CydAB
∆ CtaCIDIEI/CtaCII
∆ CtaCIDIEI/CydAB
∆ CtaCII/CydAB
∆ CtaCIDIEI/CtaCII/CydAB
∆COX
∆ARTO
∆Cyd
∆COX/ARTO
∆COX/Cyd
∆ARTO/Cyd
Triple
Reference
(1)
Oxygen consumption (nmol O2/mg
DCW/min)
5.35 ± 0.41
This study
This study
This study
This study
This study
This study
This study
4.74 ± 0.37
4.40 ± 1.38
4.31 ± 1.27
4.15 ± 1.15
1.28 ± 0.36
5.27 ± 2.27
1.98± 0.37
Supplemental Table S3
Nucleotide changes between the published Synechocystis genome and the strain used in this
study.
No nucleotide differences were observed between the laboratory wild-type strain and ΔCOX/Cyd.
Genes that are mobile elements are indicated.
Nucleotide
change(s)
T-G
C-T
G-T
C-A
T-C
A-G
multiple changes
A-G
multiple changes
multiple changes
A-T
T-C
A-G
C-A
T-A
Genome position
488230
842060
1012958
1200306
1211827
1364187
1455191-1455288
1819782
1970657-1970703
1972125-1972813
2092571
2198893
2301721
2602717
2602734
C-T
G-A
C-G
C-T
multiple changes
multiple changes
T-C
T-G
G-A G-T
A-G
3 changes
A-C
2748897
2921706
2922010
2922031
2997706-2997876
2998521-2998658
3096187
3109829
3110189 3110343
3142651
3348685-3348773
3528785
Gene
slr1609
rpl3
between repA/ftsZ
slr1862
sll1774
pyrF
slr1712
psbA3
slr1682
slr1683, slr1684
sll0422
sll0142
slr0168
slr0468
slr0468
between
slr0210/ssr0332
slr0511
slr0511
slr0511
ssl0172
sll0092
ssr1175
sll0666
sll0665
sps
slr0665
slr1187
Further
comments
mobile element
mobile element
mobile element
mobile element
mobile element
mobile element
mobile element
mobile element
mobile element
mobile element
Supplemental Table S4
Sequence of primers used in this study.
* Restriction endonuclease sites introduced into the primer are underlined.
Primer name
Sequence (5’ to 3’)
COXleftfor
GTACTCTAGAGACGGGATTCCAAGGAGAAT
COXleftrev
GATCGGATCCACACCGCCAAGGTAAAAACA
COXrightfor
GATCGAGCTCCAAACCACTGCCCTTAGCTC
COXrightrev
GACTGAATTCGAGCACTTTGAGCTTATCACCA
Ctaleftfor
GTACTCTAGATACATCCCAGACCCAAAAGG
Ctaleftrev
GATCGGATCCTTGGGGGAATATAAGCTCCA
Ctarightfor
GATCGAGCTCTCCCAAAAAGACCACGCTAC
Ctarightrev
GACTGAATTCTAACAATGCCGACCCCTAAC
Cydleftfor
GTACTCTAGAAGTGCCGGAGAGTCGTTAGA
Cydleftrev
GATCGGATCCTATGGAAAATGGCAGTGACG
Cydrightfor
GATCGAGCTCTGTGTTTGAAGGCATTACCG
Cydrightrev
GACTGAATTCGTGCTAGAGGCCAACGAAAC
COXf
CAGAAAAGGTGGATGGCATT
COXr
CCACCAGGAACAGGACAACT
Ctaf
GAGCGGTGGGTTAATTGATG
Ctar
GCCAAAGCTGGGGTAAAGAT
Cydf
TAGGGTGGGCACAACTCTCT
Cydr
GGGATCAGGAAACCAATGAA
ctaE1trxAfor
ATCCCATTTCGGGGTAGAAG
ctaE1trxArev
GGGTTTCCTGCTCAAATTCA
trxAfor
ACGCTGAATTTGAGCAGGAA
trxArev
AACTCCAGTAATTTGGGTTTGG
cruFfor
GGTAGGGAAAAGTCCGCTTC
cruFrev
TTTGGTCAGAGCCGGTTTAC
cruFctaC2for
ATCAGCAATAGCCGCTCAAT
cruFctaC2rev
TCCCACAACACCTCTGAAAA
* Restriction endonuclease sites introduced into the primer are underlined.
1.
Williams JGK (1988) Construction of specific mutations in photosystem-II photosynthetic
reaction center by genetic-engineering methods in Synechocystis-6803. (Translated from
English) Methods in Enzymology 167:766-778 (in English).