Supporting Information
Isolation and structural determination of C8-vinyl-bacteriochlorophyll d from the bciA and
bchU double mutant of the green sulfur bacterium Chlorobaculum tepidum
Jiro Harada ·Tadashi Mizoguchi ·Kota Nomura ·Hitoshi Tamiaki
Table S1 APCI-mass spectrometric data of the pigments extracted from the wild type of Cba.
tepidum
Table S2 APCI-mass spectrometric data of the pigments extracted from the tepdU strain
Table S3 APCI-mass spectrometric data of the pigments extracted from the tepdA/U strain
Figure S1 Constructions of Cba. tepidum bchU gene inactivated mutants. (A) Schematic maps show
bchU of Cba. tepidum. Genes are indicated by rectangles. The aadA gene confers resistance
to spectinomycin and streptomycin. Arrows represent the oligonucleotide primers, aadA-fortepU-F (i), aadA-for-tepU-R (ii), tepU-comf-F (iii), and tepU-comf-F (iv). (B and C) PCR
analyses using the genomic DNA extracted from wild type (lanes 1 and 5), tepdA (lanes 2 and
6), tepdU (lanes 3 and 7), and tepdA/U (lanes 4 and 8) strains. (B) Agarose gel electrophoresis
shows amplified fragment of around bchU locus using above tepU-comf-F and -R primers. The
PCR products were then digested by a restriction enzyme NdeI. The fragments yielded from
wild type (lane 1) and tepdA (lane 2) were 2.34 kbp, while those from tepdU (lane 3) and
tepdA/U (lane 4) were 1.82 and 0.61 kbp. (C) Agarose gel electrophoresis indicated the
fragments amplified bciA locus using the primer set of tepbciA-comf-F and -R. The EcoRVdigested PCR products from wild type and tepdU were 2.21 kbp, while those from tepdA and
tepdA/U were 1.44 and 0.70 kbp. Lanes M in (B) and (C) panels represent a molecular size
marker (the sizes of bands are indicated at left)
Figure S2 1H-NMR spectra of purified R[V,E]BChl d (a) and R[E,E]BChl d (b) in THF-d8 at room
temperature. Observed NOE correlations for R[V,E]BChl d are indicated by arrows in the inset
molecular structure
Figure S3 (A) Preparation of natural and synthetic R[E,E]BChl d from the Cba. parvum NCBI8327d
and Cba. tepidum tepdA/U: (i) extraction, (ii) HPLC separation, (iii) hydrogenation by rhodium
catalyst on alumina in acetone. (B) UV/Vis absorbance (upper) and CD spectra (bottom) of
natural (black lines) and synthetic (red lines) R[E,E]BChl d in THF
Figure S4 Co-chromatographic analysis of an equimolar mixture of natural and synthetic R[E,E] BChl
d. The elution of pigments was monitored at 430 nm. (a) BChl d homologs extracted from
Cba. parvum NCBI8327d; (b) natural R[E,E]BChl d isolated from Cba. parvum NCBI8327d;
(c) synthetic R[E,E]BChl d from R[V,E]BChl d isolated from Cba. tepidum tepdA/U; (d) cochromatography of (b) and (c). Peak 1, R[E,M]BChl d; Peak 2, R[P,M]BChl d; Peak 3,
R[E,E]BChl d; Peak 4, R[P,E]BChl d; Peak 5, S[P,E]BChl d; Peak 6, S[I,E]BChl d
Figure S5 Growth measurements of Cba. tepidum mutants.
(A) Growth profiles of the wild type and
mutant cells at 30 E·sec ·m . The growth rates (h ) and doubling times (h) were calculated
from these curves: wild type, 0.12 h-1 and 5.8 h; tepdA, 0.04 h-1 and 17.3 h; tepdU, 0.07 h-1 and
9.9 h; tepdA/U, 0.03 h-1 and 23.1 h. (B) The Qy absorption maxima of the wild type and mutants
-1
-2
-1
were plotted during the cell growth. Symbols: open circles, wild type; closed circles, tepdA;
open squares, tepdU; open triangles, tepdA/U. Values at each plot are averages of three
independent experiments, and shown in panel C
Table S1 APCI-mass spectrometric data of the pigments extracted from the wild type of Cba. tepidum
HPLC
peak #
a
b
Assignment
1
R[E,M]BChl c
2
R[E,E]BChl c
3
R[P,E]BChl c
4
S[P,E]BChl c
5
R[I,E]BChl c
6
S[I,E]BChl c
Observed for
[M+H]
+
793.30
(204.15) c
807.35
(204.20)
821.35
(204.15)
821.30
(204.10)
835.35
(204.15)
835.35
(204.10)
[M1+H]
+a
Calculated for
[M2+H]
+b
589.15
775.35
603.15
789.35
617.20
803.35
617.20
803.35
631.20
817.35
631.25
817.35
[M-(174-ester)+H]+
[M-(31-OH)+H]+
c
The difference between [M+H]+ and [M1+H]+
[M+H]+
[M1+H]+
793.45
(204.18)
807.47
(204.19)
821.49
(204.19)
821.49
(204.19)
835.50
(204.19)
835.50
(204.19)
589.27
603.28
617.30
617.30
631.31
631.31
Table S2 APCI-mass spectrometric data of the pigments extracted from the tepdU strain
HPLC
peak #
a
Assignment
7
R[E,M]BChl d
8
R[E,E]BChl d
9
R[P,E]BChl d
10
S[P,E]BChl d
11
S[I,E]BChl d
Observed for
[M+H]
+
779.35
(204.10) c
793.40
(204.15)
807.45
(204.20)
807.45
(204.20)
821.40
(204.10)
[M1+H]
+a
Calculated for
[M2+H]
+b
575.25
761.35
589.25
775.35
603.25
789.45
603.25
789.40
617.30
803.40
[M-(174-ester)+H]+
b
[M-(31-OH)+H]+
c
The difference between [M+H]+ and [M1+H]+
[M+H]+
[M1+H]+
779.44
(204.19)
793.45
(204.18)
807.47
(204.19)
807.47
(204.19)
821.49
(204.19)
575.25
589.27
603.28
603.28
617.30
Table S3 APCI-mass spectrometric data of the pigments extracted from the tepdA/U strain
HPLC
peak #
a
Assignment
12
R[V,M]BChl d
13
R[V,E]BChl d
14
S[V,E]BChl d
Observed for
[M+H]
+
777.35
(204.15) c
791.40
(204.20)
791.40
(204.20)
[M1+H]
+a
Calculated for
[M2+H]
+b
573.20
759.30
587.20
773.35
587.20
773.35
[M-(174-ester)+H]+
b
[M-(31-OH)+H]+
c
The difference between [M+H]+ and [M1+H]+
[M+H]+
[M1+H]+
777.42
(204.18)
791.44
(204.19)
791.44
(204.19)
573.24
587.25
587.25
Figure S1 Constructions of Cba. tepidum bchU gene inactivated mutants. (A) Schematic maps show
bchU of Cba. tepidum. Genes are indicated by rectangles. The aadA gene confers resistance to
spectinomycin and streptomycin. Arrows represent the oligonucleotide primers, aadA-for-tepU-F (i),
aadA-for-tepU-R (ii), tepU-comf-F (iii), and tepU-comf-F (iv). (B and C) PCR analyses using the
genomic DNA extracted from wild type (lanes 1 and 5), tepdA (lanes 2 and 6), tepdU (lanes 3 and 7),
and tepdA/U (lanes 4 and 8) strains. (B) Agarose gel electrophoresis shows amplified fragment of
around bchU locus using above tepU-comf-F and -R primers. The PCR products were then digested
by a restriction enzyme NdeI. The fragments yielded from wild type (lane 1) and tepdA (lane 2) were
2.34 kbp, while those from tepdU (lane 3) and tepdA/U (lane 4) were 1.82 and 0.61 kbp. (C) Agarose
gel electrophoresis indicated the fragments amplified bciA locus using the primer set of tepbciA-comfF and -R. The EcoRV-digested PCR products from wild type and tepdU were 2.21 kbp, while those
from tepdA and tepdA/U were 1.44 and 0.70 kbp. Lanes M in (B) and (C) panels represent a
molecular size marker (the sizes of bands are indicated at left)
Figure S2 1H-NMR spectra of purified R[V,E]BChl d (a) and R[E,E]BChl d (b) in THF-d8 at room temperature.
R[V,E]BChl d are indicated by arrows in the inset molecular structure
Observed NOE correlations for
Figure S3 (A) Preparation of natural and synthetic R[E,E]BChl d from the Cba. parvum NCBI8327d
and Cba. tepidum tepdA/U: (i) extraction, (ii) HPLC separation, (iii) hydrogenation by rhodium
catalyst on alumina in acetone. (B) UV/Vis absorbance (upper) and CD spectra (bottom) of natural
(black lines) and synthetic (red lines) R[E,E]BChl d in THF
Figure S4 Co-chromatographic analysis of an equimolar mixture of natural and synthetic R[E,E] BChl
d. The elution of pigments was monitored at 430 nm. (a) BChl d homologs extracted from Cba.
parvum NCBI8327d; (b) natural R[E,E]BChl d isolated from Cba. parvum NCBI8327d; (c) synthetic
R[E,E]BChl d from R[V,E]BChl d isolated from Cba. tepidum tepdA/U; (d) co-chromatography of (b)
and (c). Peak 1, R[E,M]BChl d; Peak 2, R[P,M]BChl d; Peak 3, R[E,E]BChl d; Peak 4, R[P,E]BChl
d; Peak 5, S[P,E]BChl d; Peak 6, S[I,E]BChl d
Figure S5 Growth measurements of Cba. tepidum mutants.
(A) Growth profiles of the wild type and
mutant cells at 30 E·sec-1·m-2. The growth rates (h-1) and doubling times (h) were calculated from
these curves: wild type, 0.12 h-1 and 5.8 h; tepdA, 0.04 h-1 and 17.3 h; tepdU, 0.07 h-1 and 9.9 h;
tepdA/U, 0.03 h-1 and 23.1 h. (B) The Qy absorption maxima of the wild type and mutants were
plotted during the cell growth. Symbols: open circles, wild type; closed circles, tepdA; open squares,
tepdU; open triangles, tepdA/U. Values at each plot are averages of three independent experiments,
and shown in panel C