Supplementary Information for:
Stereochemical conversion of C3-vinyl group to 1-hydroxyethyl group in bacteriochlorophyll c
by the hydratases BchF and BchV: adaptation of green sulfur bacteria to limited-light
environments
Jiro Harada1*, Misato Teramura2, Tadashi Mizoguchi2, Yusuke Tsukatani3,4, Ken Yamamoto1, and
Hitoshi Tamiaki2*
1
Department of Medical Biochemistry, Kurume University School of Medicine, Kurume, Fukuoka 830-0011,
Japan
2
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
3
Earth-Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo 152-8550, Japan
4
PRESTO Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
To whom correspondence should be addressed:
Jiro Harada, Department of Medical Biochemistry, Kurume University School of Medicine, Asahi-machi 67,
Kurume, Fukuoka 830-0011, Japan, Tel: +81 (942) 31-7544; Fax: +81 (942) 31-4377; E-mail:
jiro_harada@med.kurume-u.ac.jp.
Hitoshi Tamiaki, Graduate School of Life Sciences, Ritsumeikan University, Noji-Higashi 1-1-1, Kusatsu,
Shiga 525-8577, Japan, Tel: +81 (77) 561-2765; Fax: +81 (77) 561-3729; E-mail: tamiaki@fc.ritsumei.ac.jp.
1
Fig. S1
(A) Schematic maps for the construction of the bchF-deleted mutant of Cba. tepidum. Genes are indicated by
rectangles. The aadA gene confers resistance to spectinomycin and streptomycin. Arrows represent the
oligonucleotide primers, aadA-tepF-F (i), aadA-tepF-R (ii), bchFus-F (iii), bchFus-R (iv), bchFds-F (v),
2
bchFds-R (vi), bchF-comf-F (vii), and bchF-comf-R (viii). (B) Schematic map showing the construction of the
bchV-deleted mutant of Cba. tepidum. Genes are indicated by rectangles. The aacC1 gene confers resistance
to gentamycin. Arrows represent the oligonucleotide primers, aacC1-tepV-F (ix), aacC1-tepV-R (x), bchVus-F
(xi), bchVus-R (xii), bchVds-F (xiii), bchVds-R (xiv), bchV-comf-F (xv), and bchV-comf-R (xvi). (C) PCR
analyses using genomic DNAs extracted from the wild-type (lanes 1, 4, and 7), tepdF (lanes 2, 5, and 8), and
tepdV strains (lanes 3, 6, and 9) of Cba. tepidum. Lanes 1-3 represent PCR products when using primers,
bchF-comf-F and bchF-comf-R to amplify the bchF locus. The DNA fragment from the tepdF (lane 2) was
2.28 kbp, which is 0.62 kbp longer than those of wild-type and tepdV (lanes 1 and 3, respectively) by insertion
of the aadA gene into the bchF locus. Lanes 4-9 represent PCR products when using primers, bchV-comf-F
and bchV-comf-R to amplify the bchV locus. The DNA fragments from the wild-type and tepdF strains are
calculated to be 1.75 kbp, while that from the tepdV strain is to be 2.00 kbp, but these fragments were not
distinguishable in the agarose gel. Therefore, the PCR fragments were digested by the EcoRV. The digested
PCR fragments of the tepdV were shown to be 0.77 and 1.23 kbp (lane 9), but the PCR fragments from
wild-type and tepdF were not digested by EcoRV (lanes 7 and 8, respectively). A DNA molecular size marker
was loaded on lane M, and the numbers indicate the lengths of the DNA marker fragments in kilo-base(s).
3
Fig. S2
BChl c compositions of wild-type (A), tepdF (B), and tepdV cells (C) grown with light irradiation, 12 (blue
bars), 30 (yellow bars), and 100 E s-1 m-2 (red bars).
4
Fig. S3
BChl c (A), 3V-BChl c (B), and BChl a contents (C) in the dried cells of wild-type and mutants, grown at 30
E s-1 m-2.
5
Fig. S4
(A) HPLC analyses of the in vitro assays of BchF and BchV reactions with Chlide a monitored at 660 nm.
The substrate (i) was reacted with the cell lysate of an E. coli culture possessing empty vector, pET21(a)+ as
control (ii), and expressing BchF (iii) and BchV (iv). The inset focuses on elution profiles between 2.5 and 3.5
min. Peak 1, Chlide a; peak 2, R-Chlide a; peak 3, S-Chlide a. (B) In-line mass spectra of peaks 1 (i), 2 (ii),
and 3 (iii). The calculated molecular mass number for [M+H]+ of each pigment is as follows; Chlide a, 615.2;
R-Chlide a, 633.3; S-Chlide a, 633.3. (C) Absorption spectra of peaks 1 (solid line), 2 (dashed line), and 3
(dotted line) in the eluent.
6
Fig. S5
Dependence of growth rates (A) as well as chlorosomal Qy maxima (B) in the cells of wild-type (open circles),
tepdF (open squares), and tepdV strains (open triangles) upon irradiated light intensity.
7
Fig. S6
SDS-PAGE analysis for the protein compositions of chlorosome isolated from the wild-type (lane 1), tepdF
(lane 2), and tepdV (lane 3). The lane M indicates molecular mass standards with masses showed in
kilo-daltons on the left. Chlorosomal Csm proteins are identified at the right according to the previous reports
(Mizoguchi et al., 2013). The chlorosome samples containing 15 g BChl c were treated and loaded to lanes.
8
Fig. S7
RT-PCR analysis of bchF and bchV genes transcripts with total RNA isolated from Cba. tepidum wild-type
cells grown under light intensity of 12 or 100 E s-1 m-2. The bchF and bchV genes as well as bciC, bchQ,
bchR, bchU, and bchK genes were involved in the BChl c biosynthetic pathways, and analyzed. The sigA gene
coding the major housekeeping sigma factor was used as an internal standard. Negative-control reactions
omitted the reverse transcriptase (RT-).
9
Fig. S8
Phylogenetic analysis of C3 hydratase paralogs among photosynthetic bacteria. The phylogenetic tree was
constructed with translated sequences of BchF and BchV paralogs using ClustalW (Larkin et al., 2007) and
MEGA6 (Tamura et al., 2013) programs. Tree construction was performed by neighbor-joining method
(Saitou and Nei, 1987), applying the Kimura 2-parameter distance estimator. Gaps in sequence alignments
were pairwisely omitted in the calculation. Bootstrap values for each clade were obtained by 3000 replications,
and indicated. The accession numbers of sequences to construct the tree are as follows: Allochromatium (Alc.)
10
vinosum DSM180 Alvin_2643, BAL96684.1; Chloracidobacterium (Cab.) thermophilium Cabther_B0080,
AEP13086.1; Cba. parvum NCIB8327d Cpar_0430, ACF10852.1; Cba. parvum NCIB8327d Cpar_0743,
ACF11160.1; Cba. tepidum BchF, AAM72649.1; Cba. tepidum BchV, AAM72997.1; Chlorobium (Chl.)
chlorochromatii CaD3 Cag_0402, ABB27675.1; Chl. chlorochromatii CaD3 Cag_1644, ABB28896.1; Chl.
clathratiforme BU-1 Ppha_0981, ACF43265.1; Chl. clathratiforme BU-1 Ppha_2338, ACF44533.1; Chl.
clathratiforme BU-1 Ppha_2749, ACF44903.1; Chl. limicola DSM245 Clim_0899, ACD89978.1; Chl.
limicola DSM245 Clim_1128, ACD90197.1; Chl. limicola DSM245 Clim_1974, ACD91006.1; Chl. luteolum
DSM273 Plut_0408, ABB24280; Chl. luteolum DSM273 Plut_1421, ABB24280.1; Chl. phaeobacteroides
BS1 Cphamn1_0268, ACE03237.1; Chl. phaeobacteroides BS1 Cphamn1_0939, ACE03884.1; Chl.
phaeobacteroides DSM 266 Cpha266_0198, ABL64265.1; Chl. phaeobacteroides DSM 266 Cpha266_1790,
ABL65807.1; Chl. phaeobacteroides DSM 266 Cpha266_2005, ABL66018.1; Chl. phaeovibrioides DSM265
Cvib_0370, ABP36392.1; Chl. phaeovibrioides DSM265 Cvib_1239, ABP37251.1; Chloroflexus (Cfx.)
aggregans DSM9485 Cagg_3129, ACL25987.1; Cfx. aurantiacus J-10-fl Caur_0415, ABY33665.1;
Chloroflexus sp. Y-400-fl Chy400_0442, ACM51881.1; Chloroherpeton (Chp.) thalassium ATCC35110
Ctha_2718, ACF15167.1; Gemmatimonas sp. AP64 BchF, WP_043581454; Prosthecochloris (Ptc.) aestuarii
DSM271 Paes_1533, ACF46553.1; Ptc. aestuarii DSM271 Paes_1805, ACF46817.1; Rhodobacter (Rba.)
capsulatus SB1003 BchF, ADE84431.1; Rba. sphaeroides 2.4.1 BchF, YP_353358.1; Rhodopseudomonas
(Rps.) palustris CGA009 BchF, CAE26982.1; Rhodospirillum (Rsp.) rubrum ATCC11170 Rru_A0624,
ABC21428.1; Roseiflexus (Rfx.) castenholzii DSM13941 Rcas_3748, ABU59788.1; Roseiflexus sp. RS-1
RoseRS_3262, ABQ91623.1; Rubrivivax (Rvi.) gelatinosus IL144 BchF, BAL96684.1.
11
Table S1
Sequences of primers for construction of the plasmids and confirmation of the mutants used
in this study.
Primer name
Primer sequence
Underline
bchFus-F
CTCTAGAGGATCCCCCTTGGTTTTGAGCTCTTCGC
bchFus-R
TTACGAACCGAACAGGTGGAGATCGGTTCTGTGAT
overlapped with multi-cloning site
(MCS) of pUC118
overlapped with aadA-F primer
bchFds-F
TCGTTCAAGCCGACGGAGAAGAAACTCAAGGCCAG
overlapped with aadA-R primer
bchFds-R
TCGAGCTCGGTACCCACGAGCACTTTGCGGTTCTT
overlapped with MCS of pUC118
bchF-comf-F
ATGACTTTCGAGCGACCACA
bchF-comf-R
GGGGTTGACCTTGATATCTG
bchVus-F
CTCTAGAGGATCCCCCTCTTCGGGGTTGAACGAAA
overlapped with MCS of pUC118
bchVus-R
GTTCTGGACCAGTTGGAAAACCGAAATTGCACCG
overlapped with aacC1-F primer
bchVds-F
CAGGCATGCAAGCTTTGATCTCCGTCCTGTTTTGC
overlapped with aacC1-R primer
bchVds-R
TCGAGCTCGGTACCCCGTTGACATGGTTGCCGATA
overlapped with MCS of pUC118
bchV-comf-F
AACCACGATACAGTCTGCGT
bchV-comf-R
CGGACTCGTACATCTTTTGG
aacC1-F
CAACTGGTCCAGAACCTTGA
aacC1-R
AAGCTTGCATGCCTGCAGG
exbchF-F
exbchF-R
AAGGAGATATACATATGCCTCGTTATACACCGGAACAG overlapped with MCS of pET21(a)+
CGGAGCTCGAATTCGGATCCGAGCTCAGACGGCTCCGC overlapped with MCS of pET21(a)+
TGGCCTTGAG
pET21a-F
GGATCCGAATTCGAGCTCC
pET21a-R
CATATGTATATCTCCTTCTTAA
exbchV-F
exbchV-R
bchF RT-F
AAGGAGATATACATATGTGTTTTTCAGGCTATCCG
CGGAGCTCGAATTCGGATCCGAGCTCAGATCGCCCCCT
GCCC
GTGCAGGCTATTCTCGCCCC
bchF RT-R
GCAAGATAGGCGGTCAGAATGAG
bchV RT-F
CAGCTCGCCAAACGCAACGC
bchV RT-R
GATACTCCATCCACGCCATCAC
bchQ RT-F
CTCACGCAGACTGTATCGTGGT
bchQ RT-R
GCCTCCTCGATCTGATGCTTCA
bchR RT-F
TCGACGAGAGAACTGGCGAC
bchR RT-R
GGGCGAAGCTGAAGTCGGGC
bchU RT-F
GTTGCCAAGGCGATGGCGTTC
bchU RT-R
GGTCGATGGCGCCCGGCAG
bciC RT-F
TCATCGGGCTTGCCTTCTTTCG
bciC RT-R
CCCGTGAATCAGGCCGCTGTA
bchK RT-F
TCTACGGCCCACTTGGAACCG
bchK RT-R
CGTAGGAAAAGCCGACCGCTG
sigA RT-F
ACGAGGGCCATCAAGGAAGGT
sigA RT-R
AGCGTGCCGACGCGGTTGAG
12
overlapped with MCS of pET21(a)+
overlapped with MCS of pET21(a)+
Supplemental references
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., et al. (2007)
Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948.
Mizoguchi, T., Tsukatani, Y., Harada, J., Takasaki, S., Yoshitomi, T. and Tamiaki, H. (2013)
Cyclopropane-ring formation in the acyl groups of chlorosome glycolipids is crucial for acid resistance of
green bacterial antenna systems. Bioorg Med Chem 21: 3689-3694.
Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic
trees. Mol Biol Evol 4: 406-425.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA6: Molecular Evolutionary
Genetics Analysis version 6.0. Mol Biol Evol 30: 2725-2729.
13