Supplementary Information to:
Hu, C. et al. Functional assessment of mycosporine-like amino acids in
Microcystis aeruginosa strain PCC 7806
Table of content:
1. Supplementary Methods
2. Supplementary Figures
Table S1 Detection of mys genes in various Microcystis sp. strains by PCR
Table S2 Primers used for the detection of mys genes in Microcystis sp. strains
Table S3: Accession numbers of Microcystis 16S-23S ITS and mys sequences in
GenBank
Fig. S1 Whole cell absorption and difference spectra of M. aeruginosa WT and mys
mutants.
Fig. S2 Relative quantification of shinorine in the recently isolated FS 2 field strain
Fig. S3 Growth of M. aeruginosa WT, mysA and mysB mutants on agar plates after
exposure to different stress conditions.
Fig. S4 Semi-quantitative RT-PCR results of mysA expression in the wild type M.
aeruginosa PCC7806
Fig. S5 Relative shinorine content of M. aeruginosa PCC7806 grown under different
stress conditions
Fig. S6 Spectral characteristics of the UV lamps used for illumination of cultures
1. Supplementary Methods
Detection of the mys genes in Microcystis strains
Microcystis strains employed in this study were listed in Table S1; Four sets of specific
primers were designed to separately target mysA, B, C and D gene, the nucleotide sequence of
each primer was listed in Table S2. The amplification of each mys genetic element was
individually carried out in 20-µL PCR reaction mixture prepared according to the
manufacturer’s instruction (DreamTaq, Thermo Scientific), and the thermal cycling was
performed on Bio-Rad C1000TM Thermal Cycler (USA), and the PCR profile was one cycle of
an initial denaturation step (95 °C for 10 min); 35 cycles of 95 °C for 30 s, annealing
temperature Tm (listed in Table S2) for 30 s and 72°C for 30 s; and one cycle of final
extension (72 °C for 10 min). The 10-µL PCR products were applied to agarose gel
electrophoresis (1.0% agarose gel containing 0.2 µg/ml ethidium bromide in TAE buffer), in
which Lambda DNA/Pst I was the DNA fragment size marker, the PCR products of
Microcystis aeruginosa PCC 7806 and NIES 843 were used for the positive and negative
control, respectively. The gel photographing and documentation were carried out in Bio-Rad
ChemiDocTM XRS + imaging system (USA).
Semi-quantitative qPCR
Semi-quantitative qPCR was conducted using 50 ng of cDNA from 3 biological replicates as
template using the same primer pairs as for quantitative real-time PCR. PCR was performed
in 20 µl reaction volume containing 2 µl Buffer, 0.5 µl dNTP mix (Thermo Fisher Scientific),
0.5 µl of each primer (10 µM), 1 l cDNA (50 ng), 0.2 µl DreamTaq Polymerase (Thermo
Fisher Scientific) and water to a final volume of 20 µl. The following program was used for
PCR reactions: 98 °C for 5 min initial denaturation, 20, 25, 30 or 35 cylces of 95 °C for 30 s,
primer annealing at 60 °C for 30s and elongation at 72 °C for 30 s and the program ended
after a final step of 72 °C for 10 min.
3. Supplementary Figures
Table S1: Detection of mys genes in various Microcystis sp. strains by PCR
strain
PCC 7806
M. 130
M. 265
M. 269
M. GLS 16.9M6nT
M. GL 5tax
M. GLS428.6M6T
M. GLS510.10M2
M. HUB5.3
M. Isancya M5
M. NIES-A89
M. TuM7C
M. MRD
PCC 9806
NIES 298
NIES 100
NIES 98
MRC
PCC 9801
NIES 843
NIES 299
CBS
PCC 7005
mysA
+
+
+
+
+
+
+
+
+
+
mysB
+
+
+
+
+
+
+
+
+
+
mysC
+
+
+
+
+
+
+
+
+
mysD
+
+
+
+
+
+
+
+
+
+
Table S2: Primers used for the detection of mys genes in Microcystis sp. strains:
Gene
mysA
mysB
mysC
mysD
Primer name
DHQS429 (screening primer)
Nucleotide sequence (5-3)
aagcttgattggcttgattg
Tm (°C)
53
DHQS786(screening primer)
atctagctcatgcaggttgg
53
OMT469(screening primer)
gccggacacaaaatcgtagt
55
OMT731(screening primer)
agggtttggttgaactgtgc
55
ATPgrasp423(screening primer)
ttacgttcccgtttgtagcc
55
ATPgrasp871(screening primer)
aagacttgcagcagcagtga
55
AlaLigase649(screening primer)
tgcttgccgctagaagagta
52
Alaligase881(screening primer)
tcgatccgaaaatcaaacaa
52
Table S3: Accession numbers of Microcystis 16S-23S ITS and mys sequences in GenBank
accession number
strain
16S-23S ITS
mysA
mysB
mysC
mysD
DIANCHI905
AOCI01000133
WP_002741115
ELS49747
WP_002741118
WP_002741119
NIES 843
AP009552
-
-
-
-
PCC 7806
AM773520
CAO90104
CAO90105
CAO90106
CAO90107
PCC 7941
CAIK01000429
-
-
-
-
PCC 9432
CAIH01000397
-
-
-
-
PCC 9443
CAIJ01000268
WP_002768098
CCI02411
WP_002768100
WP_002768102
PCC 9701
CAIQ01000358
-
-
-
-
PCC 9717
CAII01000834
WP_004266794
WP_004266793
WP_004266792
WP_004266791
PCC 9806
CAIL01000263
-
-
-
-
PCC 9807
CAIM01000209
-
-
-
-
PCC 9808
CAIN01000175
WP_002794106
WP_002794105
WP_002794104
WP_002794103
PCC 9809
CAIO01000252
-
-
-
-
T1-4
CAIP01000212
-
-
-
-
TAIHU 98
ANKQ01000002
-
-
-
-
Fig. S1: Whole cell absorption and difference spectra of M. aeruginosa WT and mys
mutants. A) Normalized absorption spectra recorded at OD750 0.4 from WT and mutant cells
and difference spectra (mysA-WT and mysB-WT) calculated from respective curves. B) Detail
view of difference spectra from A). C) Integrated relative absorbance in the UV (shinorine)
and PAR wavelength range of mutant cultures compared to WT.
120.00
rel. shinorine content [%]
100.00
80.00
60.00
40.00
20.00
0.00
2
8
time [d]
Fig. S2: Relative quantification of shinorine in the recently isolated FS2 field strain.
Cultures were treated with UV-B light for 2 and 8 days. The strain still forms colonies with
extended mucilage. Values were related to the shinorine content of untreated cultures (100%).
Shown are mean values of three independent replicates.
Fig. S3: Growth of M. aeruginosa WT, mysA and mysB mutants on agar plates after
exposure to different stress conditions. Cells were applied to the agar plates in three
different dilutions and photos were taken after 14 d. For UV-A treatments, plates were grown
under medium white light and exposed to UV-A light for 1 h daily. Chemical stress factors
were added directly to agar plates in indicated concentrations.
Fig. S4: Semi-quantitative RT-PCR results of mysA expression in the wild type M.
aeruginosa PCC7806. Each sample was run with 20, 25, 30 and 35 cycles. No significant
upregulation was observed after UV-A, NaCl and H2O2 treatment for 1 h compared to the
untreated white light control. The rnpB gene served as constitutively expressed control gene.
rel. shinorine content [%]
120
100
80
60
40
20
0
NaCl
H2O2
High light
Fig. S5: Relative shinorine content of M. aeruginosa PCC7806 grown under different
stress conditions. Cultures were exposed to osmotic, oxidative and high light stress
conditions for one (dark grey) or five days (light grey). Values were related to the shinorine
content of untreated cultures (100%). Shown are mean values of three independent replicates.
Fig. S6: Spectral characteristics of the UV lamps used for illumination of cultures (from:
http://www.sankyo-denki.co.jp/).