1
Supporting information
2
3
Experimental procedures
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Strains and growth conditions
5
Strains used in this study are detailed in Table S2. Escherichia coli was
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grown at 37oC on LB (Luria-Bertano) plates set with 1 % (w/v) agar.
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Trichodesmium erythraeum IMS101 was grown in filter-sterilised (0.2 μm) YBC-II
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media (Chen et al., 1996) with modified trace metals (Table S1). Cells were grown
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in 25 cm2 sterile polystyrene cell culture flasks with 0.2 μm vent caps (Corning
10
Inc., NY, USA) at 27 °C with gentle orbital shaking (150 rpm) under a 12 hr/12 hr
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light (ca. 130 μmol photons m-2 s-1)/dark cycle. Growth was monitored through
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cell counts performed using a Sedgewick rafter counting chamber and a GX CAM-
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1.3 camera on an L1000A biological microscope (GT Vision Ltd, Suffolk, UK). Cell
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and filament lengths were identified using GX capture software (GX Optical,
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Suffolk, UK) and the number of cells per ml was calculated by dividing the total
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filament length by the average cell length. Synechocystis sp. PCC 6803 was grown
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in
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[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES)-KOH pH 8.2
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(BG11-TES) under photoautotrophic conditions. Liquid cultures were grown
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shaking (150 rpm) at 30 oC under a constant illumination of ca. 50 μmol photons
21
m-2 s- 1. Growth was monitored by OD750. For growth on plates, BG11-TES was
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supplemented with 1% (w/v) agar, 0.3% (w/v) sodium thiosulphate, 5 mM
23
glucose and kanamycin (as indicated).
BG11
media
(Rippka
et
al.,
1979)
buffered
with
10
mM
N-
1
24
For growth experiments phosphate was omitted from the above media (no
25
added P), with sodium phosphate or sodium phosphite added back to a final
26
concentration of 50 μM for YBC-II or 175 μM for BG11.
27
28
Heterologous expression of ptxABCD in Synechocystis
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Oligonucleotide primers and plasmids details are listed in Tables S3 and
30
S4, respectively. Fragments encoding ptxABCD, ptxABC or ptxD genes were
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amplified from the Trichodesmium genome by PCR with Q5 polymerase (New
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England Biolabs Ltd, Hitchin, UK), digested with NdeI and BamHI, and cloned into
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the NdeI and BglII sites of the pFLAG vector (Hollingshead et al., 2012). The
34
resulting constructs (pPtxABCD, pPtxABC and pPtxD) were sequenced and used
35
to transform wildtype Synechocystis as described previously (Cereda et al., 2014).
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Transformants were isolated on plates with 5 μg ml-1 kanamycin and genome
37
copies segregated by sequential doubling of the antibiotic concentration to 40 μg
38
ml-1. In all cases parallel transformations were performed with selective plates
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containing only sodium phosphite as a source of P. Segregation was confirmed by
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PCR with primers flanking the integration site and the region of the genome
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containing the integrated genes was confirmed to be correct by automated DNA
42
sequencing.
43
44
RNA extractions, Quantitative (q)PCR and End-point analysis
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Trichodesmium cultures were filtered onto GF/F filters and RNA was
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extracted using the RNeasy Plant Mini Kit (Qiagen, West Sussex, UK). The
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extracted RNA was DNase treated with TURBO DNA-free™ DNase (Life
48
Technologies Ltd, Paisley, UK) and converted to cDNA using the Tetro cDNA
2
49
Synthesis Kit (Bioline Reagents Limited, London, UK). Oligonucleotide primer
50
pairs for amplification of 125-189 bp products were tested using melt curve
51
analysis to confirm single product amplification. In agreement with the MIQE
52
guidelines (Bustin et al. 2009), primer sets had a qPCR reaction efficiency between
53
90 and 105% and a linearity greater than r2=0.98. The analysis was performed on
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an Mx3005P qPCR using Brilliant III Ultra-Fast SYBR® Green Master mix (Agilent
55
Technologies UK Limited, Cheshire, UK). All samples were run in three technical
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replicates and expression of genes of interest Tery_0366 (ptxB) and Tery_0368
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(ptxD) was normalised against the reference genes rnpB (Chappell and Webb,
58
2010) and rotA (Orchard et al., 2009) using qBase+ software (Biogazelle,
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Zwijnaarde, Belgium). Normalised relative quantities (NRQs) were scaled to the
60
phosphate-added treatment and presented as relative fold change (RFC) using the
61
same software. RNA was extracted, DNase treated and used for cDNA synthesis for
62
Synechocystis gene expression analysis in the same way as for Trichodesmium. End
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point RT-PCR was performed using gene-specific primers with Q5 polymerase and
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analysed on 2% agarose gels. The rnpB gene was used as a reference (Pinto et al.,
65
2012).
66
67
Bioinformatic analysis
68
Conservation of the phosphite genes within Trichodesmium species was analysed
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by searching for homologues of Tery_0365-0368 in three recently published
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genomic datasets (Walworth et al., 2015), consisting of two draft genomes
71
(T. erythraeum 21-75 and T. theibautii H9-4) and an environmental metagenome
72
from the Atlantic Ocean. Searches were performed using tBlastn (Altschul et al.,
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1997) with an e-value cutoff of 0.000001.
3
74
To investigate expression of the phosphite utilisation genes in other
75
Trichodesmium samples, four publically available transcriptomic datasets were
76
searched for reads matching Tery_0365-0368. The datasets consisted of two in situ
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meta-transcriptomes, one from the Amazon River Plume (Hilton et al., 2014) and
78
one from the South Pacific Subtropical Gyre (Hewson et al., 2009), and two
79
transcriptomes from cultured samples (Pfreundt et al., 2014; unpublished dataset,
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NCBI accession PRJNA237745). Illumina datasets were searched for reads
81
aligning to Tery_0365-0368 with >90% sequence identity over 100% of the read
82
length using MegaBlast (Morgulis et al., 2008), via the SRA Blast service from NCBI.
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454 datasets were searched for reads encoding homologues of Tery_0365-0368
84
using tBlastn with an e-value cut off of 0.000001 and a minimum percentage
85
identity of 90%.
86
87
88
89
90
91
92
93
94
95
96
97
98
4
99
Tables
100
101
Table S1. Modified YBC-II trace metal composition compared to standard YBC-II
102
(Chen et al., 1996). All additions are equal to the concentrations used by Shi et al.,
103
(2012) to supplement seawater, except molybdenum, which is increased to
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average seawater concentrations (James et al., 2005).
Trace metal
Modified YBCII
Standard YBC-II
EDTA
20 μM
2 μM
Cu
8 nM
1 nM
Zn
20 nM
4 nM
Co
8 nM
2.5 nM
Mn
18 nM
20 nM
Mo
100 nM
11 nM
Ni
20 nM
-
Se
10 nM
-
105
106
107
108
109
110
111
112
113
114
5
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Table S2. Strains used in this study.
Organism
Strain
Details1
Source
Synechocystis
PCC6803
Williams, 1988
Cereda et al., 2014
ptxABCD+
Tery_0365-0368
inserted at psbA2
This study
locus; kanR
ptxABC+
Tery_0365-0367
inserted at psbA2
This study
locus; kanR
Tery_00368
ptxD+
inserted at psbA2
This study
locus; kanR
Trichodesmium
Prufert-Bebout
NCMA,
et al., 1993
Maine, USA
IMS101
erythraeum
Agilent
Escherichia coli
XL1-blue
N/A
Technologies,
Stockport, UK
116
1 kanR
= kanamycin resistant
117
6
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Table S3. Oligonucleotide primers used in this study.
Primer
Sequence (5′-3′)
Details
ptxABCD_F
ATGCCATATGATCTCTCTAAATAAACTGGGTGTGAC
NdeI site
underlined
ptxABCD_R
ATGCGGATCCTTATAGCTTGTTAACAGCTCCTTGA
BamHI site
underlined
ptxABC_R
ATGCGGATCCTTAAATAAACTTTTGACGCAAATAGTTAC
BamHI site
underlined
ptxD_F
ATGCCATATGGATAAAAAACCATTAGTTGTTATTACC
NdeI site
underlined
psbA2_F
AAACGCCCTCTGTTTACCCA
-
psbA2_R
TCAACCCGGTACAGAGCTTC
-
ptxA_qPCR_F
TGGTGGACAACAACAACGAG
-
ptxA_qPCR_F
AAGCCCCAATTCTATCTTCTTGAC
-
ptxB_qPCR_F
GCGATCAAGCTTCTACTTCTAGTC
-
ptxB_qPCR_R
CATTGGCAACAGCAATAGCAAC
-
ptxC_qPCR_R
GCTGCTCGCAATACTACTCCT
-
ptxC_qPCR_R
ATACCCCAGGAAGTGTGCC
-
ptxD_qPCR_F
CCCGACTTATTAGCAGCACC
-
ptxD_qPCR_R AGCTTTTCCTAATTTTCCCATCCC
-
rnpB_Tery_F
GGAACCGGAAAAAGACCAACC
-
rnpB_Tery_R
GCCACAGAAAAATACCGCCAA
-
rotA_Tery_F
AAGGAGGTTGCCCAAAAGGT
-
rotA_Tery_R
GCCACCTGTATCTTTACCAGCA
-
rnpB_6803_F
CAAACTTGCTGGGTAACGCC
-
rnpB_6803_R TACTGCTGGTGCGCTCTTAC
-
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7
120
Table S4. Plasmids used in this study.
Plasmid
pFLAG
Details
Source
Integrates genes inserted into NdeI/BglII
Daniel P. Canniffe,
sites into Synechocystis genome under
University of Sheffield,
control of psbA2 promoter (Canniffe et al.,
UK
2013)
pPtxABCD
Tery_0365-0368 in pFLAG
This study
pPtxABC
Tery_0365-0367 in pFLAG
This study
pPtxD
Tery_0368 in pFLAG
This study
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125
126
127
128
129
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131
132
133
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References
136
Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., et al.
137
(2009) The MIQE guidelines: minimum information for publication of quantitative
138
real-time PCR experiments. Clin Chem. 55, 611-22.
139
140
Canniffe, D.P., Jackson, P.J., Hollingshead, S., Dickman, M.J., and Hunter, C.N.
141
(2013) Identification of an 8-vinyl reductase involved in bacteriochlorophyll
142
biosynthesis in Rhodobacter sphaeroides and evidence for the existence of a third
143
distinct class of the enzyme. Biochem J. 450, 397-405.
144
145
Chappel, P.D., and Webb, E.A. (2010) A molecular assessment of the iron
146
stress response in the two phylogenetic clades of Trichodesmium. Environ
147
Microbiol. 12, 13-27.
148
149
Chen, Y., Zehr, J.P., and Mellon, M. (1996) Growth and nitrogen fixation of
150
the
diazotrophic
filamentous
non
heterocystous
cyanobacterioum
151
Trichodesmium sp. IMS 101 in defined media: evidence for a circadian rhythm. J.
152
Phycol. 32: 916–23.
153
154
Cereda, A., Hitchcock, A., Symes, M.D., Cronin, L., Bibby, T.S., and Jones, A.K.
155
(2014) A bioelectrochemical approach to characterize extracellular electron
156
transfer by Synechocystis sp. PCC6803. PloS One. 9, e91484.
157
9
158
Hewson, I., Poretsky, R.S., Dyhrman, S.T., Zielinski, B., White, A.E., Tripp, H.J.,
159
et al. Microbial community gene expression within colonies of the diazotroph,
160
Trichodesmium, from the Southwest Pacific Ocean. ISME J. 3, 1286-300.
161
162
Hilton, J.A., Satinsky, B.M., Doherty, M., Zielinski, B., and Zehr, J.P. (2014)
163
Metatranscriptomics of N2-fixing cyanobacteria in the Amazon River plume. ISME
164
J. Epub ahead of print.
165
166
167
James, R. H. (2005) Marine biogeochemical cycles: Biogeochemical
processes in seawater. 2nd edn. Oxford London: Butterworth-Heinemann.
168
169
Morgulis, A., Coulouris, G., Raytselis, Y., Madden, T. L., Agarwala, R., and
170
Schäffer, A. A. (2008). Database indexing for production MegaBLAST searches.
171
Bioinformatics, 24(16), 1757–1764.
172
173
Orchard, E.D., Webb, E.A., and Dyhrman, S.T. (2009) Molecular analysis of
174
the phosphorus starvation response in Trichodesmium spp. Environ Microbiol.
175
11, 2400-11.
176
177
Pinto, F., Pacheco, C.C., Ferreira, D., Moradas-Ferreira, P., and Tamagnini, P.
178
(2012) Selection of Suitable Reference Genes for RT-qPCR Analyses in
179
Cyanobacteria. PLoS One. 7:e34983.
180
10
181
Pfreundt, U., Kopf, M., Belkin, N., Berman-Frank, I., and Hess, W.R. (2014)
182
The primary transcriptome of the marine diazotroph Trichodesmium erythraeum
183
IMS101. Sci Rep. 4, 6187.
184
185
Prufert-Bebout, L., Paerl, H.W., and Lassen, C. (1993) Growth, nitrogen
186
fixation, and spectral attenuation in cultivated trichodesmium species. Appl .
187
Environ. Microbiol. 59, 1367–1375.
188
189
Rippka R., Deruelles J., Waterbury J.B., Herdman M., and Stanier R.Y. (1979)
190
Generic Assignments, Strain Histories and Properties of Pure Culture of
191
Cyanobacteria. J. Gen. Microbiol. 111, 1-61
192
193
Shi, D., Kranz, S.A., Kim, J.M., and Morel, F.M. (2012) Ocean acidification
194
slows nitrogen fixation and growth in the dominant diazotroph Trichodesmium
195
under low-iron conditions. Proc. Natl. Acad. Sci. USA. 109, 3094-100.
196
197
Walworth, N., Pfreundt, U., Nelson, W.C., Mincer, T., Heidelberg, J.F., Fum F.,
198
et al. (2015) Trichodesmium genome maintains abundant, widespread noncoding
199
DNA in situ, despite oligotrophic lifestyle. Proc. Natl. Acad. Sci. USA. 112, 4251-6.
200
201
Williams J.G.K. (1988) Construction of Specific Mutations in Photosystem II
202
Photosynthetic Reaction Center by Genetic-Engineering Methods in Synechocystis
203
6803. Method. Enzymol. 167, 766-778.
204
205
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