Supplementary methods
Growth of algae
Chlamydomonas reinhardtii (wild-type strain 12) was a gift from Saul Purton, University
College London, UK. Cyanidium caldarium was a gift from David Vernon, University of Leeds,
UK. Amphidinium operculatum and A. carterae were a gift from Chris Howe, University of
Cambridge, UK. Euglena gracilis was a gift from Alan Battersby, University of Cambridge, UK.
Thlalassiosira pseudonana (CMPP 1335) was received from the Provasoli-Guillard National
Center for Culture of Marine Phytoplankton, Maine, USA. All other algal strains were received
from the Culture Collection of Algae and Protozoa (CCAP), Oban, UK.
The conditions and media used for the growth of different algal strains are summarised in the
table below. With the exception of Euglena minimal medium and Cyanidium medium1, used to
assess the vitamin B12 requirements of Euglena gracilis and Cyanidium caldarium respectively,
the recipes for all of the other media used in this study can be found on the CCAP website
(www.ife.ac.uk/CCAP/).
Species
Porphyridium purpureum (CCAP
1380/3)
Rhodella maculata (CCAP 1388/2)
Cyanidium caldarium
Cyanophora paradoxa (CCAP 981/1)
Chlamydomonas reinhardtii
Haematococcus pluvialis (CCAP 34/6)
Lobomonas rostrata (CCAP 45/2)
Mantoniella squamata (CCAP 1965/1)
Pyramimonas disomata (CCAP 67/8)
Tetraselmis verrucosa (CCAP 66/46)
Chromulina chionophila (CCAP 909/9)
Poterioochromonas malhamensis
(CCAP 933/1C)
Thalassiosira pseudonana
Skeletonema pseudocostatum (CCAP
1077/7)
Hemiselmis virescens (CCAP 984/5)
Rhodomonas maculata (CCAP 979/14)
Pavlova gyrans (CCAP 940/1B)
Pavlova sp. (CCAP 931/2)
Gymmodium catenatum (CCAP 1117/7)
Amphidinium operculatum
Amphidinium catenatum
Oxyrrhis marina (CCAP 1133/5)
Euglena gracilis
Medium
PE
Conditions
25C, shaking, continuous light
F/2 + Si
Troxler, 1972
JM
TAP
Euglena medium
JM
F/2+ Si
ASW
F/2 + Si
BB
JM:SE
18C, standing, 16h:8h light:dark
37C, shaking, dim light
18C, standing, 16h:8h light:dark
25C, shaking, continuous light
25C, shaking, continuous light
25C, shaking, continuous light
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
25C, shaking, continuous light
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
F/2 + Si
F/2 + Si
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
ASW
ASW
F/2 + Si
F/2 + Si
F/2 + Si
ASW or F/2
ASW
F/2 + Si
Euglena medium
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
25C, shaking, continuous light
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
18C, standing, 16h:8h light:dark
25C, shaking, continuous light
Media Designed in this Study
ASWH (Artificial Seawater for Halomonas)
Per litre
Sucrose
NaCl
Ultramarine synthetic sea salts
Tricine
Extra salts stock solution
10g
16.4g
33.6g (see below)
0.50g
3.75 ml (see below)
Nutrient stock solution:
Per litre
NaNO3
Na2HPO4
K2HPO4
83.4g
1.20g
1.00g
‘Ultramarine synthetic’ sea salts
Supplied by Waterlife Research Industries Ltd., 476 Bath Road, Longford West Drayton,
Middlesex, UB7 0ED, England
Euglena Minimal Medium
Per litre
Glutamic acid
Glycine
Glucose
Malic acid
MgSO4.7H2O
CaCl2.2H2O
CoCl2.6H2O
Extra salts stock solution (from ASWH)
Euglena salts
Vitamin mix
3g
3g
3g
3g
500mg
200mg
25mg
5.00ml
0.5ml
1.5ml
Adjust to pH 4.8 with 10M KOH
Vitamin Mix:
Per litre
Biotin
Thiamine HCl
40mg
40mg
Euglena Salts:
Per litre
H3BO3
CuSO4.5H2O
Fe(NH4)2SO4.6H2O
MnSO4.4H2O
ZnSO4.7H2O
(NH4)6Mo7O24.4H2O
5.7mg
15.7mg
140mg
8.1mg
79mg
200mg
Growth of algae at natural vitamin B12 concentrations
To assess whether the concentration of vitamin B12 in the natural environment was limiting for
the growth of vitamin B12-dependent freshwater and marine algae, Euglena gracilis,
Amphidinium operculatum and Porphyridium purpureum were grown in media made exactly as
described above, except that distilled water was replaced by natural filter-sterilised water. For the
growth of freshwater algae, the water was collected from the Granta pond, Cambridge, UK and
used to make the appropriate medium. The medium was then filter-sterilised through a 0.2 m
Millipore filter before being used. Exactly the same protocol was used for the growth of marine
algae, except the water used was seawater collected at West Runton, Norfolk, UK. Bacterial
growth was not observed in the filter-sterilised media unless it was inoculated with a bacterial
culture.
Supplementation of Lobomonas rostrata medium
Lobomonas rostrata was grown in 50 ml Jaworski’s medium for 7 days in continuous light with
shaking in the presence of 10 g/l vitamin B12, 10 mM methionine and 1 M folic acid. A 200 l
sample of this culture was removed and used to inoculate fresh medium, which was grown for a
further 7 days. This subculturing was repeated 6 times. After each 7 day period, a 1 ml sample of
culture was removed and the optical density measured at 600 nm.
For homocysteine analysis, a 40 ml sample of culture, grown for 7 days, was centrifuged at 5,000
g for 20 min. The supernatant was discarded, and the pellet resuspended in 1 ml distilled water.
The cells were sonicated with 6 x 20 second bursts separated by 30 s, on ice, in the presence of
100 l glass beads (213-300 m). The cell debris and glass beads were removed by centrifuging
the sample at 13,000 g for 5 min at 4 C, and the supernatant was used for homocysteine analysis
as described below.
Homocysteine analysis
Homocysteine levels were determined by HPLC after pre-column derivatisation with
monobromobimane, as previously described2, with some modifications. Cell-free extracts were
derivatised in a reaction mixture containing 0.1 M Hepes-NaOH, pH 8.0, 5 mM
monobromobimane, 2 mM DTT (final concentrations). The reaction was incubated in the dark at
room temperature for 10 min before the addition of 25 mM methanesulphonic acid. Samples
were run on an Aglient 1100 series HPLC equipped with fluorescence detector (excitation 388
nm; emission 480 nm) using an Ace 5 AQ column (4.6 x 250 mm; Advanced Chromatography
Technologies) at 37 ºC. A gradient of acetonitrile in 0.25 % acetic acid and 57 mM sodium
perchlorate at a flow rate of 1 ml min-1 was used to elute the fractions. Thiols were identified by
comparison against commercial standards. The concentration of homocysteine was standardised
against total soluble protein in the extract. The concentration of soluble protein was determined
using the Bradford assay.
Isolation of bacteria from freshwater
Samples of water were collected from the Granta pond, Cambridge, UK in sterile bottles. A 200
l sample of the water was spread onto LB agar plates, and the plates were incubated at 20 C
for 48 h. Bacterial colonies, each with different morphologies, were picked from the plates with a
sterile wire loop, re-cultured onto fresh LB agar plates, and incubated for a further 48 h.
Individual colonies were then used to inoculate E. gracilis cultures.
Porphyridium purpureum cell mass determination
P. purpureum cells were grown in 50 ml PE medium for 28 days as described above. The entire
culture was transferred to a 50 ml tube, and centrifuged at 5,000 g for 10 min. The pellet was
resuspended in 1 ml fresh PE medium, and transferred to a pre-weighed 1.5 ml tube. The cells
were centrifuged at 13,000 g for 5 min, and the supernatant discarded. The 1.5 ml tube,
containing the cell pellet was re-weighed, and the cell mass determined.
Identification of isolated bacteria
Three colonies were picked from an agar plate and used to inoculate liquid media. The cells were
grown to stationary phase. The culture was centrifuged at 4,500 g for 10 min, the pellet
resuspended in 200 l sterile distilled water and heated to 100C for 15 min. The sample was
cooled to room temperature, centrifuged at 13,000 g, and the supernatant was used as a crude cell
extract for PCR.
The 16S rRNA gene was amplified by PCR using degenerate primers as described previously3.
The amplified product was gel purified, and directly sequenced using the same primers that were
used for PCR amplification. The 16S rRNA gene sequence was then compared to other
sequences in the ribosomal database at the Michigan State University (http://rdp.cme.sus.edu).
DAPI staining
A stock solution of 1 mg/ml DAPI (4’6-diamidine-2’-phenylindole dihydrochloride) was
prepared in methanol. The stock solution was diluted to a final concentration of 1 g/ml with
sterile seawater. An aliquot of 500 l of a Porphyridium purpureum culture was placed in an
eppendorf tube, and the cells were left to settle for 30 min. The supernatant was removed, and
500 l of 1 g/ml DAPI was incubated with the cells for 15 min. The cells were washed twice
with 1 ml sterile seawater, mounted on a glass slide and visualised under an eppifluorescent
microscope at 325 nm, 400x magnification. The P. purpureum cells were not centrifuged at any
point during the staining procedure as this disrupts the clumping of cells.
Growth experiments with Halomonas sp.
A single Halomonas sp. colony picked from an ASWH agar plate was used to inoculate 10 ml
ASWH liquid medium, and the cells grown at 30 C for 16 h. A 2 ml sample of this culture was
used to inoculate 100 ml ASWH medium containing 0.01% w/v fucoidin, and the cells were
grown aerobically at 30 C. A 1 ml sample was taken every 4 h and the OD600 measured. After,
16, 24 and 36 hours a 10.1 ml sample was taken from the culture: 100 l was used for serial
dilutions on ASWH agar plate, and the remaining 10 ml of medium was centrifuged at 4,000 g
for 10 min at 20 C. The supernatant was discarded, the pellet resuspended in 500 l sterile
water, and the resulting solution assayed for vitamin B12 (as above).
Reverse Transcription – PCR
Total RNA was extracted from C. reinhardtii or T. pseudonana using the method described4 and
stored under ethanol at -80C. A 5-100 g sample of RNA was precipitated and treated with
Rnase-free DNase (Promega) for 30 min at 37 C to remove any contaminating DNA. The
cDNA was synthesized using Superscript IITM (Invitrogen) with dT17 primers exactly as
described by the manufacturers instructions. The cDNA was amplified with Taq DNA
polymerase (Bioline). The PCR conditions for the amplification of the metE fragment from C.
reinhardtii were 28 cycles at 94 C for 40 seconds, 55 C for 1 minute, and 70 C for 1 minute.
The metH and actin fragments from C. reinhardtii were amplified under exactly the same
conditions except the annealing temperature was lowered from 55 C to 45 C. The primers used
for PCR on C. reinhardtii cDNA were:
metE F, 5`-GTGGACGACCCCCGCTCTGCG-3`
metE R, 5`-CCATGCGGTCGATGGCGGGC-3`
metH F, 5`-CAAGTACAAGGCGTGGAAGG-3`
metH R, 5`-GTTGGTCTCAATGATGTCCG-3`
actin F, 5`-TATCGTGCTGGACTCTGG-3`
actin R, 5`-GATCTTCATCAGGTAATCG-3`.
The PCR conditions for the amplification of the cbiP and actin fragments from T. pseudonana
cDNA were 35 cycles at 94 C for 40 seconds, 48 C for 40 seconds, and 70 C for 1 minute.
The primers used for the PCR on T. pseudonana cDNA were:
TP cbiP F, 5`-TCTTGCTCAAGTCAGGAGGC-3`
TP cbiP R, 5`-CACGGTACCACAAACACAGG-3`
TP actin F, 5`-AACATTATTGTTATTGGGAATGAGC-3`
TP actin R, 5`-ACTCTGGAAAGTAGAAAGAGAAGCC-3`.
References
1. Troxler, R. F. Synthesis of bile pigments in plants. Formation of carbon monoxide and
phycocyanobilin in wild-type and mutant strains of the alga, Cyanidium caldarium.
Biochemistry, 11, 4235-4242 (1972)
2. Fahey, R. C. & Newton, G. L. Determination of low-molecular-weight thiols using
monobromobimane fluorescent labeling and high-performance liquid chromatography.
Methods in Enzymology 143, 85-96 (1987)
3. Lane, D. J. Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt and M.
Goodfellow, eds. N. Y., John Wiley and Sons: 115. (1991)
4. Witman, G. B., Carlson, K., Berliner, J. & Rosenbaum, J. L. Chlamydomonas flagella. I.
Isolation and electrophoretic analysis of microtubules, matrix, membranes, and
mastigonemes. J. Cell Biol. 54, 507-539 (1972)