Supplementary Information
Contents:
Fig. S1.................................................................................................................................... p. 1
Fig. S2.................................................................................................................................... p. 2
Fig. S3........................ ........................................................................................................p. 3
Materials and methods........................................................................................................p. 4-7
Syringe
Seal
Sample vessel
Filtered water sample
+ nitrate/ammonium/phosphate
Water sample
(source community)
Fig. S1 Setup of chemotaxis assays. A chemoattractant is added to a filtered volume of an
aquatic sample containing a microbial community of interest and placed in a syringe. The
syringe is then submerged in a non-filtered volume of the aquatic sample and the
chemoattractant starts to diffuse out of the syringe. Chemotactic organisms move towards the
chemoattractant and accumulate in the syringe.
1
8.E+06
8
***
Cell density (counts ml-1 x 106)
7.E+06
7
***
6.E+06
6
***
5.E+06
5
4.E+06
4
3.E+06
3
2.E+06
2
1.E+06
1
0.E+00
0
Lakelake
water
water
Control
Control
Phosphate
Phosphate
Nitrate
Ammonia
Fig. S2 Mean cell counts associated with control and chemoattractant-spiked assays. Error
bars represent standard deviations. The asterisks indicate that all chemoattractant-spiked
assays contained significantly more cells than the control assays (P < 0.001, GLM). Counts in
the assays with attractants did not differ from one another (P > 0.05, GLM).
2
80000000
8
***
70000000
7
Cell density (counts ml-1 x 106)
***
60000000
6
***
50000000
5
40000000
4
30000000
3
2
20000000
1
10000000
0
FW T0
FW T30 PO43- NO3- NH4+
Ctrl PO4 NO3 NH4
T30
T30
T30
0 min
30 min (growth stimulation)
Ctrl
FW
PO43- NO3- NH4+
Ctrl
PO
NO3 NH4
4
Chem Chem
Chem
Chem
30 min (chemotaxis)
Fig. S3 Mean cell counts associated with a) lake water immediately after filtration (0.2 µm),
b) filtered lake water with and without inorganic nutrients after 30 min incubation without the
syringe tip submerged in lake water (growth stimulation test), and c) filtered lake water with
and without inorganic nutrients after 30 min incubation with the syringe tip submerged in
lake water (chemotaxis assays). Error bars represent standard deviations. The asterisks
indicate that all inorganic nutrient-spiked chemotaxis assays contained significantly more
cells than the non-incubated control assay (Ctrl 0 min; P < 0.001, GLM). Counts in the assays
with attractants did not differ from one another (P > 0.05, GLM). Negligible growth occurred
as a result of adding the nutrients to the filtered lake water indicating that cell counts in the
chemotaxis assays reflect cells that moved into the syringes.
3
Materials and methods
Sample collection and chemotaxis assays
A 1 L water sample was collected from the top 20 cm of a eutrophic lake at The
University of Queensland. The lake holds approximately 33 ML water (pH 6) containing 1.5
ppm PO4-P, 0.76 ppm NH4-N and 0.24 ppm NO3-N. Chemotaxis assays were performed by
submerging the tips of 1 ml syringes containing 80 µl 0.1 M chemoattractant (nitrate: KNO3,
ammonium: NH4Cl, or phosphate: KH2PO4) into 20 ml lake water for 30 min (Fig. S1). Each
chemoattractant was prepared in 0.2 µm filtered lake water to ensure that the chemical
characteristics of the background solution in the syringe was similar to that in the lake.
Filtered lake water control assays with no added chemoattractant were performed in parallel
and facilitated the measurement stochastic movement of cells into our assays. All assays were
replicated six times, providing three samples for cell counting and three samples for 16S
rRNA gene amplicon sequencing. To ensure that cell counts in the assays after 30 mins
reflected chemotaxis rather than inorganic nutrient-stimulated growth of residual cells postfiltration we enumerated cells in 0.2 µm filtered lake water with and without added nutrients
before and after 30 min incubation without submerging the tip of the syringe in the lake water
(Fig. S3).
Flow cytometry – Enumeration of microbial cells
The contents of each syringe was: 1) stained with 10,000X SYBR Green I for 15 min,
2) diluted 1:10 (non-filtered lake water), or 1:2 (all other samples) with 0.22 µm filtered TE
buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and then 3) supplemented with yellow-green
carboxylate-modified microspheres (used as reference objects; Invitrogen) to a final
4
concentration of 105 beads ml-1. Cell counts were measured using a Becton Dickinson LSRII
flow cytometer (BD, Australia) equipped with an FITC 200 filter and a 5 W argon-ion laser
operated at 20 mW, 488 nm.
DNA extraction, PCR and pyrosequencing
Three replicate samples were pooled (240 µl final volume), and then centrifuged at
14,000 g, 15 min. After removing the supernatant, pelleted cells were resuspended in 10 µl
Lyse and Go PCR reagent (Thermo Scientific) and genomic DNA was extracted according
the manufacturer’s instructions. PCRs were then performed in 50 µl volumes each containing
5 µl DNA extract, molecular biology grade water, 1X PCR Buffer minus Mg2+ (Invitrogen),
50 nM of each of the dNTPs (Invitrogen), 1.5 mM MgCl2 (Invitrogen), 0.3 mg BSA (New
England Biolabs), 0.02 U Taq DNA Polymerase (Invitrogen), and 8 µM each of the primers
926F and 1392R (Engelbrektson et al., 2010) modified on the 5’ end to contain the 454 FLX
Titanium Lib L adapters B and A, respectively. The reverse primers also contained a 5-6
base barcode sequence positioned between the primer sequence and the adapter. A unique
barcode was used for each sample. Thermocycling conditions were as follows: 95°C for 1
min; then 34 cycles of 95°C for 1 min, 55°C for 45 s, 72°C for 90 s; then 72°C for 10 min.
Amplifications were performed using a Veriti® 96-well thermocycler (Applied Biosystems).
Amplicons were purified using a QIAquick PCR purification kit (Qiagen), quantified using a
NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA) and then
normalised to 25 ng µl-1 and pooled for 454 pyrosequencing. Sequencing was performed by
Macrogen Inc. (Seoul, Korea).
Data analyses
5
Data were analysed as described in Dennis et al., (2012), briefly, sequences were
quality filtered and dereplicated using the QIIME script split_libraries.py with the
homopolymer filter deactivated (Caporaso, et al., 2010) and then checked for chimeras
against the GreenGenes databases using UCHIME ver. 3.0.617 (Edgar, et al., 2011).
Homopolymer errors were corrected using Acacia (Bragg et al., 2012). Sequences were then
subjected to the following procedures using QIIME scripts with the default settings: 1)
sequences were clustered at 97% similarity, 2) cluster representatives were selected, 3)
GreenGenes taxonomy was assigned to the cluster representatives using BLAST, and 4)
tables with the abundance of different operational taxonomic units (OTUs) and their
taxonomic assignments in each sample were generated. The number of reads was then
normalised to 6500 per sample.
Variation in the composition of microbial communities between normalised samples
(beta diversity) was investigated using Principal Component Analysis (PCA). Differences in
cell counts between assays were compared using Generalised Linear Modelling (GLM). All
analyses were implemented using R 2.12.0 (R Development Core Team, 2010). The
phylogeny of certain key OTUs was inferred using Arb (Ludwig et al., 2004), which
facilitated comparisons between our query sequences and full length sequences in the
GreenGenes database.
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