Supplementary Materials:
Materials and methods:
Pseudo-nitzschia genotyping
The Pseudo-nitzschia cultures were identified by morphology using light microscopy
and genotyped by sequencing the 18S rRNA gene. Pseudo-nitzschia genomic DNA
was extracted with PowerSoil DNA Isolation Kit (MoBio Laboratories Inc., Solana
Beach, CA). P. pungens and P. fraudelenta were amplified with primer pairs 1360F
(5’-GCGTTGAT/ATACGTCCCTGCC- 3’) and ITS055R (5’CTCCTTGGTCCGTGTTTCAAGACGGG-3’), while P. australis was amplified with
primer pairs 18S-F (5’-CTGCGGAAGGATCATTACCACA-3’) and ITS055R using
EconoTaq DNA Polymerase (Lucigen, Middleton, WI) under the following PCR
conditions: 94°C for 2 min, 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for
1.5 min and 72°C for 10 min extension. Gel bands were excised and cleaned with the
GeneJet Gel Extraction Kit (Fermentas Inc, USA). The products were sent to a service
facility (Laragen Inc, Culver, CA, USA) for direct sequencing after labeling with the
Big Dye Terminator with AmpliTaq FS Sequencing Kit (Applied Biosystems, Foster
City, CA, USA) using the PCR primers as sequencing primers.
Bacteria culturing and genotyping with 16S rDNA
Bacteria cultures used for fitness assay and domoic acid assays were grown in 8-ml
sterile marine broth at room temperature (22oC) for 48 hours in a lab shaker set at 200
rpm. Bacterial cultures were all cloudy at the time of harvest. The bacterial cultures
were centrifuged at 3000 g, washed once and resuspended in sterile FSW and
measured at OD600 (0.65- 0.81) with a Biophotometer (Eppendorf AG, USA). CFU
counts using drop plate method is reported in M&M section.
A 50 ul aliquot was boiled for 10 minutes to extract bacterial DNA for 16S rDNA
genotyping. An aliquot of the sample was diluted in 1:100 and used as DNA template
for 16S rDNA PCR amplification using 27F and 1492R primer pairs under the same
PCR conditions, purification and sequencing protocol as mentioned.
Bacterial DNA extraction and 16S rDNA bacterial pyrosequencing
(Dowd et al., 2008 and Sun et al., 2011)
DNA samples were extracted using RTL buffer with β- mercaptoethanol, and lysed
with sterile beads in a tissue lyser. The samples were then processed with the Qiagen
DNA Stool Kit (Qiagen, Valencia, CA) and diluted to a final concentration of 20
ng/l. Pyrosequencing was carried out by initially generating a sequence library from
a one-step PCR of 30 cycles using a mixture of Hot Start and HotStar high fidelity
Taq polymerase.
Results
a
b
Figure S1. Rarefaction curves for each species of Pseudo-nitzschia based on a)
Phylogenetic Diversity at a sequence depth of 151 and b) Shannon Index at a
sequence depth of 6650.
Microbial composition of different Pseudo-nitzschia species
For the identification of microbial composition, the data were combined and analyzed
for each host species. Majority of the OTUs belong to Alpha-proteobacteria (8098%), Gamma-proteobacteria (0.1- 4%) and Bacteroidetes (0.7 – 2%) with a small
representation from Firmicutes (0.7%) and Actinobacteria (1%). The first three
bacterial phyla are consistent with what was found in many Pseudo-nitzschia species
using culture (Kaczmarska et al., 2005), and ARISA methods (Guannel et al., 2011).
Pyrosequencing approach (this study), however, was able to detect rare bacterial
OTUs associated with Pseudo-nitzschia such as the Firmicutes and Actinobacteria.
Figure S2 shows the relative abundance of bacterial OTUs present in each
algal sample at the family level. Six families comprise the sequences from Alphaproteobacteria, three of which are unique to P. pungens (Hyphomonadaceae, 6.2%;
Phyllobacteriaceae, 11.8%; Hyphomicrobiaceae, 1%), while two families
(Erythrobacteraceae, 0.1% and Rhizobiaceae, 0.1%) are only found in P. fraudelenta.
The family Rhodobacteraceae is highly represented (56 - 98%) in all 3 Pseudonitzschia species, while the family Sphingomonadaceae (5.3 -7.6%) is only present in
P. pungens and P. australis. It is remarkable that sequences from Gammaproteobacteria (although lowest in relative abundance compare to other phyla) are
more represented in P. australis than in P. pungens or P. fraudelenta. Three families
(Alteromonadaceae, 0.6%; Pseudoalteromonadaceae, 0.2%; Vibrionaceae, 1.1%) are
found in P. australis while the family Piscirickettsiaceae (3.8%) is only represented in
P. pungens. From the Bacteroidetes phylum, only 3 families are found to associate
with Pseudo-nitzschia. All three of these families are found in P. pungens
(Cyclobacteracea, 3.8%; Flavobacteriaceae , 3.4% and Cryomorphoceae, 8.3%). P.
australis and P. fraudelenta also contained sequences from Flavobacteriaceae (0.8% 1.9%) but at very low proportions.
All of the OTUs were searched for closest homologous sequences in GenBank
and identified at the genus level. A total of 64 genera associate with the three species
of Pseudo-nitzschia; 27 genera comprise the Alpha-proteobacteria, 17 genera
represent the Gamma-proteobacteria, 15 genera from Bacteroidetes, 4 genera from
Firmicutes and 1 genus from Actinobacteria (Figure S3). Interestingly, P. pungens
associates mostly with bacteria belonging to genera from Alpha-proteobacteria. It is
noteworthy to mention that the OTU homologous to Cellulophaga (used in the fitness
assay below) is only found in P. pungens. Moreover, even though only one genus
(Methylophaga) from Gamma-proteobacteria is found in the bacterial pyrosequences
of two P. pungens samples, we were able to culture bacteria from other P. pungens
clones whose 16S rDNA were homologous to the Gamma-proteobacteria
Marinobacter, Alteromonas, Pseudoalteromonas and Glaciecola (Table 1). Indeed,
not all possible OTUs have been sequence in these species as shown by the lack of
saturation in the rarefaction curves (Figure 1). We assume therefore that P. pungens
associates with more bacteria than can be recognized in this study. Consequently,
OTUs homologous to Sulfitobacter, Roseobacter, Phaeobacter, Rhodobacter and
Sphingophyxis are all common associates of the three Pseudo-nitzschia species
(Figure S3). Marinobacter, Alteromonas, Roseobacter and Sulfitobacter are also
commonly reported to be associated with several marine phytoplankton species
(Stewart et al., 1997, Hold et al., 2001, Alavi et al., 2001, Schäfer et al., 2002, Green
et al., 2004, Kaczmarska et al., 2005, Jasti et al., 2005, Grossart et al., 2005, Sapp et
al., 2007, Hunken et al., 2008; Guannel et al., 2011) and can be considered as the core
microbiome of marine phytoplankton. The OTU Winogradskyella and Staleya (i.e.
Sulfitobacter) that were previously found in the toxic Pseudo-nitzschia multiseries
(Kaczmarska et al., 2005, Guannel et al., 2001) are also found in the pyrosequences
of the toxic P. fraudelenta and P. australis (Figure 3) and in our 16S rDNA library of
other P. australis clones (M.P. Sison-Mangus, unpublished data). One of the
underlying goals in this study is to determine if there is a bacterial species or a
relative abundance of bacteria that is significantly associated with toxic Pseudonitzschia. We tested this hypothesis by G- test of independence, to determine which
OTU is significantly associated with toxic algae and by ANOVA, to determine if the
relative abundance of an OTU is different between the toxic and non-toxic algae,
applying Bonferroni correction to the p-value. However, none of the OTUs proved to
be statistically significant in both tests.
100
Bacteroidetes;Sphingobacteria;Cyclobacteriaceae
Bacteroidetes;Flavobacteria;Flavobacteriaceae
90
Bacteroidetes;Flavobacteria;Cryomorphaceae
Firmicutes;Clostridiaceae
80
Relative Abundance ( %)
Gammaproteobacteria;Vibrionaceae
Gammaproteobacteria;Pseudomonadaceae
70
Gammaproteobacteria;Pseudoalteromonadaceae
60
Gammaproteobacteria;Piscirickettsiaceae
Gammaproteobacteria;Oceanospirillales
50
Gammaproteobacteria;Alteromonadales genera
incertae sedis
Gammaproteobacteria;Alteromonadaceae
40
Alphaproteobacteria;Sphingomonadaceae
Alphaproteobacteria;Rhodobacteraceae
30
Alphaproteobacteria;Rhizobiaceae
20
Alphaproteobacteria;Phyllobacteriaceae
Alphaproteobacteria;Hyphomonadaceae
10
Alphaproteobacteria;Hyphomicrobiaceae
Alphaproteobacteria;Erythrobacteraceae
P. fraudelenta-8
P. fraudelenta-1
P. australis-15
P. australis-12
P. australis-B5
P. pungens-C5A
P. pungens-B2A
0
Actinobacteria;Coriobacteriaceae
Figure S2. Relative abundances of partial bacterial16S rDNA sequences from
Pseudo-nitzschia samples classified at the family level using Ribosomal Database
Project 2.2 (RDP).
Coriobacteriaceae
P. australis
P. fraudelenta
P. pungens
Collinsella 2
Bacteroides 2
Cyclobacteriaceae
Algoriphagus 11
Brumimicrobium 27
Cryomorphaceae
Fluviicola 24
Lishizhenia 21
Aequorivita 387
Bizionia 79
Bacteroidetes
Cellulophaga 5
Flavobacteriales
Croceimarina 3
Flaviramulus 205
Flavobacteriaceae
Polaribacter 44
Salegentibacter 157
Salinimicrobium 44
Tenacibaculum 76
Winogradskyella 28
Planococcus 2
Bacillales
Staphylococcus 2
Firmicutes
Clostridium 16
Clostridiales
Blautia 2
Hyphomicrobiaceae
Zhangella 3
Phyllobacteriaceae
Rhizobiales
Hoeflea 34
Agrobacterium 22
Rhizobium/Agrobacterium group
Rhizobium 3
Hyphomonas 19
Antarctobacter 13587
Citreicella 4
Jannaschia 865
Litoreibacter 4
Loktanella 3887
Marivita 4
Methylarcula 22290
Bacteria
Oceanibulbus 12
Alphaproteobacteria
Rhodobacterales
Oceanicola 749
Rhodobacteraceae 1514
Octadecabacter 2201
Phaeobacter 47
Rhodobacter 10797
Roseobacter 19974
Roseovarius 6
Ruegeria 66
Sulfitobacter 11460
Thalassobacter 71
Proteobacteria
Thalassobius 310
Erythrobacter 29
Sphingomonadales
Sphingomonadaceae
Novosphingobium 2
Sphingopyxis 524
Myxococcus 2
Alteromonadaceae
Glaciecola 588
Marinobacter 17
Gilvimarinus 20
Alteromonadales
Colwelliaceae
Colwellia 6
Thalassomonas 3
Pseudoalteromonadaceae
Pseudoalteromonas 181
Halothiobacillus 2
Enterobacteriaceae
Gammaproteobacteria
Escherichia 2
Serratia 5
Kangiella 2
Oceanospirillales
Oceanospirillaceae
Amphritea 69
Marinomonas 3
Neptumonas 7
Pseudomonadaceae
Piscirickettsiaceae
Pseudomonas 2
Methylophaga 21
Porticoccus 3
Vibrionaceae
Vibrio 443
Figure S3. Comparative taxonomic tree and the relative abundance of the 16S rDNA
sequences of the bacterial genera represented in the three Pseudo-nitzschia species
datasets. The number of OTUs were combined and summarized by diatom species.
Only bacterial genera with 2 sequences were included. The numbers next to the genus
names are cumulative numbers of sequences assigned to this taxon. Red – P.
australis; Blue – P. fraudelenta; Green – P. pungens.
Figure S4. Replication of fitness assay using different clones of P. pungens and P.
australis, co-cultured with individual members of microbiota isolated from P.
pungens. (a-c) P. pungens- B2A co-cultured with its own microbiota from Gammaproteobacteria, Alpha-proteobacteria and Bacteroidetes, respectively. (d-f) P.
australis- B6 co-cultured with P. pungens microbiota from Gamma-proteobacteria,
Alpha-proteobacteria and Bacteroidetes, respectively. Numbers after the name of
bacterial treatment in the legend are specific growth rates day-1 of the Pseudonitzschia species. Asterisks denote significant differences (p<0.05) between specific
growth rates of the bacterial treatment and axenic cultures analyzed with post hoc
Student’s T. PP – P. pungens; PA – P. australis; PF- P. fraudelenta; AX – axenic
(n=3); ALT- Alteromonas (n=3); MAR- Marinobacter (n=3); PSEPseudoalteromonas (n=3); PHAEO- Phaeobacter (n=3); ROSEO- Roseobacter
(n=3); CEL- Cellulophaga (n=3); POL- Polaribacter (n=3).
Figure S5. Cellular domoic acid concentration (g. L-1) measured from P. australis
(a) and P. fraudelenta (b) at late exponential stage after co-cultivation with individual
bacteria isolated from P. pungens and P. australis. PA – P. australis; PP – P.
pungens; AX – axenic (n= 3; 3); NON-AX - non- axenic (n= 3; 2); GLA- Glaciecola
(n= 2; 3); MAR- Marinobacter (n= 3; 2); PSE- Pseudoalteromonas (n= 2; 3); ALTAlteromonas (n= 3; 3); ROSEO- Roseobacter (n= 3; 2); PHAEO- Phaeobacter (n= 2;
2); SAL- Salegentibacter (n= 3; 2); CEL- Cellulophaga (n= 3; 2); POL- Polaribacter
(n= 2; 2); PLAN- Planococcus (n= 3; 2); BACI- Bacillus (n= 3; 3). The group mean
(line), sample mean (midline inside diamond), mean error bars and the 95%
confidence points for each group represented by the diamond’s top and bottom points
are depicted in the graph. Asterisks denote significant differences (p<0.05) between
bacterial treatment and group mean by Analysis of the Means with transformed ranks.
Number of trials, n, for P. australis and P. fraudelenta, respectively, are indicated in
parenthesis after name of bacteria with each bacterial treatment.
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