Computational protocol: Discovery of an Endosymbiotic Nitrogen-Fixing Cyanobacterium UCYN-A in Braarudosphaera bigelowii (Prymnesiophyceae)

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[…] Seawater samples collected from surface level (0m) of Tomari Port by a bucket on 22nd November 2012 and on 18th June 2013 were selected for molecular phylogenetic studies of B. bigelowii from the samples originally collected for regular floristic studies of coccolithophores in the area (Hagino, unpublished). 10 liters of seawater was pre-filtered through a 50-µm mesh-size plankton net (Sefar Inc. DIN-110) and then concentrated using a piece of 1-µm mesh-size plankton net (Sefar Inc. NY1-HD) placed on a kitchen sieve. A living cell of B. bigelowii labeled as TMR-scBb-1 was isolated from the sample collected on 22nd November 2012, and the cells labeled as TMR-scBb-7 and -8 were isolated from the sample collected on 18th June 2013 using a micropipette under an inverted light microscope (Olympus CKX41). Each cell was carefully cleaned with sterile seawater using a micropipette under the inverted light microscope. Each cell was observed (magnification x1500) and photographed using a Nikon E600POL microscope (), and then subjected to single cell PCR amplification as outlined in Takano and Horiguchi .In the beginning of this study, we used 18S and 16S rDNA primers published by previous studies for PCR amplification and sequencing (). Based on the 16S rDNA sequences obtained from the experiments, we designed new primers for bacterial and plastid16S rRNA genes of B. bigelowii and then conducted subsequent PCR amplification of 16S rDNA using published and our new primers. Details of the experiments are described below.The first reaction of PCR of the specimen TMR-scBb-1 was performed using external primers for 18S rDNA (SR1 and SR12) and for 16S rRNA primers (27F and 1492R). The first reaction of PCR of the specimens TMR-scBb-7 and -8 were performed using external primers for 18S rDNA (SR1 and SR12), for 16S rRNA primers (27F and 1492R), and nitrogenase primers (nifH4 and nifH3) (). The conditions for the first PCR round for the specimen TMR-scBb-1 were one initial denaturation step at 98°C for 60s, followed by 35 cycles of denaturation at 94°C, annealing at 50°C for 30s and extension at 72°C for 60s. The conditions for the first PCR reaction for the specimens TMR-scBb-7 and -8 were one initial denaturation step at 98°C for 60s, followed by 35 cycles of denaturation at 94°C, annealing at 47°C for 30s and extension at 72°C for 60s.In the second PCR reaction in the beginning of the experiments, short internal sequences of 18S and 16S rDNA genes were amplified using 0.5 µL of the first PCR product of the specimen TMR-scBb-1 as the DNA template with three pairs of 18S rDNA primers (SR1c-SR5tak, SR4-SR9, SR8-SR12) and three pairs of 16S rDNA primers (27F-Nitro821R and CYA359F-Nitro821R, and CYA 359F-1492R) (). In order to check possible occurrence of contamination, negative control samples without template were subjected to PCR amplification together with the samples containing template. The PCR conditions for the second reaction were one initial denaturation step at 98°C for 30s, followed by 10 cycles of denaturation at 96°C for 30s, annealing at 57°C for 30s and extension at 72°C for 30s, 10 cycles of denaturation at 96°C for 30s, annealing at 56°C for 30s and extension at 72°C for 30s, and 10 cycles of denaturation at 95°C for 30s, annealing at 55°C for 30s and extension at 72°C for 30s. The temperature profile was completed by a final extension cycle at 72°C for 4 min. In order to determine the amplification efficiency and possible occurrence of contamination, 2.5 µl of each second round PCR products were visualized by agarose electrophoresis. The successfully amplified products of the second round of PCR amplifications without contamination were sequenced directly using the ABI PRISM BigDye Terminator Cycle Sequencing Kit ver. 1.1 (Perkin-Elmer, Foster City, CA, USA) on a DNA auto sequencer ABI PRISM 3130 Genetic Analyzer (Perkin-Elmer) in the Faculty of Science, Hokkaido University, Japan. The results were confirmed by sequencing both forward and reverse strands.The 16S rRNA gene sequences obtained from the specimen TMR-scBb-1 in the experiments described above were subjected to a Blast Search. Sequences obtained by primer pairs of 27F-Nitro821R and by primer pairs of CYA359F-Nitro821 were identical to each other, and close to the bacterial16S rDNA sequences of uncultured nitrogen-fixing cyanobacterium UCYN-A. On the other hand, the sequences obtained by the primers CYA359F-1492R were close to the plastid 16S rDNA sequences of members in the class Prymnesiophyceae, Division Haptophyta. Based on the sequences obtained by the ‘CYA359F-Nitro821R’ and by ‘CYA359F-1492R’, we designed one primer for bacterial 16S RNA gene of B. bigelowii (16SBbsp726F) and four primers for plastid 16S rRNA gene of B. bigelowii (Bbplastid 445F, Bbplastid 604F, Bbplastid 628R, and Bbplastid 830R), respectively ().In the subsequent second PCR reactions for the specimen TMR-scBb-1, bacterial 16S and plastid 16S rRNA genes were amplified with three pairs of bacterial 16S rDNA primers (27F-Nitro821R, CYA359F-Nitro821R, and 16SBbsp726F-1492R), and three pairs of plastid 16S rDNA primers (27F-Bbplastid682R, Bbplastid 445F-Bbplastid830R, and Bbplastid604F-1492R), respectively. In the second PCR reactions for the specimens TMR-scBb-7 and -8, four pairs of 18S rDNA primers (SR1c-SR3, SR2tak-SR7, SR4-SR9, SR8-SR12), three pairs of bacterial 16S rDNA primers (27F-Nitro821R, CYA359F-Nitro821R, and 16SBbsp726F-1492R), three pairs of plastid 16S rDNA primers (27F-Bbplastid682R, Bbplastid 445F-Bbplastid830R, and Bbplastid604F-1492R), and one pair of nifH primers (nifH1-nifH2) were used. In all the second PCR reactions, negative control samples without template were subjected to PCR amplification together with the samples with template in order to check possible contamination. The PCR conditions for the second reaction were one initial denaturation step at 98°C for 30s, followed by 30 cycles of denaturation at 96°C for 30s, annealing at 55°C for 30s and extension at 72°C for 30s. The temperature profile was completed by a final extension cycle at 72°C for 4 min. In order to determine the amplification efficiency and possibility of contamination, 2.5 µl of each second round PCR products were visualized by agarose electrophoresis. Agarose electrophoresis suggested that fragments of 18S, plastid 16S, and bacterial 16S rRNA genes were successfully amplified without contamination, however, the nifH gene was not amplified for an unknown reason.The products of the second reaction of PCR amplifications of the 18S and 16S rRNA genes were sequenced directly using the ABI PRISM BigDye Terminator Cycle Sequencing Kit ver. 1.1 (Perkin-Elmer, Foster City, CA, USA) on a DNA auto sequencer Applied Biosystems 3130xl Genetic Analyzer in the Institute of Plant Science and Resources, Okayama University, Japan. The results were confirmed by sequencing both forward and reverse strands. Three 18S rDNA sequences (AB778293, AB847980 and AB847981), three plastid 16S rDNA sequences (AB847984, AB847985 and AB847986) and three bacterial 16S rDNA sequences (AB778292, AB847982 and AB847983) obtained in this study were deposited into the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/index-e.html).For the phylogenetic analysis of the 18S rRNA gene, a total of 44 OTUs including two members of the Pavlovophyceae as an out-group, were obtained from GenBank. The 18S rDNA sequence of C. parkeae strain Kawachi (AM490994), which was published in Medlin et al. , was originally obtained from a culture strain of C. parkeae established by M. Kawachi (Kawachi, pers. comm.). Since 18S rDNA sequences of B. bigelowii from the specimens TMR-sc-Bb-1 (AB778293), TMR-sc-Bb-7 (AB847980), and TMR-sc-Bb-8 (AB847981) obtained in this study were identical to the previously reported six sequences of B. bigelowii Genotype III (AB250784, AB847974-AB847978), we used the previously published sequence AB250784 as a representative of the Genotype III in this analysis. Taxonomic classification of the Haptophyta followed Edvardsen et al. . For the phylogenetic analysis of the plastid 16S rRNA gene, a total of 48 OTUs including a member of the Pavlovophyceae as an out-group were obtained from GenBank. Since the plastid 16S rDNA sequences of B. bigelowii from the specimens TMR-sc-Bb-1 (AB847984), TMR-sc-Bb-7 (AB847985), and TMR-sc-Bb-8 (AB847986) obtained in this study were identical to the each other, we only included the sequence from TMR-scBb-1 (AB847984) as a representative of the plastid 16S rDNA of B. bigelowii in this analysis. For the phylogenetic analysis of the bacterial 16S rRNA gene, a total of 33 OTUs, including Gloeobacter violaceus (Gloeobacterales) as an out-group, were used. Since the bacterial 16S rDNA sequences of B. bigelowii from the specimens TMR-sc-Bb-1 (AB778292), TMR-sc-Bb-7 (AB847982), and TMR-sc-Bb-8 (AB847983) were identical to the each other, we only included the sequence from TMR-scBb-7 (AB847982) as a representative of the bacterial 16S rDNA of B. bigelowii in this analysis. Each set of sequences was aligned using Clustal W (http://www.genome.jp/tools/clustalw/).For the 18S, bacterial 16S and plastid 16S rRNA genes, phylogenetic trees were constructed based on Maximum Likelihood (ML), Neighbor Joining (NJ), and Maximum Parsimony (MP) methods using PAUP version 4.0b10 , and also based on Bayesian inference (BI) using Mr. BAYES v3.1.2 . The use of Neighbor Joining and Maximum Parsimony analyses provided similar results (not shown) to those of ML analyses, therefore we describe only the method and results of ML and BI analyses in this study. Substitution models were selected using Modeltest 3.7 and MrModeltest 2.2 for ML and BI, respectively. ML was performed using the heuristic search option with a branch-swapping algorithm Tree bisection-reconnection (TBR). For the ML analyses, the NJ tree was used as a starting tree. Bootstrap analyses with 1000 replicates for ML analyses were applied to examine the robustness and statistical reliability of the topologies . Markov chain Monte Carlo iterations for the BI analysis of 18S, plastid 16S and bacterial 16S rRNA genes were carried until the average standard deviations of split frequencies below 0.01, indicated convergence of the iterations.For ML analysis of the 18S rRNA gene, a likelihood score (-lnL  =  9116.6914) was obtained under the TrN+I+G model with the following parameters: assumed nucleotide frequencies A =  0.2400, C =  0.2192, G =  0.2799, and T =  0.2608; substitution-rate AC =  1, AG =  1.4310, AT =  1, CG =  1, CT =  4.0184, GT =  1; proportion of sites assumed to be invariable  =  0.5941; rates for variable sites assumed to follow a gamma distribution with shape parameter  =  0.5669, and number of rate categories  =  4, estimated by Modeltest 3.7. For the BI analysis of the 18S rRNA gene, GTR+I+G model was selected by the MrModeltest, and the Markov chain Monte Carlo iterations were carried out until 1.5 million generations.For the ML analysis of plastid 16S rRNA gene, a likelihood score (-lnL  =  4482.6802) was obtained under the GTR+I+G model with the following parameters: assumed nucleotide frequencies A =  0.2632, C =  0.1814, G =  0.3033, and T =  0.2520; substitution-rate AC =  0.96991, AG =  7.9722, AT =  2.4899, CG =  1.7909, CT =  11.6406, GT =  1.0000; proportion of sites assumed to be invariable  =  0.3964; rates for variable sites assumed to follow a gamma distribution with shape parameter  =  0.4353, and number of rate categories  =  4, estimated by Modeltest 3.7. For the BI analysis of the plastid 16S rRNA gene, GTR+I+G model was selected by the MrModeltest, and the Markov chain Monte Carlo iterations were carried out until 16.5 million generations.For the ML analysis of the bacterial 16S rRNA gene, a likelihood score (-lnL  =  6723.6357) was obtained under the TrN+I+G model with the following parameters: assumed nucleotide frequencies A =  0.2575, C =  0.2219, G =  0.3085, and T =  0.2120; substitution-rate AC =  1, AG =  3.0334, AT =  1, CG =  1, CT =  4.1247, GT =  1; proportion of sites assumed to be invariable  =  0.6197; rates for variable sites assumed to follow a gamma distribution with shape parameter  =  0.6451, and number of rate categories  =  4, estimated by Modeltest 3.7. For the BI analysis of the bacterial 16S rRNA gene, GTR+I+G model was selected by MrModeltest, and the Markov chain Monte Carlo iterations were carried out until 1.5 million generations. […]

Pipeline specifications

Software tools Clustal W, PAUP*, ModelTest-NG, MrModelTest
Databases DDBJ
Application Phylogenetics
Chemicals Nitrogen