Supplemental Information. Acclimation of Oxygenic Photosynthesis. to Iron Starvation Is Controlled by the srna IsaR1

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1 Current Biology, Volume 27 Supplemental Information Acclimation of Oxygenic Photosynthesis to Iron Starvation Is Controlled by the srna IsaR1 Jens Georg, Gergana Kostova, Linda Vuorijoki, Verena Schön, Taro Kadowaki, Tuomas Huokko, Desirée Baumgartner, Maximilian Müller, Stephan Klähn, Yagut Allahverdiyeva, Yukako Hihara, Matthias E. Futschik, Eva-Mari Aro, and Wolfgang R. Hess

2 Figure S1. Conservation of IsaR1 homologs throughout the cyanobacterial phylum. Related to STAR Methods. (A) The locations of isar1 loci are shown alongside a phylogenetic tree based on 16S rrna sequences. The isar1 loci (blue triangles) are frequently associated with upp (orange triangles) and crth (green triangles) genes. The evolutionary distances are in the number of base substitutions per site. Evolutionary analyses were conducted using MEGA 6 [S1]. The organisms chosen for CopraRNA prediction are highlighted with a grey box, and those for which transcriptome data confirmed the presence of IsaR1 are labelled with an asterisk. (B) Multiple ClustalW sequence alignment of the 20 putative IsaR1 homologs from different cyanobacteria used for CopraRNA predictions. Experimentally verified transcripts are indicated by an asterisk. (C) IsaR1 transcript accumulation under different conditions (N-, nitrogen deficiency; Fe-, iron deprivation; cold; heat; HL, high light; exp., exponential growth; stat., stationary phase) with a probe against IsaR1.

3 Figure S2. Analysis of the IsaR1 promoter. Related to Figure 7. (A) Promoter kinetics of the isia and isar1 genes in response to iron depletion. Reporter strains carrying transcriptional fusions of the isia and isar1 upstream sequences with luxab genes were used to monitor in vivo promoter activity in Synechocystis 6803 by bioluminescence detection. The strains were grown in liquid cultures in the presence of 6 µm Fe 2+ to an optical density of 0.8. Iron depletion was then induced by adding 60 µm of the iron-specific chelator DFB at time point 0. Bioluminescence was measured as emitted light counts per second. A strain carrying promoterless luxab genes was used as negative control. Data are the mean ± SD of two replicate cultures. (B) Analysis of the binding of the recombinant FurA protein to the native P isar1 promoter (PisaR1) or the mutated version (PisaR1sub). The -35 element in P isar1 is shown in green letters and the changed nucleotides in orange letters. The red arrow indicates the mobility shifted Fur bound DNA fragment, whereas the black arrow indicates the unbound DNA fragment. (C) The IsaR1 upstream region including the first 2 transcribed nucleotides in Synechocystis 6803 is shown and compared to 31 homologs. The positions of putative -35 and -10 elements are indicated. The Fur binding sequence similar to the one defined for the isia gene in Synechocystis 6803 [S2] is shown on top of the sequence logo and its position indicated by the horizontal black lines underneath. Promoter prediction was done with the MEME webserver version [S3] and standard parameters.

4 Figure S3. Verification of IsaR1 overexpression. Related to the transcriptome analysis as shown in Figure 4. Northern hybridization was performed for triplicate samples to verify ectopic overexpression of IsaR1 (upper part). The gene isar1 was introduced into Synechocystis 6803 on the replicative vector pvz322 under control of the P pete promoter (IsaR1OE). The control strain (WT) was the WT into which plasmid pvz322 was introduced lacking the P pete-isar1 cassette (WT_pVZ). Expression of IsaR1 from this promoter was induced by the addition of 2 µm CuSO 4 in BG11 medium containing the normal iron concentration and samples were taken at the indicated time points. Below is the ethidium bromide stained gel before blotting.

5 Figure S4. Raw fluorescence data from the sgfp assays. Related to Figure 5, Figure 6 and to STAR Methods. Raw GFP Fluorescence of the respective translational 5 UTR-GFP fusion in presence of the control plasmid pjv300 or the srna (IsaR1). For each clone, the fluorescence of 50,000 events was collected by flow cytometry. The events were individually gated for each well to retain the events with a fluorescence lower than or equal to the mean of all fluorescence values plus four times the standard deviation. The mean of the gated events was averaged for 6 independent biological replicates, standard deviation was calculated from these replicates. (A) Confirmed targets with compensatory mutations. (B) Confirmed targets. (C) Negative targets. (D) Targets with a fluorescence at background or slightly above the control plasmid background levels, which made it impossible to conclude a regulatory function of IsaR1.

6 Figure S5. Fold repression data from the sgfp assays. Related to Figure 5 and Figure 6. The fold repression was calculated as the ratio of the mean sgfp fluorescence of the respective translational 5 UTR sgfp fusion in the presence of the control plasmid pjv300 and a plasmid for the overexpression of the respective srna, after the subtraction of the background fluorescence. The background fluorescence was measured with the control plasmids pxg-0 (with a luciferase gene instead of GFP) and pjv300, from which a short nonsense transcript is transcribed instead of a specific srna. The error of the fold repression was calculated considering error propagation under the assumption that the values could be correlated. (A-C) Fold-repression values corresponding to the raw fluorescence values from Figure S4A-C. (D) Fold-repression values from 5 UTRs shown in Figure S4D with a fold repression >1.5, but a large error due to fluorescence close to background. (E) Predicted interaction between IsaR1 and the sodb 5 UTR. The start codon is shown in blue and the compensatory point mutations are indicated in orange.

7 Figure S6. Western Blot of PsaB in IsaR1OE and WT_pVZ. Related to Figure S4 and Figure 7. The PsaB subunit of PSI is negatively affected by IsaR1 overexpression. Western blot with time series of induction of ectopic expression of IsaR1 by copper addition. As a control for the linear range of the detected signal, the samples from the 0 h time point were diluted to 50% of the total protein concentration. The standard deviations are based on 3 independent biological replicates. Quantification was performed separately for the wild type and the IsaR1OE strains and the 0 h intensities were set to 100%.

8 Figure S7. Predicted interaction sites for IsaR1 targets supported by the sgfp reporter gene assay (related to Figures 5, 6, S4 and S5). The secondary structure (dg = kcal mol -1 ) is predicted to consist of a major Rho-independent terminator encompassing most of the molecule and a 5 domain functioning as a single seed region. Nucleotide positions predicted by IntaRNA to be involved in the interactions are shown in red, note that these might extend into the following stem in case of sufb (Figure 6D) and petf (Figure 5D). Experimentally tested point mutations interrupting these interaction are indicated by red boxes. A weak hair pin motif in the 5 region is indicated by brackets. The mfold web server [S4] was used for the prediction.

9 Supplemental References S1. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, S2. Kunert, A., Vinnemeier, J., Erdmann, N., and Hagemann, M. (2003). Repression by Fur is not the main mechanism controlling the iron-inducible isiab operon in the cyanobacterium Synechocystis sp. PCC FEMS Microbiol. Lett. 227, S3. Bailey, T.L., and Elkan, C. (1994). Fitting a mixture model by expectation maximization to discover motifs in bipolymers. In Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology (AAAI Press, Menlo Park, California), pp Available at: [Accessed November 12, 2016]. S4. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, S5. Wright, P.R., Richter, A.S., Papenfort, K., Mann, M., Vogel, J., Hess, W.R., Backofen, R., and Georg, J. (2013). Comparative genomics boosts target prediction for bacterial small RNAs. Proc. Natl. Acad. Sci. U. S. A. 110, E S6. Wright, P.R., Georg, J., Mann, M., Sorescu, D.A., Richter, A.S., Lott, S., Kleinkauf, R., Hess, W.R., and Backofen, R. (2014). CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Res. 42, W S7. Kopf, M., Klähn, S., Scholz, I., Matthiessen, J.K.F., Hess, W.R., and Voß, B. (2014). Comparative analysis of the primary transcriptome of Synechocystis sp. PCC DNA Res. 21, S8. Hernández-Prieto, M.A., Schön, V., Georg, J., Barreira, L., Varela, J., Hess, W.R., and Futschik, M.E. (2012). Iron deprivation in Synechocystis: inference of pathways, non-coding RNAs, and regulatory elements from comprehensive expression profiling. G3 Bethesda Md 2,