N-acyl homoserine lactone derived tetramic acids impair

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Letter

N-acyl homoserine lactone derived tetramic acids impair photosynthesis in Phaeodactylum tricornutum Frederike Stock, Michail Syrpas, Shiri Graff van Creveld, Simon Backx, Lander Blommaert, Lachlan Dow, Willem Stock, Ewout Ruysbergh, Bernard Lepetit, Benjamin Bailleul, Koen Sabbe, Norbert De Kimpe, Anne Willems, Peter G. Kroth, Assaf Vardi, Wim Vyverman, and Sven Mangelinckx ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b01101 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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ACS Chemical Biology

N-acyl homoserine lactone derived tetramic acids impair photosynthesis in Phaeodactylum tricornutum Frederike Stock1, Michail Syrpas2, Shiri Graff van Creveld3, Simon Backx2, Lander Blommaert1,4, Lachlan Dow5, Willem Stock1, Ewout Ruysbergh2, Bernard Lepetit5, Benjamin Bailleul4, Koen Sabbe1, Norbert De Kimpe2, Anne Willems6, Peter G. Kroth5, Assaf Vardi3, Wim Vyverman1•, Sven Mangelinckx2•

1Protistology

and Aquatic Ecology, Department of Biology, Ghent University, Krijgslaan

281/S8, 9000 Ghent, Belgium 2SynBioC,

Department of Green Chemistry and Technology, Ghent University, Coupure Links

653, 9000 Ghent, Belgium 3Department

of Plant and Environmental Sciences, Faculty of Biochemistry, Weizmann

Institute of Science, 76100 Rehovot, Israel 4Institut

de Biologie Physico-Chimique (IBPC), UMR 7141, Centre National de la Recherche

Scientifique (CNRS), Sorbonne Université, 13 Rue Pierre et Marie Curie, F-75005 Paris, France 5Plant

Ecophysiology, Department of Biology, University of Konstanz, 68457 Konstanz,

Germany 6Laboratory

of Microbiology, Department of Biochemistry and Microbiology, Ghent

University, 9000 Ghent, Belgium • shared senior author

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Abstract Marine bacteria contribute substantially to nutrient cycling in the oceans and can engage in close interactions with microalgae. Many microalgae harbor characteristic satellite bacteria, many of which participate in N-acyl homoserine lactone (AHL) mediated quorum sensing. In the diffusion-controlled phycosphere, AHLs can reach high local concentrations, with some of them transforming into tetramic acids, compounds with a broad bioactivity. We tested a representative AHL, N-(3-oxododecanoyl) homoserine lactone, and its tetramic acid rearrangement product on the diatom Phaeodactylum tricornutum. While cell growth and photosynthetic efficiency of photosystem II were barely affected by the AHL, exposure to its tetramic acid rearrangement product had a negative effect on photosynthetic efficiency and led to growth inhibition and cell death in the long term, with a minimum inhibitory concentration between 20 and 50 . These results strengthen the view that AHLs may play an important role in shaping the outcome of microalgae - bacteria interactions.

Keywords: N-acyl homoserine lactones, quorum sensing, N-(3-oxododecanoyl) homoserine lactone, tetramic acid, photosynthesis


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Marine bacteria play a pivotal role in the nutrient cycling of the oceans.1 Many of them live in close association with microalgae, either in the phycosphere (the microscale mucus region that surrounds phytoplankton cells)2 or in phototrophic biofilms.3 Several studies showed that marine microalgae can harbor very specific bacterial communities, with the most common phyla being -Proteobacteria, Bacteroidetes and -Proteobacteria.2, 4-8 Among them the number of bacterial genera is very limited, with Roseobacter, Ruegeria, Sulfitobacter, Flavobacterium and Alteromonas representing the main groups.4-6 Interestingly, Proteobacteria are known to contain numerous species which engage in cell-cell signaling via N-acyl homoserine lactones (AHLs).9 AHLs are prominent quorum sensing (QS) compounds consisting of an -amino-butyrolactone moiety coupled via nitrogen to an acyl side chain which can reach a length of up to eighteen carbon atoms. This acyl side chain varies in its degree of saturation and in the oxidation state at the C3 position, which can be unsubstituted, hydroxylated or more oxidized as carbonyl group.10 A screening for AHL production among 67 Alphaproteobacteria, isolated from the water column, dinoflagellate and diatom cultures, picoplankton and Laminaria surfaces, revealed that more than half of the strains produced AHLs, predominantly mixtures of almost exclusively long chained AHLs (≥ eight carbon atoms).11 In bacteria attached to sinking organic carbon particles, N-(octanoyl) and N-(dodecanoyl) homoserine lactone as well as N-(3-oxohexanoyl) and N-(3-oxooctanoyl) homoserine lactone were structurally identified.12 In addition to their canonical role in intraspecific communication, AHLs have also been demonstrated to function as inter-kingdom signals between bacteria and eukaryotes.13,14 For instance, AHL molecules can be perceived by algae as a beneficial chemical cue resulting in chemotaxis by Ulva zoospores or as a threat, triggering the activation of sophisticated defense mechanisms.13, 15-17 Despite previous studies highlighting the relative ubiquity of AHL production in marine bacteria,11,

12

reports on in situ AHL concentrations in marine environments are rare.

Nevertheless, an accumulation of > 3000 M of N-(dodecanoyl) homoserine lactone was measured in a subtidal biofilm, suggesting that AHLs can accumulate to high local concentrations.18 However, AHLs also degrade rapidly, via lactonolysis, rendering the compound inactive (Figure 1, path A).19 Alternatively, 3-oxo-AHLs can also undergo rearrangement via a Claisen-like condensation reaction (Figure 1, path B), leading to the formation of tetramic acids (TAs).20 TAs possess a broad spectrum of bioactivity, including antimicrobial, antifungal and herbicidal properties.21 The TA derived from N-(3oxododecanoyl) homoserine lactone (OXO12), for instance, exhibits high antimicrobial activity 3 ACS Paragon Plus Environment


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against Gram-positive bacteria, thereby providing OXO12 producing bacteria such as Pseudomonas aeruginosa with an additional chemical weapon against competing microorganisms.20, 22 In this study, we envisioned that AHLs and TAs play a role in diatom-bacteria interactions and hypothesized that these compounds could negatively affect diatom physiology. OXO12 and its tetramic acid rearrangement product, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine2,4-dione (TA12) were synthesized as examples of a 3-oxo-AHL and its respective TA derivative. OXO12 was selected as its physicochemical properties and the kinetics of its decomposition into TA have been described earlier.20 Moreover, a preliminary screen with different 3-oxo-AHLs revealed OXO12 to exert a growth retarding effect on Phaeodactylum tricornutum when growth was followed for one week (SI 1). To this end, we investigated how the marine model diatom P. tricornutum responds during short and long-term exposure to OXO12 and TA12, via photo-physiology, growth and viability experiments. In addition, we explored the generality of our findings by testing its effect on the diatom Cylindrotheca closterium as well as on natural marine diatom communities. In fact, both OXO12 and TA12 inhibited diatom growth (Figure 2-A and B). When P. tricornutum was treated with TA12, cell growth was inhibited with concentrations as low as 20 M and was completely inhibited at 50 M (Figure 2-B). At 50 M TA12, SYTOX staining revealed that approximately 25% of the cell population was dead after six days. Starting from 80 M TA12, cell death increased to approximately 90% of the population and at 150 M TA12 cell lysis was observed (SI 2). Exposure of P. tricornutum to OXO12 also induced growth inhibition (Figure 2-A), however, at higher concentrations compared to TA12. Starting from 150 M, a significant reduction in cell abundance was observed and exposure to 300 M led to a complete growth arrest. Importantly, cell death did not exceed 5% of the population in any of the tested OXO12 concentrations. These findings prompted us to investigate the short-term physiological response of P. tricornutum upon treatment with OXO12 and TA12. To this end, chlorophyll fluorescence was measured by pulse amplitude modulated (PAM) fluorometry, a non-destructive way to characterize photosynthesis. A decrease in the quantum efficiency of photosystem II (PSII), F/Fm’, measured in cultures exposed to light, reflects an inhibition of photosynthetic electron transport.23 The inhibition of PSII quantum efficiency increased with rising TA12 concentrations (Figure 2-D), a trend which was not observed in the OXO12 treatment (Figure 2-C). At the highest TA12 concentration tested (300 M), PSII quantum efficiency was 4 ACS Paragon Plus Environment

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completely inhibited after approximately 1.5 hours, coinciding with a strong decrease in maximum fluorescence, Fm’ (not shown). This inhibition suggests that the photosynthetic electron transport of P. tricornutum was severely impaired. Below 50 M TA12, cells recovered within hours of exposure to TA12. In contrast, cells treated with up to 300 M OXO12 remained largely unaffected in the first hours (SI 3). The inhibition of F/Fm’ in the TA12 treatment pointed towards a blockage of the photosynthetic electron transport chain but did not indicate where it occurs. To gain insight into the location of the blockage, we measured chlorophyll a fluorescence induction kinetics of P. tricornutum cells exposed to TA12. As a comparison, P. tricornutum was treated with the PSII inhibitor DCMU, which is known to occupy the QB niche of PSII at low concentrations.24 After ten minutes of incubation, the DMSO control showed a typical O-J-I-P transient upon exposure to a saturating light pulse (Figure 3) whereas DCMU treatment changed the fluorescence transient in such a way that maximum fluorescence (Fm) was already reached at the J-step (2 ms) of the curve, reflecting that QA is entirely reduced.24 Similarly, we observed a concentration dependent increase in the curve at the J-step when P. tricornutum was treated with TA12, indicating a blockage between the primary electron acceptor site in PSII, QA, and the secondary acceptor QB. However, we did not reach Fm at the J-step, indicating that electron transport was not entirely blocked (Figure 3). Based on these results we claim that the effect of TA12 is similar to tenuazonic acid (SI 4), a herbicidal TA which inhibits electron transport from QA to QB in the unicellular green alga Chlamydomonas reinhardtii.24 In addition, we could show that TA12 also inhibits photosynthesis in a similar way in natural marine diatom communities (SI 5–7). This is revealed by the strong decrease of the quantum efficiency of PSII at all light irradiances (SI 5), which is translated into a decrease in relative electron transport rate through PSII (rETRPSII) (SI 6). In parallel, the fluorescence induction curve in TA12 conditions strongly resembles that of DCMU (SI 7A and B), confirming that TA12 inhibits the QA to QB transfer in natural diatom communities. The overall growth pattern of P. tricornutum treated with TA12 indicates that the minimum inhibitory concentration (MIC) of TA12 on P. tricornutum is between 20 and 50 M (Figure 2-B), which is comparable to the previously reported MIC of TA12 on Gram-positive bacteria (8–55 M).20 In comparison, the half-inhibitory concentration of the herbicidal TA tenuazonic acid on PSII in C. reinhardtii was found at 192.94 g/mL (0.98 mM) which is more than one order of magnitude higher.24 In addition to its strong inhibitory effect on photosynthesis, it is possible that TA12 has an additional mode of action as concentrations of 150 M and higher 5 ACS Paragon Plus Environment


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caused cell lysis. TA12 was shown to dissipate membrane potential and pH gradient in Grampositive bacteria22, a mode of action which is similar to the structurally related tetramic acid reutericyclin, which has been shown to act as an ionophore in Gram-positive bacteria, with MIC’s between 0.06 and 2.6 mg/L (0.17–7.44 M).25 It is conceivable that bacterial signal molecules, and thus their tetramic acid transformation products, can accumulate to high local concentrations in the diffusion-controlled phycosphere and biofilm matrix where diatoms and bacteria interact. We measured the rearrangement of 100 M OXO12 into TA12 in artificial sea water (average pH 8.4) via HPLC-MS analysis over the course of six days (SI 8). During this period the concentration of TA12 increased to a maximum of nearly 20 M during the first 24 hours. Consequently, extrapolation of this result suggests that the MIC for TA12 can be obtained from starting concentrations of OXO12 of ~250 M, ten-fold below the concentration measured in a subtidal biofilm.18 Moreover, we observed that the TA12 concentration decreased after 24 hours, which could be explained by the formation of untraceable water-soluble products from TA12. In addition, we detected the highest concentration of the lactonolysis product (not quantified) at day three. In conclusion, we show that the tetramic acid derived from OXO12 can negatively affect photosynthesis in P. tricornutum and natural diatom communities, and provide a model showing that TA12 is likely to bind at the QB quinone binding site in PSII (Figure 4). Moreover, we observed that the effect of TA becomes more potent with an increase in acyl chain length at the C3 position (TA14) and that different diatom species respond differently to TAs with shorter acyl chain length (TA6–TA10) (SI 9). While TA12 and TA14 led to a strong decrease in growth (probed with culture fluorescence) starting from 20 and 10 M, respectively, in P. tricornutum and Cylindrotheca closterium, TA6–TA10 hardly affected growth in C. closterium and decreased growth by only 20% in P. tricornutum (SI 9). Taken together, these data suggest that AHL-derived tetramic acids may play a significant role in bacteria – diatom interactions in the global oceans.


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METHODS Culture conditions Wildtype P. tricornutum (Pt1) Bohlin Strain 8.6 was obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP2561). C. closterium VD05 was obtained from the Belgian Coordinated Collections of Microorganisms (BCCM, http://bccm.belspo.be). Cells were cultured in artificial seawater (ASW) (34.5 g/L Tropic Marin, 0.08 g/L NaHCO3) supplemented with Guillard’s f/2 (Sigma-Aldrich) at 18°C. For Pulse Amplitude Modulated (PAM)-fluorometry experiments P. tricornutum was grown in 12:12 h light:dark cycles at 25 mol photons m-2 s-1. Due to different culturing facilities, P. tricornutum was grown under 16:8 h light:dark cycles and a light intensity of 80 mol photons m-2 s-1 produced by cool fluorescent tl-tubes for flow cytometry experiments and for measuring OJIP fluorescence transients. All experiments were performed with exponentially growing cultures (if not stated otherwise approximately 106 cells/mL). Pulse Amplitude Modulated (PAM) Fluorometry For the short-term response, compounds were added to the cell suspension in a 48-well plate (Greiner Cellstar, Sigma). The plate was placed under a PAM-fluorometer (MAXI Imaging PAM M-series, Walz Mess-und Regeltechnik). The light adapted equivalent of minimal fluorescence (F0’) and maximal fluorescence (Fm’) were measured over a time course of five hours under actinic light (15 mol photons m-2 s-1), provided by the MAXI Imaging PAM blue LED-panel.26 Every three minutes an 800 ms saturating pulse was applied. Sequential data of F’ and Fm’ were subsequently averaged over triplicates. The photosynthetic efficiency of PSII was calculated as F/Fm’, with F = Fm’– F’.23 Inhibition of photosynthetic efficiency in each sample was calculated according to Schreiber et al.27: inhibition of

ΔF/F'm

(

(%) = 1 –

)

∆F/F'm sample ∆F/F'm control

* 100%

For natural samples, chlorophyll fluorescence was measured with a Joliot-type spectrophotometer (JTS-10, Biologic). The excitation light was obtained with a white probing LED and a blue/green filter. Actinic light and saturating pulses (5000 mol photons m-2 s-1, 250 ms) were obtained with a 629 nm LED crown. A high-pass filter (Schott RG-695) in front of the photodiode detector was used to cut the actinic light. The relative electron transport rate through PSII (rETRPSII) versus intensity (I) curves were constructed by exposing the same 7 ACS Paragon Plus Environment


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sample to six different light intensities until steady state photosynthesis and calculated as (Fm’– F’)/Fm’ * I. For long-term growth experiments, cells were dark-adapted for 15 min prior to the fluorescence measurement. Minimum fluorescence, F0, served as a proxy for biomass and was measured every day for one week. For graph SI 9, F0 values measured at the last day of the experiment were used and a relative fluorescence was calculated as: rel. F0 (%) =

(

)

F0 sample F0 control

* 100%

Cell counts and cell death analysis by flow cytometry Flow cytometry analysis was done on either an Eclipse (Sony Biotechnology Inc.) or a LSRII (Becton Dickinson) flow cytometer, both equipped with 405 and 488 nm solid-state air- cooled lasers and with standard optic filter set-up. For each sample 10,000 events were acquired. For cell viability, and cell counts, samples were stained with a final concentration of 1 M Sytox Green (Invitrogen) and incubated in the dark for 30 min at 20 °C. Samples were then analyzed on an Eclipse Flow cytometer (ex: 488 nm and em: 500–550 nm). An unstained sample was used as a control to eliminate the background signal. Cells were identified by plotting the chlorophyll fluorescence (663–737 nm) versus green fluorescence (500–550 nm). OJIP fluorescence transients An axenic P. tricornutum culture was adjusted to approx. 2 x 106 cells/mL and supplemented with 40 M sodium bicarbonate. TA12 stocks were prepared in DMSO and added to the diatom culture in 1.5 mL cuvettes (Brand) such that the final DMSO volume was 0.5% (v/v). DCMU stock was prepared in ethanol and added to the culture at a final concentration of 1 M. After compound addition the treated cultures were incubated in the dark for ten minutes. Finally, the OJIP fluorescence transients of the cultures were measured with an Aqua Pen (AP-C 100, Photon Systems Instruments). OJIP fluorescence transients were normalized according to Strasser et al.28: Vt = (Ft– F0)/(Fm– F0) Vt = relative variable fluorescence; Ft = fluorescence at time t; F0 = minimum fluorescence at 50 s; Fm = maximum fluorescence. Statistical analysis 8 ACS Paragon Plus Environment

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Flow cytometry data were analyzed using One-way ANOVA and Dunett’s multiple comparisons test. In this analysis, all treatments were compared to their corresponding DMSO control. Statistically significant differences are indicated with asterisks: * = p ≤ 0.05. All statistical analyses were performed with GraphPad Prism Software (GraphPad Prism Software version 6.01 for Windows, www.graphpad.com). For more details on the methods used in this study and compound spectra, see supporting information.

ACKNOWLEDGEMENT F. Stock would like to thank E. Bonneure for providing technical advice on the organic synthesis of OXO12 and TA12. L. Blommaert and B. Bailleul thank L. Guillou, A. Peltekis, S. Ferté, G. Maron and L. Crand for their help regarding experiments on diatom communities. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 642575, the “Research Foundation Flanders (FWO)” (G:0374.11N) and Ghent University (BOF-GOA 01G01911). L. Blommaert and B. Bailleul also acknowledge the support by the European Research Council (ERC) PhotoPHYTOMIX project (grant agreement No. 715579). B. Lepetit thanks the DFG (grant no. LE 3358/3-1) and the Elite program of the Baden-Württemberg foundation.

Supporting Information Available: This material is available free of charge via the internet at http://pubs.acs.org. Supporting information includes method descriptions of phytoplankton cultivation and organic synthesis of OXO12 and TA12 as well as HPLC and NMR spectra of OXO12 and TA12. Moreover, supporting figures include growth data on P. tricornutum treated with different homologues of OXO12, light microscopy pictures of P. tricornutum after TA12 treatment, raw Fm/F’ data on which Figures 2-C and D are based, structure of tenuazonic acid, Fm/F’ data of natural phytoplankton communities treated with TA12, relative electron transport rate of natural phytoplankton communities treated with TA12, fluorescence induction curves of natural phytoplankton communities treated with TA12, HPLC measurements of OXO12 rearrangement into TA12 and fluorescence measurements of P. tricornutum and C. closterium after six days of exposure to different TA homologues. 9 ACS Paragon Plus Environment


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[18] Huang, Y. L., Dobretsov, S., Ki, J. S., Yang, L. H., and Qian, P. Y. (2007) Presence of acyl-homoserine lactone in subtidal biofilm and the implication in larval behavioral response in the polychaete Hydroides elegans, Microb. Ecol. 54, 384-392. [19] Yates, E. A., Philipp, B., Buckley, C., Atkinson, S., Chhabra, S. R., Sockett, R. E., Goldner, M., Dessaux, Y., Camara, M., Smith, H., and Williams, P. (2002) N-acylhomoserine Lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa, Infect. Immun. 70, 5635-5646. [20] Kaufmann, G. F., Sartorio, R., Lee, S. H., Rogers, C. J., Meijler, M. M., Moss, J. A., Clapham, B., Brogan, A. P., Dickerson, T. J., and Janda, K. D. (2005) Revisiting quorum sensing: Discovery of additional chemical and biological functions for 3-oxo-Nacylhomoserine lactones, Proc. Natl. Acad. Sci. U. S. A. 102, 309-314. [21] Petermichl, M., and Schobert, R. (2017) 3-Acyltetramic Acids: A Decades-Long Approach to a Fascinating Natural Product Family, Synlett 28, 654-663. [22] Lowery, C. A., Park, J., Gloeckner, C., Meijler, M. M., Mueller, R. S., Boshoff, H. I., Ulrich, R. L., Barry, C. E., Bartlett, D. H., Kravchenko, V. V., Kaufmann, G. F., and Janda, K. D. (2009) Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals, J. Am. Chem. Soc. 131, 14473-14479. [23] Genty, B., Briantais, J.-M., and Baker, N. R. (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence, Biochim. Biophys. Acta, Gen. Subj. 990, 87-92. [24] Chen, S., Xu, X., Dai, X., Yang, C., and Qiang, S. (2007) Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii, Biochim. Biophys. Acta, Bioenerg. 1767, 306-318. [25] Gänzle, M. G. (2004) Reutericyclin: biological activity, mode of action, and potential applications, Appl. Microbiol. Biotechnol. 64, 326-332. [26] Consalvey, M., Perkins, R. G., Paterson, D. M., and Underwood, G. J. C. (2005) Pam fluorescence: A beginners guide for benthic diatomists, Diatom Research 20, 1-22. [27] Schreiber, U., Quayle, P., Schmidt, S., Escher, B. I., and Mueller, J. F. (2007) Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence imaging, Biosens. Bioelectron. 22, 2554-2563. [28] Strasser, R., Srivastava, A., and Tsimilli-Michael, M. (2000) The fluorescence transient as a tool to characterize and screen photosynthetic samples, In Probing Photosynthesis: Mechanism, Regulation and Adaptation (Yunus, M., Pathre, U., and Mohanty, P., Eds.), pp 445-483, CRC Press.



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FIGURE LEGENDS Figure 1. Reaction of N-(3-oxododecanoyl) homoserine lactone (OXO12) into its lactone hydrolysis product (Path A) and its tetramic acid transformation product TA12 (Path B) (adapted from Kaufmann et al.).20 Figure 2. Long- and short-term response of P. tricornutum to treatment with OXO12 and TA12. A) and B) Cell density (black bars, left y-axis) and percentage of dead cells (grey bars, right yaxis) of P. tricornutum at day six after compound addition of A) OXO12 and B) TA12. C) and D) inhibition of PSII of P. tricornutum treated with C) OXO12 and D) TA12. Measurement was started within five minutes after compounds were added to the culture and monitored over five hours. Values represent averages of three replicates. Figure 3. OJIP fluorescence transients of P. tricornutum treated with increasing TA12 concentrations and DCMU as positive control. The J-step of the curve at 2 ms is indicated with a dotted line. Vt was calculated as described in Methods. Figure 4. Schematic overview of the electron transport chain and the proposed site of blockage of TA12 at the electron acceptor site of PSII (red line). PQ = plastoquinone; PQH2 = plastoquinol; Cyt b6f = Cytochrome b6f complex; Fd = ferredoxin; FNR = ferredoxin-NADP+ oxidoreductase. Black arrows indicate electron transport and blue dotted arrows highlight proton flow.


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Graphical abstract

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Figure 1. Reaction of N-(3-oxododecanoyl) homoserine lactone (OXO12) into its lactone hydrolysis product (Path A) and its tetramic acid transformation product TA12 (Path B) (adapted from Kaufmann et al.).20 166x74mm (300 x 300 DPI)


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Figure 2. Long- and short-term response of P. tricornutum to treatment with OXO12 and TA12. A) and B) Cell density (black bars, left y-axis) and percentage of dead cells (grey bars, right y-axis) of P. tricornutum at day six after compound addition of A) OXO12 and B) TA12. C) and D) inhibition of PSII of P. tricornutum treated with C) OXO12 and D) TA12. Measurement was started within five minutes after compounds were added to the culture and monitored over five hours. Values represent averages of three replicates. 140x105mm (300 x 300 DPI)



Figure 3. OJIP fluorescence transients of P. tricornutum treated with increasing TA12 concentrations and DCMU as positive control. The J-step of the curve at 2 ms is indicated with a dotted line. Vt was calculated as described in Methods. 70x49mm (300 x 300 DPI)


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Figure 4. Schematic overview of the electron transport chain and the proposed site of blockage of TA12 at the electron acceptor site of PSII (red line). PQ = plastoquinone; PQH2 = plastoquinol; Cyt b6f = Cytochrome b6f complex; Fd = ferredoxin; FNR = ferredoxin-NADP+ oxidoreductase. Black arrows indicate electron transport and blue dotted arrows highlight proton flow.


N-acyl homoserine lactone derived tetramic acids impair

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