# Singlet oxygen-induced alteration of bacteria associated with phytodetritus: effect of bacterial pigments

Gwénola Simon, Laurie Casalot, Corinne Valette, Christopher Burot, Jean-François Rontani, Patricia Bonin

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Gwénola Simon, Laurie Casalot, Corinne Valette, Christopher Burot, Jean-François Rontani, et al. Singlet oxygen-induced alteration of bacteria associated with phytodetritus: effect of bacterial pigments. 2023. hal-04286634

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# Singlet oxygen-induced alteration of bacteria associated with phytodetritus: effect of bacterial pigments

**Gwénola Simon**

Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO

**Laurie Casalot**

Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO

**Corinne Valette**

Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO

**Christopher Burot**

Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO

**Jean-François Rontani**

Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO

**Patricia Bonin**

(✉ patricia.bonin@mio.osupytheas.fr)

Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO

## Article

**Keywords:** Type II photosensitized oxidation, Singlet oxygen, Phytodetritus, Pigmented and unpigmented bacteria, Carotenoids, Vaccenic acid, DNA repair system

**Posted Date:** September 12th, 2023

**DOI:** https://doi.org/10.21203/rs.3.rs-3294986/v1

**License:** © 🌐 This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** No competing interests reported.
Singlet oxygen-induced alteration of bacteria
associated with phytodetritus: effect of
bacterial pigments.

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Gwénola Simon, Laurie Casalot, Corinne Valette, Christopher Burot, Jean-

François Rontani, Patricia Bonin*

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Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO, Marseille, France.

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<footnote>* Corresponding author: patricia.bonin@mio.osupytheas.fr</footnote>
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**Abstract.**

We sought to determine whether the presence of carotenoids actively protects bacteria associated with senescent phytoplankton cells from damage caused by singlet oxygen (¹O₂) resulting from the cessation of photosynthetic reactions. We irradiated senescent cells of the diatom *Thalassiosira sp.* associated with the bacterial strains *Pseudomonas stutzeri* (a ubiquitous planktonic unpigmented heterotrophic bacterium) or *Dinoroseobacter shibae* (an aerobic anoxygenic bacterium often associated with phytoplankton rich in the spheroidenone, carotenoid). The analysis focused on the photooxidation of bacterial membrane lipids (vaccenic acid) and the expression of genes involved in DNA repair systems. In *P. stutzeri* cells, ¹O₂ transferred from senescent algae was not completely scavenged by bacterial membrane MUFAs and reached the cytoplasm, allowing both ¹O₂ and UV radiation to cause DNA damage. In *D. shibae*, scavenging by bacterial membrane MUFAs quenching by spheroidenone allowed only a small fraction of ¹O₂ to reach the cytoplasm, where the efficient detoxifying activity of mutY limited their impact on the DNA of this strain, thus preventing oxidative stress. The fact that Rhodobacteriales is the dominant order in bacterioplankton communities associated with algal blooms could thus be due to the protective effect of its constituent carotenoids against ¹O₂- and UV-induced damage.

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**Keywords.** Type II photosensitized oxidation; Singlet oxygen; Phytodetritus; Pigmented and unpigmented bacteria; Carotenoids; Vaccenic acid; DNA repair system.

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Introduction

Photosynthetic microorganisms (phytoplankton) such as diatoms, dinoflagellates and coccolithophores are the main primary producers in marine ecosystems, capable of producing organic matter using light energy and inorganic nutrients. This organic matter is heavily processed by bacteria, which mineralize approximately 50% of oceanic primary production (PP) ¹,². The other 50% of PP either enters the marine food web or gets buried into seafloor sediments through a process called the ‘biological pump’ ³. Bacterial activity in the oceans (secondary production) is therefore highly dependent on the abundance of primary producers ¹, and varies with phytoplankton blooms at large or small scales ⁴.

Phytoplankton-bacteria interactions are highly intertwined, and changes in composition of phytoplankton blooms (in terms of abundance and species assemblages) lead to changes in bacterial-community composition ⁵⁻⁷. As this microbial secondary production is a key process to better understand the functioning of marine ecosystems, there has been intensive research into the phylogenetic composition of bacterioplankton communities (i.e. free-living planktonic bacteria in marine ecosystems).

Singlet oxygen (¹O₂), which is produced by type II photosensitized oxidation processes in senescent phototrophic organisms ⁸, is strongly electrophilic and lacks spin restriction ⁹ and therefore readily reacts with phytoplanktonic unsaturated lipids ¹⁰,¹¹, proteins ¹², and nucleic acids ¹³,¹⁴. ¹O₂ can also react with the octadec-11-enoic (vaccenic) acid of the bacteria attached to this algal material, affording isomeric *trans* allylic hydroperoxyacids ¹⁵,¹⁶. Recently, it was demonstrated that high solar irradiances strongly favor this transfer of ¹O₂ to bacteria ¹⁷. The effect of exogenous pure ¹O₂ on Gram-negative and Gram-positive bacteria has already been investigated ¹⁸,¹⁹, but the transfer of ¹O₂ from senescent phytoplankton to their attached bacteria has been only demonstrated by Rontani et al. in 2003 ¹⁵. Further studies demonstrated: (i) the good correlation between the photodegradation state of bacteria attached to phytodetritus and
the photooxidation state of phytoplanktonic chlorophyll ¹⁶, and (ii) inhibition of ¹O₂ transfer by the silica matrix of diatom frustules ²⁰. This transfer of ¹O₂ could strongly decrease the mineralization potential of bacteria attached and thus favor the preservation of phytoplanctonic material. The bacterial resistance to its deleterious effect could be a determining factor in the selection of microorganisms capable of interacting with senescent phytoplankton at the end of the bloom.

It is still hard to generalize bacterioplankton-community structures, but some important bacterial groups, such as *Bacteroidetes* and *Proteobacteria*, are typically abundant during bloom events, regardless of phytoplankton species ⁶,²¹,²². Amongst them, *Roseobacter*, a genus that falls within the *Alphaproteobacteria* class, are often dominant in marine phytoplankton-associated bacterial communities ²³⁻²⁷. Many studies investigating this compositional balance have given explanations for why strains belonging to the *Roseobacter* genus are so efficient at colonizing phytoplankton, including their high colonization capabilities ²⁸, their production of quorum-sensing molecules ²⁹, and their antimicrobial compounds ³⁰. Another physiological characteristic of the *Roseobacter* genus that could explain their high abundance in bacterioplankton assemblages is the particular aerobic anoxygenic phototrophic (AAP) metabolism that occurs among this taxon ³¹. AAP bacteria are capable of photoheterotrophy, a metabolic pathway that enables them to produce energy from light, using photosynthetic reaction centers composed of bacteriochlorophyll-a (BChl *a*; ³². AAP bacteria are also known to contain high amounts of pigments such as carotenoids ³³, which may protect these organisms against oxidative damage such as type II photosensitized oxidation, a process that can happen during late bloom phases ³⁴.

It has already been hypothesized that bacteria with high amounts of pigments such as carotenoids may be able to tolerate exposure to ¹O₂ ¹⁹,³⁵. This resistance to oxidative stress
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could explain why the *Roseobacter* genus is highly represented among bacterioplankton
communities during late bloom phases when the phytoplankton cells are senescent.

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Here, we compared the effect of ¹O₂ produced during the senescence of a diatom (*Thalassiosira* sp.) on the lipids and DNA of two Gram-negative bacteria widely found in marine environments, one pelagic ubiquitous unpigmented bacterium, *Pseudomonas stutzeri* ³⁶ and one pigmented bacterium, *Dinoroseobacter shibae* ³⁷ frequently associated with phytodetritus. For the first time, the impact of ¹O₂ produced during algal senescence on the physiology of associated bacteria was studied at the membrane-lipid and DNA levels.

## Material and methods

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**Phytodetritus production** : The non-axenic diatom *Thalassiosira* sp. RCC1714 was obtained from the Roscoff Marine station culture collection. Diatoms were grown in F/2 medium at 17°C in an environment-controlled cabinet under an irradiance of 83 µmol photon.m⁻².s⁻¹ (Osram, Lumilux, 12 h:12 h light/dark cycles). After two months of growth, cell density was measured by counting under microscope. *Thalassiosira* sp. Cell death was induced by sonication on ice using a Branson Sonifier 450 ultrasound generator equipped with a Branson S450A cell disruptor probe at 240 W for 10 minutes (50% duty cycle). The sonicated cells were then aliquoted in 50-mL Falcon tubes and stored at -20 °C for later photooxidation experiments.

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**Bacterial cultures** : *P. stutzeri* ATCC 14405 and *D. shibae* DSM 16493 were grown aerobically overnight on a rotary shaker in the dark at 17°C on marine broth medium (US Biological™) diluted 1:10 with sterilized artificial sea water ³⁸. These pre-cultures were used to inoculate the same medium to obtain under the same conditions an initial OD₆₂₀nm of about 0.020. In the middle of the exponential growth phase, the cultures were stopped and cell
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abundances were counted based on epifluorescence in the presence of fluorochrome (4P,6-diamidine-2P-phenylindole dihydrochloride; DAPI) as previously described in Otto ³⁹.

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**Photooxidation experiments:** Based on the recent results of Burot et al. ¹⁷ showing enhanced $¹O_2$ transfer to bacteria at high irradiances, experiments were carried out in 100-mL sterilized quartz tubes using an Altas Suntest CPS + solar simulator with a Xenon NXE 1500 lamp producing a solar irradiance (2285 µmol photon.m⁻².s⁻¹) in the 300-800 nm range close to the levels generally observed at noon at the surface of Mediterranean Sea ⁴⁰. To limit irradiation to visible range, we also simultaneously carried out experiments in 100-mL glass (opaque to UV) tubes with irradiance reduced to 2038 µmol photon.m⁻².s⁻¹ in this case.

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Phytodetritus and bacteria were mixed (at a 1:5 algal:bacterial cells ratio) in K medium⁴¹ (K 25 × diluted with sterilized artificial sea water) at a final volume of 100 mL, and immediately exposed to light in the solar simulator. Tubes were held at 17°C by immersion in a water bath connected to a cryothermostat. Four sampling times were defined: 0.5, 2, 3.5 and 5 h. At each sampling time, 15 mL and 10 mL samples were collected in triplicate for q-RT PCR and lipids analysis, respectively. The 15-mL samples were filtered on 0.8-µm polycarbonate filters (to collect only attached bacteria on particles and not the free-living organisms) and stored at -80°C until further RNA extraction. The 10-mL samples were filtered on 0.8-µm glass fiber filters (GF/F) and stored at -20°C until further lipid extraction. Each experiment was conducted in four separate tubes prepared simultaneously in the same conditions. A single tube was sacrificed at each sampling timepoint.

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Control experiments were conducted without phytodetritus. Bacteria were mixed with sterilized artificial sea water added with 0.1 g. L⁻¹ of lactate/acetate (50:50) as carbon source, then placed in 100 mL sterilized quartz tubes and immediately exposed to light in the solar simulator. Three sampling times for the controls were defined: 0, 2 and 5 h. At each sampling
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time, a 15 mL sample was filtered on 0.2-µm polycarbonate filters (to recover bacteria that are all free-living in this case) and stored at -80°C until further RNA extraction. For the lipid analysis, the samples were centrifuged with a Beckman & Coulter Allegra X-22-series centrifuge at 20,000 g for 30 min, then pelleted and stored at -20°C. Here again, the three samplings were conducted in separate control tubes prepared in the same conditions.

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## Lipid analyses

**Lipid extraction:** Samples (GF/F filters) were reduced with excess NaBH₄ after adding MeOH (25 mL, 30 min) to reduce labile hydroperoxides (resulting from photo- or autoxidation) to alcohols, which are more amenable to analysis using gas chromatography-mass spectrometry (GC-MS) as described elsewhere ⁴². Briefly, Water (25 mL) and KOH (2.8 g) were then added, and the resulting mixture was saponified by refluxing (2 h), then cooled, acidified with HCl (2 N) to pH 1, and extracted with dichloromethane (DCM; 3 × 20 mL).

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**Derivatization:** Dry TLEs were silylated in a mixture of pyridine/bis-(trimethylsilyl) trifluoroacetamide (BSTFA; Supelco; 2:1, v/v) as previously described ⁴² and analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS).

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**Gas chromatography-tandem mass spectrometry (GC-MS/MS):** GC-MS and GC-MS/MS analyses were performed on an Agilent 7890A/7010A tandem-quadrupole gas chromatograph system (Agilent Technologies, Les Ulis, France) using a cross-linked 5% phenyl-methylpolysiloxane (Agilent; HP-5MS ultra inert, 30 m × 0.25 mm, 0.25-µm film thickness) capillary column. The operating conditions used have been described previously by Rontani et al. ⁴². Vaccenic acid photooxidation products were quantified in multiple-reaction-monitoring (MRM) mode using the transitions *m/z* 339 → 131 for the parent vaccenic acid and *m/z* 199 →

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129, *m/z* 213 → 129, *m/z* 357 → 129 and *m/z* 371 → 129 for its oxidation products ¹¹. Precursor ions were selected from the most intense ions (and specific fragmentations) observed in electron ionization (EI) mass spectra. Collision-induced dissociation (CID) was optimized by using collision energies at 5, 10, 15 and 20 eV. Quantification involved peak integration and determination of individual response factors using standards and Mass Hunter software (Agilent Technologies, Les Ulis, France).

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*Standard compounds:* Vaccenic acid was obtained from Sigma-Aldrich and oxidized using Fe²⁺/ascorbate ⁴³. Subsequent reduction of the resulting hydroperoxides in methanol with excess NaBH₄ afforded the corresponding hydroxyacids.

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**Quantitative RT-PCR** : RNA extractions were performed with the High Pure RNA Isolation Kit (Roche Applied Science, United States) as per the supplier's instructions. After extraction, any potential DNA contaminations were removed using the TURBO DNA-free™ kit (Ambion® by Life Technologies™) as per the manufacturer's instructions. RNA quantification and 260/230 and 260/280 ratio measurement were performed by spectrophotometry (NanoDrop 2000c, Thermo Scientific). Absence of DNA contamination was verified on RNA samples for each targeted gene (as described below) by PCR without the reverse transcription step. Reverse transcription (RT) reactions were carried out using random primers with the PrimeScript™ RT reagent Kit (TaKaRa) as per the supplier's instructions.

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To plot the standard curves, fragments of the targeted genes were amplified and cloned into pGEMT-easy vectors (Promega) following the supplier's instructions. The primers used to amplify and clone the targeted genes for *P. stutzeri* and *D. shibae* are reported in Table S1. The PCR reactions (20 µL) contained 50 ng of genomic DNA from *P. stutzeri* or *D. shibae*, 0.5 µM of each primer, and 10 µL of GoTaq® G2 Hot Start Master Mix (Promega). The PCR program

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consisted in an initial denaturation step of 5 min at 98°C to activate the polymerase, then cycling (30 repetitions) with a 30 s denaturation step at 98°C, a 30 s hybridization step at the appropriate temperature for the selected primer pair, and an elongation phase of 20 s at 72°C. A final elongation phase at 72°C for 2 min was added for easy cloning in a T vector. The cloned DNA fragments were checked by sequencing. After purification, the plasmid concentrations were determined by spectrophotometry and used to produce the standard curves for qPCR allowing quantification of the synthetized cDNAs.

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The various target cDNAs were quantified by qPCR (qPCR Master Mix, Promega) as per the supplier's instructions. Each qPCR run performed used 1 µL of the obtained cDNA and 0.5 µM of each primer in a final 20 µL reaction volume. The qPCR program consisted in an initial denaturation step of 2 min at 98°C, then cycling (40 repetitions) with a 20 s denaturation step at 98°C, a 20 s hybridization step at the appropriate temperature for the selected primer pair, and an elongation phase of 20 s at 72°C. Experiments were done using the CFX Opus Real-Time PCR system (Bio-Rad) and the associated software. The starting quantity was determined using Bio-Rad CFX manager 2.1 software and transformed into copies per µL of cDNA. The quantified expression level of the housekeeping gene (rpoD) encoding for a σ<sup>70</sup> subunit of the RNA polymerase was used to normalize gene expression. Each extracted RNA was analyzed in 6 technical replicates (two independent reverse transcriptions and three qPCR analyses per cDNA).

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**Data Availability** : The sequences generated during the current study are available in the Genbank repository, the accession numbers are : #2741504, #2741532, #2741540, #2741543, #2741547, #2741570, #2741574, #2741584

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**Ethical approval.** This article does not contain any studies with human participants performed by any of the authors.

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**Results and discussion**

The aim of this work was to determine whether bacterial pigmentation plays a protective role against the ¹O₂ damage when the bacteria are attached to lightened phytodetritus. To do so, we irradiated senescent cells of the diatom *Thalassiosira* sp. associated with the bacterial strains *P. stutzeri* (a ubiquitous heterotrophic bacterium without carotenoids) and *D. shibae* (an AAP containing a high proportion of the carotenoid spheroidenone). Experiments were carried out under two radiative fluxes (1) at high solar irradiance (PAR, Photosynthetically active radiation, + UV) close to that generally observed at noon at the surface of the Mediterranean Sea ⁴⁰ and (2) after limiting irradiation to visible range (PAR only) to determine the effect of ¹O₂ only. Analysis focused on the effects of ¹O₂ on photooxidation of the major MUFA of these bacteria (vaccenic acid) and on activation of their DNA repair systems.

**Type II photosensitized oxidation of bacterial vaccenic acid:** Oxidative damage in attached cells of *P. stutzeri* and *D. shibae* was monitored using the concentration of photooxidation products of vaccenic acid (pmol.L⁻¹). Indeed, vaccenic acid is found in high proportions in *P. stutzeri* and *D. shibae* ³⁷,⁴⁴ but is only present in trace amounts in the bacterial community associated with the non-axenic strain of *Thalassiosira* sp. Employed ¹⁷. Type-II photosensitized oxidation of vaccenic acid involves the addition of ¹O₂ to the two carbon atoms of its Δ¹¹ double bond to afford 11-hydroperoxyoctadec-12(*trans*)-enoic and 12-hydroperoxyoctadec-10(*trans*)-enoic acids ¹¹. These hydroperoxides can be easily converted to 13-hydroperoxyoctadec-11(*trans*)-enoic and 10-hydroperoxyoctadec-11(*trans*)-enoic acids, respectively, via stereoselective radical allylic rearrangement ⁴⁵. We used stable allylic hydroxyacids arising from NaBH₄ reduction of these hydroperoxyacids as tracers of photooxidation of bacterial membranes.
For both bacterial strains, only trace amounts of vaccenic acid photooxidation products were detected before irradiation and in the different algae-free controls (Fig. 1). As previously observed ¹⁵⁻¹⁷, oxidation of vaccenic acid strongly increased when irradiation was carried out in the presence of senescent phytoplankton cells (Fig. 1). The production of vaccenic acid oxidation products appeared to be maximal at 803 KJ.m⁻², which corresponds to 30 min of exposure only.

In the case of *P. stutzeri*, the concentration of vaccenic acid oxidation products was significantly higher in the presence of solar light (PAR+UV) radiation compared with non-UV levels (Figs. 1A and 1B, t-test, n=18, p=0.0005< 0.05). The sensitizer (chlorophyll of senescent *Thalassiosira* sp. cells), which catalyzes type-II photosensitized oxidation of bacterial vaccenic acid, absorbs in both the PAR ⁸ and UV ⁴⁶ ranges. Consequently, an additional amount of ¹O₂ is produced during PAR+UV irradiation, thus inducing higher oxidation of vaccenic acid in membranes of *P. stutzeri*. In contrast, there was no significant difference in concentrations of vaccenic acid photooxidation products in *D. shibae* cells after irradiation by solar (PAR+UV) or PAR-only irradiance (Figs. 1C and 1D, t-test, n=18, p=0.203>0.05). This observation clearly shows that the additional amount of ¹O₂ produced by UV radiations is quenched by the carotenoid spheroidenone in the membranes of *D. shibae*. Although this carotenoid cannot totally prevent type-II photosensitized oxidation of MUFAs in *D. shibae* membranes (Fig. 1C and 1D), it seems to limit the damage caused by overproduction of ¹O₂ in the presence of UV.

Carotenoids containing two polar hydroxy groups (e.g. zeaxanthin), which lie across the monolayer membrane ⁴⁷, can efficiently protect against ¹O₂-mediated bacterial killing ⁴⁸. In contrast, carotenoids such as spheroidenone that lack hydroxy groups can become completely embedded in the lipid layer of the membrane ⁴⁹. This embedment makes them less likely to react with ¹O₂, which can therefore go on to rapidly damage unsaturated molecules in its vicinity. This can explain why spheroidenone cannot totally prevent vaccenic acid oxidation in
membranes of *D. shibae*. Note that spheroidenone may play a key role in photoprotection of *D. shibae* against UV radiation-induced damage: as spheroidenone absorbs strongly between 300 and 400 nm<sup>50</sup>, it can absorb harmful UV radiation.

**Effect of ¹O₂ on the DNA repair system genes:** The other aim of this work was to monitor the response to ¹O₂ of the DNA repair system in pigmented and non-pigmented bacteria. ¹O₂ is known to react with a wide range of cellular compounds, such as lipids, proteins, and DNA<sup>12–14</sup>.

Bacteria respond to adverse conditions by activating regulation pathways that increase their chances of survival. One such pathway—the aptly named SOS response—is induced by genome damage or disruption of DNA replication, and results in the synthesis of DNA repair enzymes and the inhibition of bacterial cell division. Bacterial cells use different strategies to protect themselves against the toxicity of ¹O₂. These strategies usually involve ‘preventive’ detoxification enzymes, such as superoxide dismutase (SOD) or catalase (CAT) <sup>51,52</sup>, an efficient “Go system” with three detoxification proteins, i.e. MutM, MutT and MutY <sup>53</sup> assigned to DNA repair, or the quenching of ¹O₂ with antioxidant compounds such as carotenoid pigments <sup>54</sup>.

Amongst the nucleotides, ¹O₂ preferentially reacts with guanine (Fig. 2) as it has the lowest oxidation potential<sup>55,56</sup>. This leads to the formation of d-8-oxoGTP<sup>57</sup>. This modified nucleotide has high mutagenic potential, as it can mispair with adenine, leading to G → T transversion<sup>58</sup>, which is the main mutation observed. To monitor activation of the DNA repair system in response to ¹O₂ exposure, we measured the relative expression of several genes related to DNA repair after ¹O₂-related damage as well as several stress-response transcription factors. In the genomes of both strains, three genes code for the detoxifying proteins of the “Go system”: *mutM* (or *fpG*), *mutY* and *mutT*. As shown in Fig. 2, MutM and MutY proteins are
glycosylases. MutM removes cytosine-paired d-8-oxoG while MutY removes adenine mispaired with d-8-oxoG <sup>59-61</sup>. MutT is a nucleotide triphosphatase that detoxifies the nucleotide pool, hydrolyzing d-8-oxoGTP into d-8-oxoGMP <sup>62</sup>.

<sup>1</sup>O<sub>2</sub> is also involved in regulation of the expression of genes associated with the bacterial SOS response to oxidative stress. Knowledge of the <sup>1</sup>O<sub>2</sub>-specific regulatory mechanisms in microorganisms is based on only a few model systems, including the anoxygenic bacterium *Rhodobacter sphaeroides* and several heterotrophic species such as *Escherichia coli*. In *R. sphaeroides*, stress response involves gene regulation by various alternative sigma factors RpoE and RpoH <sup>52,63,64</sup>. The sigma factor RpoE is maintained in an inactive state by forming a complex with its cognate anti-sigma factor ChrR <sup>52</sup>. Recent studies have shown that under exposure to <sup>1</sup>O<sub>2</sub>, RpoH becomes activated due to the rapid proteolysis of the RpoE:ChrR complex. RpoH (σ32) is the primary sigma factor controlling the heat shock response, but it is also known to be induced by other conditions (for a review, see <sup>65</sup>).

Amongst all the genes involved in the potential response to <sup>1</sup>O<sub>2</sub> exposure, we chose to focus on one representative of each type of response: *mutY*, *rpoH* (involved in stress response), and *chrR* (involved in <sup>1</sup>O<sub>2</sub>-induced response and *rpoH* regulation). Fig. 3 shows the relative expression of these genes in *P. stutzeri* and *D. shibae* after exposure to artificial (PAR) and solar (PAR + UV) light in the presence of senescent *Thalassiosira* cells. Note, however, that 8-oxoDG is not specific to 1O2-induced alteration of DNA, but is also produced during the oxidation of DNA by other reactive oxygen species (ROS) (e.g., peroxyl or hydroxyl radicals arising from hydroperoxide homolysis or H2O2). Consequently, the alteration of DNA observed in bacterial cytoplasm may also result from the action of these ROS (notably by H2O2, which is known to readily cross cell membranes <sup>66,67</sup>).

In *P. stutzeri*, *mutY* and *rpoH* were expressed from the beginning of exposure and reached peak expression (803 KJ.m<sup>-2</sup>) from 30 minutes (the experimental conditions did not allow
samples to be taken earlier) (Fig. 3A). In addition to being very fast, the response did not seem to be influenced by algae nor by UV light (Table S2), and so the slightest trace of singlet oxygen in an unpigmented organism leads to the expression of *mutY* and *rpoH*. The lower expression at 5 h under PAR + UV exposure (8028 kJ.m⁻²) could be explained by cells entering the stationary phase of growth, which arrives earlier than during exposure to PAR. *chrR* showed no difference in expression level under UV exposure but showed a very strong decrease in expression under PAR conditions.

In *D. shibae*, the presence of UV induced a significant increase of *mutY* and *chrR* expressions which is only observed in the presence of senescent *Thalassiosira* cells (Fig. 3B, Table S2). In the same way, *rpoH* expression showed completely different patterns between solar and PAR conditions but was not influenced by the presence of the algae.

These observations suggest that ¹O₂ arising from the algae has more difficulty penetrating the pigmented cells (comparison between pigmented and unpigmented cells in response to PAR exposure; Fig. 3, dotted bars). When UV is absent, only ¹O₂-related oxidation remains. In this case, carotenoids are efficient enough to fight against this oxidant, therefore not allowing the ¹O₂ to enter the cell, and all three genes are expressed at a lower level. When algae and UV are present, leading to the production of more ¹O₂, there is not enough carotenoid in the pigmented cell to fight against the increased oxidative stress, and so *mutY* and *chrR* show higher expression. However, relative expression levels increased more with the unpigmented than the pigmented bacteria, showing some kind of protection due to the presence of spheroidenone. This protection is evidenced by a lower expression of *rpoH* observed in *D. shibae* (Fig. 3B) indicative of the lack of oxidative stress in this pigmented attached bacterium.

## Conclusion
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In this work, we studied the impact of type II photooxidation processes on different bacteria attached to senescent phytoplanktonic cells, with analysis focused on the photooxidation of bacterial membrane lipids (vaccenic acid) and the expression of genes involved in DNA repair systems. The aim was to determine whether the presence of carotenoids can actively protect bacteria against the damaging effects of ¹O₂ under PAR and PAR+UV irradiations. Comparison of the results obtained with the bacteria *D. shibae* (pigmented) and *P. stutzeri* (not pigmented) showed that under high irradiance conditions (that strongly favor the transfer of ¹O₂ from senescent algae to bacterial cells; ¹⁷), the presence of spheroidenone in *D. shibae* cannot totally prevent ¹O₂-induced oxidative alterations of unsaturated membrane lipids. However, the carotenoid spheroidenone appears to limit ¹O₂ damage, and it can absorb damaging UV radiation.

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We also investigated the activation and regulation of the DNA repair system in response to exposure to ¹O₂ and UV radiation. In *P. stutzeri* cells, the ¹O₂ produced in senescent algae is not entirely scavenged by bacterial membrane MUFAs and so some ¹O₂ is able to reach the cytoplasm (Fig. 4A). Due to the lack of photoprotection across the membranes, DNA is thus damaged by both ¹O₂ and UV radiations. (Fig. 4A). However, in *D. shibae*, scavenging by bacterial membrane MUFAs quenching by spheroidenone limit the effects of ¹O₂ on bacterial membranes. Only a small fraction of UV radiation reaches the cytoplasm, where the efficient detoxifying activity of *mutY* limits its impact on the DNA of this strain, thus preventing oxidative stress (Fig. 4B). This limited impact is supported by the apparent inactivity of the *rpoH* gene responsible for oxidative stress response in photoheterotrophic bacteria.

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Based on these results, the dominance of the *Roseobacter* genus in bacterioplankton communities associated to algal blooms could be attributed in part to the protective action of their carotenoids against ¹O₂ and UV-driven damage.
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**Acknowledgements.** The project leading to this publication has received funding from European FEDER Fund under project 1166-39417. The laboratories, where all the experiments were performed, are in compliance with ISO9001-2015. The authors wish to thank the OMICS platform of MIO for providing the technical means and skills of its personnel and making available equipment and protocols.

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**Originality-Significance Statement.** For the first time, the impact of ¹O₂ produced during algal senescence on the physiology of associated bacteria was studied at the membrane-lipid and DNA levels which places this current work in the top 10% of current research in environmental microbiology.

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**References**

1. Cole, J., Findlay, S. & Pace, M. Bacterial production in fresh and saltwater ecosystems: a cross-system overview. *Mar. Ecol. Prog. Ser.* **43**, 1–10 (1988).

2. Ducklow, H. W., Kirchman, D. L., Quinby, H. L., Carlson, C. A. & Dam, H. G. Stocks and dynamics of bacterioplankton carbon during the spring bloom in the eastern North Atlantic Ocean. *Deep Sea Research Part II: Topical Studies in Oceanography* **40**, 245–263 (1993).

385

3. Eppley, R. W. & Peterson, B. J. Particulate organic matter flux and planktonic new production in the deep ocean. *Nature* **282**, 677–680 (1979).

390

4. Azam, F. & Malfatti, F. Microbial structuring of marine ecosystems. *Nat Rev Microbiol* **5**, 782–791 (2007).
5. Riemann, L., Steward, G. F. & Azam, F. Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom. *Applied and Environmental Microbiology* **66**, 578-587 (2000).

395

6. Pinhassi, J. et al. Changes in Bacterioplankton Composition under Different Phytoplankton Regimens. *Applied and Environmental Microbiology* **70**, 6753-6766 (2004).

7. Teeling, H. et al. Substrate-Controlled Succession of Marine Bacterioplankton Populations Induced by a Phytoplankton Bloom. *Science* **336**, 608-611 (2012).

400

8. Nelson, J. R. Rates and possible mechanism of light-dependent degradation of pigments in detritus derived from phytoplankton. *Journal of Marine Research* **51**, 155-179 (1993).

9. Dmitrieva, D. & Romasheva, N. Sustainable Development of Oil and Gas Potential of the Arctic and Its Shelf Zone: The Role of Innovations. *Journal of Marine Science and Engineering* **8**, 1003 (2020).

405

10. Rontani, J.-F. & Belt, S. T. Photo- and autoxidation of unsaturated algal lipids in the marine environment: An overview of processes, their potential tracers, and limitations. *Organic Geochemistry* **139**, 103941 (2020).

11. Rontani, J.-F. *Lipid Oxidation Products: Useful Tools for Monitoring Photo- and Autoxidation in Phototrophs*. (2021).

410

12. Morgan, P. E., Dean, R. T. & Davies, M. J. Protective mechanisms against peptide and protein peroxides generated by singlet oxygen. *Free Radical Biology and Medicine* **36**, 484-496 (2004).

13. Agnez-Lima, L. F., Mascio, P. D., Napolitano, R. L., Fuchs, R. P. & Menck, C. F. M. Mutation Spectrum Induced by Singlet Oxygen in Escherichia coli Deficient in Exonuclease III. *Photochemistry and Photobiology* **70**, 505-511 (1999).

415
14. Ravanat, J.-L., Mascio, P. D., Martinez, G. R., Medeiros, M. H. G. & Cadet, J. Singlet Oxygen Induces Oxidation of Cellular DNA *. *Journal of Biological Chemistry* **275**, 40601-40604 (2000).

15. Rontani, J.-F., Koblížek, M., Beker, B., Bonin, P. & Kolber, Z. S. On the origin of cis-vaccenic acid photodegradation products in the marine environment. *Lipids* **38**, 1085-1092 (2003).

16. Petit, M., Sempéré, R., Vaultier, F. & Rontani, J.-F. Photochemical Production and Behavior of Hydroperoxyacids in Heterotrophic Bacteria Attached to Senescent Phytoplanktonic Cells. *International Journal of Molecular Sciences* **14**, 11795-11815 (2013).

17. Burot, C., Bonin, P., Simon, G., Casalot, L. & Rontani, J.-F. Singlet oxygen-induced alteration of bacteria associated to phytodetritus: effect of irradiance. *Journal of Phycology* (2023).

18. Dahl, Thomas A., Robert Middenand, W. & Hartman, Philip E. Pure Singlet Oxygen Cytotoxicity for Bacteria. *Photochemistry and Photobiology* **46**, 345-352 (1987).

19. Dahl, T. A., Robert Midden, W. & Hartman, P. E. Pure exogenous singlet oxygen: Nonmutagenicity in bacteria. *Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis* **201**, 127-136 (1988).

20. Petit, M. *et al*. Transfer of singlet oxygen from senescent irradiated phytoplankton cells to attached heterotrophic bacteria: Effect of silica and carbonaceous matrices. *Marine Chemistry* **171**, 87-95 (2015).

21. Fandino, L. B., Riemann, L., Steward, G. F., Long, R. A. & Azam, F. Variations in bacterial community structure during a dinoflagellate bloom analyzed by DGGE and 16S rDNA sequencing. *Aquatic Microbial Ecology* **23**, 119-130 (2001).
22. Rink, B. *et al.* Effects of phytoplankton bloom in a coastal ecosystem on the composition of bacterial communities. *Aquatic Microbial Ecology* **48**, 47–60 (2007).

23. Rüger, H. J. & Höfle, M. G. Marine star-shaped-aggregate-forming bacteria: Agrobacterium atlanticum sp. nov.; Agrobacterium meteori sp. nov.; Agrobacterium ferrugineum sp. nov., nom. rev.; Agrobacterium gelatinovorum sp. nov., nom. rev.; and Agrobacterium stellulatum sp. nov., nom. rev. *Int J Syst Bacteriol* **42**, 133–143 (1992).

24. González, J. M. *et al.* Bacterial Community Structure Associated with a Dimethylsulfoniopropionate-Producing North Atlantic Algal Bloom. *Applied and Environmental Microbiology* **66**, 4237–4246 (2000).

25. Alavi, M., Miller, T., Erlandson, K., Schneider, R. & Belas, R. Bacterial community associated with Pfiesteria-like dinoflagellate cultures. *Environmental Microbiology* **3**, 380–396 (2001).

26. Buchan, A., González, J. M. & Moran, M. A. Overview of the Marine Roseobacter Lineage. *Applied and Environmental Microbiology* **71**, 5665–5677 (2005).

27. Jasti, S., Sieracki, M. E., Poulton, N. J., Giewat, M. W. & Rooney-Varga, J. N. Phylogenetic Diversity and Specificity of Bacteria Closely Associated with Alexandrium spp. and Other Phytoplankton. *Applied and Environmental Microbiology* **71**, 3483–3494 (2005).

28. Dang, H. & Lovell, C. R. Bacterial Primary Colonization and Early Succession on Surfaces in Marine Waters as Determined by Amplified rRNA Gene Restriction Analysis and Sequence Analysis of 16S rRNA Genes. *Applied and Environmental Microbiology* **66**, 467–475 (2000).

29. Wagner-Döbler, I. *et al.* Discovery of Complex Mixtures of Novel Long-Chain Quorum Sensing Signals in Free-Living and Host-Associated Marine Alphaproteobacteria. *ChemBioChem* **6**, 2195–2206 (2005).
30. Long, R. A. & Azam, F. Antagonistic Interactions among Marine Pelagic Bacteria. *Applied and Environmental Microbiology* **67**, 4975-4983 (2001).

31. Allgaier, M., Uphoff, H., Felske, A. & Wagner-Döbler, I. Aerobic Anoxygenic Photosynthesis in Roseobacter Clade Bacteria from Diverse Marine Habitats. *Applied and Environmental Microbiology* **69**, 5051-5059 (2003).

32. Yurkov, V. & Csotonyi, J. T. New Light on Aerobic Anoxygenic Phototrophs. in *The Purple Phototrophic Bacteria* (eds. Hunter, C. N., Daldal, F., Thurnauer, M. C. & Beatty, J. T.) 31-55 (Springer Netherlands, 2009). doi:10.1007/978-1-4020-8815-5_3.

33. Petit, M. et al. Dynamic of bacterial communities attached to lightened phytodetritus. *Environ Sci Pollut Res* **22**, 13681-13692 (2015).

34. Rontani, J.-F. et al. Intense photooxidative degradation of planktonic and bacterial lipids in sinking particles collected with sediment traps across the Canadian Beaufort Shelf (Arctic Ocean). *Biogeosciences* **9**, 4787-4802 (2012).

35. Glaeser, S. P., Grossart, H.-P. & Glaeser, J. Singlet oxygen, a neglected but important environmental factor: short-term and long-term effects on bacterioplankton composition in a humic lake. *Environmental Microbiology* **12**, 3124-3136 (2010).

36. Lehmann, K. & Neumann, R. Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik. in (1896).

37. Biebl, H. et al. Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated from dinoflagellates. *International Journal of Systematic and Evolutionary Microbiology* **55**, 1089-1096 (2005).

38. Kester, D. R., Duedall, I. W., Connors, D. N. & Pytkowicz, R. M. Preparation of Artificial Seawater1. *Limnology and Oceanography* **12**, 176-179 (1967).
490

39. Otto, F. Chapter 11 DAPI Staining of Fixed Cells for High-Resolution Flow Cytometry of Nuclear DNA. in *Methods in Cell Biology* (eds. Darzynkiewicz, Z. & Crissman, H. A.) vol. 33 105–110 (Academic Press, 1990).

495

40. Sarra, A. di et al. Assessing the Quality of Shortwave and Longwave Irradiance Observations over the Ocean: One Year of High-Time-Resolution Measurements at the Lampedusa Oceanographic Observatory. *Journal of Atmospheric and Oceanic Technology* **36**, 2383–2400 (2019).

41. Keller, M. D., Selvin, R. C., Claus, W. & Guillard, R. R. L. Media for the Culture of Oceanic Ultraphytoplankton **1,2.** *Journal of Phycology* **23**, 633–638 (1987).

500

42. Rontani, J.-F., Lalande, C., Vilgrain, L., Vaultier, F. & Amiraux, R. Control of the preservation of sympagic algal material in surficial sediments of central and eastern Baffin Bay by bactericidal hydroperoxides and free fatty acids. *Marine Chemistry* **247**, 104177 (2022).

43. Loidl-Stahlhofen, A., Hannemann, K. & Spiteller, G. Generation of α-hydroxyaldehydic compounds in the course of lipid peroxidation. *Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism* **1213**, 140–148 (1994).

505

44. Rainey, P. B., Thompson, I. P. & Palleroni, N. J. Genome and Fatty Acid Analysis of Pseudomonas stutzeri. *International Journal of Systematic and Evolutionary Microbiology* **44**, 54–61 (1994).

45. Porter, N. A., Caldwell, S. E. & Mills, K. A. Mechanisms of free radical oxidation of unsaturated lipids. *Lipids* **30**, 277–290 (1995).

510

46. Quesada, A. & Vincent, W. F. Strategies of adaptation by Antarctic cyanobacteria to ultraviolet radiation. *European Journal of Phycology* **32**, 335–342 (1997).

47. Ourisson, G. & Nakatani, Y. Bacterial Carotenoids as Membrane Reinforcers: A General Role for Polyterpenoids: Membrane Stabilization. in *Carotenoids: Chemistry and*
*Biology* (eds. Krinsky, N. I., Mathews-Roth, M. M. & Taylor, R. F.) 237–245 (Springer US, 1989). doi:10.1007/978-1-4613-0849-2_16.

48. Tatsuzawa, H., Maruyama, T., Misawa, N., Fujimori, K. & Nakano, M. Quenching of singlet oxygen by carotenoids produced in Escherichia coli – attenuation of singlet oxygen-mediated bacterial killing by carotenoids. *FEBS Letters* **484**, 280–284 (2000).

49. Strzałka, K. & Gruszecki, W. I. Effect of β-carotene on structural and dynamic properties of model phosphatidylcholine membranes. I. An EPR spin label study. *Biochimica et Biophysica Acta (BBA) - Biomembranes* **1194**, 138–142 (1994).

50. Niedzwiedzki, D. M. *et al*. Carotenoid-induced non-photochemical quenching in the cyanobacterial chlorophyll synthase-HliC/D complex. *Biochimica et Biophysica Acta (BBA) - Bioenergetics* **1857**, 1430–1439 (2016).

51. Kim, S.-Y., Kim, E.-J. & Park, J.-W. Control of Singlet Oxygen-induced Oxidative Damage in Escherichia coli. *BMB Reports* **35**, 353–357 (2002).

52. Glaeser, J., Nuss, A. M., Berghoff, B. A. & Klug, G. Chapter 4 - Singlet Oxygen Stress in Microorganisms. in *Advances in Microbial Physiology* (ed. Poole, R. K.) vol. 58 141–173 (Academic Press, 2011).

53. Michaels, M. L. & Miller, J. H. The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). *J Bacteriol* **174**, 6321–6325 (1992).

54. Conn, P. F., Schalch, W. & Truscott, T. G. The singlet oxygen and carotenoid interaction. *Journal of Photochemistry and Photobiology B: Biology* **11**, 41–47 (1991).

55. Cadet, J. & Teoule, R. Comparative Study of Oxidation of Nucleic Acid Components by Hydroxyl Radicals, Singlet Oxygen and Superoxide Anion Radicals. *Photochemistry and Photobiology* **28**, 661–665 (1978).
56. Steenken, S. & Jovanovic, S. V. How Easily Oxidizable Is DNA? One-Electron Reduction Potentials of Adenosine and Guanosine Radicals in Aqueous Solution. *J. Am. Chem. Soc.* **119**, 617-618 (1997).

57. Ravanat, J.-L., Martinez, G. R., Medeiros, M. H. G., Di Mascio, P. & Cadet, J. Mechanistic aspects of the oxidation of DNA constituents mediated by singlet molecular oxygen. *Archives of Biochemistry and Biophysics* **423**, 23-30 (2004).

58. Shibutani, S., Takeshita, M. & Grollman, A. P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. *Nature* **349**, 431-434 (1991).

59. Tchou, J. et al. 8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity. *Proceedings of the National Academy of Sciences* **88**, 4690-4694 (1991).

60. Boiteux, S., Gajewski, E., Laval, J. & Dizdaroglu, M. Substrate specificity of the Escherichia coli Fpg protein formamidopyrimidine-DNA glycosylase: excision of purine lesions in DNA produced by ionizing radiation or photosensitization. *Biochemistry* **31**, 106-110 (1992).

61. Agnez-Lima, L. F. et al. DNA damage by singlet oxygen and cellular protective mechanisms. *Mutation Research/Reviews in Mutation Research* **751**, 15-28 (2012).

62. Costa, R. M. A., Chiganças, V., da Silva Galhardo, R., Carvalho, H. & Menck, C. F. M. The eukaryotic nucleotide excision repair pathway. *Biochimie* **85**, 1083-1099 (2003).

63. Anthony, J. R., Newman, J. D. & Donohue, T. J. Interactions Between the Rhodobacter sphaeroides ECF Sigma Factor, σE, and its Anti-sigma Factor, ChrR. *Journal of Molecular Biology* **341**, 345-360 (2004).

64. Anthony, J. R., Warczak, K. L. & Donohue, T. J. A transcriptional response to singlet oxygen, a toxic byproduct of photosynthesis. *Proceedings of the National Academy of Sciences* **102**, 6502-6507 (2005).
65. Yura, T. Regulation of the heat shock response in Escherichia coli: history and perspectives. *Genes Genet Syst* **94**, 103–108 (2019).

66. Omar, N. M., Prášil, O., McCain, J. S. P. & Campbell, D. A. Diffusional Interactions among Marine Phytoplankton and Bacterioplankton: Modelling H2O2 as a Case Study. *Microorganisms* **10**, 821 (2022).

67. Omar, N. M., Fleury, K., Beardsall, B., Prášil, O. & Campbell, D. A. Genomic capacities for Reactive Oxygen Species metabolism across marine phytoplankton. *Plos one* **18**, e0284580 (2023).

565

570
<table><caption>**Table S1.** Sequences and hybridization temperatures (°C) of primers used for the cloning and amplification of targeted genes in *P. stutzeri* (*Ps*) and *D. shibae* (*Ds*).</caption><thead><tr><th>Target Gene</th><th>Primer Name</th><th>Primer sequence 5' → 3'</th><th>Fragment size (pb)</th><th>PCR hybridization temperature (°C)</th></tr></thead><tbody><tr><td rowspan="2">16s Ps</td><td>PS_16S-F</td><td>AGC-TAG-TTG-GTG-AGG-TAA-AGG</td><td rowspan="2">291</td><td rowspan="2">60.0</td></tr><tr><td>PS_16S-R</td><td>GTA-TTA-CCG-CGG-CTG-CTG-GCA</td></tr><tr><td rowspan="2">mutY Ps</td><td>PS栏 mutY-F</td><td>GGC-TAC-TTC-GAC-CGC-TTC-AT</td><td rowspan="2">74</td><td rowspan="2">60.0</td></tr><tr><td>PS栏 mutY-R</td><td>AGC-ACT-TCA-TCT-TCC-TCG-GC</td></tr><tr><td rowspan="2">rpoH Ps</td><td>PS栏 rpoH-F</td><td>GGT-CTG-ATG-AAG-GCG-GTC-AA</td><td rowspan="2">209</td><td rowspan="2">60.0</td></tr><tr><td>PS栏 rpoH-R</td><td>ACC-TCA-TCG-TTG-TTC-AGC-CA</td></tr><tr><td rowspan="2">chrR Ps</td><td>PS栏 chrR-F</td><td>CGA-GTT-GGA-ATT-ACG-TCG-CG</td><td rowspan="2">109</td><td rowspan="2">60.0</td></tr><tr><td>PS栏 chrR-R</td><td>CGG-CTC-GAT-CAG-TTC-CTT-GT</td></tr><tr><td rowspan="2">rpoD Ps</td><td>PS栏 rpoD-F</td><td>CAG-TCC-CGC-CTC-AAA-GAG-TT</td><td rowspan="2">148</td><td rowspan="2">60.0</td></tr><tr><td>PS栏 rpoD-R</td><td>CGT-TGA-TGC-CCA-TGT-CGT-TG</td></tr><tr><td rowspan="2">16s Ds</td><td>Rhodo 240F</td><td>GGC-CCG-CGT-TGG-ATF-AGR-TA</td><td rowspan="2">276</td><td rowspan="2">62.0</td></tr><tr><td>Univ 516R</td><td>GTD-TTA-CCG-CGG-CKG-CTG-RCA</td></tr><tr><td rowspan="2">mutY Ds</td><td>DS栏 mutY-F</td><td>GGC-TCT-CCG-AGG-TGA-TGC-TG</td><td rowspan="2">179</td><td rowspan="2">62.0</td></tr><tr><td>DS栏 mutY-R</td><td>CGC-GCA-TTT-CAG-CAG-GTT-G</td></tr><tr><td rowspan="2">rpoH Ds</td><td>DS栏 rpoH-F</td><td>ATA-TAC-CAA-TCT-TCC-GGC-CCC-C</td><td rowspan="2">106</td><td rowspan="2">60.0</td></tr><tr><td>DS栏 rpoH-R</td><td>GGC-CAG-CAT-GTA-TTC-CTC-TTC-G</td></tr><tr><td rowspan="2">chrR Ds</td><td>DS栏 chrR-F</td><td>AGC-TGC-CCA-AAT-CCA-TGA-CA</td><td rowspan="2">97</td><td rowspan="2">62.0</td></tr><tr><td>DS栏 chrR-R</td><td>TTC-ACA-TCC-TCC-ACA-TCG-CC</td></tr><tr><td rowspan="2">rpoD Ds</td><td>DS栏 rpoD-F</td><td>GCA-AGA-CCG-ATG-ACC-AGG-AC</td><td rowspan="2">150</td><td rowspan="2">60.0</td></tr><tr><td>DS栏 rpoD-R</td><td>TCT-GTT-CCG-AGC-TGA-CCT-GA</td></tr></tbody></table>

All the primers have been designated from the complete genome of *P. stutzeri* (ACCESSION NUMBER CP002881) and *D. shibae* (ACCESSION NUMBER CP000830).
<table><caption>Table S2: ANOVA analyses of data comparing the effects of *Thalassiosira* sp. input with solar irradiance, UV, solar light and PAR light on the vaccenic-acid oxidation product and the level of expression of *mutY*, *chrR* and *rpoH* (threshold, p< 0.005).</caption><thead><tr><th></th><th>Source</th><th>DDL</th><th>Somme des carrés</th><th>Moyenne des carrés</th><th>F</th><th>Pr > F</th></tr></thead><tbody><tr><td rowspan="4">Vaccenique<br>oxidation product</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>1</td><td>9615978,78</td><td>9615978,78</td><td>82,91</td><td>0,00000</td></tr><tr><td>specific effect of UV</td><td>1</td><td>19299253,72</td><td>19299253,72</td><td>23,47</td><td>0,00008</td></tr><tr><td>solar Light dose</td><td>1</td><td>5143869,38</td><td>5143869,38</td><td>9,13</td><td>0,01287</td></tr><tr><td>PAR Light dose</td><td>1</td><td>2366453,91</td><td>2366453,91</td><td>4,78</td><td>0,05358</td></tr><tr><td rowspan="8">P Stutzeri</td><td rowspan="4">mutY</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>2</td><td>12,30</td><td>6,15</td><td>2,81</td><td>0,08675</td></tr><tr><td>specific effect of UV</td><td>3</td><td>0,76</td><td>0,25</td><td>0,17</td><td>0,91785</td></tr><tr><td>solar Light dose</td><td>1</td><td>14,41</td><td>14,41</td><td>12,99</td><td>0,00482</td></tr><tr><td>PAR Light dose</td><td>1</td><td>0,94</td><td>0,94</td><td>1,99</td><td>0,18852</td></tr><tr><td rowspan="4">chrR</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>2</td><td>1,30</td><td>0,65</td><td>0,33</td><td>0,72188</td></tr><tr><td>specific effect of UV</td><td>3</td><td>34,81</td><td>11,60</td><td>6,37</td><td>0,00331</td></tr><tr><td>solar Light dose</td><td>1</td><td>4,46</td><td>4,46</td><td>1,27</td><td>0,28665</td></tr><tr><td>PAR Light dose</td><td>1</td><td>0,94</td><td>0,94</td><td>1,99</td><td>0,18852</td></tr><tr><td rowspan="4">rpoH</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>2</td><td>23,80</td><td>11,90</td><td>4,46</td><td>0,02664</td></tr><tr><td>specific effect of UV</td><td>3</td><td>0,99</td><td>0,33</td><td>0,19</td><td>0,90177</td></tr><tr><td>solar Light dose</td><td>1</td><td>10,79</td><td>10,79</td><td>5,39</td><td>0,04269</td></tr><tr><td>PAR Light dose</td><td>1</td><td>0,78</td><td>0,78</td><td>1,96</td><td>0,19213</td></tr><tr><td rowspan="8">D. Shibae</td><td rowspan="4">Vaccenique<br>oxidation product</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>1</td><td>1142347,63</td><td>1142347,63</td><td>16,31</td><td>0,00237</td></tr><tr><td>specific effect of UV</td><td>1</td><td>833878,21</td><td>833878,21</td><td>0,73</td><td>0,40090</td></tr><tr><td>solar Light dose</td><td>1</td><td>1557444,57</td><td>1557444,57</td><td>2,00</td><td>0,18718</td></tr><tr><td>PAR Light dose</td><td>1</td><td>5287382,51</td><td>5287382,51</td><td>5,09</td><td>0,04769</td></tr><tr><td rowspan="4">mutY</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>2</td><td>16,22</td><td>8,11</td><td>24,56</td><td>0,00001</td></tr><tr><td>specific effect of UV</td><td>3</td><td>129,74</td><td>43,25</td><td>151,30</td><td>0,00000</td></tr><tr><td>solar Light dose</td><td>1</td><td>2,99</td><td>2,99</td><td>12,68</td><td>0,00517</td></tr><tr><td>PAR Light dose</td><td>1</td><td>0,00</td><td>0,00</td><td>0,00</td><td>0,98159</td></tr><tr><td rowspan="4">chrR</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>2</td><td>9,61</td><td>4,81</td><td>23,14</td><td>0,00001</td></tr><tr><td>specific effect of UV</td><td>3</td><td>828,10</td><td>276,03</td><td>4090,43</td><td>0,00000</td></tr><tr><td>solar Light dose</td><td>1</td><td>0,60</td><td>0,60</td><td>6,99</td><td>0,02458</td></tr><tr><td>PAR Light dose</td><td>1</td><td>0,01</td><td>0,01</td><td>0,22</td><td>0,65006</td></tr><tr><td rowspan="4">rpoH</td><td>Thalassiosira sp. effect with solar Irradiance</td><td>2</td><td>1,10</td><td>0,55</td><td>1,96</td><td>0,17018</td></tr><tr><td>specific effect of UV</td><td>3</td><td>117,53</td><td>39,18</td><td>236,09</td><td>0,00000</td></tr><tr><td>solar Light dose</td><td>1</td><td>0,06</td><td>0,06</td><td>0,24</td><td>0,63195</td></tr><tr><td>PAR Light dose</td><td>1</td><td>0,35</td><td>0,35</td><td>4,73</td><td>0,05478</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 1: Production of vaccenic acid oxidation products (pmol. L-1) during irradiation of Thalassiosira sp. cells associated with bacterial strains Pseudomonas stutzeri (A & B) or Dinoroseobacter shibae (C & D) with PAR and PAR + UV radiation, respectively.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 2: The different scenarios related to 1O2 DNA damage and the role of the "Go system" proteins in repair. (°G = 8-oxo-7,8-dihydro-2'-deoxyguanosine = d-8-oxoG).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Fig. 3: Log2 of the relative expression of the targeted genes in Pseudomonas stutzeri (A) or Dinoroseobacter shibae (B) with PAR (plain bars) and PAR + UV (dotted bars) radiation. The hatched bars represent the controls, conducted without Thalassiosira cells. The results are relative to the expression of the rpoD gene and relative to that of the controls (0). Means ± SD (n=3).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 4: Conceptual scheme showing the effect of singlet oxygen photochemically produced in phyötodetritus on membrane components and DNA of unpigmented (A) and pigmented (B) associated bacteria.</figcaption></figure>
**Supplementary Files**

This is a list of supplementary files associated with this preprint. Click to download.

* allTableS.pdf