Functional potential and evolutionary response to long-term heat selection of bacterial associates of coral photosymbionts

ABSTRACT Corals rely on a wide range of microorganisms for their functioning, including intracellular dinoflagellates (Symbiodiniaceae) and bacteria. Marine heatwaves trigger the loss of Symbiodiniaceae from coral tissues–coral bleaching–often leading to death. While coral-bacteria interactions are widely studied, Symbiodiniaceae-bacteria interactions have remained largely uninvestigated. Here, we provide a genomic analysis of 49 bacteria, spanning 16 genera, that are closely associated with six cultured Symbiodiniaceae species. We analyzed bacterial functional potential by focusing on potentially beneficial functions for the Symbiodiniaceae host, including B vitamin synthesis and antioxidant abilities, which may be crucial for Symbiodiniaceae heat tolerance and, in turn, coral resistance to thermal bleaching. These analyses suggest a wide potential for B vitamin synthesis and the scavenging of reactive oxygen species (through the production of carotenoids or antioxidant enzymes), and possibly the transfer of organic carbon to host cells. Single nucleotide polymorphism analysis between bacteria isolated from wild-type and heat-evolved Symbiodiniaceae cultures revealed that exposure to long-term elevated temperature has resulted in mutations in genes known to be involved in host-symbiont interactions, such as secretion systems. Climate change may therefore modify how Symbiodiniaceae and bacteria interact. This study provides an overview of the possible roles of Symbiodiniaceae-associated bacteria in Symbiodiniaceae functioning and heat tolerance, reinforcing the need for further studies of such interactions to fully understand coral biology and climate resilience. IMPORTANCE Symbiotic microorganisms are crucial for the survival of corals and their resistance to coral bleaching in the face of climate change. However, the impact of microbe-microbe interactions on coral functioning is mostly unknown but could be essential factors for coral adaption to future climates. Here, we investigated interactions between cultured dinoflagellates of the Symbiodiniaceae family, essential photosymbionts of corals, and associated bacteria. By assessing the genomic potential of 49 bacteria, we found that they are likely beneficial for Symbiodiniaceae, through the production of B vitamins and antioxidants. Additionally, bacterial genes involved in host-symbiont interactions, such as secretion systems, accumulated mutations following long-term exposure to heat, suggesting symbiotic interactions may change under climate change. This highlights the importance of microbe-microbe interactions in coral functioning.

studied member of this microbial consortium (6).By providing photosynthate to their host, Symbiodiniaceae meet most of the energy needs of corals (7) and are essential for the health of coral reefs.Unfortunately, this symbiotic association is extremely fragile and is particularly sensitive to temperature fluctuations.Increases in sea surface temperatures can result in the loss of Symbiodiniaceae from coral tissues, a process known as coral bleaching, which can result in coral death (8).The main mechanism thought to trigger coral bleaching is an increase in the production of reactive oxygen species (ROS) by Symbiodiniaceae, which leak into coral cells, overwhelm the coral's antioxidant response, and trigger a cellular cascade that results in Symbiodiniaceae loss (9).Mass coral bleaching events are increasing in frequency and intensity because of anthropogenic climate change (10), thus, highlighting the need to increase knowledge on coral and Symbiodiniaceae biology and to develop novel coral conservation and restoration methods, while greenhouse gas emissions are curbed.
Corals also house a wide diversity of bacteria, which colonize all microhabitats within their host (4,(11)(12)(13).Putative bacterial functions include nutrient cycling and protection against pathogens (4,12).Recently, Symbiodiniaceae were also found to associate with bacteria, both extra and intracellularly (14)(15)(16).Symbiodiniaceae-associated bacterial communities are highly diverse and change after both short-term thermal stress (17)(18)(19) and long-term heat selection (19), pointing at a potential bacterial involvement in Symbiodiniaceae thermal tolerance.Two studies have shown that inoculation of Symbiodiniaceae cultures with a single bacterial strain (belonging to the Muricauda and Roseovarius genera, respectively) can enhance Symbiodiniaceae (Durusdinium sp. and Breviolum minutum, respectively) performance under temperature and/or light stress (18,20).In the case of Durusdinium/Muricauda, this was attributed to the production of a carotenoid by Muricauda, a pigment able to scavenge ROS (20).Nevertheless, the functions and functional potential of most Symbiodiniaceae-associated bacteria remain mostly unexplored (13,21).
Here, we provide the first comparative genomic analysis of Symbiodiniaceae-associ ated bacteria.From a culture collection of more than 200 bacterial isolates, cultured from six Symbiodiniaceae species, we sequenced the genomes of 49 representative isolates.We present an in-depth analysis of bacterial functional potential that may be beneficial for Symbiodiniaceae hosts, such as B vitamin synthesis and ROS scavenging.We also analyze the effect of Symbiodiniaceae thermal selection on bacterial genomic evolution.This study provides an important step to understanding the contribution of bacteria to Symbiodiniaceae functioning, and, in turn, their roles in coral health and climate resilience.

Isolation of bacteria from Symbiodiniaceae cultures
In a previous study, 141 pure bacterial isolates, spanning 20 genera, were obtained from six Symbiodiniaceae species (15).We recently showed that long-term heat selection of the species Cladocopium proliferum affected the composition of Symbiodiniaceae-asso ciated bacteria (19), both at the extracellular and intracellular levels (22).Thus, we expanded our culture collection by pure culturing bacteria from one wild-type (WT10) and two heat-evolved (SS03 and SS08) C. proliferum strains.For each C. proliferum strain, samples were washed and either bead-beat, in order to release potentially intracellular bacteria or not.A total of 65 pure bacterial isolates were obtained and identified through 16S rRNA gene sequence analysis (Table S1).These newly obtained isolates spanned 10 genera, 5 of which were previously isolated from Symbiodiniaceae cultures (15).Four genera (Marinobacter, Muricauda, Mamelliela, and Roseitalea) were cultured from all three C. proliferum strains.
Six genera (Janibacter, Aestuariicoccus, Muricauda, Roseibium, Roseitalea, and Oceaniradius) were isolated exclusively from C. proliferum samples that were bead-beaten prior to spreading on agar plates, suggesting they may be intracellular associates.Nonetheless, Muricauda, Roseitalea, and Roseibium (previously referred to as Labrenzia) were previously isolated from C. proliferum WT10 samples that were not bead beaten (15), suggesting they can be extracellular.Therefore, only Janibacter, Aestuariicoccus, and Oceaniradius are strong candidates for being intracellular symbionts of C. proliferum, while all others are likely extracellular, though still closely associated.

Genome sequencing of 49 Symbiodiniaceae-associated bacteria
To explore the genomic abilities of bacteria closely associated with Symbiodiniaceae, the whole genome of 49 isolates was sequenced, assembled, and annotated.The 49 isolates were chosen to span both the bacterial and Symbiodiniaceae diversity of our culture collection, by including isolates from all six Symbiodiniaceae host species, and the majority of the bacterial orders recovered.When possible, we sequenced bacterial species that were present in all Symbiodiniaceae host species to allow for comparative analyses.All 49 assembled genomes exhibited completeness above 98% and contamina tion below 2.5%, and ranged from 2.98 to 6.88 Mb in size (Table S2).The taxonomy of these genomes was confirmed with the GTDB-Tk tool, the construction of a phyloge netic tree, and the calculation of average nucleotide and amino acid identity (ANI and AAI, respectively; Fig. 1; Fig. S1; Table S2).This corroborated the taxonomy previously obtained through 16S rRNA gene sequence analysis (see "Taxonomy notes" in Table S2).It is worth noting that, within one genus, isolates from distinct Symbiodiniaceae hosts often show >99.9%ANI and 100% AAI with each other (e.g., Muricauda, Mameliella, Roseitalea, and Marinobacter; Fig. S1), suggesting a lack of host-symbiont specificity.
The metabolic abilities of the 49 isolates were reconstructed using a KEGG annotation (Table S3), and pathways of interest are summarized in Fig. 2. Because our genomes were not 100% complete and lacked contiguity, pathways that were more than 75% complete were considered functional, a threshold commonly used in similar analyses (23)(24)(25).Again, no host specificity or co-diversification was observed; isolates of a given genus all had almost identical metabolic potentials, regardless of the Symbiodiniaceae host they were cultured from.This is intriguing because the study utilized a range of Symbiodinia ceae strains originally isolated from four coral species, one sea anemone species, and one giant clam species.This is consistent with the fact that the community composition of intracellular bacteria was highly similar across these Symbiodiniaceae cultures (15).Therefore, these bacteria may be opportunistic associates (as opposed to long-term symbionts), perhaps selected during the initial isolation of Symbiodiniaceae or acciden tally introduced in Symbiodiniaceae cultures later on, which have been maintained over the years, presumably because they were not detrimental and potentially beneficial, to their Symbiodiniaceae host.Almost all isolates possessed all genes for glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, the biosynthesis of all nucleotides, and the biosynthesis of several amino acids (threonine, valine, isoleucine, leucine, lysine, and histidine).Only two Roseibium isolates had the genes to perform denitrification, while all four Roseibium isolates had the genetic potential for dissimilatory nitrate reduction.Denitrifying potential is commonly observed in other Roseibium strains (sometimes referred to as Labrenzia) (26,27).Several isolates also possessed complete secretion systems, which may assist in host-bacteria and bacteria-bacteria interactions, including Symbiodiniaceae infection; a type II secretion system (T2SS) was found in three isolates (belonging to Marinobacter); a type IV secretion system (T4SS) was found in 11 isolates (belonging to Roseovarius, Roseivivax, Mameliella, and Aquicoccus); a type VI secretion system (T6SS) was found in seven isolates (belonging to Halomonas, Marinobacter, and Roseibium).We also interrogated the presence of eukaryotic-like proteins, which may facilitate host-bacteria interactions by mediating bacterial protein-eukaryotic host-protein interactions (28), but did not detect any in any of the 49 genomes reported here.

Potential for B vitamin synthesis
B vitamins are required by most microalgae (29)(30)(31), including Symbiodiniaceae (21,32), and promote algal growth.For example, biotin (vitamin B 7 ) is essential for fatty acid metabolism, while thiamine (vitamin B 1 ) is important for carbohydrate and amino acid metabolism (30).Commonly used Symbiodiniaceae culture media, such as Daigo's IMK medium used in this study, contain biotin, thiamine, and cobalamin (vitamin B 12 ), although some B vitamins have been shown to be provided by bacteria in some cases.For example, Halomonas sp. and Sinorhizobium meliloti provide cobalamin to the unicellular algae Porphyridium purpureum and Chlamydomonas reinhardtii, respectively (29,33).Therefore, we interrogated the completeness of B vitamin biosynthesis pathways in Symbiodiniaceae-associated bacteria (Fig. 2; Tables S3 and S4).
All but one isolate were found to be able to synthesize riboflavin (vitamin B 2 ), while 43 possessed the genes for tetrahydrofolate (vitamin B 9 ) synthesis (only missing in one Oceaniradius, two Roseibium, and three Roseovarius), 28 possessed the genes for pantothenate (vitamin B 5 ) synthesis (belonging to Bacillus, Roseivirga, Muricauda, Halomonas, Marinobacter, Oceanicaulis, and Mameliella), and 23 isolates showed potential to synthesize cobalamin (belonging to Halomonas, Oceaniradius, Roseitalea, Stappia, Roseovarius, Mameliella, Aquicoccus, Roseibium, and Nioella).The potential for the synthesis of other vitamins was scarcer: 12 isolates for thiamine synthesis (belonging to Bacillus, Halomonas, Qipengyuania, Roseibium, Oceanicaulis, Aquicoccus, and Roseovarius); four isolates for pyridoxal (vitamin B 6 ) synthesis (belonging to Halomonas and Marino bacter); five for pimeloyl-ACP and biotin synthesis, including synthesis of the biotin precursor pimeloyl-ACP (belonging to Bacillus, Halomonas, and Marinobacter).Only MMSF_3323 (Halomonas sp.) had the potential to synthesize the seven B vitamins that were investigated, while MMSF_3353 (Bacillus sp.) and all three Marinobacter isolates could synthesize five (Table S4).Provisioning of cobalamin by a separate Halomonas strain was demonstrated in the red alga P. purpureum (29).Therefore, there is high potential for B vitamin provisioning from Symbiodiniaceae-associated bacteria.How these nutrients are exchanged between the partners remains unknown, although some isolates did possess ABC transporters for thiamine that may facilitate its export.Intracel lular bacteria may be able to directly export the vitamins in the Symbiodiniaceae cell's cytoplasm, making it readily available.
The potential for bacterial B vitamin provisioning is important for several reasons.First, as shown in several other microalgae, B vitamin supplementation, specifically cobalamin, can increase algal growth (29,33,34).Second, cobalamin supplementa tion by bacteria was linked to increased thermal tolerance in C. reinhardtii (33), which is relevant to Symbiodiniaceae in the context of thermal stress and coral See Table S3 for the full list of pathways.
bleaching.Third, coral hosts of Symbiodiniaceae cannot synthesize B vitamins either.Some coral-associated bacteria, such as Endozoicomonas, have been hypothesized to synthesize B vitamins for their coral hosts (35)(36)(37).Symbiodiniaceae-associated bacteria may also provide B vitamins to the wider coral holobiont, rather than just Symbiodiniaceae.

Potential for ROS scavenging during thermal stress
ROS have been implicated in the thermal stress response of Symbiodiniaceae and, in turn, in coral bleaching (8,9,38).Because of this, the production of ROS is a key trait in the design of probiotics for improving thermal tolerance in Symbiodiniaceae and corals (18,20,(39)(40)(41)(42).While Symbiodiniaceae continuously produce ROS [such as singlet oxygen ( 1 O 2 ), superoxide (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH − )], thermal stress results in an increased production of ROS that may ultimately leak into coral cells and trigger coral bleaching (9).Thus, we looked at pathways and proteins potentially involved in ROS scavenging and/or the production of antiox idants.Pathways for the synthesis of glutathione and ubiquinone, two antioxidants, were complete in 30 and 4 isolates, respectively (Fig. 2; Table S3).All Proteobacteria were able to produce glutathione, while only the four Gammaproteobacteria isolates (Halomonas and Marinobacter) were able to produce ubiquinone.Additionally, specific protein families (Pfam) or their synthesis pathways related to ROS and RNS scaveng ing were manually searched in the 49 genomes (Table 1): synthesis of zeaxhanthin, a carotenoid that quenches singlet oxygen and can scavenge other ROS (43); dimethyl sulfonioproprionate (DMSP) and dimethylsulfate (DMS), OH − scavengers (44); mannitol, another OH − scavenger (45); ROS-scavenging enzymes, including superoxide dismutases (O 2 − scavengers), peroxiredoxins and peroxidases (H 2 O 2 scavengers), glutaredoxins and thioredoxins (general ROS scavengers).These are summarized in Fig. 3.
Only 14 isolates, all belonging to Muricauda, were found to be able to synthesize zeaxanthin.This is consistent with a previous study showing that Durusdinium-associated Muricauda can produce zeaxanthin and contributes to Symbiodiniaceae tolerance to thermal and light stress (20).Twenty-two isolates possessed the dsyB gene, involved in the production of DMSP (belonging to Roseovarius, Mameliella, Roseovivax, Roseibium, Stappia, Roseitalea, Oceaniradius, and Marinobacter), while all isolates except the two Roseivirga isolates were able to synthesize DMS, through DMSO reduction and/or DMSP cleavage.Interestingly, a Roseovarius isolate, MMSF_3448, which enhanced thermal tolerance in Breviolum minutum cultures (18) had the genes to produce both DMSP and DMS, which may be part of the mechanism underlying the benefit found in that study.Additionally, bacterial DMSP and DMS metabolism was shown to increase in the proximity of Symbiodiniaceae cells (46), suggesting sulfur-based interactions between Symbiodiniaceae and their associated bacteria.A gene encoding for a mannitol-1-phos phate dehydrogenase, necessary for mannitol production, was found in 22 isolates (belonging to Bacillus, Halomonas, Qipengyuania, Oceaniradius, Aquicoccus, Mameliella, Roseivivax, Nioella, Roseibium, Stappia, and Roseitalea).Mannitol has been shown to mitigate thermal bleaching in corals and sea anemones (47,48).Finally, while all isolates possessed ROS-scavenging enzymes, some isolates had particularly high numbers, including isolates belonging to Halomonas, Muricauda, Roseibium, and Roseivirga.Isolates of the Muricauda and Roseitalea genera were the only bacteria possessing genes encoding thioredoxins, while only Roseitalea isolates possessed all six queried enzymes.Hence, there is a high potential for Symbiodiniaceae-associated bacteria to assist with ROS removal during thermal stress via various mechanisms.This is particularly important for intracellular bacteria, as they may be able to export ROS-scavenging compounds directly into the Symbiodiniaceae cytoplasm, strengthen the antioxidant response, and minimize ROS leakage out of the Symbiodiniaceae cells.Most of the bacterial genera studied here are commonly detected in and cultured from cnidarian hosts (11,49,50) and may therefore participate in the in hospite Symbiodiniaceae response to thermal stress, and thereby influence the outcome of coral thermal stress.Nonetheless, whether these bacteria truly associate with Symbiodiniaceae in hospite remains unknown.

Potential for carbon exchange
Finally, we investigated the presence of carbon exporters in the bacterial genomes.The "sugars will eventually be exported transporters" (SWEET) are bidirectional sugar transporters found in eukaryotes and are hypothesized to regulate carbon exchange between Symbiodiniaceae and corals (51).SemiSWEET proteins are the bacterial homologues of SWEET proteins, although sugar export has not yet been confirmed in bacteria (52).Five isolates were found to have SemiSWEET genes (Fig. 3): two Roseivirga isolates, one Aquicoccus isolate, and two Roseovarius isolates.In a healthy state, it is unlikely that bacteria would provide glucose to Symbiodiniaceae, which can make its own through photosynthesis, and the bacterial transporters may instead be used to import photosynthate.However, sugars may be exported via the same transporter, which may help compensate for the decrease in photosynthesis, and thereby glucose production, by Symbiodiniaceae during thermal stress.This is the first report of genes encoding for SemiSWEET proteins in Symbiodiniaceae-associated bacteria, and their functional analysis is needed to shed light on their role in Symbiodiniaceae functioning and thermal tolerance.

Genomic evolution in response to Symbiodiniaceae experimental evolution
Among the bacterial genomes sequenced from C. proliferum cultures, we obtained a representative from each of the three C. proliferum strains (WT10, SS03, and SS08) for four bacterial species: Marinobacter sp.(MMSF_3506, MMSF_3519, and MMSF_3550), Muricauda sp.(MMSF_3501, MMSF_3509, and MMSF_3543), Mameliella sp.(MMSF_3510, MMSF_3537, and MMSF_3552), and Roseitalea sp.(MMSF_3504, MMSF_3516, and MMSF_3546).Within a given bacterial species, all three genomes were >99.9% identi cal (based on ANI and AAI calculations, Fig. S1), suggesting they belong to the same strain.Therefore, we assumed that the different bacterial isolates were present in the original C. proliferum culture, before experimental evolution started, and independently evolved in the three C. proliferum strains under elevated temperature (SS strains) versus ambient temperature (WT10 strain).We performed a single nucleotide polymorphism (SNP) analysis to uncover which bacterial genes, if any, were impacted by host heat selection.
Across the four bacterial strains, we detected 32 SNPs between bacteria isolated from WT10 C. proliferum and the corresponding bacteria isolated from SS C. proliferum (Table S5).Six SNPs were synonymous mutations and therefore not expected to bear pheno typic effects.For each given bacterial strain, we next chose to focus on the non-synony mous mutations that were present in both bacteria isolated from SS C. proliferum (Table S5, bolded rows), as there is a higher chance these would be true SNPs and may point to convergent evolution under heat selection.No non-synonymous SNPs were detected in Roseitalea sp.
In Mameliella sp., a missense mutation affected the chvI gene, a transcriptional regulator known to be essential for the virulence of the plant pathogen Agrobacterium tumefaciens (53), as well as the successful establishment of symbiosis in the nodule-form ing and nitrogen-fixing plant associates Rhizobium leguminosarum and S. meliloti (54,55).
In Muricauda sp., missense mutations affected genes encoding a heme A synthase, a histidine kinase, and a DNA gyrase.Histidine kinases have a broad range of actions in signal transduction, while DNA gyrases are involved in DNA replication.The impact of mutations on those two genes is therefore hard to assess.Heme A synthases are involved in heme synthesis, a cofactor involved in many cellular processes, including antioxidant functions through its incorporation into hemoproteins, as well as infectivity and virulence.Intracellular Brucella abortus are dependent on heme production for their survival (56), while heme deficiency negatively impacts mouse tissue colonization by Staphylococcus aureus (57).Additionally, excess free heme catalyzes the formation of ROS, resulting in oxidative stress (58).If this mutation resulted in decreased heme synthesis, it may contribute to lower ROS production during heat stress and thereby participate in the heat tolerance of SS C. proliferum.
In Marinobacter sp., a missense mutation affected gspE, which encodes an ATPase involved in the T2SS.As is the case with most secretion systems, the T2SS is required for the virulence of a variety of pathogens (59), as well as the colonization of the leech gut by the mutualistic symbiont Aeromonas veronii (60).Additionally, a frameshift mutation affected pilO, while a mutation in pilM resulted in the loss of a stop codon; both genes are involved in the type IV pilus assembly.Type IV pili are involved in the formation of biofilms but also support bacterial motility and may play a role in host colonization (61).
In all three cases, the detected mutations in response to heat selection affected genes potentially involved in host-symbiont interactions, such as host infection or symbiosis establishment.Whether they enhance or are detrimental to these interactions remains unknown, and functional studies will be needed to uncover the phenotypic effects of these mutations.

Conclusion
We provide the first extensive genomic analysis of Symbiodiniaceae-associated bacteria, by presenting the genomes of 49 bacteria isolated from six Symbiodiniaceae genera.Most bacteria have the potential to be beneficial for their hosts, through the production of B vitamins or antioxidants.Halomonas sp.MMSF_3323 is particularly interesting as it was predicted to synthesize seven essential B vitamins.Isolates of the Muricauda genus may also be crucial, as they were the only bacteria in our data set that were able to produce antioxidant carotenoids.Marinobacter sp. are also likely important for Symbiodi niaceae health as they can produce four B vitamins, as well as many ROS-scavenging compounds, such as ubiquinone and glutathione.It is noteworthy that the functional potential of these bacteria was independent of their Symbiodiniaceae host.Given that these Symbiodiniaceae were initially obtained from corals, clams, and anemones, it is unclear whether the bacteria analyzed here would interact directly with an animal host in hospite, or rather only with Symbiodiniaceae.Genomic analyses of Symbiodinia ceae-associated bacteria in hospite are therefore needed to assess potential lifestyle differences.
Finally, we analyzed the effect of heat selection on genome evolution in four bacterial species.In Muricauda sp., Marinobacter sp., and Mameliella sp., mutations observed in bacteria isolated from heat-evolved Symbiodiniaceae were observed in genes poten tially involved in host-symbiont interactions.While the effect of the mutations was not investigated, it is possible that future climate conditions will reinforce or damage interactions between bacteria and Symbiodiniaceae.Whether this may affect coral climate resilience remains to be investigated.More extensive genomic comparisons, using long-read sequencing for increased accuracy, are needed to fully understand the response of Symbiodiniaceae-associated bacteria to host heat selection.Overall, our study reveals the beneficial potential of Symbiodiniaceae-associated bacteria.

Cultures of bacteria from three C. proliferum strains
Three C. proliferum cultures were selected to isolate pure bacteria from: SCF055-01.10(WT10), SCF055-01.03(SS03), and SCF055-01.08(SS08).These three C. proliferum strains originated from a monoclonal culture isolated from Acropora tenuis (62).SS03 and SS08 have been evolving in the laboratory after ratcheting to 31°C since 2011, while WT10 was kept at 27°C.At the time of sampling for this study (August-October 2022), cultures were experimentally evolved for an estimated 300-330 generations (based on the generation time of the WT cultures).Symbiodiniaceae cultures were maintained in 15 mL of Daigo's IMK medium (1X), prepared with filtered Red Sea Saltwater (fRSSW, 34 ppt) in sterile 50 mL polypropylene culture flasks where the media was changed fortnightly.These flasks were kept in a 12 h light:12 h dark incubator with lighting at 50-60 µmol pho tons m −2 s −1 of photosynthetically active radiation (Taiwan Hipoint Corporation, model 740FHC LED, light chambers) and temperature at either 27°C (WT) or 31°C (SS).
Symbiodiniaceae cultures cell counts were quantified (Life Technologies Countess II FL) and an aliquot of 10 6 cells was deposited in a 5 µM mesh size strainer (pluriSelect, Germany) to separate the Symbiodiniaceae (ranging from 6 to 9 μm in diameter) and their closely associated (physically attached/intracellular) bacterial cells from loosely associated (planktonic) bacterial cells.Strainers were sealed with parafilm and centri fuged at 10,000 × g for 3 min.A volume of 500 µL of fRSS was added to the strainers, which were sealed and centrifuged at 10,000 × g for 3 min.Symbiodiniaceae cells and their associated bacteria were resuspended from the filter in 1 mL of fRSS.For each culture, one aliquot was not processed any further and one other aliquot was beadbeaten with 100 mg of sterile glass beads (400-600 nm) for 30 s at 30 Hz in a Tissue-lyser II (Qiagen, Germany), in order to open up Symbiodiniaceae cells and release intracellular bacteria to facilitate their growth on agar plates.Both aliquots were serially diluted and spread onto BD Difco Marine Agar 2216 (MA) and three Oxoid R2A Agar (R2A) prepared with fRSS, culture plates.Plates were incubated at 27°C (for the WT10 samples) or at 31°C (SS03 and SS08 samples) for 7 d to facilitate bacterial colony growth.Representatives of all morphologies were subcultured to attain cultural purity.Pure cultures were stored in sterile 40% glycerol at −80°C.

16S rRNA gene sequencing from cultured bacteria
Individual, pure freshly grown bacterial colonies were suspended in 20 µL of sterile Milli-Q water, incubated for 10 min at 95°C then used as templates in PCRs.PCR amplification of the bacterial 16S rRNA gene was performed with primers 27F and 1492R (63) in reactions containing 2 µL template DNA, 1 µL of each primer at a 10 µM concentration (0.25 µM final concentration), 20 µL of 2X Mango Mix (Bioline 25034), and 16 µL of PCR grade water.The amplification cycle was: 5 min at 94°C; 30 cycles of 60 s at 94°C, 45 s at 50°C, and 90 s at 72°C; 10 min at 72°C; with a final holding temperature of 4°C.The PCR products were purified Sanger sequenced with primer 1492R at Macrogen (South Korea).Raw sequences were trimmed and proofread in Geneious Prime v2019.1.3.Sequences were analyzed in BLAST.For each sequence, the closest hit and percent identity were recorded in Table S1.

DNA extraction and whole-genome sequencing
For each isolate selected for whole-genome sequencing, a single colony was picked with an inoculation loop and snap-frozen.DNA extraction was performed on the QIAsymphony using the DSP Virus/Pathogen Mini Kit (Qiagen).Library preparation was performed using Nextera XT (Illumina Inc.) according to the manufacturer's instructions.Whole-genome sequencing was performed on NextSeq 500/550 with a 150-bp PE kit.
SNP detection between WT and SS samples was then performed using snippy v4.6.0 (83), where both WT and SS samples were inputted as the reference genome in turn.Variants were then selected if they were found in both reciprocal snippy analyses.

FIG 1
FIG 1 Maximum-likelihood phylogenetic tree of the 49 pure-cultured Symbiodiniaceae-associated bacteria based on 120 marker genes (GTDB-Tk).The 49 isolates were assigned to 16 bacterial genera, as stipulated on the right-hand side.Bootstrap support values based on 1,000 replications are provided.

FIG 2
FIG 2 Metabolic potential of 49 Symbiodiniaceae-associated bacteria.The completeness of metabolic pathways (KEGG module database) of interest was annotated using METABOLIC-G and pathway completeness (in %) was estimated in EnrichM.

FIG 3
FIG 3 Putatively beneficial functions in Symbiodiniaceae-associated bacterial genomes.Protein families (Pfam) IDs were retrieved in each genome based on an InterProScan annotation.SeeTable 1 for full details on each Pfam ID.DMSP, dimethylsulfonioproprionate; DMS, dimethylsulfate; ROS, reactive oxygen species.

TABLE 1
Genes and respective protein families (Pfam) involved for which Symbiodiniaceae-associated bacterial genomes were screened a a DMSP, dimethylsulfonioproprionate; DMS, dimethylsulfate; DMSO, dimethylsulfoxide; ROS, reactive oxygen species; SWEET, sugars will eventually be exported transporter.