Integrated proteome and pangenome analysis revealed the variation of microalga Isochrysis galbana and associated bacterial community to 2,6-Di- tert-butyl-p-cresol (BHT) stress

doi:10.21203/rs.3.rs-4932929/v1

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Integrated proteome and pangenome analysis revealed the variation of microalga Isochrysis galbana and associated bacterial community to 2,6-Di- tert-butyl-p-cresol (BHT) stress

https://doi.org/10.21203/rs.3.rs-4932929/v1

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published 24 Oct, 2024

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The phenolic antioxidant 2,6-Di-tert-butyl-p-cresol (BHT) has been detected in various environments and is considered a potential threat to aquatic organisms. Algal-bacterial interactions are crucial for maintaining ecosystem balance and elemental cycling, but their response to BHT remains to be investigated. This study analyzed the physiological and biochemical responses of the microalga Isochrysis galbana and the changes of associated bacterial communities under different concentrations of BHT stress. Results showed that the biomass of I. galbana exhibited a decreasing trend with increasing BHT concentrations up to 40 mg/L. The reduction in chlorophyll, carotenoid, and soluble protein content of microalgal cells was also observed under BHT stress. The production of malondialdehyde and the activities of superoxide dismutase, peroxidase, and catalase were further determined. Scanning electron microscopy analysis revealed that BHT caused surface rupture of the algal cells and loss of intracellular nutrients. Proteomic analysis demonstrated the upregulation of photosynthesis and citric acid cycle pathways as a response to BHT stress. Additionally, BHT significantly increased the relative abundance of specific bacteria in the phycosphere, including Marivita, Halomonas, Marinobacter, and Alteromonas. Further experiments confirmed that these bacteria had the ability to utilize BHT as the sole carbon resource for growth, and genes related to the degradation of phenolic compounds were detected through pangenome analysis.

Isochrysis galbana

6-Di-tert-butyl-p-cresol

Proteomics

Pangenome༛Phycosphere bacteria

Microalgae are the important producers in aquatic ecosystems and contribute to approximately half of the globe’s primary productivity (Lu et al. 2021b). Phycosphere is a diffusion-limited layer which was enriched in amino acids, small molecule compounds and complex macromolecules secreted by microalgae (Shibl et al. 2020). Phycosphere bacteria can receive these various organic compounds as the energy source and enhance microalgal growth by removing excess O₂ and producing CO₂, as well as releasing inorganic salts, vitamins, trace elements, and growth promoters (Kamalanathan et al. 2019; Seymour et al. 2017). Therefore, the coexistence of algae and bacteria in aquatic environments plays a significant role in maintaining the biogeochemical cycles of elements, such as carbon, nitrogen, sulfur, and phosphorus (Cirri and Pohnert, 2019; Pomeroy et al. 2007). However, the rapid development of modern industrial production led to the discharge of industrial and domestic pollutants into water bodies. Aquatic environmental toxicology studies have found that the long-term presence of pollutants in water bodies poses a serious threat to the survival of aquatic organisms and health of people (Qu et al. 2020; Xiao et al. 2022). In aquatic habitats, the effect of pollutants on the microalgae growth and the dynamic changes of phycosphere bacterial communities have attracted more attentions.

Synthetic phenolic antioxidants (SPAs) are a family of low-cost, high efficiency, and excellent antioxidants, and have been widely used in the many of foods and industrial field (Du et al. 2023; Xu et al. 2021). SPAs have been detected in dust, rivers, seabed sediments, soil, and wastewater treatment plants, especially in water bodies (Liu et al. 2017; Ji et al. 2023; Liu and Mabury, 2018). Studies found that nine kinds of SPAs were in the surface sediments of northern coastal regions of China (Wang et al. 2018). Further researches proved that SPAs can continuously accumulate in aquatic organisms and even in humans through the food chain. SPAs have been detected in human urine, serum, and the daily intake of SPAs through mollusks has exceeded 600 ng/kg (Murawski et al. 2021; Liu et al. 2018). Food contact materials release chemical substances into food, and the migration of SPAs occurred under certain conditions (Wang et al. 2021). Moreover, the vitro and vivo toxicity of SPAs demonstrated that these compounds have an obvious estrogen effect and exhibited teratogenic and carcinogenic effects at high concentrations (Wang et al. 2021; Fries and Püttmann, 2004). The most widely used SPA is 2,6-di-tert-butyl-p-cresol (BHT) which has been listed by the European Union as a ‘high production volume chemical’ with global production exceeding 60,000 tons in 2000 (Lanigan et al. 2002). BHT undergoes a series of physicochemical reactions in the natural environment and its metabolites have been detected in various aquatic system (Sarmah et al. 2020). Due to the widespread distribution and characteristics of BHT, potential effects of BHT on the aquatic organisms are worthy of attention.

In recent years, more studies have been conducted to analyze the potential ecological risks of pollutants to algae and phycosphere bacteria (You et al. 2021; Lu et al. 2021a). Prior researches have demonstrated that the changes in algal biomass, cell structure, reactive oxygen species balance and metabolism progress were caused by various pollutants. Azoxystrobin was found to inhibit the growth of Chlorella pyrenoidosa by inducing oxidative damage and perturbing amino acid metabolism, ascorbate synthesis, fatty acid metabolism, and RNA translation (Lu et al. 2018). In addition, the presence of pollutants also led to significant changes in the phycosphere bacterial communities (Xiao et al. 2023). Generally, phycosphere bacteria have different sensitivities to changes in environmental factors, resulting in various responses. Following exposure to antibiotics of tetracycline and ciprofloxacin, the growth of Chlorella vulgaris was obviously inhibited. Simultaneously, a relative abundance decreasing of α-proteobacteria and Bacteroidetes was observed, while Cyanobacteria, β- and γ-proteobacteria increased in response to antibiotic exposure in the phycosphere bacterial community (Xue et al. 2023). However, the responses mechanism of microalgae and phycosphere bacteria in the phycosphere to BHT are poorly understood.

Pan-genomics is emerging as a powerful computational paradigm in bioinformatics and classifies the complete set of genes in a microbial community, such as different strains within the same genus or species, including core genes, dispensable genes, and strain-specific genes (Tettelin et al. 2005). Dispensable genes and strain-specific genes may confer specific survival advantages to microorganisms, such as ecological adaptation, virulence mechanisms, antibiotic resistance, etc. Therefore, pan-genomic provides crucial data for discovering individual-specific microbial genomes and offer key molecular targets for molecular identification across different lineages (Zhong et al. 2021; Zhong et al. 2018). By comparing the diversity and differences in genomic composition among different strains of the same microorganism, pan-genomics analysis displays an effective means to explore the environmental adaptive mechanisms of bacteria within microbial environmental communities at the gene level (Sibbesen et al. 2023).

In this study, the changes in biomass, chlorophyll a, carotenoids, and soluble protein content of microalga Isochrysis galbana under different concentrations of BHT stress were determined. Proteomics technology was employed to investigated the response mechanism of microalgal cells under the BHT exposure. Phycosphere bacterial communities change at the phylum and genus levels were explored with high-throughput sequencing. Pan-genomics analysis was executed to investigate the molecular mechanisms of BHT-tolerance bacteria by identifying the genes related to phenol and BHT degradation.

Culture and growth inhibition test

Microalgae strain of I. galbana, receiving from Institute of Oceanography, Chinese Academy of Sciences, was inoculated into f/2 liquid medium. Microalgal cells were placed in a light incubator at 25 ± 1 ℃ with a light intensity of 3800 Lux and a light/dark cycle of 12/12 h for cultivation. The conical flasks were shaken three times every day. The growth status of I. galbana was measured using a visible spectrophotometer under the optical density (OD) value of 680 nm. BHT mother solution was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1 g/L. To investigate the effect of BHT on microalgae, the exposure time was 8 days and the concentration gradient of BHT for subsequent experiments was 0, 10, 20, and 40 mg/L. Three parallel samples were set up for each group of experiments.

Photosynthetic pigment and soluble protein determination

The content of chlorophyll a and carotenoid were determined after an 8-day cultivation according to the previous study. 10 mL of algal solution was collected and centrifuged at 8,000 rpm for 10 min. The supernatant was discarded and algal cells were resuspended in 10 mL of 95% ethanol solution. The absorbance of the supernatants was measured at wavelengths of 440 nm, 645 nm, and 663 nm using 95% ethanol as the blank. The pigment content was calculated according to following formulas:

$$\:\text{c}\text{h}\text{l}\text{o}\text{r}\text{o}\text{p}\text{h}\text{y}\text{l}\text{l}\:\text{a}\:\left(\text{mg/L}\right)\text{=2.7×}{\text{OD}}_{\text{663}}\text{-2.69×}{\text{OD}}_{\text{645}}\:（2-1）\:\text{c}\text{h}\text{l}\text{o}\text{r}\text{o}\text{p}\text{h}\text{y}\text{l}\text{l}\text{b}\left(\text{mg/L}\right)\text{=2}\text{2}\text{.}\text{9}\text{×}{\text{OD}}_{\text{6}\text{45}}\text{-}\text{4}\text{.6}\text{8}\text{×}{\text{OD}}_{\text{6}\text{63}}\:（2-2）$$

$$\:\text{c}\text{a}\text{r}\text{o}\text{t}\text{e}\text{n}\text{o}\text{i}\text{d}\:\left(\text{mg/L}\right)\text{=}\text{4.7}\text{×}{\text{OD}}_{\text{440}}\text{-}\text{0.27}\text{×}\text{(}\text{\:}\text{chlorophyll\:}\text{a}\text{-}\text{\:}\text{chlorophyll\:}\text{b}\text{)}$$

2-3

For soluble protein analysis, algal cells were collected by centrifugation and resuspend with phosphate buffer (PBS, pH 7.8). The cells were then lysed by sonication for 5 min, followed by centrifugation at 4,000 rpm for 20 min to retain the crude enzyme solution. Coomassie Brilliant Blue solution was used to analyze content of soluble protein of I. galbana under the absorbance at 595 nm.

Oxidative stress analysis

Malondialdehyde (MDA) is an indicative of biological membranes peroxidation which was usually caused by ROS. MDA content and antioxidant enzyme activities were analyzed with the supernatant extracted from the I. galbana cells. After extraction with 5% (w/v) trichloroacetic acid, the MDA content was measured as an indicator of lipid peroxidation, following by thiobarbituric acid fluorescence assay (Xiao et al. 2023). Activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) in the supernatant was assayed according to a previously described method (Xiao et al. 2023).

Scanning electron microscope (SEM) analysis

To explore the effect of BHT on the microalgae, a JEOL JSM-6700F scanning electron microscope (SEM) instrument was used to analyze the microstructure change of the I. galbana cells under the 20 mg/L and 40 mg/L BHT exposure. Algal cell pellets were obtained by centrifugation and resuspend in 2.5% glutaraldehyde solution. After refrigerating for 12 h, algal cells were washed three times with PBS (pH 7.8) buffer and then dehydrated by sequentially adding 30%, 50%, 70%, and 90% ethanol solutions. Algal cells were used to vacuum freeze-drying and their morphological structure was observed at an acceleration voltage of 25 kV.

Proteomics analysis

I. galbana cells in different concentration of BHT groups were extracted by sonication in the lysis buffer and centrifugation at 8,000 rpm for 20 min. The collected supernatants were frozen at -80°C with liquid nitrogen for further processing. Total protein concentration was quantified by the Bradford method and the absorbance at 562 nm using a SpectraMax microplate reader. Total protein extraction was prepared and digested into peptides which were labeled with TMT reagent. Proteomics sequencing was completed by Shanghai Meiji Biomedical Technology Co., Ltd (Shanghai, China). Student’s t-test was used to analyze the differentially expressed proteins (DEPs), according to the following standards: |log2FC|≥1 and p-value < 0.05. Gene ontology (http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) database were used for functional annotation and metabolic pathways analysis of DEPs.

DNA extraction and High-throughput sequencing

The algal solution treated with 0, 20 mg/L, and 40 mg/L of BHT was filtered through a 0.22 µm membrane. Total bacterial DNA was extracted and purified using a column bacterial DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.) following the standard operating procedure. High-throughput sequencing of the 16S rRNA gene was performed by Shanghai Meiji Biomedical Technology Co., Ltd (Shanghai, China). Trimmomatic software (version 0.35) was utilized to trim raw data sequences using a sliding window approach, cutting off bases. The FLASH software was used to merge the qualified paired-end raw data with a maximum overlap of 200 bp. The split libraries function of QIIME removed sequences containing N bases and UCHIME software was employed to eliminate chimeras from the clean tags for OTU clustering. Based on the standardized OTU and species annotation information provided by the sequencing company, OTUs were clustered at 97% similarity using the RDP Bayesian algorithm on the QIIME (version 1.9.1) platform, and sequences were compared with those in the Greengene database (version 13.8). Microbial α-diversity indices at different sequencing depths for each sample were calculated using Mothur software, including the Shannon, Chao1, and Simpson index.

Isolation and determination of phycosphere bacteria

The dilution separation method was used to purify the phycosphere bacteria and 2216E agar medium was employed for bacteria cells culture. The plates containing bacteria were placed in a biochemical incubator at 28°C for culture of 3–4 days. Bacterial genomic DNA was extracted using a genomic DNA kit and used as a template for PCR-based bacterial identification, with the PCR primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1541R (5’-AAGGAGGTGATCCACCC-3’). PCR amplification products were checked by 1% agarose gel electrophoresis and sequenced. A BLAST search is performed for DNA sequence comparison and the phylogenetic evolutionary tree of the phycosphere bacteria is constructed using MEGA 6.0 software.

BHT tolerance analysis of phycosphere bacteria

BHT tolerance of eight different phycosphere bacteria strains were executed. BHT with concentration of 20 and 40 mg/L solved with the flasks containing 50 mL of filtrated seawater and 200 µL bacterial solution into the flasks, cultures were incubated at 28°C for 5 days with agitation of 150 rpm. After every 24h, cultures were collected and OD₆₀₀ was measured to reflect the bacteria growth. In parallel, a bacterial culture with the same volume of sterile seawater served as the control group.

Pangenome analysis

The genomic sequencing data used in this study were downloaded from the NCBI database and the detailed information was listed in the Fig. S1. Pangenome analysis of strains affiliated to genera Marivita, Halomonas, Marinobacter, Alteromonas, Microbacterium and Planococcus were performed with selected 35, 30, 30, 24, 11 and 23 genomes using the Bacterial Pan Genome Analysis (BPGA) software and Integrated Prokaryotes Genome and pangenome Analysis (IPGA) website (https://nmdc.cn/ipga/), respectively (Chaudhari et al. 2016; Liu er al. 2022) (Fig. S1). To depict the pangenome (core and accessory genome) assessment of strains included in the comparison, a reciprocal best hit search was also performed.

Statistical Analysis

Data processing was executed with GraphPad Prism 8.0 software and expressed as mean ± standard deviation. LSD analysis of variance was used for statistical analysis, and p < 0.05 was considered significant.

I. galbana growth analysis

The study revealed a growth inhibitory effect of BHT on I. galbana during an 8-day cultivation period. Figure 1a demonstrated that the cell growth of I. galbana was notably inhibited with increasing BHT concentrations. At the 8th day, when the exposure concentrations of BHT were 5 and 40 mg/L, the biomass of microalgae decreased by 13.62% and 25.08% compared to the control group, respectively. Figure 1b-d illustrated that the chlorophylls a, carotenoid, and soluble protein contents of I. galbana exhibited a decreasing trend as the concentration of BHT increased during the different exposure periods. The results indicate that the increase in BHT concentration not only impacted microalgal biomass accumulation but also suppressed the synthesis of soluble proteins. Effect of BHT on oxidative stress

Figure 2a showed that different concentrations of BHT induced an excessive amount of reactive oxygen species (ROS) in I. galbana cells at the 4th and 8th day. For instance, malondialdehyde (MDA) levels under 5 mg/L BHT increased by 21.4% and 27.2% on the 4th and 8th day compared to the control group, respectively. The variation in MDA content suggested that BHT caused oxidative stress in I. galbana. As depicted in Fig. 2b-d, the activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) significantly increased in the BHT-treatment group compared with the control experiment group on the 4th day. On the 8th day, no obvious variations in SOD, CAT, and POD enzymatic activity were observed between the control and 20 mg/L BHT-treatment groups. Although a similar change between the control and the 40 mg/L BHT-treatment group in CAT activity was not obvious, 40 mg/L BHT significantly facilitated the production of SOD and POD, which was helpful for eliminating excess ROS. This result suggested that the presence of the three antioxidant defense enzymes had a significant effect on reducing oxidative stress in I. galbana algal cells during short-term BHT exposure. However, SOD and POD played an important role in scavenging ROS when I. galbana algal cells were exposed for longer periods and at higher BHT concentrations.

SEM analysis

As depicted in Fig. 3a, the cells of I. galbana without BHT treatment exhibited an intact structure with a clean and smooth surface. In contrast, Fig. 3b and c show that I. galbana cells with BHT treatment displayed coarse, swollen surfaces and were attached with particles. At a BHT concentration of 40 mg/L, cellular rupture was observed in the I. galbana, indicating that BHT induced intracellular nutrient leakage.

Comparative proteomics analysis

In order to investigate the molecular mechanisms related to different concentrations of BHT stress in I. galbana, comparative proteomics were performed on algae treated with 0, 20, and 40 mg/L BHT, respectively. The volcano plots for differentially expressed proteins (DEPs) of each comparison group are illustrated in Fig. 4a and b. In comparison to the control group, 63 and 68 DEPs were identified in the 20 and 40 mg/L BHT-treatment groups, respectively. Specifically, 40 upregulated and 23 downregulated proteins were identified between the 20 mg/L BHT-treatment group versus the control, while 38 upregulated and 31 downregulated proteins were found between the 40 mg/L BHT-treatment group versus the control. The GO and KEGG functional annotations showed that the DEPs were allocated into 17 and 13 different pathways at 40 mg/L BHT-treatment versus the control, respectively (Fig. 4c and d). These pathways include alanine, aspartate, glutamate metabolism, the citric acid (TCA) cycle, oxidative phosphorylation, and nitrogen metabolism. Proteins associated with nitrogen metabolism and the TCA cycle were all upregulated, whereas those in the phagosome were predominantly downregulated. Furthermore, a majority of the proteins in oxidative phosphorylation were upregulated.

Diversity Analysis of Bacterial Communities

The filtered raw data from Illumina sequencing yielded 224,762 effective representative bacterial sequences across 60 OTUs, categorized into 6 phyla, 10 classes, 23 orders, 29 families, 45 genera, and 51 species in the samples. A Venn diagram was employed to demonstrate the distribution and relationship of differential species between groups (Fig. 5a). It showed that control and 20 mg/L BHT-treatment group shared 10 species, control and 40 mg/L BHT-treatment shared 10 species, 20 mg/L BHT-treatment and 40 mg/L BHT-treatment shared 12 species, and 7 species were shared by all samples. α-Diversity analysis presented in Fig. 5b was used to assesses the richness and diversity of phycosphere microbial communities. The Sobs, Ace, and Chao indices reveal that community abundance under BHT stress (20 mg/L and 40 mg/L) is significantly greater than that of the control group, indicating increased species richness. The Shannon index correlates positively with BHT concentration, while the Simpson index correlates negatively, suggesting that BHT in the algal environment has modified microbial diversity, leading to significant shifts in the microbial community structure.

BHT-tolerance analysis of phycosphere bacteria

Total 12 different phycosphere bacteria, Planococcus sp. IGS-1, Exiguobacterium mexicanum sp. IGS-3, Bacillus megaterium sp. IGS-4, Bacillus megaterium sp. IGS-5, Halomonas cupida IGS-6, Halomonas sp. IGS-8, Halomonas qaidamensis IGS-9 Microbacterium paraoxydans sp. IGS-10, Alteromonas sp. IGS-15, Marinobacter sp. IGS-16, Sulfitobacter sp. IGS-18 and Marinobacter sp. IGS-21, were obtained from the culture solution of the I. galbana (Fig. S3a). Fig. S3b showed that all of bacterial cells in the various genus had a marked increase on the 72h culture period when different concentration of BHT was added into the culture. This result indicated that these bacteria can all utilize BHT as the sole carbon source or energy for growth. Among them, the biomass of Halomonas, Marinobacter and Alteromonas were significantly more pronounced, exceeding six-fold times that of the control group, while Planococcus sp. IGS-1 exhibited the lowest increase in biomass and only had a 1.35-fold times increasing compared to the control group.

Pangenomic analysis

To gain insight into the BHT metabolism response mechanism of phycosphere bacteria, the pangenomic data of six different bacteria taxa genus, including Marivita, Halomonas, Marinobacter, Alteromonas, Planococcus and Microbacterium, which had marked increasing in the relative abundance under the BHT stress, were analyzed. KEGG annotation indicated Halomonas had a higher proportion of genes involved in xenobiotic degradation pathways compared to other bacteria, indicating that it may have significant advantage in degrading xenobiotic substances (Fig. 6). It can be seen that most of genes locating in xenobiotic degradation pathways of Marinobacter were flexible gene components that were not necessary for some of these species. These newly obtained genes in flexible genome represented the capability of microorganisms to adapt to environmental changes and may greatly enhance the physiological and ecological adaptability.

Phenol and its derivatives can be degraded by a diverse range of bacteria which were classified predominantly into Gram-negative proteobacteria and Gram-positive actinobacteria (e.g., Rhodococcus, Arthrobacter). The individual phenolic compounds are transformed by peripheral pathways to a limited number of central intermediates that are further converted into intermediates of the citrate cycle via a few central pathways. Therefore, the genes involved in the phenol compound metabolism pathway in the Halomonas and Marinobacter was investigated through pangenome analysis. As shown in Fig. 7, phenol and its derivative BHT in Halomonas and Marinobacter was initiated by hydroxylation of the aromatic ring using phenol hydroxylase (pheA). The hydroxylated product catechol was then degraded by the enzyme catechol 1,2-dioxygenase (CatA) or protocatechuate 3,4-dioxygenase (proD) through ortho-cleavage pathway, or catechol 2,3-dioxygenase (dmpB) through meta-cleavage pathway. It is worth noting that both types of bacteria possess the cis, cis-muconate cycloisomerase (CatB), muconolactone delta-isomeras (CatC) and fatty acid desaturase (fadA), while the additional genes pcaG/pcaH and pcaB were only found in the Halomonas. This result indicated that Halomonas had two different ortho-cleavage pathways for phenol or BHT degradation, suggesting its strong phenol metabolism capability. In addition, only a very small number of genes dmpD, mhpD and mhpE were found, indicating that meta-cleavage pathway may not exist in these two bacteria. These research findings were consistent with previous studies and gained a deeper understanding at the genetic level of phenol metabolic pathways of different phycosphere bacteria.

This study has shown that algal growth was inhibited in 5–40 mg/L treatment before 8 days, and the decrease of photosynthetic pigment synthesis and soluble protein content in the I. galbana was also found. Studies have shown that phenolic substances exhibited inhibitory effects on the growth of the green alga Dunaliella salina and the diatom Skeletonema costatum, with 96-h EC₅₀ values of 72.29 and 27.32 mg/L, respectively, at the sublethal concentrations. Another study has found that high concentrations of paraquat and sodium bis(2-ethylhexyl) sulfosuccinate can reduce the soluble protein content and inhibited the growth of Chlorella pyrenoidosa in the single-species toxicity and combined toxicity assays (Shen et al., 2019). SEM analysis showed BHT can be absorbed by the binding site of cellular surface and caused intracellular nutrient leakage of algal cells. The absorption of the phenol compound on the binding sites of the algal cells has been observed in other algae, which is consistent with the findings of this study (Fawzy and Alharthi, 2021; Saravanan et al., 2021).

Excess production of MDA induced by BHT was observed and antioxidant defense enzymes SOD and POD played an important role in the ROS scavenging during the various exposure time. Proteomic analysis revealed that upregulation of photosynthesis, oxidative phosphorylation and TCA pathways as the response to the BHT stress. Photosynthesis is the most extensive process of biological carbon fixation with the light reactions and an essential step for organismal growth. Photosystem II (PS II), situated within the thylakoid membrane and comprised of multiprotein complexes, is a photosynthetic unit crucial for phototrophic organisms. It can utilize light energy to split water molecules, providing electrons for the reduction of NADP⁺ in plastoquinone or for non-cyclic photophosphorylation. The core component D1 protein encoded by PsbA gene could receive excitation energy from photons and drive redox reactions in the oxygen evolving complex to the cytochrome b6/f complex. D1 protein has a high sensitivity to ROS and is easily damaged by oxidative when cellular ROS levels are excessive (Huo et al., 2016). Under BHT stress, the increased ROS in the chloroplasts of I. galbana led to acceleration degradation of D1 protein.

Cytochrome b-559 in Photosystem II is composed of subunit α encoded by PsbE and subunit β encoded by PsbF in the chloroplast. PsbE, PsbF, PsbL, and PsbJ genes form an operon, with the removal of the entire operon or individual PsbE (F) genes leading to destabilization of the complex Photosystem II reaction center (Shinopoulos and Brudvig, 2012). Under BHT stress, the rise in ROS levels caused the upregulation of PsbE, which possesses superoxide dismutase activity, to mitigate the destructive impact of ROS. Photosystem I (PS I) is a multiprotein complex on the thylakoid membrane of algae, functioning as a light-driven plastocyanin: ferredoxin oxidoreductase, containing five chloroplast-encoded subunits and six nuclear-encoded peptides. The largest subunits, encoded by chloroplast PsaA and PsaB, are integral to the reaction center, essential for the complex's stable assembly (Bateman and Purton, 2000). BHT stress partially disrupted the stable assembly of I. galbana photosynthetic systems, resulting in the downregulation of PsaB in PS I (Fig. S2a). Oxidants can denature [4Fe-4S] centers, with superoxide proven to deactivate several [4Fe-4S]-containing enzymes (Fischer et al., 1997). As BHT induced excess production of ROS which led to the denaturation and inactivation of enzymes related to the terminal centers, PsaC was upregulated to preserve PS I electron transfer stability.

TCA cycle is an essential metabolic system that generates a significant amount of free energy, producing ATP through the electron transport chain and oxidative phosphorylation to supply energy for cellular physiological functions. TCA cycle powers the antioxidant system, with aconitase serving as a site of action for ROS and isocitrate dehydrogenase regulating the activity of thiol-dependent and glutathione antioxidant systems. Acetyl-coenzyme A synthetase and hydrolase participate in reducing ROS toxicity, making the TCA cycle a vital locus for ROS homeostasis. The bifunctional enzyme aconitate hydratase 2/2-methylisocitrate dehydrogenase (acnB), an iron-sulfur (4Fe-4S) protein, is the main enzyme catalyzing citrate to isocitrate conversion through cis-aconitate and plays an important role in the TCA cycle metabolic pathway (Weber et al., 2022). The instability of acnB indicates its suitability as a primary metabolic enzyme, swiftly modulating energy metabolism in response to oxidative or pH stress. The presence of BHT led to increased intracellular ROS, causing oxidative stress, and stimulates the upregulation of acnB in response to the levels of intracellular O²⁻. Succinate dehydrogenase, a critical enzyme in the TCA cycle, catalyzes the conversion of succinate to fumarate and is pivotal for intermediary metabolism and aerobic energy production in living cells (Kregiel, 2012). Its regulation significantly influences succinate levels in the TCA cycle, affecting the rate of the TCA and glyoxylate cycles, and consequently the growth metabolism and protein synthesis in microalgae. Thus, the upregulation of succinate dehydrogenase provides necessary reductive equivalents to mitigate both exogenously induced by BHT and respiratory chain-derived superoxide anions.

Oxidative phosphorylation, occurring in the mitochondrial inner membrane, synthesizes ATP from the energy derived from fatty acids, sugars, and other substrates, with its complexes oxidizing NADH to supply electron acceptors for metabolic processes such as the TCA cycle and glycolysis (Fig. S2b and c) (Wilson, 2017). The homeostasis of oxidative phosphorylation is vital for various intracellular activities. Ubiquinone oxidoreductase (Complex I), the largest multimeric complex in the electron transport chain (ETC), catalyzes the initial step of mitochondrial oxidative phosphorylation (OXPHOS). The activity of Complex I is linked to three primary core subunits: NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFs2), NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFs7), and NADH dehydrogenase 1 (ND1), which transfer electrons from NADH to ubiquinone via flavin mononucleotide (FMN) and iron-sulfur clusters (He et al., 2024). BHT-induced ROS elevation could inactivate terminal iron-sulfur cluster enzymes. To preserve the stability of the electron transport system, NDUFs2 and NDUFs7 are upregulated. Cox1 contains a low-spin heme a site and a binuclear center composed of high-spin heme a3 and a copper atom (CuB). Cox2 interacts with cyt c and includes a mixed-valence binuclear copper center (CuA). The four-electron oxygen reduction process begins at the CuA site in Cox, proceeds to heme a, and concludes at the binuclear active site where O₂ is bound and reduced. The effective assembly of the Cox1 subunit, containing heme a, can prevent oxidative stress (Je et al., 2021). With BHT stress elevating ROS levels, Cox1 is upregulated to manage oxidative stress.

Additionally, BHT obviously increased the relative abundance of BHT tolerance bacteria in the phycosphere with Illumina sequencing, including Marivita, Halomonas, Marinobacter, and Alteromonas. The relative abundance of microbial communities at the phylum level showed that Proteobacteria had the highest relative abundance in the all samples. Research found that Proteobacteria were most abundant in other I. galbana, with relative abundance of bacteria affiliated with α-proteobacteria and γ-proteobacteria accounted for a predominant proportion in I. galbana 3009, I. galbana 3010 and I. galbana 3011, consistent with the conclusions of this experiment (Ling et al., 2020). Figure 5c revealed the significant differences in the structure of bacterial communities at the genus level. The primary bacterial genera contained Marivita, Halomonas, Oceanicaulis, Marinobacter, Alteromonas, Roseitalea and unclassified Rhodobacteraceae. It can be seen that the relative abundance of the genus Marivita did not change significantly under 20 mg/L BHT concentration but increased by 18.46% under high BHT concentration. Marivita, as the most abundant genus within the Rhodobacteraceae family, is widely distributed in the phycosphere. Marivita can absorb and metabolize organic compounds containing carbon, nitrogen, and phosphorus in the water, and possess the ability to degrade organic pollutants (Wei et al., 2023). The genus Halomonas, belonging to the abundantly enriched Proteobacteria, increased by 21.37% and 0.92% under 20 and 40 mg/L BHT concentrations, respectively. Compared to the control group, the relative abundances of the genera Oceanicaulis, Marinobacter and Alteromonas significantly increased under BHT stress, while Roseitalea and unclassified Rhodobacteraceae decreased.

Our experimental results revealed that Halomonas exhibited the highest multiplication in growth compared to other bacteria under the presence of BHT. Pangenome analysis demonstrated that Halomonas had a higher proportion of genes involved in xenobiotic degradation pathways compared to other bacteria and two different ortho-cleavage pathways for phenol or BHT degradation which was helpful for BHT or phenol metabolism capability. Studies reported that Halomonas sp. can degrade various organic pollutants such as polycyclic aromatic hydrocarbons, phenols, and hydrocarbons in crude oil (Peng et al., 2020). The presence of genes encoding ring-cleavage enzymes in the β-ketoadipate pathway in Halomonas sp. FP35T indicated the ability to metabolize aromatic compounds and exhibited chemotaxis towards environmental pollutants phenol (100-1,000 ppm) and naphthalene (100–500 ppm) (Tena-Garitaonaindia et al., 2019). Halomonas sp. PH2-2 degraded phenol at concentrations as high as 1,100 mg/L within 168 h (Haddadi, 2013). Marinobacter is crucial in the aerobic degradation of various polycyclic aromatic hydrocarbons and alkanes in the organic carbon cycle on environmentally polluted areas (Matturro et al., 2023). It was found that Marinobacter sp. used biphenyl as the sole carbon source on agar plates, and after a 250-d of aerobic culture, there was a significant 28.7% reduction in polycyclic aromatic hydrocarbons in marine sediments (Rani et al., 2017). Marinobacter sp. SJ18 completely degraded cyclohexanecarboxylate within 60 h and 84 h under aerobic and anaerobic conditions, respectively (Zan et al., 2023). Therefore, the increase in relative abundance of BHT-tolerance bacteria in the phycosphere can reduce the stress of BHT on the I. galbana.

Author contributions

Linke Guo: Writing–original draft. Shuangwei Li, Dongle Cheng: Methodology. Xiao Lu and Xinying Gao: Conceptualization. Linlin Zhang and Jianjiang Lu : Writing–review & editing, Supervision.

Funding

This work was supported by the Taishan scholars Program of Shandong Province (tsqn202211157); Shandong Excellent Youth Science Fund Project (Overseas) (2023HWYQ-077).

Data availability

Data will be made available on request.

Conflict of interest The authors declare no competing interests.

Ethical approval Not applicable.

Supplementary data Supplementary data to this article can be found online at the website.

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Integrated proteome and pangenome analysis revealed the variation of microalga Isochrysis galbana and associated bacterial community to 2,6-Di- tert-butyl-p-cresol (BHT) stress

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Abstract

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Introduction

Methods and Materials

Culture and growth inhibition test

Photosynthetic pigment and soluble protein determination

Oxidative stress analysis

Scanning electron microscope (SEM) analysis

DNA extraction and High-throughput sequencing

Isolation and determination of phycosphere bacteria

BHT tolerance analysis of phycosphere bacteria

Pangenome analysis

Statistical Analysis

Results

SEM analysis

Comparative proteomics analysis

Diversity Analysis of Bacterial Communities

BHT-tolerance analysis of phycosphere bacteria

Pangenomic analysis

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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