Interactions between the nitrogen-fixing cyanobacterium Trichodesmium and siderophore-producing cyanobacterium Synechococcus under iron limitation

Abstract As diazotrophic cyanobacteria of tremendous biomass, Trichodesmium continuously provide a nitrogen source for carbon-fixing cyanobacteria and drive the generation of primary productivity in marine environments. However, ocean iron deficiencies limit growth and metabolism of Trichodesmium. Recent studies have shown the co-occurrence of Trichodesmium and siderophore-producing Synechococcus in iron-deficient oceans, but whether siderophores secreted by Synechococcus can be used by Trichodesmium to adapt to iron deficiency is not clear. We constructed a mutant Synechococcus strain unable to produce siderophores to explore this issue. Synechococcus filtrates with or without siderophores were added into a Trichodesmium microbial consortium consisting of Trichodesmium erythraeum IMS 101 as the dominant microbe with chronic iron deficiency. By analyzing the physiological phenotype, metagenome, and metatranscriptome, we investigated the interactions between the nitrogen-fixing cyanobacterium Tricodesmium and siderophore-producing cyanobacterium Synechococcus under conditions of iron deficiency. The results indicated that siderophores secreted by Synechococcus are likely to chelate with free iron in the culture medium of the Trichodesmium consortium, reducing the concentration of bioavailable iron and posing greater challenges to the absorption of iron by Trichodesmium. These findings revealed the characteristics of iron-competitive utilization between diazotrophic cyanobacteria and siderophore-producing cyanobacteria, as well as potential interactions, and provide a scientific basis for understanding the regulatory effects of nutrient limitation on marine primary productivity.


Introduction
Cyanobacteria are the oldest oxygen-releasing photosynthetic microorganisms on Earth and are at the foundation of all material and energy in the marine food chain [1,2].Cyanobacteria are dominated by Prochlorococcus and Synechococcus genera and are widespread in the ocean, contributing up to 50% of primary productivity in oligotrophic open ocean [3][4][5][6].Some cyanobacteria not only perform photosynthetic carbon fixation, but also biological nitrogen (N) fixation, thereby reducing inert atmospheric N 2 to ammoniacal nitrogen, which participate in the metabolism of countless organisms and provide a major N source in the oceans [6][7][8][9][10].
Trichodesmium organisms appear as typical diazotrophic cyanobacteria, with widespread occurrence in vast oligotrophic subtropical and tropical oceans, where they supply a considerable percentage of the bioavailable N input to the global ocean and are particularly important in oligotrophic ocean ecosystems [11][12][13][14].Cyanobacterial growth and metabolism are limited by various physical factors, such as temperature and light, and other than these two factors are mainly limited by the bioavailability of N, phosphorus (P), and iron (Fe) [15][16][17].Cyanobacteria may cooperate or compete with each other under nutrient limitation, but we know little about the interactions between different cyanobacteria [18][19][20].
The capability of diazotrophic cyanobacteria to fix atmospheric N 2 overcomes the scarcity of other inorganic N sources and is usually limited by the concentration of bioavailable P and Fe in the oligotrophic ocean [21][22][23][24][25][26].Trichodesmium has employed various strategies to meet Fe and P demands over the long period of its evolution [27][28][29][30].These strategies include modification of its metabolic and physiological characteristics, such as expressing phosphatases to cleave phosphate groups from organic P, using phosphite as a sole phosphorus source for growth, restructuring the proteome broadly, and reducing cell size to adapt to P limitation [22,29,31].Under Fe-deficient conditions, Trichodesmium cyanobacteria can regulate cellular Fe quotas, take up different Fe molecules, modulate genes related to Fe uptake, reduce trichome length and growth rates, and increase their surface area to become spherical, thus capturing Fe contained in dust to ameliorate Fe deficiency [27,[32][33][34][35][36].
Trichodesmium exists in the ocean as single filaments and macroscopic colonies formed by aggregating filaments (trichomes) [37].The colonies are usually symbiotic with heterotrophic epibionts and other microorganisms, including diatoms, proteobacteria, and other cyanobacteria [38][39][40].Nitrogen, vitamins, trace elements, and other metabolites are exchanged between symbionts and Trichodesmium, thus forming diverse interactions to optimize the growth of the entire microbial consortium and contribute to success in oligotrophic systems [41][42][43][44][45].For example, alkaline phosphatases and acylhomoserine lactones can be produced by Trichodesmium consortia members and ultimately assist Trichodesmium with P acquisition under Plimited conditions [46,47].Trichodesmium symbionts also assist in Fe uptake.According to recent reports, 23 bacterial strains symbiotic with Trichodesmium can produce siderophores in culture to enhance Fe mineral dissolution and bioavailability by assisting in Fe utilization from dust [48,49].However, genomic analyses indicate that Trichodesmium does not possess known pathways for siderophore synthesis but does have TonB-ExbB-ExbD transporter components that share homology with Synechocystis sp.PCC 6803 and provide the possibility of Fe uptake chelated by siderophores secreted by symbionts [18,50,51].Moreover, some symbionts of Trichodesmium can utilize more diverse Fe sources than Trichodesmium, and some of these symbionts produce siderophores with different chemical properties.It has been reported that these siderophores mainly include hydroxamates, phenolatescatecholates, carboxylates, and others, and they differ in structure, hydrophobicity, and Fe-binding affinity [52].Trichodesmium may differ in terms of acquisition strategies of siderophores with different chemistries [48,53].These findings collectively suggest more sophisticated interactions in Trichodesmium consortia than we were previously aware of, which provide mutual advantages for the entire consortium and deserve to be explored more deeply.
In the present study, based on metagenomic sequencing, various Synechococcus species that can produce synechobactin (a siderophore with Fe-binding functionalities and two αhydroxamate groups), were found to be symbionts of Trichodesmium that were originally isolated from the North Atlantic Ocean and cultured under Fe-deficient conditions in the laboratory.The interaction of Synechococcus with Trichodesmium under Fe deficiency, whether assisting each other or competing with each other and whether siderophores secreted by Synechococcus can be utilized by Trichodesmium, is not yet clear.Therefore, to investigate this issue in detail, a Synechococcus sp.PCC 7002 mutant lackingsiderophore biosynthesis capability was constructed to explore the interactions under Fe-deficient conditions between Synechococcus, which may or may not secrete siderophores, and Trichodesmium..
It has been well reported that the siderophore that Synechococcus sp.PCC 7002 produces is an amphiphilic synechobactin with a chemical structure that is already clear, consisting of two αhydroxamate groups, one of which contains a fully saturated fatty acid tail in order of decreasing hydrophobicity [52].The length of the fatty acid tail varies, with the most common C12, C10, and C8 varieties being named synechobactin A, B, and C, respectively [54].Previous studies have reported that a gene cluster of G0025 to G0018 is responsible for encoding a series of enzymes that catalyze the biosynthesis of siderophores [55].In this study we constructed and used a mutant by knocking out G0025 (sidA), G0024 (sidB), and G0023 (sidC).Except for the interaction between Trichodesmium and Synechococcus, the role of another major symbiont, Marinobacter, in the adaptation of Trichodesmium to Fe deficiency was also analyzed.We found that Trichodesmium Fe deficiency was aggravated after treatment with Synechococcus filtrate.Metagenomic and metatranscriptomic analyses have demonstrated that the Synechococcus filtrate containing siderophores is likely to inhibit the growth of Trichodesmium but promotes the growth of Marinobacter.Our results revealed a new pattern of Fe competitive utilization between Synechoccocus and Trichodesmium in Trichodesmium consortia.

Cyanobacteria culture and general methods
A Trichodesmium consortium originating from the North Atlantic Ocean was obtained from the laboratory of David A. Hutchins at the University of Southern California.This consortium consists of Trichodesmium erythraeum IMS 101 as the dominant microbe as well as Synechoccocus, Marinobacter, and some other microbes.Stationary culture of the Trichodesmium consortium was conducted in YBC-II medium with 250 nmol/L of Fe (Fe replete) or 5 nmol/L of Fe (Fe depleted) and semicontinuously grown at 28

Fe-deficient Trichodesmium treated with the filtrates secreted from Synechococcus wild type and Mut-G0023-25 mutant
The Trichodesmium consortium was semicontinuously cultured under Fe-depleted conditions (5 nmol/L Fe) for more than 2 months.The Mut-G0023-25 knockout mutant and wild-type Synechococcus sp.PCC 7002 were respectively cultured under Fe-depleted conditions (5 nmol/L Fe) both without kanamycin for 1 week, and their filtrates were collected by filtering the culture twice through a 0.22μm filter membrane.Then the collected filtrates were added into the Trichodesmium culture, which had been chronically grown in Fe-depleted medium for more than 2 months.Meanwhile, a blank control was set by adding the same solution of Fe-depleted A + medium (5 nmol/L Fe) into Fe-depleted Trichodesmium culture.To determine whether the dominant species of Trichodesmium consortium changes, metagenomic sequencing was conducted after Fe-depleted Trichodesmium being treated with A + medium and Synechococcus filtrate for 5 days.

Trace-metal clean techniques
All materials used in the Fe-limited cultivation, including conical bottles, PC bottles, and tips, were soaked in 6 mol/L HCl at least 12 hours and rinsed six times with Milli-Q water (18.2MΩ/cm) to remove residual metal ions.All relevant solutions were using analytical pure reagents without Fe contamination, and all solutions were passed through the chelating column of Chelex-100 to remove the residual Fe before use.Filtering instead of autoclaving was used to avoid Fe contamination in the process of sterilization.

Chlorophyll a content determination
Cultured algae were gathered by centrifugation at 12 000 rpm for 5 min, the precipitate was collected, and 1 mL of 100% methanol was added to the precipitate and gently mixed.The solution was then kept out of light and incubated at 4 • C overnight to settle.Centrifugation at 12 000 rpm for 5 min after blending was followed by supernatant absorbance value measurement at 665 nm.The chlorophyll a content (μg/mL) was equal to 12 times OD 665 /OD 730 .

Algal cell content measurement
100 μL of cultured Trichodesmium were added to the plankton counting frame (XBC-100CSL) and rested for 1-2 min until all the algal cells settled.A cell was first found at low magnification, then the view was converted to high magnification and the corresponding scale was used (positive differential interference microscope BX53) (Olympus, Tokyo, Japan).The length of the whole algal filament and single cell was determined using measuring software, and the cell number in the counting box was the length of whole algal filament or the length of a single cell.

Low-temperature fluorescence emission spectrum determination at 77 K
Approximately 400 μL of exponential phase cells were added to glass tubes then pre-cooled and dark-adapted at 4 • C, followed by liquid nitrogen f lash-freezing until being measured.Cell f luorescence emission spectra were measured using an F-4500 f luorescence spectrophotometer (Hitachi, Tokyo, Japan).According to a previously described study, the excitation wavelength was set to 430 nm and spectra were normalized at an intensity of 720 nm [51].Results were normalized to cell number.

RNA extraction and real-time quantitative PCR
Total RNAs of exponential phase cells were extracted as described previously [57].First-strand cDNA was synthesized by reverse transcription with a HiScript II Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China).Real-time quantitative PCR was conducted with gene-specific primers (isiA, 5 -CTGCTCGTTGTTGGTTGACA-3 and 5 -TCTGCTACGCCATTCAA AGC-3 ; rnpb, 5 -TGGTAACAGGCATCCCAGATAGATA-3 and 5 -CG GGTTCTGTTCTCTCAACTCAA-3 ).The RT-PCR reaction mixture and process were conducted following instructions in the SYBR Green PCR Master Mix manual (Vazyme, Nanjing, China).Gene expression levels were normalized to rnpb.The 2 − Ct method was used to calculate the relative level of mRNA expression [57].

Metagenomic sequencing
Total DNA was extracted using a FastPure ® Bacteria DNA Mini kit (Vazyme, Nanjing, China) following the manufacturer's protocols.Then the metagenomic DNA was using an Illumina NovaSeq PE150.Duplicate reads, adapter reads, and reads of length <75 bp were first removed.Then the reads were assembled into contigs using MEGAHIT and the assembly data were aligned with sequences (E value, ≤1e −10 ) in the NCBI (National Center for Biotechnology Information) non-redundant protein database [58].

Metatranscriptome sequencing
The Trizol method was used to extract total RNAs and the Epicentre Ribo-Zero rRNA Removal Kit was used to remove ribosomal RNA [57].The library was constructed using a NEBNext ® Ultra II™ Directional RNA Library Prep Kit for Illumina followed by pairedend sequencing using Illumina HiSeq.After filtering out lowquality reads with fastp and removing ribosome RNAs by alignment with the Rfam database, Hisat2 was used to align the filtered reads to the reference genome and obtain sam/bam files for each sample [59].RSEM, edgeR, clusterProfiler, Rockhopper were used to calculate the read count for each sample, analyze differential gene expressions, identify functional and pathway enrichment in differential genes, analyze operon and transcription start sites, respectively [60][61][62].

Statistical tests
Values were presented as the mean (SD).For 2 group comparison, p values were derived from 2-tailed Student t tests or Paired Wilcoxon test to determine statistical significance.For all comparisons, p < 0.05 was considered statistically significant difference, while p < 0.01 was considered highly significant difference.All experiments were biologically repeated three times.

A stable Trichodesmium Fe-deficient culture
Trichodesmium erythraeum IMS 101 was originally isolated from the North Atlantic Ocean and semi-continuously cultured with YBC-II medium under Fe-depleted condition (5 nmol/L Fe) for more than 2 months (about 15 cycles) in our laboratory (see Methods and materials section).Trichodesmium erythraeum IMS 101 formed single filament (trichome) morphology under laboratory conditions (Figure S1A), which has been reported to be symbiotic with microorganisms such as Synechococcus [18].Physiological phenotype measurements showed that the Trichodesmium biomass grew in Fe-depleted YBC-II medium, but significantly slower than those grown in Fe-replete medium (250 nmol/L Fe) (Figure S1B).Furthermore, the Trichodesmium room temperature chlorophyll a fluorescence absorption peak of those grown in Fe-depleted medium shifted from 680 to 675 nm compared with those grown in Fereplete medium (Fig. 1A), indicating the Trichodesmium showed an Fe-deficient phenotype.The 77 K low-temperature f luorescence emission spectroscopy assay shows the characteristic photosystem II complex peak (685 nm and 695 nm) and a photosystem I complex peak (715 nm).The major 77 K emission peak at 685 nm indicates the production of IsiA, which is typically characteristic of Fe deficiency and homologous to PsbC (the CP43 of PS II).Trichodesmium grown in Fe-depleted medium show an increase in the relative peak at 685 nm and a decrease in the relative peak at 715 nm compared with Trichodesmium grown in Fe-replete medium, thus revealing that isiA expression is higher under Fedepleted conditions ( Fig. 1B).These data collectively suggest that Trichodesmium consortia adapt to a Fe-deficient physiological phenotype after long periods of Fe-depleted cultivation.

Synechococcus and Marinobacter are the primary Trichodesmium symbionts
As Trichodesmium is a microorganism that originated from the wild, there were diverse microbe symbiosis between Trichodesmium and its consortium.To explore the microbial community composition of the Trichodesmium consortium that grew in Fe-depleted YBC-II medium for long periods in our laboratory condition, metagenomic sequencing was conducted.Results show Trichodesmium to be the dominant genus with about 45.3% relative abundance in the Trichodesmium consortium (Fig. 2A).Two other genera of high abundance are Synechococcus, which is a genus of photosynthetic autotrophic carbon-fixing cyanobacteria, and the heterotrophic bacteria Marinobacter, with the relative abundance of 4.6% and 3.6%, respectively.Relative abundance was 4.6% and 3.6%, respectively (Fig. 2A).At a species taxonomic level, only one species of Trichodesmium existed in the consortium, while most Synechococcus and Marinobacter species were unclassified and unknown in our analysis.Apart from these unknown species, there were 17 Synechococcus species and 38 Marinobacter species (Fig. 2B) in the consortium.These data suggest that the Trichodesmium consortium originally isolated from the North Atlantic Ocean formed a complex symbiotic microorganism community together with the dominant Trichodesmium species in in Fe-depleted YBC-II medium under the conditions in our laboratory.

Siderophores secreted by Synechococcus induced more severe Fe deficiency of Trichodesmium
Interestingly, Synechococcus sp.PCC 7002, a siderophore-producing strain, was found to be a symbiont of Trichodesmium.Synechococcus sp.PCC 7002 was previously believed to mainly inhabit offshore regions, but recent analysis revealed that it is also distributed in the open ocean [56,63].When facing Fe limitation, Synechococcus sp.PCC 7002 can biosynthesize an amphiphilic siderophore, synechobactin, to chelate Fe, which aids in adaptation to Fe deficiency [64].The siderophores can be secreted to the extracellular filtrate through the type I secretion system HlyB-HlyD-TolC, and the siderophore-chelated Fe can be transported into cells through TonB-ExbB-ExbD transporter components and utilized by Synechococcus sp.PCC 7002 in Fe-deficient environments.However, the effect of Synechococcus sp.PCC 7002 siderophore secretion on Trichodesmium and its consortium is not clear.Some genes encoding TonB-ExbB-ExbD transporter components are present in Trichodesmium genomes, while TBDT, the outer membrane TonBdependent transporter, is absent [50,51].
To detect whether siderophores secreted by Synechococcus can be utilized by Trichodesmium to assist in Fe-deficient environmental adaptation, a Synechococcus strain knocking out mutant G0023-G0025 (Mut-G0023-G0025) was constructed (Figure S2).We explored the interactions between wild-type or mutant Synechococcus strains and Trichodesmium in Fe-deficient environments.The Mut-G0023-25 knockout mutant (unable to biosynthesize siderophores) and wild-type Synechococcus sp.PCC 7002 were cultured under Fe-depleted conditions (5 nmol/L Fe) for 1 week.The Synechococcus culture systems were then filtered through a 0.22-μm filter membrane twice to collect filtrate (exudates of Synechococcus sp.PCC 7002), followed by the addition of this filtrate into Trichodesmium grown in Fe-depleted medium.Meanwhile, a control was set by adding the same solution of Fedepleted A + medium (5 nmol/L Fe) into Fe-depleted Trichodesmium culture.Results showed no significant difference in Trichodesmium chlorophyll a content among the three different treatments (Fig. 3A).Other physiological parameters, including cell contents, chlorophyll a f luorescence, and Trichodesmium isiA expression showed that the Synechococcus filtrate from either wild-type or mutant strains resulted in even more severe Fe-deficiency of Trichodesmium.Comparing the effects of wild-type filtrate and mutant filtrate on Trichodesmium culture, it can be found that the addition of wild-type Synechococcus sp.PCC 7002 filtrate resulted in a more severe Fe-deficiency to Trichodesmium, as shown in the cell counts.This finding suggested that the siderophores secreted by Synechococcus cannot aid Trichodesmium to adapt to Fe deficiency, and the Fe chelated by siderophores secreted by Synechococcus is likely not to be used by Trichodesmium (Fig. 3).Given that the Trichodesmium culture includes multiple symbionts rather than a single species of pure culture, we speculate that the siderophores produced by Synechococcus may exert an overall inhibitory inf luence on the Trichodesmium consortium.

Addition of Synechococcus filtrate results in a decrease of relative Trichodesmium abundance
To determine whether the Synechococcus filtrate inhibited the Trichodesmium consortium as a whole or merely inhibited Trichodesmium, metagenomic sequencing was conducted after Fe-depleted Trichodesmium was treated with A + medium and Synechococcus filtrate for 5 days.Generally, cyanobacteria secrete numerous extracellular substrates, including proteins, lipids, short-chain fatty acids, and others via eff lux systems [65][66][67][68].We speculated that the Synechococcus exudates at late logarithmic growth (cultured under Fe-depleted conditions for 1 week) were of higher concentrations, including organic matter in addition to inorganic ions, than exudates at the beginning of the exponential phase of growth.Addition of the filtrate induced significant changes in the relative abundance of dominant organisms compared with the control group (Fig. 4).For example, the relative abundance of Trichodesmium decreased, while the abundance of one of the main symbionts, Marinobacter, increased (Fig. 4A and B).Compared to the blank control treated with A + medium, the filtrate secreted by both wild-type and the mutant Synechococcus contained more organic matter, which might cause a great advantage over heterotrophic microorganisms such as Marinobacter rather than photoautotrophic Trichodesmium ( Fig. 4A and B).The increase in the relative abundance of Marinobacter inevitably led to a decrease in the abundance of other related organisms within the community, including Trichodesmium.In addition, the change in the abundance of Trichodesmium induced by wild-type filtrate was more significant than the change induced by the mutant filtrate ( Fig. 4B).Siderophores contained in wild-type filtrate are likely beneficial for the growth of symbionts which complete with siderophore transporter components [69] such as Marinobacter.Furthermore, siderophores contained in the filtrate of wild-type Synechococcus may competitively adsorb the free inorganic Fe required for Trichodesmium in the medium and result in more severe Trichodesmium Fe deficiency.Trichodesmium consortia and Synechococcus filtrate contents and inhibitory mechanisms need to be further explored to explain these phenomena.

Global biological community processes are promoted by Synechococcus filtrate
We explored the changes in functional gene abundance by analyzing metagenomic sequencing data to ascertain the reasons for the increased severity of Trichodesmium Fe deficiency after adding wild-type Synechococcus filtrate.As shown in the heatmap (Fig. 5), almost all functional categories within the consortium increased in the samples treated with Synechococcus filtrate regardless of the presence of siderophores compared with the samples treated with A + medium.These data suggest that complex substrates contained in Synechococcus filtrate may be the major reason for the promotion of global biological community processes (Fig. 5), which is consistent with the observed increase in Synechococcus and Marinobacter relative abundances (Fig. 4A).Additionally, filtrate promotion was more pronounced for some basal consortium metabolisms, such as amino acid metabolism, carbohydrate metabolism, membrane transport, and replication and repair (Fig. 5); however, these results cannot explain why Trichodesmium Fe deficiency is more severe, as the data from metagenomic sequencing cannot ref lect which organisms encode for those functional genes whose abundances have changed.

Synechococcus filtrate
To further explore changes in the expression abundance of functional Trichodesmium genes, we conducted metatranscriptome sequencing after treatment with Synechococcus filtrate or A + medium for 5 days.The Trichodesmium erythraeum IMS 101 genome was used as a reference genome to analyze the sequencing data.By analyzing the expression abundance of genes that relate to Fe absorption and transport, photosynthesis and respiration, and other essential metabolic pathways of Trichodesmium, we found that for most of these genes the expression abundances decreased after Synechococcus filtrate treatment compared with treatment with A + medium, demonstrating that the substrates in filtrate inhibited Trichodesmium (Fig. 6A, B and C).Genes related to Trichodesmium Fe uptake, transport, and storage were downregulated after treatment with wild-type and mutant strain filtrates (Fig. 6A).Comparing the effects of the filtrates from the wild-type and mutant strains, we found that the expression of the genes related to Fe-deficiency adaptation was much lower in the wild-type-treated group, including tonB, exbB, exbD, sufB, sufC, sufD, sufF, futA, and futB (Fig. 6A).Furthermore, genes related to Trichodesmium photosynthesis and respiration (such as psaA, psaF, and NADH-ubiquinone/plastoquinone oxidoreductase genes) and genes related to various essential metabolic pathways (including nitrogen fixation, RNA modification, and amino acid synthesis) were also downregulated after Synechococcus secretion filtrate treatment (Fig. 6B and C), suggesting the filtrate affects Trichodesmium photosynthesis, respiration, and global metabolism, consistent with observed physiological phenotype.
Because Marinobacter abundance increased in the sample treated with Synechococcus filtrate, the Marinobacter adhaerens genome was used as a reference genome, as was the Trichodesmium erythraeum IMS 101 genome, in our metatranscriptome analysis.Results showed that Marinobacter genes related to nitrogen utilization (e.g., urease, nitrite reductase, and urea transport) were significantly downregulated in the sample when Synechococcus filtrate was added (Fig. 6D).This finding illustrated a reduction of directly available nitrogen sources in the environment and was consistent with decreased Trichodesmium abundance.In contrast, genes relevant to Marinobacter TonB-dependent siderophore receptors, sugar utilization, and amino acid utilization are significantly upregulated (Fig. 6D), indicating that the rich organic matter and siderophore chelated Fe in the filtrate are beneficial for Marinobacter growth and lead to an increase in its relative abundance.Marinobacter consumed Fe and further reduced the concentration of Fe.In addition, siderophores secreted by the Synechococcus wild-type strain are likely to chelate with free Fe in the medium of the Trichodesmium consortium, reducing the

Discussion
Recent research revealed that the habitats of major nitrogenfixing and siderophore-producing cyanobacteria are gradually overlapping [56].In addition, the presence of closely related Synechococcus species in environmental Trichodesmium samples from both the Sargasso Sea and the southwest Pacific Ocean indicated that Synechococcus can be symbiotic with Trichodesmium, which as a nitrogen-fixing cyanobacterium and is known for its widespread occurrence in vast oligotrophic subtropical and tropical oceans in these habitats [11,[70][71][72].In particular, our recent analysis of Tara ocean data shows the widespread distribution of siderophore-producing Synechococcus species both offshore and in pelagic ocean (which were previously thought to be mainly distributed offshore) [56,73].In Fe-deficient ocean, some Synechococcus such as Synechococcus sp.PCC 7002 can synthesize and secrete siderophores, which can bind inorganic free Fe 3+ in the environment and then be transported back into the cells through TonB-dependent transporters (TBDTs) mediated by TonB-ExbB-ExbD components [56,64].The special Fe uptake mechanism provides siderophore-producing Synechococcus a high advantage in Fe-deficient ocean [56].The interactions between nitrogen-fixing and siderophore-producing cyanobacteria in oceans may have profound implications for the contribution of primary productivity and elemental cycling in the oceans.
Fe deficiency is one of the limiting factors of Trichodesmium in oligotrophic open ocean [17].As a diazotrophic cyanobacterium that cannot produce siderophore but has TonB-ExbB-ExbD transporter components, Trichodesmium may adapt to Fe deficiency by absorbing siderophore secreted by its epibionts [50]. of the study by Basu et al. [48] revealed enhanced Fe uptake by Trichodesmium in the presence of desferrioxamine B (DFO-B) and desferrioxamine E (DFO-E), while the Fe uptake of Trichodesmium was inhibited when only DFO-B was provided [48,53].The various utilization of different siderophores may be related to the different chemistries of siderophores as well as the utilization of different forms of Fe by symbionts.Given the specific Fe uptake mechanism, Synechococcus may have a higher competitive advantage in Fe-deficient pelagic ocean, and the interaction between Synechococcus and Trichodesmium may have a great impact on future marine primary productivity.To simulate the interaction between Trichodesmium and Synechococcus in Fe-deficient ocean water, Trichodesmium have been chronically grown in Fe-depleted medium for more than 2 months before treated with Synechococcus filtrate.By comparing the responses of Trichodesmium to the filtrates with or without synechobactin or not, we found that the growth rate of Trichodesmium slows down, photosynthetic efficiency is decreased, and the expression of isiA is increased-all demonstrating that the filtrates that contained synechobactin were likely to inhibit the growth of Trichodesmium under Fe-deficient conditions and induced a more severe Fe deficiency in Trichodesmium.Simultaneously, the relative abundance of Trichodesmium decreased, especially in the group treated with the wild-type Synechococcus filtrate that contains siderophores.The possible reason for this finding can be attributed to the observation that the siderophores produced by Synechococcus are absorbed by other symbionts with complete siderophore transporter components or stimulate the symbionts to produce siderophores and other products, which in turn exerted further survival pressure on Trichodesmium under Fedeficient conditions.Furthermore, Synechococcus abundance variation is opposite that of Trichodesmium abundance variation, indicating a negative correlation between them in this consortium, which is similar to a recent discovery [74].Synechococcus is outcompeted when diazotrophs increase the NH 4 + /NO 3 − ratio (the main nitrogen produced by the fixation of N 2 by Trichodesmium is NH 4 + ), which favors Prochlorococcus, suggesting a different competitive pattern between nitrogen-fixing cyanobacterium and Synechococcus [74].Interactions between cyanobacteria and associated symbionts are quite complex [75,76], and there is a lot of work to be done to figure out the complex interactions.Metagenomic and metatranscriptomic analyses show that treatment with both Synechococcus filtrates reduces the abundance of Trichodesmium but promotes global Trichodesmium consortium metabolism, especially in samples treated with wild-type Synechococcus filtrate containing siderophores.This finding may be due to the filtrate being rich in siderophores and organic matter that can be utilized directly by heterotrophic Marinobacter [54,77,78].Functional gene expression abundance analyses in our study indicate that the metabolism of the symbiotic microorganisms is enhanced, while the metabolism of Trichodesmium, including its Fe transport and utilization, photosynthesis, respiration, and other essential metabolic mechanisms are actually downregulated, which is consistent with the physiological phenotype seen in Fe-deficient Trichodesmium.These data collectively demonstrate that the siderophores produced by Synechococcus chelate most of the available free Fe in the medium, which is then used by the symbionts such as Synechococcus and heterotrophic microorganisms ( Fig. 7).The cell size of Trichodesmium is large, thus the specific surface area is smaller than that of Synechococcus and heterotrophic microorganisms.The smaller specific surface area offers them quite an advantage for absorbing Fe and adapting to an environment with low Fe concentrations when the concentration of Fe in the environment decreases [57].Once other organisms are taken up and lower the concentration of Fe, it becomes harder for Trichodesmium to take in Fe and makes Fe deficiency in Trichodesmium even worse, inhibiting the growth of Trichodesmium (Fig. 7).This phenomenon is consistent with previous research demonstrating that Trichodesmium can most readily access inorganic Fe or Fe in association with weak organic ligands but not strongly bound organic complexes under Fe-limited culture conditions [ 35].In addition to siderophores, Synechococcus filtrate also contains various organic matters and extracellular proteins [77,79].These can serve as Fe, carbon, and other nutrient sources to be directly used by heterotrophic symbionts, such as Marinobacter, and serve as a primer to stimulate and expedite Marinobacter growth (Fig. 7).Within this context, increased Marinobacter and Synechococcus abundances further compete with Trichodesmium, which poses a disadvantage for Trichodesmium and deccreases its relative abundance.The production of other siderophores and products from symbionts after treating with Synechococcus filtrate is another possibility to explain the inhibition of Trichodesmium growth, while the way these substrates function in Trichodesmium consortium is unknown and makes the interaction more complex.
Our findings revealed the inhibition of Synechococcus siderophore to Trichodesmium using culture interaction experiments in the laboratory.This finding highlighted the observation that Fe chelated by siderophores secreted by Synechococcus exerts an inhibitory inf luence on Trichodesmium and defines the competitive relationship in Fe utilization between Synechococcus and Trichodesmium in Fe-deficient habitats.These microorganisms have been in culture for long time and whether these interactions will be sustained in the more complex and dynamic ocean environment needs to be investigated in the future.But at least microorganisms such as Synechococcus and Marinobacter can be found in many field metagenome data [18].Since interactions between different species in the consortium are quite complex, more field work is needed to elucidate the interaction between Trichodesmium and symbionts in nutrient acquisition and environmental adaptation.

Figure 1 .
Figure 1.Trichodesmium phenotype under Fe-deficient cultivation.(A) Trichodesmium absorption spectra at room temperature.The Trichodesmium chlorophyll a f luorescence absorption peak when cultured under Fe-depleted conditions shifted from 680 to 675 nm compared to that under Fe-replete conditions.(B) 77 K low-temperature f luorescence emission spectroscopy (excitation wavelength at 430 nm) shows the induction level of IsiA.

Figure 2 .
Figure 2. Trichodesmium consortium microbial composition under Fe deficiency in the laboratory.(A) Relative abundance of microorganisms in a Trichodesmium consortium under Fe-deficient culture."Others" represent microbial genera with less than 1% of reads.(B) Predominant symbiotic microbial composition at the species taxonomic level.the gray boxes indicate unclassified or unknown microorganisms.

Figure 4 .
Figure 4. Differences in Trichodesmium colony community composition between treatments.(A) Relative abundance of microorganism in Trichodesmium colonies."Others" represent microorganisms with relative abundance levels below 1%.(B) Difference between the addition of wild-type Synechococcus (with siderophores) and mutant filtrate (without siderophores) in the abundance of dominant microorganisms compared with the control, that is, the treatment with added a + medium.

Figure 5 .
Figure 5. Changes in the relative abundance of genetic functions among different treatments as shown with a heatmap.The top 25 functional categories in the sample in terms of average abundance are shown, with abundance characterized by color depth, the darker the color, the larger the value.

Figure 6 .
Figure 6.Expression changes of Trichodesmium-related and Marinobacter-related genes.Heatmap of the expression abundance of genes involved in Fe absorption and utilization (A), photosynthesis and respiration (B), and other essential metabolic pathways (C) in Trichodesmium treated by A + medium, Synechococcus wild-type filtrate, and mutant filtrate.Expression abundance of genes is indicated by color depth.(D) Changes in expression abundance of related genes of Marinobacter under treated with filtrate from wild type compared to the A + medium treatment.The size and the color of the dot represents the fold change and p value, respectively.