Unearthing the genetic resources of Arabian sea seamount and metagenomic insights into phosphate cycling genes for next generation plant biostimulants

The field of metagenomics has been revolutionized by technological advancements in synthetic biology, bioinformatics, and sequencing which could help to unravel microbiomes and their functional interaction in deep-sea ecosystem with significant bio-prospection perspectives. Functional and sequence-based metagenomics have been recently adopted towards understanding microbiomes of different ecological niche and bio-prospecting novel biocatalysts from uncultured microbes in environmental sources including water and soil¹. Recent studies proposed that sequence-based metagenomics could enable the identification of potential genes involved in biogeochemical cycling, whereas function-based screening involves the development of clones from metagenomic libraries of various environmental niche to bio-prospect enzymes with a specific function². Hence, it would be essential to combine both approaches for conservation of deep-sea microbiome through developing functional metagenome libraries of extreme environments, which would result in preserving biodiversity hotspots and better characterization of their enzymes for industrial application.

Oceans, as a habitat, provide a complex and extraordinary physical and chemical environment that is essential for all types of microorganisms, particularly marine bacteria, viruses and protists to thrive in specific ecological niche³. Deep-sea sediments, within marine habitats, constitute a harsh environment for microbial populations due to high pressure, low temperature, lack of light, poor nutrient availability and are recognized as well adapted versatile environment with understudied oceanic ecosystems, which imparts the significant necessity for conservation strategies in this fragile ecosystem to ascertain their future sustainability⁴. Seamounts are typically regarded as hotspots of species diversity and oases of biomass abundance due to their distinct ecosystems in the deep ocean^5,6. Considerable efforts have been undertaken in the last few years to investigate the diversity, ecology, and function of the prokaryotes that live in seamount habitats through the use of metagenomics⁷, which has made it possible to record the diversity of microbes and investigate their functional potential in the ocean, where more than 99% of microbes remain yet uncultured⁸. Recent bathymetric surveys report on many of the selected seamount region in the Laxmi basin of Arabian Sea indicating their volcanic origin, which suggests that sea mounts of Arabian Sea could be potential hotspots for bio-prospection of industrially important enzymes from marine microbes with extremophilic properties^9,10. Hence, it is proposed that metagenomic clone libraries could be used for conserving unculturable microorganisms as living repository of Indian deep-sea seamounts samples for a longer period towards addressing solutions through exploring sustainable biotechnological applications from recombinant microbes without exploiting the Oceanic environment for routine sampling and collection of samples.

A microbial community as a whole can be treated as a super- or mega-organism, whose metabolic pathways and networks are accessible with the use of metagenomics approaches¹¹. Metagenome sequencing analysis has emerged as a potent technique for comprehending the biogeochemical cycling (e.g., phosphorus, nitrogen, carbon, sulphur, and metals) driven by microbes in different environments^3,12. Oxford Nanopore Technologies is a powerful metagenomics tool, producing long reads that resolve complex genomic regions like repetitive or conserved elements which effectively recovers complete metagenome assembled genomes from various samples, including sludge, water, sediments, and feces, demonstrating its versatility¹³. Its ability to deliver fast results at a lower cost makes it an invaluable tool for diverse applications, from agricultural studies to medical research¹⁴. Nanopore sequencing based method has recently emerged as significant metagenomics technique, which has been implemented in our earlier studies towards understanding microbial diversity and harnessing potential of deep-sea environment including Bay of Bengal¹⁵ and Arabian Sea sediment¹⁶. Several detailed studies have been conducted on the cycling and fractionation of phosphate in the deep ocean of Arabian Sea. Recent research showed that the formation of a coastal upwelling zone in the Southeastern Arabian Sea during southwest and northeast monsoons results in the cycling of Phosphorus¹⁷. The genes responsible for phosphorus cycling (PCGs) and the microorganisms involved in the process are not thoroughly studied¹⁸. Alkaline phosphatases (EC 3.1.3.1) are the most investigated phosphoric monoester hydrolases and its genes phoD and phoX are shown to be more prevalent in marine environments than phoA^19,20,21. The gene encoding a new alkaline phosphatase was discovered in a metagenomic library constructed from ocean-tidal flat sediments of Korea’s west coast which was identified to be a part of a newly discovered PhoX family, different from the well-researched classical PhoA family²². There has been limited research on the gene clusters that play a role in the cycling of phosphorus in deep-sea sediments, leaving a significant gap in our understanding of this crucial process.

Recently, several sequencing techniques have been applied in soil metagenomes to predict the entire sequence of biosynthetic gene clusters in silico^23,24. Metagenomic methods have also been adopted to clone and sequence soil genome for screening specific enzymes⁷ where its functional analysis could lead to more accurate metagenome annotation than sequence-based analysis alone²⁵. Functional screening of enzymes through metagenomics could find potential implication to address the challenges associated with low enzyme expression and complex purification processes²⁶. The most appropriate vector must be chosen to insert the metagenomic DNA depending on the objective of functional screening wherein expression vectors like fosmids, cosmids (< 40 kb), bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs) (> 40 kb) are useful for larger biosynthetic gene clusters^27,28. Recent reports unveil that soil metagenomes could be cloned using cosmid or fosmid library to identify the biosynthetic gene clusters efficiently²⁹ which is adopted in the present study for constructing metagenomic libraries from Arabian Sea seamount sediments.

In general, it is necessary to confirm functional screening of any enzyme from metagenome clone library by sequencing-based methods^30,31. Recent reports on sequencing of fosmid clone libraries with large inserts revealed that functional screening of fosmids clones with specific enzyme activity must be de novo sequenced which is more significant towards identifying specific gene targets³¹. Earlier reports reveal that numerous novel genes that encode variant enzymes from marine environments have been discovered by functional metagenomics³², which include lipase from marine sponge³³, cellulase from Coral, Siderastrea stellata³⁴ and DNA polymerase from glacial ice³⁵. Fosmid metagenome library from ocean tidal flat sediments has been previously reported to have undergone fluorescence-based screening for lipase, cellulase, and phosphatase through GESS-FACS^22,36. Recent progress in functional metagenomics screening methods combined with advances in next generation sequencing technologies could result in efficient bio-prospection of significant enzymes and metabolites from specific environmental niche³¹. These coupled methodologies have been adopted for mining industrial biocatalyst esterase from deep-sea samples including thermal water of alkaline hot spring at Lobios (Ourense, Spain)³⁷ and Atlantis IIbrinepool, Red Sea(Indic Ocean)³⁸ even amylase from Arabian Sea sediment^39,40.

Several recent reports reveal the significance of Phosphate-solubilizing bacteria (PSB) as resourceful biofertilizers for sustainable agriculture due to its ability to secrete alkaline phosphatase (phoD)⁴¹, acid phosphatase (phoC)⁴² and phytase enzymes⁴³ that can solubilise insoluble phosphorus in the soil for efficient absorption, uptake and utilization by plants⁴⁴. Currently, only a small number of such plant growth-promoting bacteria are used in agriculture^45,46 wherein our recent findings revealed two potential phosphate solubilising bacteria Priestia megaterium and Bacillus velezensis, from seamount sediments of Arabian Sea which promotes plant growth⁴⁷. Recent approaches target the use of synthetic synergistic communities, obtained from one or multiple soil microbiomes that show best growth conditions even under biotic or abiotic stress, aiming at a broader target range, higher robustness and a better plant-beneficial efficacy^48,49. Furthermore, advanced nanopore sequencing methods is emerging as a potent tool to generate plant biostimulants that could help to increase crop yield⁵⁰, thereby resulting in development of next generation fertilizers to overcome harmful impact of synthetic molecules on environment⁵¹. However, there are no reports on bio-prospection of plant biostimulating enzymes from biodiversity hotspots including deep-sea sediment which could serve as a living metagenomic repository of extremophilic enzymes and biomolecules that could be used for sustainable agriculture with implication in environmental restoration.

This study aimed to bio-prospect phosphatase enzyme using functional metagenomics approach by constructing a metagenomics clone library using fosmid vector from seamount sediments of Arabian Sea as methodology to develop biological innovations through conservation of deep-sea microbes. Further, the potential metagenomic clones producing maximum phosphatase were sequenced by nanopore long read technology to understand the microbiome hidden in metagenomic clones of dark seamount sediment and to confirm functional potential of PSB towards identifying phosphate cycling gene expression variation conferring phosphate solubilisation. This study focused to correlate the abundance of PSB from seamount sediment with plant growth promoting properties towards understanding its implication in sustainable agriculture.

Collection of sample

Marine sediment samples were collected from Arabian Sea on board NIOT-Ocean Research vessel, Sagar Manjusha (SAMA 10) cruise in November 2022. About 30 g of sediment was collected and transferred to a labelled sterile bag, then stored at 4 °C on board before being transferred to − 40 °C at the laboratory facility for further analysis. This study focused on Arabian Sea sediment collected from bottom of seamount M-ASM2B (Station 5–12.988333°N, 73.600283°E) at a depth of 850 m. Bathymetric mapping for the sample location of station 5 (ST5) was illustrated using ArcGIS 10.8 and bathymetric data acquired from https://gebco.net/ (Fig. 1). Environmental DNA from this sediment sample was utilized to construct a fosmid metagenomic library, subsequently screened and sequenced to characterize the phosphatase enzyme from the recombinant clones.

Construction of fosmid metagenomic library from seamount sediment

High molecular weight environmental DNA extraction method was adopted from Patin and Goodwin⁵² and further modified for seamount sediment samples to yield high quality DNA, where optimized lysis buffer was used for extraction⁵³ followed by Polyethylene glycol (PEG-6000) for precipitation of high molecular weight DNA⁵⁴. Multiple extractions were carried out and pooled together to attain a higher concentration of metagenomic DNA for cloning. The quality of DNA was analysed using agarose gel electrophoresis and quantified using Nanodrop Spectrophotometer (DS-11 Spectrophotometer, Denovix). The extracted DNA was purified using a silica-based column as per the kit protocol (Qiagen DNeasyPowerSoil Pro kit). Metagenomic library was constructed using Copy Control Fosmid Library Production Kit with pCC1FOS Vector from Epicentre Biotechnologies (CopyControlFosmid Library Production Kit, Lucigen, USA). Five micrograms of extracted metagenomic DNA were end-repaired and size selected according to the manufacturer protocol. The required amount of DNA was ligated, packed in lambda phages and were transformed into EPI300 Escherichia coli cells⁵⁵. The recombinant clones were selected on LB agar plates with chloramphenicol (12.5 µg/mL). The clones obtained were stored as pooled clones in 20% glycerol at -80 °C in 96 well plates for further functional screening and sequencing of potential clones.

Confirmation of recombinant clones

To confirm the insertion of metagenomic DNA in the recombinant clones, fosmid DNA was extracted from the random clones by alkaline lysis method with some modifications⁵⁶. Fosmid DNAs were purified with 2.5 M Ammonium acetate precipitation followed by ethanol wash and eluted in nuclease-free water. Restriction digestion was carried out with Not I enzyme (10 U) in fosmid DNA (500 ng) to confirm the size of inserted metagenomic DNA in the clones. PCR amplification of fosmid DNA was also carried out using pCC1FOS Sequencing Primers (as suggested by the manufacturer), 5′-GGATGTGCTGCAAGGCGATTAAGTTGG-3′ (Forward) and 5′-CTCGTATGTTGTGTGGAATTGTGAGC-3′ (Reverse) to confirm the insert size of metagenomic DNA. PCR amplification was carried out using fosmid DNA (200 ng), PrimeSTAR GXL DNA Polymerase (1.25 U) from Takara which is specifically used to amplify long amplicons. PCR reaction cycle was carried out in 50 µL reaction volume on 96 well thermocycler: initial denaturation was carried out at 95 ⁰C for 5 min, 35 cycles of amplification as follows: denaturation at 95 ⁰C for 30 s, annealing at 50 ⁰C for 30 s, elongation at 72 °C for 30 s and final elongation was performed at 72 °C for 5 min. The size of the PCR products was analyzed using agarose gel electrophoresis.

Screening of phosphatase enzyme from metagenome clone library

The fosmid metagenome library could be screened for phosphatase using either function-based screening or sequence-based screening. Function-based screening helped to identify the potential metagenomic clones producing required enzymes using a specific substrate, while sequence-based screening locates the biosynthetic gene clusters in the library and efficiently identify the microbiome present in metagenomic DNA. The methodology for combination of functional and sequence-based screening approach followed in the present study for bio-prospecting phosphatase enzyme from metagenome clone libraries of Arabian Sea sediment is illustrated in Supplementary Fig. 1.

Agar based primary screening for phosphatase

The pooled clones were subjected to substrate-based screening for phosphatase enzyme on NBRIP agar media containing tricalcium phosphate (TCP) as substrate⁵⁷ and bromophenol blue as pH indicator which was incubated at 37 °C for 48 h. The recombinant clones with halozones around it were selected as phosphate solubilisers with phosphatase activity.

Quantification of phosphatase

Phosphatase enzyme was quantified for the positive clones using p-nitrophenyl phosphate (p-NPP) as substrate by spectrometric method. The recombinant clones with halozones were inoculated in 200 µL of NBRIP broth in 96 well plates while EPI300 E. coli served as negative control. The plate was incubated at 37 °C for 48 h. The phosphatase activity of recombinant clones was measured with a colorless substrate p-nitrophenyl phosphate (p-NPP), which yielded yellow p-nitrophenol product upon phosphate solubilisation (p-NP)⁵⁸. The reaction mixture consisted of 200 µL 1 mM p-NPP (dissolved in 0.1 M Glycine-NaOH buffer) and 200 µL of cell free extract, made up to a fixed final volume of 1300 µL with nuclease free water. After a reaction time of 60 min at 25 °C in dark, the reaction was stopped by adding 200 µL of 0.05 M NaOH. The generated product p-nitrophenol (p-NP) was measured by spectrophotometry (UH5300 Spectrophotometer, HITACHI) at 405 nm⁵⁹. The concentration of phosphate released was estimated from the standard graph of para-nitrophenol (p-NP). GraphPad Prism 7 and SPSS 29.0 were used, respectively, for the graphical depiction and statistical analysis of phosphatase activity of recombinant clones. Test data were subjected to a one-way ANOVA using SPSS 29.0 whereas differences between means at p < 0.05 were evaluated by Tukey HSD test.

Extraction of phosphatase enzyme and its partial purification

The potential recombinant clone with maximum phosphatase activity was selected for extraction of phosphatase enzyme. The selected clones were inoculated in specific media containing peptone 5 g; yeast extract 2.5 g; glucose 1 g; Mg₂SO₄ 0.05 g; K₂HPO₄ 0.2 g; distilled water 1 L⁶⁰ and incubated at 37℃ for 2 days to estimate the protein content. The cells were suspended in 50 mM Tris HCl buffer (pH 7.4), sonicated for 10s, and then cooled on ice for 15 s. This cycle was repeated 10 times after which the cells were collected at 10,000 g for 15 min. The cell free supernatant was precipitated by 80% saturated ammonium sulfate and dialyzed (membrane cut off 12,000Da, Sigma) against the same buffer at 4 °C overnight⁶¹. Using bovine serum albumin (BSA) as a standard (Sigma, Germany), the protein content of crude and partially purified phosphatase was quantified⁶². The activity of phosphatase enzyme in both the crude and partially purified protein was assayed using p-nitrophenyl phosphate (p-NPP) and measured spectrophotometrically (UH5300 Spectrophotometer, HITACHI) at 405 nm⁵⁹.

Estimation of organic acid

Organic acid quantification was performed for the five potential recombinant clones with maximum phosphatase activity using a Shimadzu HPLC system with a quaternary pump, auto-sampler, and programmable UV-Vis detector. Organic acid separation was performed using a Phenomenex Luna c-18 column (4.6 *150 mm, 5 μm particle size), protected by a guard column (Phenomenex, c-18, 5 μm particle size) and detected at 214 nm. The data was collected in three dimensions – absorbance, time, and wavelength – using LC Solutions software. A 100% isocratic mobile phase of 50 mM potassium phosphate buffer with a pH of 2.80⁶³ was used. The flow rate was set to 0.5 mL/min, and the column was kept at 35 °C using a column oven. Prior to injection, the sample and standard were passed through a 0.22 μm PTFE syringe filter (Acrodisc, Pal German, Germany). Calibration curves were developed for organic acid standards, which include lactic acid, oxalic acid, succinic acid, citric acid, gluconic acid, acetic acid, and formic acid.The maximum phosphate solubilising clones were inoculated in 30 mL NBRIP broth medium. The flasks were incubated at 37 °C in an orbital shaker at 100 rpm for 48 h. About 5mL of bacterial culture was centrifuged at 10,000 rpm for 15 min and filtered through 0.22 μm nylon membranes (acrodisc, Pal German, Germany) to obtain cell-free supernatant. Twenty microlitres of filtered supernatant were injected into the HPLC system (LC2010, Shimadzu). Identification and quantification of organic acids in the recombinant clones were performed by comparing retention times and peak areas of respective standards (Supplementary Table 1). All the samples were analyzed in duplicates.

Sequencing of potential recombinant clones encoding maximum phosphatase

Fosmid DNA was extracted from the five phosphatase positive clones using same methodology as described. The concentration and purity of DNA was measured using Nanodrop Spectrophotometer (DS-11 Spectrophotometer, Denovix). The integrity of DNA was observed on agarose gel electrophoresis and the DNA concentration was quantified using Qubit dsDNA HS assay kit in Qubit fluorometer (Qubit 4 Flourometer, Invitrogen Thermo Fischer Scientific). DNA samples were end-repaired (NEBnext ultra II end repair kit, New England Biolabs, MA, USA), cleaned up with 1X AmPure beads (Beckmann Coulter, USA). Native barcode ligation was performed with NEB blunt/ TA ligase (New England Biolabs, MA, USA) using SQKNBD114.96 and cleaned with 1xAmPure beads. Qubit quantified barcode ligated DNA samples were adapter ligated for 15 min using NEBNext Quick Ligation Module (New England Biolabs, MA, USA). Library was cleaned up using 0.6X AmPure beads (Beckmann Coulter, USA) and finally sequencing library was eluted in 15 µL of elution buffer and sequenced on Nanopore PromethION system, (PromethION P24 and Data Acquisition Unit, ONT, Oxford, UK) using PromethION flow cell (FLO-PRO114M) according to effective library concentration and data amount required. NGS library preparation and sequencing was carried out at Genotypic Technology Pvt. Ltd., Bangalore, India.

Sequence pre-processing and taxonomic analysis

Nanopore sequenced reads were de-multiplexed using Guppy v2.3.4 and the quality was verified using Nanostat tool⁶⁴. Data pre-processing includes removal of adapters, de-replication of reads and quality control⁶⁵. Metagenome Assembly was performed using the Flye tool with meta-option and the processed high-quality reads were assembled into contigs. Contigs were compared against NT Database (non-redundant nucleotide seq) using Centrifuge³⁶, which contain reference sequences from a wide range of organisms, including bacteria, Archaea, viruses and eukaryotes classifying from kingdom to higher taxonomic ranks. Pavian tool was further used to estimate and interactively explore the taxonomic content⁶⁶. Sankey plot is used for representing taxa abundance and connections between different taxonomic ranks. Comparative taxonomic analysis was carried out using relative abundance values for top 10 taxa at species level which are represented by heat map generated using R-statistical vegan package Non-negative Matrix Factorization (NMF)⁶⁷.

Functional annotation for phosphate cycling genes

Functional annotation was carried out using KEGG database⁶⁸, where three level classifications was carried out to predict the number of reads involved in the enzyme hydrolysis. Further pathway annotation was carried out using PCycDB which is recently developed database specifically for prediction and fast analysis of phosphorus cycling genes from metagenome datasets¹⁸. The genes involved in oxidative phosphorylation, transporters, two-component system, and organic phosphoester hydrolysis are major processes for microbes to regulate, transport, and uptake P sources from the environment which were predicted from PCycDB using BLAST analysis¹⁸. R-statistical package NMF was used to generate heat map based on gene expression count for each samples.

Bio-priming and pot assay for plant growth application

Bio-primed tomato seeds (5 seeds per pot, in duplicates) were sown in each experimental pot of sterilised garden soil. This pot experiment was conducted over a 14-day period in a plant house located near the Department of Marine Biotechnology at the National Institute of Ocean Technology in Chennai where temperature was maintained between 25 ºC and 36 ºC which is optimal for plant growth. Throughout the study, humidity levels were kept between 75% and 80%, and standard daylight duration was measured at 12 h, with a light intensity ranging from 600 to 1000 µmol/m²/s. Fosmid recombinant cultures were sprayed to their respective pots, while double distilled water served as the negative control, and Paenibacillus pseudetheri (a phosphobacterium obtained from Manidharma Biotech Private Limited in Chennai) was used as the positive control. After 14 days of growth, the plants were uprooted to measure vegetative parameters such as root length, shoot length, and the weight of both roots and shoots (measured for both fresh and dry weight).

Fosmid metagenomic library construction from seamount sediment sample

Metagenomic DNA of high concentration was obtained from seamount sediment of Arabian Sea using modified metagenomic DNA extraction method and visualised by agarose gel electrophoresis, which showed an intense band approximately 40 kb as shown in Fig. 2a. The quantification results indicated high molecular weight DNA of concentration1026 ng/µL with low A260/A280 value (1.4) and A260/A230 value (0.9) suggesting that some contaminants still remained in the modified protocol. The extracted DNA was purified using QIAGEN Power Soil Pro Kit (Qiagen Inc., USA) wherein purified DNA was recovered with concentration of 903.3 ng/µL, A260/A280 value of 1.8 and an A260/A230 value of 1.9 indicating its suitability for constructing fosmid metagenomic libraries. About 571 ng of end-repaired metagenomic DNA was used for ligation and its subsequent transformation in host cell E.coli which resulted in 9068 clones with an efficiency of 1.5 × 10⁴ clones per µg of DNA (Fig. 2b). The library was constructed with an average insert size of 40 kb comprising about 362 Mbp of metagenomic DNA from the Arabian Sea seamount sample. These recombinant clones were stored in 20% glycerol as 1024 pooled clones which would enable efficient functional screening of enzymes from large number of soil metagenomics clones²⁹.

Extraction of fosmid DNA and confirmation of recombinant clones

Fosmid DNA from randomly selected metagenomic clones was extracted to confirm the efficiency of cloning. Alkaline lysis method followed by purification yielded a fosmid DNA of intact quality. Cloning efficiency was confirmed by restriction digestion and amplification of fosmid DNA. Four hours of restriction digestion of fosmid DNA with restriction enzyme NotI released 3 fragments as confirmed through gel electrophoresis wherein one of band was found to be approximately 40 kb in size, indicating the presence of metagenomic DNA in the recombinant clones. Fosmid DNA was extracted from random clones and amplified using PCC1FOS sequencing primers and long Taq polymerase which resulted in high molecular weight amplified product of approximately 40 kb indicating successful insertion of metagenomic DNA into the vector and demonstrating cloning efficiency of fosmid vectors(Supplementary Fig. 2).

Functional screening and quantification of phosphatase in recombinant clones

Out of 1024 pooled clones, 42 clones showed halozone formation around colonies on NBRIP with bromophenol blue agar plates indicating its phosphatase activity by primary screening method (Supplementary Fig. 3). All those 42 clones were considered positive for phosphatase production and further quantified for phosphatase enzyme by spectro-photometric method. Compared to EPI300 E. coli (negative control), all recombinant clones showed significantly increased phosphatase activity. Among them, NIOT F41 (330.06 µM/mL, p < 0.001) exhibited maximum phosphatase activity followed by NIOT F42 (277.93 µM/mL, p < 0.001), NIOT F10 (272.89 µM/mL, p < 0.001), NIOT F13 (261.11 µM/mL, p < 0.001) and NIOT F24 (230.85 µM/mL, p < 0.001) (Fig. 3). These five potential recombinant clones with maximum activity were selected for further downstream analysis.

Extraction and partial purification of phosphatase

The enzyme concentration was measured using para-nitrophenyl phosphate (pNPP) as substrate for both crude and partially purified enzyme and the total protein content was also measured to determine the specific activity of the phosphatase enzyme. In the present study, it is evident that partially purified phosphatase enzyme from NIOT F41 exhibited the maximum specific activity of 41.2 U/mg (Table 1). On the other hand, the specific activity of the crude enzyme for the same clone NIOT F41 was lower (5.2 U/mg) compared to partially purified enzyme. It was also observed that fold of purity was increased for partially purified enzyme of NIOT F41 (7.95) compared to crude enzyme (1.0). These results indicate that specific activity of the enzyme increased after partial purification by dialysis method in all the five potential recombinant clones.

Estimation of organic acid produced by recombinant clones

The organic acids produced by the recombinant clones were identified by HPLC from the standard curves of pure organic acids (Supplementary Table 1). Among these, three different organic acids were detected in higher concentration (gluconic acid, oxalic acid and succinic acid) by the recombinant clones as shown in Fig. 4. Gluconic acid was produced in higher concentration by NIOT F41 (637 mg/L), NIOT F13 (1041 mg/L) and NIOT F24 (997 mg/L). Oxalic acid and succinic acid were produced in higher concentration by the recombinant clones NIOT F42 (327 mg/L) and NIOT F10 (610 mg/L) respectively. A very few unknown acids were also detected, but none of them did match with the standards used for the identification. These results suggest that the production of organic acid could be attributed to the phosphate solubilisation by these recombinant clones.

Pre-processing and assembly of sequenced fosmid reads

Fosmid sequencing was carried out for the five potential positive clones which showed synthesis of maximum phosphatase enzyme in the quantification analysis. Sequencing of five clones resulted in an average of 6,00,786 reads using long read sequencing technology and further processed to remove adapter sequence which generated the high-quality processed data for microbiome identification. The cloning vector sequence of pCC1FOS (Genbank accession EU140751) matching with the processed reads and also with the E. coli genome (NC_000913) were trimmed and removed by Deconseq tool (version 0.4.3) [filter parameters- 90% coverage and 94% identity]⁶⁵. The assembled reads had an average contig length of 25,104 bp at an average of 1259 contigs across the sample and detailed statistics is represented in Table 2. The processed reads were used in further downstream analysis for taxonomic and functional annotation of all the samples.

Sequence annotation for taxonomic analysis

Taxonomic analysis of the processed reads was performed against centrifuge indexed NT database. The assignment of individual reads to taxa based on their hits were analyzed and summarized from kingdom to species level along with read counts (Supplementary Fig. 4). Taxonomic analysis of the five phosphatase positive clones revealed that the major phylum was found to be Firmicutes for NIOT F10 (50.82%) and NIOT F42 (81.82%), while Proteobacteria was represented higher in NIOT F41 (72%), NIOT F13 (71.28%) and NIOT F24 (84.72%). The bacterial classification from kingdom to species level is shown as Sankey vvisualization plot for each sample which revealed that class Bacilli was found abundant in NIOT F10 (50.88%) and NIOT F42 (81.86%) at class level (Supplementary Fig. 4a). On the other hand, class Gammaproteobacteria was represented high in NIOT F24 (80%), NIOT F41 (66.85%) and NIOT F13 (39.63%) as shown in Supplementary Fig. 4b. Among the genus, Pseudomonas was found to be abundant in NIOT F41 clone (64.86%) and NIOT F24 (78.7%), while Bacillus was found to be relatively higher in NIOT F10 (30.12%) and Staphylococcus was comparatively abundant in NIOT F42. At the species level, Pseudomonas aeruginosa was found to be majorly represented in three of the sequenced clones, NIOT F24 (43.46%), NIOT F41 (37.74%) and NIOT F13 (25.16%) whereas Bacillus subtilis (17.77%) and Staphylococcus warneri (43.46%) was highly represented in NIOT F10 and NIOT F42 respectively. Taxonomic analysis of microbiome in sediment metagenome results suggest that major genus including Pseudomonas, Bacillus and Staphylococcus identified from Nanopore sequencing of potential phosphatase producing clones harbored already reported phosphate solubilising bacterium in literature^59,69.

Comparative taxonomic analysis

The relative abundance of taxonomic classification by heat map results confirmed that different phylum was majorly represented among the five clones indicating distinct properties of each clone with insertion of DNA from various organisms in the metagenomic DNA. The results of heat map generated at species level (Fig. 5) clearly showed that Paenibacillus sp. Y412MC10 and Bacillus subtilis was found to be relatively high in NIOT F10, while Pseudomonas aeruginosa was abundant in NIOT F13, NIOT F24 and NIOTF41 which were previously reported to be phosphate solubilising microbes in earlier studies^59,69. On the other hand, Staphylococcus warneri was found to be represented high in NIOTF42 suggesting that all these five potential fosmid clones harbor phosphate solubilising microbes attributing to their maximum phosphatase activity^59,70. Statistical diversity analysis results showed that among the five clones, Shannon index for NIOT F10 was found to be high (2.79) indicating diversity richness in this specific clone compared to others. On the other hand, Simpson index representing the diversity measure within a single ecosystem or sample was calculated as value close to 1 for all the five samples (Supplementary Table 2) indicating that there are several species in the community, and also the proportion of species population is evenly composed of all the representative species.

Functional classification

Major gene ontology classifications in all samples were represented in gene ontology distribution at level three (Supplementary Fig. 5). KEGG pathway analysis results showed that metabolism category was highly represented in all the five clones indicating their role in phosphate metabolism (Excel S1). Three level classifications were carried out by KEGG analysis for enzymes involved in phosphate cycling pathways. Hydrolases enzyme class was analyzed in detail since the phosphatase enzyme belongs to this class of enzymes (Excel S2). PCycDB analysis was carried out for functional analysis of phosphate cycling genes responsible for cellular P metabolic processes. Comparative analysis of gene expression count for phosphate cycling genes across five samples showed that each clone was unique wherein phoD and phoX gene that encode for alkaline phophatase was high in NIOT F24 while PhoA was relatively high in NIOT F13 (Fig. 6). On the other hand, different cluster of genes involved in synthesis of non-specific acid phosphatase, olpA and phoN was relatively abundant in NIOT F41 and NIOT F10 respectively but the major cluster of gene phoC encoding for same enzyme was produced high in NIOT F42. Notably, opd gene which encode for phosphotriesterase was majorly represented with high intensity in NIOT F13. Similarly, phy encoding for Phytase was also high in NIOT F13 but appA encoding for same enzyme was found to be very high in NIOTF10. These results suggest that the gene for alkaline phosphatase was encoded high in recombinant metagenome clones of NIOT F13 and NIOT F24 whereas the gene cluster for acid phosphatase were abundant in NIOT F41, NIOT F10 and NIOT F42 metagenomic clones. On the contrary, the genes encoding for phytases are high in NIOT-F13 and NIOT-F10 clones suggesting the unique characteristics of each clone for enhanced phosphatase activity (Fig. 6).

Seed priming and pot assay for Plant growth application

Seed priming assay using tomato seeds treated with fosmid recombinant clones NIOT F10, NIOT F13, NIOT F24, NIOT F41 and NIOT F42 showed 70%, 70%, 60%, 80% and 80% seed germination, respectively, which were greater than that of negative control (50%) which is represented in Table 3. Whilst seeds primed with positive control showed 70% which is lower than two clones (NIOT F41 and NIOT F42) indicating the efficiency of metagenomic clones from Arabian Sea. In the pot assay, NIOT F24 exhibited statistically significant effect on shoot length, measuring 9.1 ± 1.1 cm (p < 0.05), which is 1.44 times longer than that of negative control. In contrast, the highest root length was observed in NIOT F10 (2.05 ± 0.05 cm, p < 0.05), representing a 1.46-fold increase compared to the negative control. Furthermore, plants treated with the recombinant clones showed a significant increase (p < 0.05) in both wet and dry biomass relative to the negative control. Notably, the wet biomass was highest in NIOT F10, at 39.3 ± 0.65 mg, which corresponds to a 1.99-fold increase compared to negative control. Moreover, the dry biomass of NIOT F42, reaching 5.1 ± 1.15 mg, was significantly higher than that of the negative control, showing an increase of 1.52-fold (Table 3). These results indicate that all the recombinant clones were shown increased plant growth attributes than negative control and comparable results with positive control.

Metagenome libraries aid in the recovery and expression of biosynthetic gene clusters⁷¹. It is reported that metagenome libraries with large insert size might contain polycistronic genes where the complete metabolic pathways could be analyzed and studied⁷². Even though several vectors are available for cloning of large insert, fosmid vectors are the most prominent one which can produce a large number of clones by inserting poly-cistronic genes thereby increase the screening of functional molecules⁷³. Fosmid vector is already reported as an effective vector for the metagenome library construction from ocean tidal flat sediment and water column^22,74. As the metagenome fosmid library discussed in this study was constructed from an extreme environment, it was anticipated that novel biocatalysts with diverse biochemical and physiological characteristics would be bio-prospected from Arabian Sea seamount libraries.

The construction of large insert environmental metagenome libraries relies on the effective recovery and purification of high-quality environmental DNA. Although many extraction procedures have been developed to isolate nucleic acids directly from environmental samples, the post-extraction purification procedures can often be time-consuming and laborious⁷⁵. In this study, modified metagenomic DNA extraction followed by silica-based column purification yielded high quality DNA. A metagenome fosmid library consisting of 9068 recombinant clones was constructed from environmental DNA of Arabian Sea seamount sediment. Reports highlight the need to conserve fragile ocean ecosystems⁴ emphasising the need for protecting biodiversity in extreme environment, which was carried out in the present study to unveil the importance of preserving the genetic diversity of microbial communities of ocean seamount sediments. Hence, fosmid metagenomic library constructed from Arabian Sea seamount could serve as a genetic resource or living repository enabling the utilization of functional genes of extremophiles even if the original microbial population becomes threatened or inaccessible in the future.

A recent study identified marine microbial genomes as a key resource for gene mining offering significant potential for discovering novel enzymes⁷⁶, which was earlier reported as significant method for unraveling biocatalysts including lipolytic enzymes, proteases, oxido-reductases, glycosyl hydrolases, phosphatases/phytases from unexplored environment of soil and water¹. Following prior findings on enzyme screening from fosmid clones, this study envisaged to screen phosphatase enzyme from metagenomics clone libraries of seamount sediment. One effective way to identify desirable enzymes from metagenomic libraries that are expressible in the cloning host is by activity-based plate screening method⁷⁷ which was implemented in the present study for functional screening of metagenomics libraries. Prior studies on biosynthetic gene cluster identification from soil metagenome were carried out in pools of 2000 clones from a total of 83,700 recombinant fosmid clones indicating the efficient functional screening using pooled clone strategy²⁹, which was adapted in this study to effectively screen a large number of clones because it is challenging to screen every recombinant clone for specific enzymes with desired activity. In this study, about 9068 clones were stored in pools of 1024 clones as glycerol stocks for efficient functional screening of enzymes and biomolecules from seamount habitats. Primary agar-based functional screening resulted in the identification of 42 recombinant clones from a total of 1024 pooled clones exhibiting phosphatase activity by halozone formation around colonies indicating their ability to solubilise phosphate from the media. Earlier studies reported about positive fosmid clones for phosphatase through agar-based screening from functional metagenome libraries of decomposing leaf litter⁷⁸ and also in hydrocarbon-polluted soil⁷⁹. Furthermore, alkaline phosphatase synthesised by phosphate solubilising bacteria has also been reported in our recent study from deep-sea sediment samples of Arabian Sea⁴⁷. These studies support the rationale behind prospection of phosphatase enzyme from the metagenomic library of Arabian Sea seamounts.

The positive clones underwent further quantitative screening for phosphatase activity using pNPP as a substrate. pNPP has already been reported as a screening substrate for phosphatase in the fosmid library constructed from the metagenome of tidal flat sediments²². In this study, the maximum activity was shown by the metagenomic clone NIOT F41 (330.06 µM/mL, p < 0.001) which is higher than the activity shown by already reported PSB Pseudomonas aeruginosa from Nigeria⁸⁰. These results suggest the potential of recombinant clones from Arabian Sea sediment for enhanced phosphatase activity which could be useful in phosphate solubilisation for agricultural soil⁸¹.

In this study, partially purified phosphatase enzyme from NIOT F41 recombinant clone showed the highest specific activity of 41.2 U/mg. Comparing this finding to similar studies from Serratia sp. exhibited a specific activity of 9.58 U/mg⁸² and Metarhizium anisopliae with 8.45 U/mg⁸³, both of which was found to have lower specific activity than that observed in the recombinant clones from seamount sediment. The maximum specific activity exhibited by the clone NIOT F41 might be due to the combination of extremophilic PSBs in the fosmid clones of Arabian Sea sediment. Notably, it was observed that specific activity of clone NIOT F41 was 5.2 U/mg for crude enzyme when compared to 41.2 U/mg for the partially purified enzyme. The present findings were found to be consistent with the previous reports where the purified enzyme was found to demonstrate better specific activity than the unpurified one⁸⁴. According to Razali et al.⁸⁵, increasing enzyme purity leads to an increase in specific activity which suggests that antagonist enzymes such as proteases are eliminated during purification allowing the protein of interest to be in the best possible form to produce its actual catalytic activity.

According to Prasad et al.⁸⁶, the primary mechanism forsolubilization of inorganic phosphates is through the action of organic acids. The release of soluble orthophosphate from tricalcium phosphate (TCP) by microorganisms typically involves the production of organic acids^87,88. In order to solubilize inorganic phosphate compounds, PSBs have been found to produce some organic acids such as acetic, formic, lactic, gluconic, glycolic, oxalic, succinic, malic and maleic acid⁸⁹. In the present findings, gluconic acid was found in higher concentration for NIOT F41 (637 mg/L), NIOT F13 (1041 mg/L) and NIOT F24 (997 mg/L). It was observed that oxalic acid and succinic acid were produced in higher concentration by the recombinant clones NIOT F42 (327 mg/L) and NIOT F10 (610 mg/L) respectively. According to the findings of Behera et al.⁹⁰, PSB isolated from mangrove soil produced highest concentration of oxalic acid (289 mg/L), followed by succinic acid (0.5 mg/L), acetic acid (0.4 mg/L), malic acid (0.3 mg/L), and citric acid (0.2 mg/L). Organic acids production by PSB strains isolated from Moroccan mines was investigated by Mardad et al.⁹¹in NBRIP broth culture, where results revealed that gluconic acid was the majorly produced organic acid by all PSB strains, reaching concentrations of up to 83.287mM. These results of previous reports on organic acid production by PSBs are in congruence with the present findings on recombinant clones producing gluconic acid as the major organic acid during phosphate solubilization. These organic acids are reported to aid in phosphate solubilization thereby enhance plant growth in vitro^92,93.

Fosmid sequencing using Nanopore technology is reported to be a cost-effective, high-throughput approach that facilitates the rapid discovery of enzymes³¹. Function based screening for phosphatase was carried out to identify clones showing maximum phosphatase activity which was confirmed by sequencing of five potential recombinant clones in Nanopore platform towards prediction of phosphate cycling genes encoded by the metagenomic clones. Nanopore-based metagenome sequencing has proven effective in recent studies towards assessing microbial community composition in agricultural soil which helped to correlate specific taxa with its functional properties⁹⁴. Our study applied this long read sequencing method to explore the bacterial taxa involved in phosphate solubilisation from Arabian Sea towards understanding its plant growth promoting properties. Taxonomic findings from metagenome sequencing of five positive fosmid clones revealed major representation from Pseudomonas, Bacillus and Staphylococcus genera. These results were in congruence with previous studies which revealed that most dominant phosphate solubilizing bacterial genera were Pseudomonas, Bacillus, Vibrio, Alcaligenes, Micrococcus, Corynebacterium and Flavobacterium isolated from deep-sea sediments in the Bay of Bengal which contributed significantly to biogeochemical cycles and benthic productivity in the Exclusive Economic Zone (EEZ)⁹⁵. Earlier studies have also shown that Staphylococcus was also found to be best phosphate solubilizing bacteria in addition to Bacillus, Pseudomonas and Paenibacillus isolated from tropical rainforest soil^59,69. These results attribute to the fact that bacterial genus identified from sequencing of seamount sediment metagenomic clones provides strong evidence with earlier studies on PSB from different ecological nichewhich would be useful to develop plant biostimulants for sustainable agriculture.

Further insight into the species level classification revealed key phosphate-solubilising bacteria, including Pseudomonas aeruginosa, Bacillus subtilis, Paenibacillus sp. Y412MC10, and Staphylococcus warneri. Among this, P. aeruginosa has been identified as a phosphate solubilizer from Ganga River⁹⁶, while B. subtilis and P. aeruginosa have been recognized as phosphate-solubilizing rhizobacteria⁹⁷ and also exhibit stress-tolerant activity in agricultural fields⁹⁸. Further, S. warneri was also found to solubilize tricalcium phosphate in oil palm rhizospheres in Colombia⁷⁰. These results implicate that the species identified from metagenomic clone libraries of Arabian Sea sediment also involve in phosphorus solubilization and could enhance plant growth in phosphate deficit soil.

Recent reports reveal that two-component system, oxidative phosphorylation, transporters, and organic phosphoester hydrolysis are major processes for microbes to regulate, transport, and uptake P sources from the environment whereinphosphoester hydrolysis pathway was emphasized in this study to understand various cluster of genes encoding acid and alkaline phosphatase enzymes¹⁸. Among the phosphate cycling genes, phoA, phoD, and phoX encode for alkaline phosphatases while phoN, aphA, phoC, and olpA encode acid phosphatases. On the other hand, opd gene encodes phosphor-triesterase, while phy and appA code for phytases as reported in PcycDB, a database recently reported for prediction of these genes from metagenomic datasets¹⁸. Comparative taxonomic analysis of five potential metagenomic clones revealed that Pseudomonas aeruginosa was found to be majorly represented in NIOT F24 and functional analysis of phosphate cycling genes by heat map results showed that phoD, and phoX encoding for alkaline phosphates were relatively high in same clone. Literature studies of several Pseudomonads also showed conservation of key pho regulators in Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas aureofaciens where PhoD and phoX was found to be expressed under Pho regulon control⁹⁹. On the other hand, Bacillus was found to be relatively abundant in NIOT F13 where comparative analysis also demonstrated that PhoA gene was found to be abundant in same clone, which could be correlated with reports on Bacillus subtilis for synthesizing alkaline phosphatase (PhoA) and various other enzyme and act as cell factory for agriculture, medicine and biomaterials¹⁰⁰. A recent study emphasizes the complex interactions between microbial species in sediment communities and their cooperative role in nutrient cycling within deep-sea ecosystems¹⁰¹. This is relevant to the current study utilizing fosmid clones derived from microbial communities of the Arabian Sea seamount for understanding the phosphate cycling genes. Thus, the functional genes identified from microbiome of the seamount can be used for understanding biogeochemical cycles of marine hotspots.

Recent studies on tomato seeds treated with plant growth promoting bacterial strains revealed significant increase in germination¹⁰² which was correlated to current findings of seed priming results where highest germination index was shown by tomato seeds treated with recombinant clones NIOT F41 and NIOT F42 compared to negative control. Pot Assay results for Plant growth application showed that shoot and root length of tomato seedlings were also highest in the recombinant clones than positive control and other plant growth attributes showed comparable results with positive control. There are earlier studies on significant plant growth when tomato seeds treated with tri-species consortia of PSBs including B. gladioli, Pseudomonas sp., and B. subtilis for tomato seed germination and positive effects on plant growth¹⁰³. Comparative results in the current findings from potential metagenomics clones producing phosphatase with enhanced plant growth promoting properties might be due to the synergistic effect of phosphate solubilising bacteria in different combinations from metagenomic DNA of Arabian Sea sediment. Next-generation sequencing technologies, such as Nanopore, have already been employed to understand the role of plant biostimulants for sustainable agriculture⁵⁰. In this study, nanopore based long-read metagenomic sequencing of Arabian Sea fosmid clones was utilized to identify the deep-sea microbiome responsible for phosphate solubilisation and to functionally annotate the phosphatase gene potentially promoting plant growth. Recent reports on biofertilizers, especially using phosphate-solubilizing bacteria highlight their ability to enhance nutrient availability, leading to increased crop yields, resilience, and stress tolerance, all of which are crucial for soil health and could help to adapt to changing climate conditions¹¹. The significance of the recent findings aligns with the objectives of current study on unculturable bacterial communities from the Arabian Sea seamount known for their extremophilic properties along with phosphate solubilising ability which could result in promising plant biostimulants with bio-fertilizers application, thereby contributing to sustainable agriculture.

This study highlights the potential use of functional and sequence-based metagenomics for bio-prospecting phosphatase gene clusters from dark sea sediment samples collected from Arabian Sea seamount. About 9068 recombinant clones of metagenome fosmid library was constructed from Arabian Sea seamount sediment which could be a living repository of uncultured deep-sea microbiome thus contributing towards understanding conservation biology of extreme environment and bio-prospecting its functional genes for biotechnological innovations. Nanopore-based metagenomic analysis of five potential clones revealed the presence of phosphate cycling genes hidden in the seamount sediment metagenome which helped to understand the taxonomic diversity and biogeochemical cycling of phosphate solubilising microbes in deep-sea. These recombinant clones that can utilize P sources and secrete phosphatase enzyme along with organic acid production could be a novel approach to enhance soil quality by solubilizing the phosphate in an eco-friendly manner for enhancing Plant growth. This study also highlights the potential of advanced nanopore sequencing techniques to improve agricultural practices by identifying key phosphate cycling genes contributing to soil fertility and plant growth using deep-sea microbes which could be potent plant biostimulants. This approach could pave the way for developing next-generation biofertilizers thereby replacing the chemical fertilisers and restoring the ecosystem health. By leveraging the potential of biotechnological innovations, this study brings an insight to unlock a new era of sustainable agriculture that mitigates climate change and help in addressing global challenges for environmental sustainability using deep-sea microbes.

Statistical analysis

All collected data in this study represent the means of two replicates, which were analysed using one-way analysis of variance (ANOVA). To separate the means, we applied Tukey’s post-hoc test along with a least significant difference (LSD) test (P < 0.05). All statistical analyses were conducted using SPSS version 29.0.

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Author notes

1. These authors contributed equally: Karpaga Raja Sundari Balachandran and Greeshma Mani.

Authors and Affiliations

1. Marine Biotechnology Group, National Institute of Ocean Technology (NIOT), Ministry of Earth Sciences (MoES), Government of India, Pallikaranai, Chennai, 600100, India

   Karpaga Raja Sundari Balachandran, Greeshma Mani, J. T. Mary Leema, Roobamathi Senthilkumar & Dharani Gopal

2. Marine Biology, Kerala University of Fisheries and Ocean Studies (KUFOS), Panangad, Kochi, Ernakulam, 682506, Kerala, India

   Akhil Thayyil Sidharthan

Contributions

Karpaga Raja Sundari Balachandran: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing, Validation Greeshma M: Conceptualization, Data curation; Formal analysis, Methodology, Software, visualization, Writing- original draft Akhil Thayyil Sidharthan: Conceptualization, Formal analysis, Methodology Mary Leema J T: Resources, Software, Visualization Roobamathi Senthilkumar: Methodology, Formal analysis, Dharani Gopal: Resources, Project administration, Supervision, Funding acquisition.

Corresponding author

Correspondence to Karpaga Raja Sundari Balachandran.

Competing interests

The authors declare no competing interests.

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