3.1. Isolation and Identification of Halophilic Archaeal Strains
A total of 24 halophilic archaeal isolates were characterized and identified from the rhizospheric and non-rhizospheric soil samples ofS. stocksii and A. amnicola (Table 1; Fig. 1 ). These isolates were identified based on 16S rRNA gene analysis. Four strains showed more than 99% homology withHalobacterium spp., four strains were related toHalomicrobium spp., three strains were identified asHalococcus spp., three strains were related to Natrinemaspp., two strains NRS3HaP17 and LK4HAP18 were belonging toNatrialba spp., one strain HL1RS17 was identified asHalolamina sediminis , one strain AT3RS21 was identified asHaloferax denitrificans and one strain AT4RS18 was identified asHalalkalicoccus jeotgali (Table 1; Fig. 1 ).
Phenotypic Characterization of Haloarchaea
The halophilic archaeal cultures were grown on MGM agar. On incubation, translucent and opaque colonies with orange, red or pink pigmentation were observed. As shown in Fig. 2A , haloarchaeal strains were extremely halophiles and only few strains were able to grow under 2M of total salt but grew optimally at 2.5 - 4.0M of salt (NaCl). These strains were able to grow at a wide range of temperature with 37-42 °C optimum temperature (Fig. 2B ). Mostly strains grew well at pH range from 6 to 8, however, some strains did grow at 55 °C and the pH of 9.5 (Fig. 2C ).
Plant Growth Promoting Potential of Halophilic Archaea
Halophilic archaeal strains were screened for various plant growth promoting (PGP) abilities such as IAA production, P-solubilization, nitrogen fixation and siderophore production. Most of the strains showed more than two PGP abilities (Table 2; Fig. S1 ). Twenty-one strains showed P-solubilization activity with a range from 8.15 to 87.77 µg/mL. Natrinema gari strain HL1RP1 had the maximum P-solubilization activity (87.77 µg/mL). Fifteen strains showed production of IAA with a range from 2.11 to 63.42 µg/mL. Only 12 strains showed nitrogen fixation ability and 8 strains showed positive results for siderophore production assay (Table 2; Fig. S1 ).
Heavy metal resistance profile of bacterial strain
More than 90% of the rhizospheric archaeal strains showed heavy metal tolerance for Cd, Ni, Cr and Zn at a concentration of 1 mM, 71-85% archaeal strains showed heavy metal tolerance at a concentration of 2.5 mM, 40-63% archaeal strains showed heavy metal tolerance at a concentration of 5.0 mM, and only a few archaea strains (0-19%) showed tolerance at a concentration of 10 mM (Fig. 3 ).
3.5. Features of Genomes ofHalorubrum lacusprofundi HL1RP11 and Halobacterium noricense NRS2-HaP9
The size of genomes of Halorubrum HL1RP11 andHalobacterium NRS2-HaP9 was 3,550,491 and 3,037,489 bp, respectively (Fig. S1 ). In the genome of HalorubrumHL1RP11 strain, a total of 3517 genes were predicted with 3,431 protein coding sequences (CDs) and RNA related genes were 55 (Table S1 ). In the genome of Halobacterium NRS2-HaP9 strain, a total of 3228 genes were predicted with 3155 protein coding sequences (CDs) and RNA related genes were 51 (Table S1 ). Plasmid sequence was not identified in both strains.
Functional Annotation of Halorubrum lacusprofundiHL1RP11 and Halobacterium noricense NRS2-HaP9 Genomes
Most of the unique genes were predicted to code hypothetical proteins in both genomes. In the genome of Halorubrum lacusprofundi HL1RP11, out of 3,431 proteins, 1354 (49.7%) were assigned to COG functional categories while in case of Halobacterium noricense NRS2-HaP9 genome, out of 3155 proteins, 1419 (44%) were assigned to COG functional categories (Fig. 4 ). The functional analysis of these genes using KEGG pathway database showed that they have an important role in various metabolic pathways including plant growth promotion, environmental adaptation, bioremediation of different toxic compounds, heavy metals, and other abiotic stresses. The functional analysis of CDSs showed that they could be classified into 19 general COG categories including the metabolism of carbohydrates, amino acids, lipids, transcription, energy, cofactors and vitamins, inorganic ions, signal transduction and cellular processes, glycan biosynthesis and metabolism, nucleotide metabolism, secondary metabolites, Iron acquisition and metabolism and xenobiotics biodegradation (Fig. 4 ).
Prediction of Genes Related to Plant Growth Promotion
The functional analysis of Halorubrum HL1RP11 and NRS2HaP9 genomes identified different genes involved in plant growth promotion of halophytes. The presence of genes involved in tryptophan biosynthesis (indole acetic acid production), and tyrosine such as trpA, trpB, trpC, trpD, trpE, trpG, tyrA2 and pheA2 was confirmed by genome analysis of both HL1RP11 and NRS2HaP9 strains. Eleven genes includingafuB, fbpB, afuA, fbpA, menF, ABC.FEV.S, ABC.FEV.P, ABC.FEV.A, feoA, FRD and adhB-1 involved in iron metabolism and siderophore production were identified in the genomes of both haloarchaeal strains (Table S2; Fig. 5, 6A and 6B ). Presence of genes includingPstA, PstB, gcd, pqq_1, PiT and phoU related to P-solubilization (PQQ-dependent alcohol dehydrogenase) were predicted in HL1RP11 and NRS2HaP9 genomes. Genes related to nitrogen fixation, nitrogenase protective and regulatory proteins such as nifU, nif3, nifU_N, glnK2 and glnB were also detected in genomes of both HL1RP11 and NRS2HaP9 strains. Putative NAD(P)H nitroreductase geneydfN was detected in genome of NRS2HaP9 strain (Table S2; Fig. 5, 6A and 6B ). Few genes including pgdA, dgoD, EOI, rhaMand uxuA related to production of exopolysaccharides were also predicted in this study (Table S2; Fig. 5, 6A and 6B ). When PGP genes from the genomes of HL1RP11 and NRS2HaP9 were compared with their reference strains, there was a difference in the number of genes especially siderophore production, nitrogen fixation and phosphate solubilization related genes (Fig. 6A and 6B ).
Prediction of Genes Related to Environmental Adaptation, Glycerol Metabolism and Membrane Transport
Genes potentially involved in environmental adaptation, glycerol metabolism and membrane transport have been identified in the genomes of halophilic archaea. Genes for environmental adaptation (ACSL, fadD, COX10, ctaB, cyoE, COX15, ctaA, RP-S6e, RPS6 and psd ), glycerol metabolism (pssA, gldA, ALDH, dhaK, dhal, dhaM, SQD1, sqdB, gck, gckA, dgs, bgsA, mgs, bgsB, araM, egsA, glpA, glpD, glpB, glpC, pgsA, PGS1 and carS ) and 63 genes related to amino acids, sugar molecules, iron, phosphate, thiamine, biotin, zinc, sulfonate and arabinogalactan transportation and signal recognition proteins were identified in the genome of Halorubrum lacusprofundi HL1RP11 (Tables S3 & S4 ). Based on functional analysis ofHalobacterium noricense NRS2-HaP9 genome, genes for environmental adaptation (ACSL, fadD, COX10, ctaB, cyoE, COX15, ctaA, RP-S6e, RPS6 and psd ), glycerol metabolism (pssA, gldA, ALDH, gck, gckA, dgs, bgsA, bgsB, araM, egsA, glpA, glpD, glpB, glpC, andcarS ) and 44 genes related to amino acids, sugar molecules, iron, phosphate, thiamine, biotin, copper, molybdate, sulfonate, nucleotides and urea transportation and some signal recognition proteins were identified (Tables S3 and S4 ). When genomes of HL1RP11 and NRS2HaP9 were compared with their reference strains, there was a difference in the number of genes especially related to environmental adaptation, and membrane transport (Fig. 6C and 6D ).
Prediction of Genes Related to Heavy Metal Resistance
Based on functional analysis of Halorubrum lacusprofundi HL1RP11, 159 genes related to heavy metal resistance including Nickel, Cadmium, Antimony, Arsenic, Iron, Chromium and Zinc
were identified (Tables S5; Fig. 7 ) while in the genome ofHalobacterium noricense NRS2-HaP9, 171 genes related to heavy metal resistance were identified (Tables S5; Fig. 7 ). When genomes of HL1RP11 and NRS2HaP9 were compared with their reference strains, we observed a clear difference in number of genes related to heavy metal resistance (Fig. 7 ).
Production of Secondary Metabolites
In the genome of Halorubrum lacusprofundi HL1RP11, antimicrobial gene clusters including thiopeptides (thiazolyl peptides), bacteriocins and terpenes were identified (Fig. S3 ). Secondary metabolites encoding gene clusters such as siderophore, thiopeptides and terpenes were identified in the genome of Halobacterium noricenseNRS2-HaP9 (Fig. S3 ). These genes might be involved in plant growth improvement and biocontrol mechanisms.
Discussion
Rhizosphere microbial communities from extreme environments such as arid, saline, alkaline and acidic are more complex than soils having a neutral pH and exhibiting moderate salinity. The rich microbial diversity of halophyte rhizospheres helps these plants cope with high salinity and drought [6]. In the current study, culturable halophilic archaeal diversity was analyzed from the rhizosphere and non-rhizospheric soils of halophytes including S. stocksii andA. Amnicola . Plant growth promoting traits of the isolated archaeal strains were screened using different selective media and genes related to plant growth promotion, secondary metabolism and osmoregulation were identified through whole genome sequence analysis ofHalorubrum HL1RP11 and Halobacterium NRS2-HaP9 strains.
Based on 16S rRNA gene analysis, a total of 24 halophilic archaeal isolates were identified from the rhizospheric and non-rhizospheric soil samples of S. stocksii and A. amnicola. In this study, 9 halophilic genera including Halobacterium, Halomicrobium, Halorubrum, Halococcus, Haloferax and Halalkalicoccus were identified. Halobacterium, Halomicrobium, Halococcus andNatrinema were more abundant as compared to other genera from the rhizosphere of halophytes. A number of previous studies have reported the halophilic archaeal genera such as Halobacterium,Halococcus, Halomicrobium, Haloferax and Halalkalicoccusfrom various aquatic hypersaline environments [44-47] but only two studies including Yadav et al. [20] and Dubey et al. [48] reported the PGP halophilic archaea from the rhizosphere of halophytes. Haloarchaeal strains isolated in this study were extremely halophiles and a few strains were able to grow under 2M of total salt but grew optimally at 2.5-4.0M of salt (NaCl).
Halophilic archaeal strains were orange to red in color because of red pigmented protein bacterioruberin. Haloarchaeal enzymes usually use KCl ions to work at high salinity levels [4, 14,15]. Haloarchaeal strains from the genera Halobacterium and Halococcus can synthesize L-glutamate as osmolyte. They have intracellular gas vesicles which are usually filled with different gases and provide buoyance and enable cells to regulate their position in the aquatic environments [15, 49]. Halobacterium salinarum contains fibrillary structures (fibrocrystalline bodies) which represent the presence of cytoskeleton-like organelle in haloarchaeal cells [50].
In this study, most of the strains showed more than two PGP abilities. About 87.5% of haloarchaeal strains showed P-solubilization activity with a range from 8.15 to 87.77 µg/mL, 62.5% of strains showed production of IAA with a range from 2.11 to 63.42 µg/mL, 50% of strains showed nitrogen fixation ability, and 33% strains showed positive results for siderophore production assay. From the rhizosphere of halophytes, halophilic archaea such as Natrialba, Natrinema, Haloarcula, and Halococcus with the ability to solubilize phosphate, produce IAA, and siderophores have been previously reported. These archaeal strains showed mineral phosphate solubilization by the production of organic acids such as oxalic, acetic, citric, and succinic acid [20, 51].
A recent study also showed that haloarchaea have the ability to produce indole acetic acid and other phytohormones [52]. Some ammonia-oxidizing archaea, e.g., Nitrosopumilus maritimus and halophilic archaea, e.g., Halobacterium salinarum andHaloferax volcanii are capable of siderophore production. Archaea use iron acquisition and produce siderophores [53-55]. Some archaeal groups play an important role in nitrogen cycle. A number of previous studies have reported the ammonia oxidizing methanogens [24, 56].
The whole genome analysis of Halorubrum HL1RP11 andHalobacterium NRS2-HaP9 revealed that there were 3431 and 3155 protein coding sequences respectively. A large number of proteins were annotated as hypothetical proteins. The functional analysis ofHalorubrum HL1RP11 and Halobacterium NRS2-HaP9 genomes using KEGG pathway database showed that it has an important role in metabolism of carbohydrates, amino acids, lipids, energy, cofactors and vitamins, inorganic ions, glycan biosynthesis and metabolism, secondary metabolites, signal transduction and cellular processes, DNA replication and repair, cell motility, transcription, translation, ribosomal biogenesis, abiotic stresses and bioremediation of different toxic compounds. Some recent studies on genome sequence analysis of haloarchaea revealed that these microorganisms have a large diversity of proteins and enzymes involved in different metabolic pathways, abiotic stress management and plant growth promotion [57-59].
The functional analysis of Halorubrum HL1RP11 andHalobacterium NRS2HaP9 genomes revealed the identification of various genes involved in plant growth promotion. The presence of genes including PstA, PstB, gcd, pqq_1, PiT and phoU related to P-solubilization (PQQ-dependent alcohol dehydrogenase) were predicted in this study. Genes involved in tryptophan biosynthesis (indole acetic acid production), and tyrosine were also identified by genome analysis of both HL1RP11 and NRS2HaP9 strains. Eleven genes involved in iron metabolism and siderophore production were identified in the genomes of both haloarchaeal strains. Genes related to nitrogen fixation, nitrogenase and related regulatory proteins (nif3, nifU, nifU_N, glnB and glnK2 ) were also detected in genomes of both HL1RP11 and NRS2HaP9 strains.
Some previous studies also reported the role of archaea in plant growth promotion with their ability to solubilize inorganic phosphate and produce phytohormones and siderophores [20, 60,61]. The ammonia monooxygenase genes were identified in ammonia-oxidizing archaea that were isolated from the rhizosphere of Littorella uniflora[24]. This study is the first report of nitrogen-fixing halophilic archaea based on biochemical detection as well as identification of genes related to nitrogen fixation by using whole genome analysis.
Halophilic archaeal genera including Haloarcula, Halococcus, Haloferax, Halobacterium and Natronococcus have the ability to produce exopolysaccharides. Various sugars such as glucose, rhamnose, galactose, mannose, galactosamine, and glucopyranosiduronic acid are involved in the biosynthesis of exopolysaccharides in archaea [62, 63]. Identification of genes related to PGP traits has been previously reported in some archaea, however, this study is the first report that described the characterization of PGP haloarchaeal strains from the rhizosphere of halophytes and identification of related genes through whole genome analysis of two haloarchaeal strains HalorubrumHL1RP11 and Halobacterium NRS2HaP9.
About 40-63% of archaeal strains showed heavy metal tolerance for Cd, Ni, Cr and Zn at a concentration of 5.0 mM. In this study, haloarchaea showed more tolerance for chromium as compared to other metals. A number of previous studies also showed that halophilic archaea includingHaloferax , Halobacterium, HalococcusHaloarcula , and Halorubrum have heavy metal resistance genes on their plasmids and chromosomal DNA. These microorganisms help plants to grow under saline polluted soils [27-29].
Genome annotation analysis showed that siderophore, thiopeptides and terpenes were commonly identified from both Halorubrum HL1RP11 and Halobacterium NRS2-HaP9. Thiopeptides (thiazolyl peptides) are a class of pretentious antibiotics produced by bacteria and archaea. These antibiotics usually show positive activity against Gram-positive bacteria such as Bacillus and Staphylococcus genera [59, 64]. Terpenes and terpenoids are important antimicrobial compounds identified and characterized in bacteria, archaea, and plants. Terpenes and terpenoids play a role in biosynthesis of the cell membrane and cell wall, electron transport and conversion of light into chemical energy including chlorophylls, bacteriochlorophylls, rhodopsins, and carotenoids [65, 66]. Some recent studies reported the identification of gene clusters related to secondary metabolism in archaea [67], however, this study is the report of identification of gene clusters related to secondary metabolism in halophilic archaea.
In summary, our results suggest that Halobacterium, Halococcus, Halorubrum, Halobacterium and Natrinema were dominant in all soils. Most of the strains identified in this study showed more than two PGP traits. More than 60% strains demonstrated positive results for P-solubilization, IAA production and heavy metal resistance. The genomic annotation of Halorubrum HL1RP11 and HalobacteriumNRS2HaP9 revealed the identification of genes involved in PGP, e.g., phosphate solubilization, IAA production, nitrogen fixation, and exopolysaccharides production; heavy metal resistance and secondary metabolism, e.g., phenazine, siderophore production and terpene related gene cluster. We suggest that PGP haloarchaeal strains may be used as an eco-friendly biofertilizer that will be a better alternative to chemical fertilizers for improving plant growth under salinity affected agricultural lands.