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, Halococcus , Haloarcula ,
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.