A novel halophilic archaeon, designated NK23
T
, was isolated from an inland saline soil sampled from Xinjiang, China. The cells of strain NK23
T
were observed to be pleomorphic, to stain ...Gram-negative and form red-pigmented colonies on agar plates. The strain can grow at 25–50 °C (optimum 37 °C), at 0.9–4.8 M NaCl (optimum 2.1 M), at 0–1.0 M MgCl
2
(optimum 0.05 M) and at pH 6.5–9.5 (optimum pH 7.0). The polar lipids were found to be phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, mannosyl glucosyl diether, sulfated mannosyl glucosyl diether, and three minor unidentified glycolipids, which were chromatographically identical to those detected in
Haloprofundus (Hpf.) marisrubri
CGMCC 1.14959
T
. On the basis of 16S rRNA gene and
rpoB′
gene sequence similarities and phylogenetic analysis, strain NK23
T
was found to be related to
Hpf. marisrubri
CGMCC 1.14959
T
(97.8% and 94.1% similarities, respectively). The average nucleotide identity values and in silico DNA–DNA hybridization values between strain NK23
T
and
Hpf. marisrubri
SB9
T
were 85.22% and 29.3%, respectively. The DNA G+C content of the novel strain was determined to be 65.29 mol%. Based on the phenotypic and chemotaxonomic data, together with phylogenetic relationships, strain NK23
T
(= CGMCC 1.14944
T
= JCM 30670
T
) is considered to represent a new species of the genus
Haloprofundus
, for which the name
Haloprofundus halophilus
sp. nov. is proposed.
Two halophilic archaeal strains, SHR37
T
and NEN6, were isolated from salt lakes located in the Tibet and Xinjiang regions of China. The two strains were found to form a single cluster (99.9% and ...99.3% similarity, respectively) separating them from the six current members of
Natronorubrum
(94.7–96.9% and 86.1–90.8% similarity, respectively) on the basis of the 16S rRNA and
rpoB′
gene sequence similarities and phylogenetic analysis. Diverse phenotypic characteristics differentiate strains SHR37
T
and NEN6 from current
Natronorubrum
members. Their polar lipids are C
20
C
20
and C
20
C
25
glycerol diether derivatives of PG, PGP-Me, and a major gycolipid chromatographically identical to disulfated mannosyl glucosyl diether (S
2
-DGD). Four minor unidentified gycolipids are also present. The OrthoANI and
in silico
DDH values of the two strains were 97.3% and 76.1%, respectively, which were much higher than the threshold values proposed as a species boundary (ANI 95–96% and
in silico
DDH 70%), which revealed that the two strains represent one species; the two values (ANI 79.0–81.9% and
in silico
DDH 23.5–25.7%) of the strains examined in this study and the current members of
Natronorubrum
are much lower than the recommended threshold values, suggesting that strains SHR37
T
and NEN6 represent a genomically different species of
Natronorubrum
. These results showed that strains SHR37
T
(= CGMCC 1.15233
T
= JCM 30845
T
) and NEN6 (= CGMCC 1.17161) represent a novel species of
Natronorubrum
, for which the name
Natronorubrum halophilum
sp. nov. is proposed.
A halophilic archaeal strain, designated C46
, was isolated from an inland salt lake in Qinghai Province, PR China. Results of phylogenetic analysis based on 16S rRNA gene sequences indicated that ...strain C46
belongs to the genus
, and the closest phylogenetic relative is
DSM 9297
with 97.7 % similarity. Despite this, strain C46
was more related to
WSA2
than other members of the genus
based on genome comparison and analysis, and the average nucleotide identity,
DNA-DNA hybridization, amino acid identity and percentage of conserved protein values between the two strains were 89.1, 53.3, 89.2 and 75.6 %, respectively, which are lower than the cutoff values proposed for species delimitation. The physiological, biochemical, genetic and genomic characteristics of strain C46
were different from those of its closest phylogenetic neighbours, which indicated that this strain represents a novel species of the genus
, for which the name
sp. nov. is proposed. The type strain is C46
(=CGMCC 1.13737
=JCM 32959
).
•The hlyA gene encoded an active halolysin.•The three cysteine residues in the catalytic domain were important for the activity.•The entire CTE domain was important for the activity.•CTE can be ...functionally interchangeable among halolysins.•HlyA displayed a significant effect on fish sauce fermentation.
Extracellular proteases from haloarchaea (halolysins) can resist high salt conditions. In this study, the gene encoding a halolysin from Halococcus salifodinae was identified. The hlyA gene encoded an active halolysin with the classical Asp-His-Ser catalytic triad of serine proteases. Site-directed mutagenesis showed that the three cysteine residues in the catalytic domain were important for the extracellular proteolytic activity and displayed an additive effect on the activity. Truncation mutants of the C-terminal extension (CTE) domain displayed very low or almost no extracellular protease activity towards milk and small peptide substrates, indicating its importance for the function of HlyA. CTE can be functionally interchangeable among halolysins. Additionally, the HlyA expressing strain as a starter culture for fish sauce fermentation significantly increased the peptide release and total free amino acid content in fish sauce. This study enriches our knowledge of the key amino acid residues and domains of halolysins, and provides an opportunity for applications of halolysins in fish sauce fermentation.
As a group, the halophilic archaea (class
Halobacteria
) are the most salt-requiring and salt-resistant microorganisms within the domain
Archaea
. Halophilic archaea flourish in thalassohaline and ...athalassohaline environments and require over 100–150 g/L NaCl for growth and structural stability. Natural hypersaline environments vary in salt concentration, chemical composition and pH, and occur in climates ranging from tropical to polar and even under-sea. Accordingly, their resident haloarchaeal species vary enormously, as do their individual population compositions and community structures. These diverse halophilic archaeal strains are precious resources for theoretical and applied research but assessing their taxonomic and metabolic novelty and diversity in natural environments has been technically difficult up until recently. Environmental DNA-based high-throughput sequencing technology has now matured sufficiently to allow inexpensive recovery of massive amounts of sequence data, revealing the distribution and community composition of halophilic archaea in different hypersaline environments. While cultivation of haloarchaea is slow and tedious, and only recovers a fraction of the natural diversity, it is the conventional means of describing new species, and provides strains for detailed study. As of the end of May 2020, the class
Halobacteria
contains 71 genera and 275 species, 49.8% of which were first isolated from the marine salt environment and 50.2% from the inland salt environment, indicating that both thalassohaline and athalassohaline environments contain diverse halophilic archaea. However, there remain taxa that have not yet been isolated in pure culture, such as the nanohaloarchaea, which are widespread in the salt environment and may be one of the hot spots in the field of halophilic archaea research in the future. In this review, we focus on the cultivation strategies that have been used to isolate extremely halophilic archaea and point out some of the pitfalls and challenges.
The current species of
Halosegnis
and
Salella
within the class
Halobacteria
are closely related based on phylogenetic, phylogenomic, and comparative genomic analyses. The
Halosegnis
species showed ...99.8–100.0% 16S rRNA and 96.6–99.6%
rpoB′
gene similarities to the
Salella
species, respectively. Phylogenetic and phylogenomic analyses showed that
Salella cibi
CBA1133
T
, the sole species of
Salella
, formed a single tight cluster with
Halosegnis longus
F12-1
T
, then with
Halosegnis rubeus
F17-44
T
. The average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH), and average amino acid identity (AAI) values between
Salella cibi
CBA1133
T
and
Halosegnis longus
F12-1
T
were 99.2, 94.2, and 98.6%, respectively, much higher than the thresholds for species demarcation. This genome-based classification revealed that the genus
Salella
should be merged with
Halosegnis
, and
Salella cibi
should be a later heterotypic synonym of
Halosegnis longus
. Halophilic archaeal strains DT72
T
, DT80
T
, DT85
T
, and DT116
T
, isolated from the saline soil of a tidal flat in China, were subjected to polyphasic taxonomic characterization. The phenotypic, chemotaxonomic, phylogenetic, and phylogenomic features indicated that strains DT72
T
(= CGMCC 1.18925
T
= JCM 35418
T
), DT80
T
(= CGMCC 1.18926
T
= JCM 35419
T
), DT85
T
(= CGMCC 1.19049
T
= JCM 35605
T
), and DT116
T
(= CGMCC 1.19045
T
= JCM 35606
T
) represent four novel species of the genera
Halorussus
,
Halosegnis
and
Haloglomus
, respectively, for which the names,
Halorussus caseinilyticus
sp. nov.,
Halorussus lipolyticus
sp. nov.,
Halosegnis marinus
sp. nov., and
Haloglomus litoreum
sp. nov., are proposed.
Two novel halophilic archaeal strains (XZGYJ-43
and ZJ1
) were isolated from Mangkang ancient solar saltern (Tibet, PR China) and Zhujiang river inlet (Guangdong, PR China), respectively. The ...comparison of the 16S rRNA gene sequences revealed that strain XZGYJ-43
is related to the current species of the family
(89.2-91.7% similarity) and strain ZJ1
showed 94.7-98.3% similarity to the current species of the genus
. Phylogenetic analyses based on 16S rRNA genes,
' genes and genomes indicated that strain XZGYJ-43
is separate from the related genera,
,
and
of the family
, and strain ZJ1
tightly clusters with the current species of the genus
. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between strain XZGYJ-43
and the current species of the family
were 71-75, 20-25 and 59-68 %, and these values between strain ZJ1
and the current species of the genus
were 77-81, 27-32 and 76-82 %, respectively, clearly below the thresholds for prokaryotic species demarcation. These two strains could be distinguished from their relatives according to differential phenotypic characteristics. The major polar lipids of strain XZGYJ-43
were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), mannosyl glucosyl diether (DGD-1; DGD-PA) and sulphated mannosyl glucosyl diether (S-DGD-1; S-DGD-PA), and those of strain ZJ1
were PA, PG, PGP-Me, DGD-PA, S-DGD-1 (S-DGD-PA) and sulphated galactosyl mannosyl glucosyl diether. Based on phenotypic, phylogenetic and genomic data, strain XZGYJ-43
(=CGMCC 1.13890
=JCM 33735
) represents a novel species of a new genus within the family
, and strain ZJ1
(=CGMCC 1.18785
=JCM 34917
) represents a novel species of the genus
, for which the names
gen. nov., sp. nov. and
sp. nov. are proposed, respectively.
Marine microorganisms have long been acknowledged as a significant reservoir of enzymes required for industrial use. In this study, a novel extracellular protease
Hsl
HlyB derived from ...marine-originated haloarchaeon
Halostella pelagica
DL-M4
T
was identified.
Hsl
HlyB contained polycystic kidney disease (PKD) domain and pre-peptidase C-terminal (PPC) domain at the C-terminus. Truncation and replacement of the C-terminal extension (CTE) of
Hsl
HlyB demonstrated the importance of the CTE in maintaining the protease activity secreted by haloarchaeon.
Hsl
HlyB and
Hsl
HlyBΔCTE were expressed in
Escherichia coli
BL21(DE3), and purified by high-affinity column refolding and gel filtration chromatography. The molecular masses of
Hsl
HlyB and
Hsl
HlyBΔCTE were 42 kDa and 20 kDa, respectively. The optimum catalytic reaction conditions were 50°C, pH 8.5, NaCl 3.5 M and 50°C, pH 7.5, NaCl 3 M, respectively. They showed good stability and hydrolysis capabilities towards a wide range of protein substrates.
Hsl
HlyBΔCTE showed higher catalytic reaction rate and better thermal stability than the wild type against azocasein and tetrapeptide substrate. The hydrolysates of soybean protein hydrolyzed by
Hsl
HlyBΔCTE had smaller average molecular masses and shorter average peptide chain lengths than those by
Hsl
HlyB. These results indicated the diversity of halolysins from marine-originated haloarchaea to harness organic nitrogen in the marine environment and provided promising candidates for application in various industries.
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