Currently, there are four mainstream taxonomic opinions on the classification of the class
at the family and order levels. The International Committee on Systematics of Prokaryotes Subcommittee on ...the Taxonomy of
(ICSP), List of Prokaryotic names with Standing in Nomenclature (LPSN) and National Centre for Biotechnology Information (NCBI) adopted taxonomies have three to four orders and up to eight families, while the Genome Taxonomy Database (GTDB) taxonomy proposes only one order with nine families. To resolve the taxonomic inconsistency, phylogenomic analyses based on concatenated single-copy orthologous proteins and 122 concatenated conserved single-copy marker proteins were conducted to infer the taxonomic status of the current representatives of the class
at the family and order levels. The current 76 genera with validly published names of the class
were able to be assigned into eight families in one order. On the basis of these results, it is proposed that the current species with validly published names of the class
should be remerged into the order
, then assigned to eight families,
,
,
,
,
,
,
and
. Thus,
fam. nov. is described based on
,
and
and
fam. nov. is proposed incorporating
and
, respectively.
Halophilic archaea represent a promising natural source of carotenoids. However, little information is available about the biological effects of carotenoids from halophilic archaea. In this study, ...the carotenoids produced by seven halophilic archaeal strains
Halogeometricum rufum, Halogeometricum limi, Haladaptatus litoreus, Haloplanus vescus, Halopelagius inordinatus, Halogranum rubrum
, and
Haloferax volcanii
were identified by ultraviolet/visible spectroscopy, thin-layer chromatography, and high-performance liquid chromatography-tandem mass spectrometry. The C
50
carotenoids bacterioruberin and its derivatives monoanhydrobacterioruberin and bisanhydrobacterioruberin were found to be the predominant carotenoids. The antioxidant capacities of the carotenoids from these strains were significantly higher than β-carotene as determined by 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. The antihemolytic activities of these carotenoid extracts against H
2
O
2
-induced hemolysis in mouse erythrocytes were 3.9–6.3 times higher than β-carotene. A dose-dependent
in vitro
antiproliferative activity against HepG2 cells was observed for the extract from
Hgm. limi
, while that from
Hpn. vescus
exhibited a relatively high activity in a dose-independent manner. These results suggested that halophilic archaea could be considered as an alternative source of natural carotenoids with high antioxidant, antihemolytic, and anticancer activity.
Halophilic extracellular proteases offer promising application in various fields. Information on these prominent proteins including the synthesizing organisms, biochemical properties, domain ...organisation, purification, and application challenges has never been covered in recent reviews. Although extracellular proteases from bacteria pioneered the study of proteases in halophiles, progress is being made in proteases from halophilic archaea. Recent advances in extracellular proteases from archaea revealed that archaeal proteases are more robust and applicable. Extracellular proteases are composed of domains that determine their mechanisms of action. The intriguing domain structure of halophilic extracellular proteases consists of N-terminal domain, catalytic domain, and C-terminal extension. The role of C-terminal domains varies among different organisms. A high diversity of C-terminal domains would endow the proteases with diverse functions. With the development of genomics, culture-independent methods involving heterologous expression, affinity chromatography, and in vitro refolding are deployed with few challenges on purification and presenting novel research opportunities. Halophilic extracellular proteases have demonstrated remarkable potentials in industries such as detergent, leather, peptide synthesis, and biodegradation, with desirable properties and ability to withstand harsh industrial processes.
Key points
•
Halophilic extracellular proteases have robust properties suitable for applications.
•
A high diversity of C-terminal domains may endow proteases with diverse properties.
•
Novel protease extraction methods present novel application opportunities.
Graphical abstract
Three rod-shaped halophilic archaeal strains, DL-M4
, LYG-109 and DLLS-108
, were isolated from the salted brown alga
produced in different marine areas of PR China. Cells of strains were motile, ...formed red-pigmented colonies on agar and lysed in distilled water. The three strains grew optimally with 2.6 M NaCl, with 0.05-0.3 M MgCl
, at 37 °C and at pH 7.0-7.5. The results of phylogenetic analyses based on the 16S rRNA and
genes differentiated these strains into two clusters belonging to the genus
, which currently contains
CBA1114
and
LT12
. Strains DL-M4
and LYG-109 formed a single cluster separate from the current two members of
(94.4-95.7 and 90.0-90.9 % similarities, respectively) while strain DLLS-108
had
CBA1114
as its nearest neighbour (97.7-97.8 and 95.9 % similarities, respectively) and was separated from
LT12
(94.4-95.8 and 93.4 % similarities, respectively). These clusters represented two distinct novel species as indicated by phenotypic characteristics, polar lipid compositions and whole-genome comparisons. Diverse phenotypic characteristics, morphology and growth characteristics, nutrition and miscellaneous biochemical tests differentiate strains DL-M4
, LYG-109, DLLS-108
from
LT12
and
CBA1114
. Strains DL-M4
and LYG-109 contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three unidentified glycolipids, while strain DLLS-108
contained these polar lipids and two unidentified phospholipids. The major respiratory quinones detected in the three isolates were menaquinone MK-8 and MK-8(H
). The average nucleotide identity (ANI) and
DNA-DNA hybridization (isDDH) values between the isolated strains and the current two members of
were found to be 79.3-86.6 (ANI) and 22.9-49.8 % (isDDH). All these results showed that the three isolates represent two novel species of the genus
for which the names
sp. nov. type strain dl-M4
(=CGMCC 1.13603
=JCM 32954
) and
sp. nov. type strain DLLS-108
(=CGMCC 1.13610
=JCM 32955
) are proposed.
Five halophilic archaeal strains (AGai3-5
T
, KZCA101
T
, CGA3
T
, WLHS1
T
and WLHSJ1
T
) were isolated from salt lakes and soda lakes in PR China. These strains had low 16S rRNA gene similarities ...(91.3–96.0 %) to closely related species of the family
Natrialbaceae
and may represent a new genus of the family. Phylogenetic and phylogenomic analyses revealed that these strains formed a distinct clade, separate from the nearby genera
Natronobiforma
and
Saliphagus
. The average nucleotide identity, digital DNA–DNA hybridization and average amino acid identity (AAI) values among these five strains and the current members of the family
Natrialbaceae
were 72–90, 20–42 and 62–91 %, respectively, clearly below the threshold values for species demarcation. According to the critical value of AAI (≤76 %) proposed to differentiate genera within the family
Natrialbaceae
, it was further indicated that these strains represented a novel genus within the family. These strains could be distinguished from the related genera according to differential phenotypic characteristics. The major lipids of these strains were phosphatidic acid (PA), phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, mannosyl glucosyl diether (DGD-PA), sulphated DGD-1 (S-DGD-PA) and sulphated galactosyl mannosyl glucosyl diether. The phenotypic, chemotaxonomic, phylogenetic and phylogenomic features indicated that strains AGai3-5
T
(=CGMCC 1.16078
T
=JCM 33549
T
), KZCA101
T
(=CGMCC 1.17431
T
=JCM 35074
T
), CGA3
T
(=CGMCC 1.17463
T
=JCM 34318
T
), WLHS1
T
(=CGMCC 1.13780
T
=JCM 33562
T
) and WLHSJ1
T
(=CGMCC 1.13784
T
=JCM 33563
T
) represent five novel species of a new genus within the family
Natrialbaceae
, named
Natronosalvus halobius
gen. nov., sp. nov.,
Natronosalvus caseinilyticus
sp. nov.,
Natronosalvus vescus
sp. nov.,
Natronosalvus rutilus
sp. nov. and
Natronosalvus amylolyticus
sp. nov., respectively.
Four halophilic archaeal strains, designated HD8-83
, LYG-36
, DLLS-82 and RC-68
, were isolated from the salted brown alga
of three different origins (Dalian, Lianyungang, Dalian and Rongcheng) in ...PR China. All strains had pleomorphic rod cells that were motile, lysed in distilled water, stained Gram-negative, and formed red-pigmented colonies on agar plate (except for DLLS-82, which formed white colonies). Based on phylogenetic analyses of the 16S rRNA genes, strain HD8-83
was closely related to
HD8-51
(97.9 % similarity), strain LYG-36
and DLLS-82 to
TBN4
(94.4 % and 94.7 % similarities, respectively), and strain RC-68
to
YJ-37-H
(96.9 % similarity). Results of phylogenetic analyses based on
genes and 728 concatenated single-copy orthologous clusters also showed that these strains formed three different branches and clustered tightly with the
members. The average nucleotide identity, average amino acid identity and
DNA-DNA hybridization values between strains LYG-36
and DLLS-82 were 98.9, 98 and 92.4%, showing that they were different strains of the same species. While those values between the isolates and other
members were below 84.7, 82.9 and 28.9 %, respectively. Based on the phenotypic, chemotaxonomic and phylogenetic properties, strains HD8-83
, LYG-36
, DLLS-82 and RC-68
represent three novel species of the genus
for which the names
sp. nov. (type strain: HD8-83
=CGMCC 1.15334
=JCM 31110
),
sp. nov. (type strain: LYG-36
=CGMCC 1.13606
=JCM 32952
; reference strain: DLLS-82=CGMCC 1.13604=JCM 32951) and
sp. nov. (type strain: RC-68
=CGMCC 1.13609
=JCM 32953
) are proposed.
Two extremely halophilic archaeal strains, GSLN9
and XZYJT29
, were isolated from the saline soil in different regions of western China. Both strains GSLN9
and XZYJT29
have two 16S rRNA genes with ...similarities of 95.1 and 94.8 %, respectively. Strain GSLN9
was mostly related to the genus
based on 16S rRNA (showing 91.0-96.0 % identities) and
genes (showing 92.0 % identity). Strain XZYJT29
showed 92.1-97.6 % (16S rRNA gene) and 91.4-93.1 % (
gene) sequence similarities to its relatives in the genus
, respectively. The polar lipid profile of strain GSLN9
included phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulphate (PGS), sulphated mannosyl glucosyl diether (S-DGD-1) and sulphated galactosyl mannosyl glucosyl diether (S-TGD-1), mostly similar to that of
H22
. PA, PG, PGP-Me, S-DGD-1 (S-DGD-PA), S
-DGD, S-TGD-1 and an unidentified glycolipid were detected in strain XZYJT29
; this polar lipid composition is similar to those of members of the genus
. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between these two strains and their relatives of the genera
and
were no more than 82, 27 and 80 %, respectively, much lower than the thresholds for species demarcation. Other phenotypic characterization results indicated that strains GSLN9
and XZYJT29
can be differentiated from the current species of the genera
and
, respectively. These results revealed that strains GSLN9
(=CGMCC 1.15215
=JCM 30842
) and XZYJT29
(=CGMCC 1.15828
=JCM 31853
) represent novel species of
and
, for which the names
sp. nov. and
sp. nov. are proposed.
A novel Gram-stain-negative, aerobic and rod-shaped halophilic archaeon, designated HD8-45
, was isolated from the red brine of salted brown alga
produced at Dalian, PR China. According to the ...results of 16S rRNA gene and
gene sequence comparisons, strain HD8-45
showed the highest sequence similarity to the corresponding genes of
YGH44
(95.1 and 85.2 % similarities, respectively),
EB27
(91.2 and 86.0 % similarities, respectively). The low sequence similarity and the phylogeny implied the novel generic status of strain HD8-45
. Genomic relatedness analyses showed that strain HD8-45
were clearly distinguished from other species in the order
, with average nucleotide identity, amino acid identity and
DNA-DNA hybridization values not more than 75.1, 65.6 and 21.5 %. The polar lipid pattern contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids and two minor glycolipids. The two major glycolipids and a minor glycolipid were chromatographically identical to disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. The major respiratory quinones were menaquinone MK-8 and MK-8(H
). The DNA G+C content was 62.0 mol% (
) and 61.9 mol% (genome). All these results showed that strain HD8-45
represents a novel species of a new genus in the order
, for which the name
gen. nov., sp. nov. is proposed. The type strain of
is HD8-45
(=CGMCC 1.15328
=JCM 31107
).
A halophilic archaeal strain, designated HD8-51
, was isolated from the salted brown alga Laminaria. Cells of strain HD8-51
were motile, pleomorphic coccoid or ovoid, and formed red-pigmented ...colonies on agar plates. Strain HD8-51
grew optimally at 3.1 M NaCl, 0.03 M MgCl2, 30 °C and pH 7.0. Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 0.85 M. Based on phylogenetic analyses of the 16S rRNA and rpoB' genes, strain HD8-51
was most closely related to members of the genus Halorussus (92.3-95.6 % and 89.2-91.7% similarities, respectively). The average nucleotide identity values and in silico DNA-DNA hybridization values between strain HD8-51
and Halorussus rarus TBN4
were 81.69 and 24.5 %, respectively. The major polar lipids of strain HD8-51
were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS) and five glycolipids, sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), galactosyl mannosyl glucosyl diether (TGD-1), sulfated mannosyl glucosyl diether (S-DGD-1), mannosyl glucosyl diether (DGD-1) and diglycosyl diether (DGD-2). The DNA G+C content was 65.9 mol%. Based on phenotypic, chemotaxonomic and phylogenetic properties, strain HD8-51
represents a novel species of the genus Halorussus, for which the name Halorussus litoreus sp. nov. is proposed. The type strain is HD8-51
(=CGMCC 1.15333
=JCM 31109
).
The halophilic archaeal strain ZS-3
T
(= CGMCC 1.12866
T
= JCM 30239
T
) was isolated from a sediment sample of Zhoushan marine solar saltern, P. R. China. Phylogenetic analyses based on 16S rRNA,
...rpoB′
genes and the concatenation of 738 protein sequences reveal that strain ZS-3
T
was related to members of the genus
Halorussus
. The OrthoANI and in silico DDH values between strain ZS-3
T
and the current
Halorussus
members are much lower than the threshold values proposed as the species boundary (ANI 95–96% and in silico DDH 70%), suggesting that strain ZS-3
T
represents a novel species of
Halorussus
(
Halorussus halophilus
sp. nov.). Diverse phenotypic characteristics differentiate strain ZS-3
T
from current
Halorussus
members. Since the strain expressed diverse hydrolyzing enzyme activity, its complete genome was sequenced. The genome of strain ZS-3
T
was found to be 4,450,731 bp with total GC content of 61.51%, and comprises one chromosome and three plasmids. A total of 4694 protein coding genes, 43 tRNA genes and 6 rRNA genes were predicted. A CRISPR–Cas system was also detected. The genome encodes sixteen putative glycoside hydrolases, nine extracellular proteases, seventeen aminopeptidases, seven carboxypeptidases, one esterase and one nitrite reductase. The exploration of the hydrolase genes may expand our understanding of adapted mechanism of halophilic archaea surviving optimally in hypersaline environments where containing organic matter. Meanwhile, various hydrolyzing enzymes may extend this microorganism for further applications in salt-based fermentation.
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