All members of the genera Haloarcula and Halomicrobium whose names have been validly published were surveyed for 16S rRNA gene polymorphism, and the transcription of the genes from two species was ...investigated during growth at different NaCl concentrations. The species of Haloarcula and Halomicrobium harbour at least two different 16S rRNA gene copies, and 18 new sequences of 16S rRNA genes were obtained. The type I and type II 16S rRNA genes of Haloarcula are divergent at 4.8-5.6% of their nucleotide positions. The type III and type IV 16S rRNA genes from Halomicrobium mukohataei JCM 9738T are 9.0% divergent, which represents the highest intraspecific divergent 16S rRNA genes so far seen. Phylogenetic analysis based on 16S rRNA genes indicated that all type I 16S rRNA genes were clustered, and the same was true for the type II 16S rRNA genes of Haloarcula species. The two clusters, respectively generated from type I and type II 16S rRNA genes, were sharply separated and their divergences (4.8-5.6%) are in the range of various divergence usually found between genera in the order Halobacteriales (about 5-10%). Results from reverse transcription-PCR showed that the type I and type II copies of Har. amylolytica BD-3T and type III and type IV copies of Hmc. mukohataei JCM 9738T were all transcribed to 16S rRNA molecules under different salt concentrations (15-28% NaCl).
Two halophilic archaeal strains TS33T and KZCA124 were isolated from two distant salt lakes on the Qinghai-Xizang Plateau, respectively. Culture-independent analysis indicated that these two strains ...were original inhabitants but low abundant taxa in respective salt lakes. Strains TS33T and KZCA124 were able to grow at 20–60 °C (optimum were 42 and 35 °C, respectively), with 0.9–4.8 M NaCl (optimum were 3.0 and 2.6 M, respectively), with 0–0.7 M MgCl2 (optimum, 0.3 M) and at pH 5.0–9.5 (optimum were pH 7.5 and pH 7, respectively). The 16S rRNA and rpoB' gene similarities between these two strains were 99.7% and 99.4%, and these two similarities among strains TS33T, KZCA124, and existing species of the family Natrialbaceae were 90.6–95.5% and 84.4–89.3%, respectively. Phylogenetic and phylogenomic analyses indicated that strains TS33T and KZCA124 formed an independent branch separated from neighboring genera, Saliphagus, Natronosalvus, and Natronobiforma. The averagenucleotideidentity (ANI), digital DNA-DNAhybridization (dDDH), and average amino acid identity (AAI) values between strains TS33T and KZCA124 were 96.4%, 73.1%, and 96.7%, respectively, higher than the thresholds for species demarcation. The overall genome-related indexes between these two strains and existing species of family Natrialbaceae were 73–77%, 21–25%, and 63–70%, respectively, significantly lower than the species boundary thresholds. Strains TS33T and KZCA124 may represent a novel species of a new genus within the family Natrialbaceae judged by the cutoff value of AAI (≤76%) proposed to differentiate genera within the family Natrialbaceae. The major polar lipids of strains TS33T and KZCA124 were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated mannosyl glucosyl diether, and sulfated galactosyl mannosyl glucosyl diether. These two strains could be distinguished from the related genera according to differential phenotypic characteristics. These phenotypic, phylogenetic, and genomic analyses revealed that strains TS33T (=KCTC 4310T = MCCC 4K00132T) and KZCA124 (=CGMCC 1.17432 = JCM 34316) represent a novel species of a new genus of the family Natrialbaceae and were named Halomontanus rarus gen. nov., sp. nov.
Halophilic archaea of the class
are the most salt-requiring prokaryotes within the domain
. In 1997, minimal standards for the description of new taxa in the order
were proposed. From then on, the ...taxonomy of the class
provides an excellent example of how changing concepts on prokaryote taxonomy and the development of new methods were implemented. The last decades have witnessed a rapid expansion of the number of described taxa within the class
coinciding with the era of genome sequencing development. The current members of the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of
propose these revisions to the recommended minimal standards and encourage the use of advanced technologies in the taxonomic description of members of the
. Most previously required and some recommended minimal standards for the description of new taxa in the class
were retained in the present revision, but changes have been proposed in line with the new methodologies. In addition to the 16S rRNA gene, the
gene is an important molecular marker for the identification of members of the
. Phylogenomic analysis based on concatenated conserved, single-copy marker genes is required to infer the taxonomic status of new taxa. The overall genome relatedness indexes have proven to be determinative in the classification of the taxa within the class
. Average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values should be calculated for rigorous comparison among close relatives.
Lysophospholipase_carboxylesterase (LPCE) has highly conserved homologs in many diverse species ranging from bacteria to humans, as well as substantial biological significance and potential ...therapeutic implications. However, its biological function and catalytic mechanism remain minimally investigated because of the lack of structural information. Here, we report the crystal structure of a bacterial esterase PE8 belonging to the LPCE family. The crystal structure of PE8 was solved with a high resolution of 1.66 Å. Compared with other homologs in the family, significant differences were observed in the amino acid sequence, three-dimensional structure, and substrate-binding pattern. Residue Arg79 undergoes configuration switching when binding to the substrate and forms a unique wall, leading to a relatively closed cavity in the substrate-binding pocket compared with the relatively more open and longer clefts in other homologs. Moreover, the mutant Met122Ala showed much stronger substrate affinity and higher catalytic efficiency because less steric repulsion acted on the substrates. Taken together, these results showed that, in PE8, Arg79 and Met122 play important roles in substrate binding and the binding pocket shaping, respectively. Our study provides new insight into the catalytic mechanism of LPCE, which may facilitate the development of structure-based therapeutics and other biocatalytic applications.
Two halophilic archaeal strains, R2T and R27T, were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Both had pleomorphic cells that lysed in distilled water, ...stained Gram-negative and formed red-pigmented colonies. They grew optimally at 42 °C, pH 7.0 and in the presence of 3.1–3.4 M NaCl and 0.03–0.5 M Mg2+. The major polar lipids of the two strains were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and four major glycolipids chromatographically identical to those of Halosimplex carlsbadense JCM 11222T. 16S rRNA gene analysis revealed that each strain had two dissimilar 16S rRNA genes and both strains were phylogenetically related to Halosimplex carlsbadense JCM 11222T (92.7–98.8 % similarities). The rpoB′ gene similarities between strains R2T and R27T and between these strains and Halosimplex carlsbadense JCM 11222T were 95.7 %, 96.1 % and 95.8 %, respectively. The DNA G+C contents of strains R2T and R27T were 62.5 mol% and 64.0 mol%, respectively. The DNA–DNA hybridization values between strains R2T and R27T and between the two strains and Halosimplex carlsbadense JCM 11222T were 43 %, 52 % and 47 %, respectively. It was concluded that strain R2T ( = CGMCC 1.10586T = JCM 17263T) and strain R27T ( = CGMCC 1.10591T = JCM 17268T) represent two novel species of the genus Halosimplex, for which the names Halosimplex pelagicum sp. nov. and Halosimplex rubrum sp. nov. are proposed. An emended description of the genus Halosimplex is also presented.
Three novel filamentous halophilic archaea, strains DFN5
T
, RDMS1, and QDMS1, were isolated from the coastal saline soil samples of the intertidal zones located in different regions of Jiangsu ...Province, China. The colonies of these strains were pinkish-white due to the presence of white spores. These three strains are extremely halophilic and grew optimally at 35–37 °C and pH 7.0–7.5. Based on 16S rRNA and
rpoB′
gene analysis, strains DFN5
T
, RDMS1, and QDMS1 gathered together in phylogenetic trees and then clustered with the current species of the genus
Halocatena
showing 96.9–97.4% and 82.2–82.5% similarities, respectively. Both the 16S rRNA gene-based and
rpoB'
gene-based phylogenies were fully supported by the phylogenomic analysis, and the overall genome-related indexes indicated that strains DFN5
T
, RDMS1, and QDMS1 should be a novel species of the genus
Halocatena
. Genome mining revealed that there are considerable differences in the genes related to β-carotene synthesis among these three strains and the current species of
Halocatena
. The major polar lipids of strains DFN5
T
, RDMS1, and QDMS1 are PA, PG, PGP-Me, S-TGD-1, TGD-1, and TGD-2. The minor polar lipids, S-DGD-1, DGD-1, S
2
-DGD, and S-TeGD may be detected. According to the phenotypic characteristics, phylogenetic analysis, genomic and chemotaxonomic features, strains DFN5
T
(= CGMCC 1.19401
T
= JCM 35422
T
), RDMS1 (= CGMCC 1.19411) and QDMS1 (= CGMCC 1.19410) were classified as a novel species of the genus
Halocatena
with the proposed name,
Halocatena marina
sp. nov. This is the first report of the description of a novel filamentous haloarchaeon isolated from marine intertidal zones.
A halophilic archaeal strain, YGH44T, was isolated from the Yinggehai marine solar saltern in Hainan Province of China. Cells were rod-shaped, stained Gram-negative and formed red-pigmented colonies ...on agar plates. Optimal growth was obtained with 3.4 M NaCl (range: 2.6-4.8 M), 0.5 M MgCl2 (range: 0.005-1.0 M), at 37 °C (range: 25-55 °C) and at pH 7.0 (range: pH 5.0-9.0). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell lysis was 1.7 M. Phylogenetic tree reconstructions based on 16S rRNA genes and rpoB' genes revealed that strain YGH44T was distinct from the related genera, Halovenus, Halapricum, Halorientalis, Halorhabdus and Halosimplex of the order Halobacteriales. The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three unidentified glycolipids. The DNA G+C content of strain YGH44Twas 69.0 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain YGH44T (=CGMCC 1.12234T=JCM 18646T) represents a novel species of a new genus within the order Halobacteriales, for which the name Salinirussus salinus gen. nov., sp. nov. is proposed.
Two halophilic archaeal strains TBN4T and TBN5 were isolated from Taibei marine solar saltern in Jiangsu, China. Both strains showed light red-pigmented colonies and their cells were rod, motile and ...Gram-stain-negative. They were able to grow at 25-50°C (optimum 37°C), at 1.4-4.3 M NaCl (optimum 2.1 M NaCl), at 0-1.0 M MgCl₂ (optimum 0.005 M MgCl₂) and at pH 6.0-9.0 (optimum pH 7.0). Their cells lyse in distilled water and minimal NaCl concentration to prevent cell lysis is 8% (w/v). The major polar lipids of the two strains were PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphate methyl ester), PGS (phosphatidylglycerol sulfate) and five glycolipids chromatographically identical to S-TGD-1 (sulfated galactosyl mannosyl glucosyl diether), S-DGD-1 (sulfated mannosyl glucosyl diether), TGD-1 (galactosyl mannosyl glucosyl diether), DGD-1 (mannosyl glucosyl diether) and DGD-2 (an unknown diglycosyl diether). Phylogenetic analysis revealed that TBN4T and strain TBN5 formed a distinct clade with genus Haladaptatus (showing 90.0-90.9% 16S rRNA gene similarities). The DNA G + C content of strain TBN4T and strain TBN5 are 66.1 and 65.4 mol%, respectively. The DNA-DNA hybridization value between strain TBN4T and strain TBN5 was 94.3%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain TBN4T and strain TBN5 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Halorussus rarus gen. nov., sp. nov. is proposed. The type strain is TBN4T (=CGMCC 1.10122T = JCM 16429T).
The deep-sea environment harbors a vast pool of novel enzymes. Owing to the limitations of cultivation, cultivation-independent has become an effective method for mining novel enzymes from the ...environment. Based on a deep-sea sediment metagenomics library, lipolytic-positive clones were obtained by activity-based screening methods.
Two novel esterases, DMWf18-543 and DMWf18-558, were obtained from a deep-sea metagenomic library through activity-based screening and high-throughput sequencing methods. These esterases shared 80.7% amino acid identity with each other and were determined to be new members of bacterial lipolytic enzyme family IV. The two enzymes showed the highest activities toward p-nitrophenyl (p-NP) butyrate at pH 7.0 and 35-40 °C and were found to be resistant to some metal ions (Ba
, Mg
, and Sr
) and detergents (Triton X-100, Tween 20, and Tween 80). DMWf18-543 and DMWf18-558 exhibited distinct substrate specificities and preferences. DMWf18-543 showed a catalytic range for substrates of C2-C8, whereas DMWf18-558 presented a wider range of C2-C14. Additionally, DMWf18-543 preferred p-NP butyrate, whereas DMWf18-558 preferred both p-NP butyrate and p-NP hexanoate. To investigate the mechanism underlying the phenotypic differences between the esterases, their three-dimensional structures were compared by using homology modeling. The results suggested that residue Leu199 of DMWf18-543 shortens and blocks the substrate-binding pocket. This hypothesis was confirmed by the finding that the DMWf18-558-A199L mutant showed a similar substrate specificity profile to that of DMWf18-543.
This study characterized two novel homologous esterases obtained from a deep-sea sediment metagenomic library. The structural modeling and mutagenesis analysis provided insight into the determinants of their substrate specificity and preference. The characterization and mechanistic analyses of these two novel enzymes should provide a basis for further exploration of their potential biotechnological applications.
Halophilic archaeal strain TGN-42-S1(T) was isolated from the Tanggu marine solar saltern, China. Cells from strain TGN-42-S1(T) were observed to be pleomorphic rods, stained Gram-negative, and ...formed red-pigmented colonies on solid media. Strain TGN-42-S1(T) was found to be able to grow at 20-50 °C (optimum 35-37 °C), at 1.7-4.8 M NaCl (optimum 3.1 M), at 0-1.0 M MgCl2 (optimum 0.1 M), and at pH 5.0-9.0 (optimum pH 7.0-7.5). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell-lysis was found to be 10 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, galactosyl mannosyl glucosyl diether (TGD-1), sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), sulfated galactosyl mannosyl galactofuranosyl glucosyl diether (S-TeGD), and three unidentified glycolipids which were chromatographically identical to those of the Halobacterium species. The 16S rRNA gene and rpoB' gene of strain TGN-42-S1(T) were phylogenetically related to the corresponding genes of Halobacterium jilantaiense CGMCC 1.5337(T) (98.8 and 93.5 % nucleotide identity, respectively), Halobacterium salinarum CGMCC 1.1958(T) (98.4 and 91.9 %), and Halobacterium noricense JCM 15102(T) (96.9 and 91.1 %). The DNA G + C content of strain TGN-42-S1(T) was determined to be 69.2 mol %. Strain TGN-42-S1(T) showed low DNA-DNA relatedness with Hbt. jilantaiense CGMCC 1.5337(T) and Hbt. salinarum CGMCC 1.1958(T), the most closely related members of the genus Halobacterium. The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain TGN-42-S1(T) (=CGMCC 1.12575(T) =JCM 19908(T)) represents a new species of Halobacterium, for which the name Halobacterium rubrum sp. nov. is proposed.
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