Aerobic Anoxygenic Phototrophic Bacteria in the Marine Environments Revealed by Raman/Fluorescence-Guided Single-Cell Sorting and Targeted Metagenomics.

Aerobic anoxygenic phototrophic bacteria (AAPB) contribute profoundly to the global carbon cycle. However, most AAPB in marine environments are uncultured and at low abundance, hampering the recognition of their functions and molecular mechanisms. In this study, we developed a new culture-independent method to identify and sort AAPB using single-cell Raman/fluorescence spectroscopy. Characteristic Raman and fluorescent bands specific to bacteriochlorophyll a (Bchl a) in AAPB were determined by comparing multiple known AAPB with non-AAPB isolates. Using these spectroscopic biomarkers, AAPB in coastal seawater, pelagic seawater, and hydrothermal sediment samples were screened, sorted, and sequenced. 16S rRNA gene analysis and functional gene annotations of sorted cells revealed novel AAPB members and functional genes, including one species belonging to the genus Sphingomonas, two genera affiliated to classes Betaproteobacteria and Gammaproteobacteria, and function genes bchCDIX, pucC2, and pufL related to Bchl a biosynthesis and photosynthetic reaction center assembly. Metagenome-assembled genomes (MAGs) of sorted cells from pelagic seawater and deep-sea hydrothermal sediment belonged to Erythrobacter sanguineus that was considered as an AAPB and genus Sphingomonas, respectively. Moreover, multiple photosynthesis-related genes were annotated in both MAGs, and comparative genomic analysis revealed several exclusive genes involved in amino acid and inorganic ion metabolism and transport. This study employed a new single-cell spectroscopy method to detect AAPB, not only broadening the taxonomic and genetic contents of AAPB in marine environments but also revealing their genetic mechanisms at the single-genomic level.

Euzebya pacifica sp. nov., a novel member of the class Nitriliruptoria.

A Gram-stain-positive, aerobic, chemo-organotrophic, rod-shaped, non-spore-forming strain, which produced convex, circular, pink-pigmented colonies, designated as DY32-46T, was isolated from seawater collected from the Pacific Ocean. DY32-46T was found to grow at 20-40 °C (optimum, 30-35 °C), pH 6.0-8.0 (optimum, pH 6.5) and with 0-5 % (w/v) NaCl (optimum, 1-2 %). The results of chemotaxonomic analysis indicated that the respiratory quinone of DY32-46T was MK-9(H4), and major fatty acids (>10 %) were C17 : 1 ω8c, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C15 : 1 ω6c. The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, one unidentified aminophospholipid, three unidentified glycolipids, three unidentified phospholipids, one unidentified phosphoglycolipid and five unidentified lipids. The DNA G+C content of DY32-46T was 70.6 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences and genomic data indicated that DY32-46T should be assigned to the genus Euzebya. ANI and in silico DNA-DNA hybridization values between strain DY32-46T and type strains of Euzebya species were 73.1-87.2 % and 20.2-32.4 %, respectively. Different phenotypic properties, together with genetic distinctiveness, demonstrated that strain DY32-46T was clearly distinct from recognized species of the genus Euzebya. Therefore, DY32-46T represents a novel species within the genus Euzebya, for which the name Euzebya pacifica sp. nov is proposed. The type strain is DY32-46T (=MCCC 1K03476T=KCTC 49091T).

A novel SGNH family hydrolase Ali5 with thioesterase activity and a GNSL motif but without a classic GDSL motif from Altererythrobacter ishigakiensis.

OBJECTIVE: We aimed to characterize a novel SGNH (Ser-Gly-Asn-His) family hydrolase from the annotated genome of marine bacteria with new features. RESULTS: A novel esterase Ali5 from Altererythrobacter ishigakiensis has been identified and classified into SGNH family. Ali5 presented a novel GNSL (Gly-Asn-Ser-Leu(X)) motif that differs from the classic GDSL (Gly-Asp-Ser-Leu(X)) motif of SGNH family. The enzyme has esterase and thioesterase activity and exhibited apparent temperature and pH optima of 40 °C and pH 7.5 (in phosphate buffer), respectively. Ali5 was found to be halotolerant and thermostable, and exhibited strong resistance to several organic solvents and metal ions. The residue Tyr196 has a great influence on the catalytic activity, which was proved by site-directed mutagenesis and subsequent kinetic characterization. CONCLUSION: The esterase Ali5 with esterase and thioesterase activities, salt and metal ions resistance and unique structural features was identified, which holds promise for research on the SGNH family of hydrolases.

Two novel deep-sea sediment metagenome-derived esterases: residue 199 is the determinant of substrate specificity and preference.

BACKGROUND: 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. RESULTS: 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 (Ba2+, Mg2+, and Sr2+) 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. CONCLUSIONS: 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.

Characterization of a novel alkaline esterase from Altererythrobacter epoxidivorans CGMCC 1.7731T.

A novel esterase gene (e25) was identified from Altererythrobacter epoxidivorans CGMCC 1.7731T by genome sequence screening. The e25 gene is 948 nucleotides in length and encodes a 315 amino acid protein (E25) with a predicted molecular mass of 33,683 Da. A phylogenetic tree revealed that E25 belongs to the hormone-sensitive lipase (HSL) family of lipolytic enzymes. An activity assay of E25 showed that it exhibited the highest catalytic efficiency when using p-nitrophenyl caproate (C6) as a substrate. The optimum pH and temperature were determined to be approximately pH 9 and 45°C, and the Km and Vmax values were 0.12 mM and 1,772 µmol/min/mg, respectively. After an incubation at 40°C for 80 min, E25 retained 75% of its basal activity. The enzyme exhibited good tolerance to metal cations, such as Ba2+, Ca2+, and Cu2+ (10 mM), but its activity was strongly inhibited by Co2+, Ni2+, Mn2+, and Zn2+. The E25 enzyme was stimulated by glycerol and retained over 60% of its basal activity in the presence of 1% Tween-80 and Triton X-100. Overall, the activity of E25 under alkaline conditions and its organic solvent and detergent tolerance indicate that E25 could be useful as a novel industrial catalyst in biotechnological applications.

Pseudohongiellanitratireducens sp. nov., isolated from seawater, and emended description of the genus Pseudohongiella.

Two Gram-stain-negative, aerobic, motile by a single polar flagellum and rod-shaped strains, designated SCS-49T and SCS-111, were isolated from seawater of the South China Sea. The two strains grew at 4-35 °C, with 0.5-7.5 % (w/v) NaCl and at pH 6.5-9.0 and were able to reduce nitrate. Q-8 was the sole ubiquinone. The major fatty acids of the two strains were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospoglycolipid, three unidentified glycolipids, five unidentified phospholipids and two to three unidentified lipids. The isolates formed a stable clade with Pseudohongiella acticola and Pseudohongiella spirulinae based on phylogenetic analysis of 16S rRNA gene sequences. Strains SCS-49T and SCS-111 exhibited 16S rRNA gene sequence similarity values of 97.2 and 96.0 % with respect to the type strains of P. acticola and P. spirulinae, respectively. The average nucleotide diversity and in silico DNA-DNA hybridization values between strain SCS-49T and P. acticola KCTC 42131T were 71.4 and 25.1 %, respectively and the values between strain SCS-49T and SCS-111 were 99.9 and 99.2 %, respectively. Based upon the phenotypic, chemotaxonomic and genetic data, strains SCS-49T and SCS-111 represent a novel species in the genus Pseudohongiella, for which the name Pseudohongiella nitratireducens sp. nov. is proposed. The type strain is SCS-49T (=CGMCC 1.15425T=KCTC 52155T=MCCC 1K03186T).

Kordiimonas lipolytica sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, rod-shaped bacterium, designated M41T, was isolated from a surface seawater sample collected from the western Pacific Ocean. The isolate grew in medium containing 0.5-10.0 % (w/v) NaCl (optimally at 1.0-3.0 %) at 15-45 °C and pH 5.5-9.5. Positive for oxidase, catalase and nitrate reduction. The respiratory quinone is Q-10. The major fatty acids (>10 %) are iso-C15:0, iso-C17:1ω9c and summed feature 3 (comprising C16:1ω7c and/or iso-C15:0 2-OH). The major polar lipids are phosphatidylethanolamine, one unidentified phospholipid, one unidentified aminolipid, and three unidentified glycolipids.The genomic DNA G+C content is 56.3 mol %. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M41T should be assigned to the genus Kordiimonas. The 16S rRNA gene sequence similarities between the isolate and the type strains of species of the genus Kordiimonas with validly published names were in the range 96.2- 98.6 %. Strain M41T exhibited average nucleotide identity (ANI) values of 81.7 and 72.3 % with respect to Kordiimonas. lacus S3-22T and Kordiimonasgwangyangensis JCM 12864T, respectively. The genome-to-genome distance analysis revealed that strain M41T shared 51.4 % DNA-DNA relatedness with K. lacus S3-22T and 16.3 % with K. gwangyangensis JCM 12864T. On the basis of phenotypic and genotypic characteristics, strain M41T represents a novel species of the genus Kordiimonas, for which the name Kordiimonas lipolytica sp. nov. is proposed. The type strain is M41T (=CGMCC 1.15304T=JCM 30877T). An emended description of Kordiimonas lacus is also provided.

Pontibacter amylolyticus sp. nov., isolated from a deep-sea sediment hydrothermal vent field.

A Gram-stain-negative, short rod-shaped bacterium, designated 9-2T, was isolated from a sediment sample collected from a hydrothermal vent field on the south-west Indian Ridge. It formed red colonies, produced carotenoid-like pigments and did not produce bacteriochlorophyll a. Strain 9-2T was positive for hydrolysis of DNA, gelatin and starch, but negative for hydrolysis of aesculin and Tween 60. The sole respiratory quinone was menaquinone-7 (MK-7). The main polar lipids consisted of phosphatidylethanolamine, one unidentified phospholipid and two unidentified polar lipids. The principal fatty acids (>5%) were summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B), iso-C15:0 and iso-C17:0 3-OH. The genomic DNA G+C content was 49.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 9-2T should be assigned to the genus Pontibacter. Levels of 16S rRNA gene sequence similarity between the new isolate and the type strains of Pontibacter species with validly published names were in the range 94.0-96.5%. On the basis of phenotypic and genotypic data, strain 9-2T represents a novel species of the genus Pontibacter, for which the name Pontibacter amylolyticus sp. nov. is proposed. The type strain is 9-2T (=CGMCC 1.12749T=JCM 19653T=MCCC 1K00278T).

Aquaticitalea lipolytica gen. nov., sp. nov., isolated from Antarctic seawater.

A Gram-stain-negative, rod-shaped bacterium, designated Ar-125T, was isolated from Antarctic seawater. It produced carotenoid-like pigments and did not produce Bchl a. Ar-125T was positive for hydrolysis of DNA, aesculin, gelatin, starch, Tween 40 and Tween 60. The sole respiratory quinone was MK-6. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, one unidentified glycolipid and two unidentified lipids. The principal fatty acids were branched-chain fatty acids, including iso-C15 : 0, iso-C15 : 1 G, summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C16 : 0, iso-C17 : 0 3-OH, iso-C16 : 0 3-OH and iso-C15 : 0 3-OH, as well as C15 : 0. The genomic DNA G+C content was 31.8 mol%. On the basis of 16S rRNA gene sequence analysis, Ar-125T is closely related to the species of the genera Bizionia(with 16S rRNA gene pairwise sequence similarity of 93.7-96.5 %), Formosa(94.3-95.8 %), Gaetbulibacter(94.2-95.7 %), Geojedonia(95.5 %), Gelidibacter (93.3-95.4 %), Meridianimaribacter(95.3 %) and Psychroserpens (94.8-95.3 %), of the family Flavobacteriaceae. Phylogenetic analysis indicated that it represented an independent lineage and that the closest relatives were members of the genus Gelidibacter. Differential phenotypic properties and chemotaxonomic differences, together with phylogenetic distinctiveness, revealed that Ar-125T could be differentiated from members of closely related genera. Therefore, it is proposed that Ar-125T represents a novel species in a new genus, for which the name Aquaticitalea lipolytica gen. nov., sp. nov. (type strain Ar-125T =CGMCC 1.15295T =JCM 30876T) is proposed.

Croceicoccus pelagius sp. nov. and Croceicoccus mobilis sp. nov., isolated from marine environments.

Strain Ery9T, isolated from surface seawater of the Atlantic Ocean, and strain Ery22T, isolated from deep-sea sediment of the Indian Ocean, were subjected to a taxonomic study using a polyphasic approach. Cells of the two strains were Gram-stain-negative, aerobic and rod-shaped. They produced yellow pigments and lacked bacteriochlorophyll a. On the basis of 16S rRNA gene sequence analysis, strain Ery9T was closely related to Croceicoccus naphthovorans PQ-2T (with 16S rRNA gene sequence similarity of 97.7 %), and strain Ery22T was closely related to Croceicoccusmarinus E4A9T (98.3 %). The 16S rRNA gene sequence similarity between strain Ery9T and strain Ery22T was 96.6 %. Phylogenetic analyses revealed that strains Ery9T and Ery22T fell within the cluster of the genus Croceicoccus and represented two independent lineages. The average nucleotide identity (ANI) values and the genome-to-genome distances between strains Ery9T and Ery22T and the type strains of species of the genus Croceicoccus with validly published names were 73.7-78.4 % and 20.1-22.3 %, respectively. The major respiratory quinone of the two isolates was ubiquinone-10 (Q-10). The DNA G+C contents of strains Ery9T and Ery22T were 62.8 and 62.5 mol%, respectively. Differential phylogenetic distinctiveness and chemotaxonomic differences, together with phenotypic properties, revealed that strains Ery9T and Ery22T could be differentiated from their closely related species. Therefore, it is concluded that strains Ery9T and Ery22T represent two novel species of the genus Croceicoccus, for which the names Croceicoccus pelagius sp. nov. (type strain Ery9T=CGMCC 1.15358T=DSM 101479T) and Croceicoccus mobilis sp. nov. (type strain Ery22T=CGMCC 1.15360T=DSM 101481T), are proposed.

A Novel Halotolerant Thermoalkaliphilic Esterase from Marine Bacterium Erythrobacter seohaensis SW-135.

A novel esterase gene, e69, was cloned from Erythrobacter seohaensis SW-135, which was isolated from a tidal flat sediment of the Yellow Sea in Korea. This gene is 825 bp in length and codes for a 29.54 kDa protein containing 274 amino acids. Phylogenetic analysis showed that E69 is a new member of the bacterial lipolytic enzyme family IV. This enzyme exhibited the highest level of activity toward p-nitrophenyl (NP) butyrate but little or no activity toward the other p-NP esters tested. The optimum temperature and pH of the catalytic activity of E69 were 60°C and pH 10.5, respectively. The enzyme exhibited stable activity over a wide range of alkaline pH values (7.5-9.5). In addition, E69 was found to be a halotolerant esterase as it exhibited the highest hydrolytic activity in the presence of 0.5 M NaCl and was still active in the presence of 3 M NaCl. Moreover, it possessed some degree of tolerance to Triton X-100 and several organic solvents. Through homology modeling and comparison with other esterases, it was suggested that the absence of the cap domain and its narrow substrate-binding pocket might be responsible for its narrow substrate specificity. Sequence and structural analysis results suggested that its high ratio of negatively to positively charged residues, large hydrophobic surface area, and negative electrostatic potential on the surface may be responsible for its alkaline adaptation. The results of this study provide insight into marine alkaliphilic esterases, and the unique properties of E69 make it a promising candidate as a biocatalyst for industrial applications.