Pelagibacterium Halotolerans Research Articles

A NADPH-Dependent Aldo/Keto Reductase Is Responsible for Detoxifying 3-Keto-Deoxynivalenol to 3-epi-Deoxynivalenol in Pelagibacterium halotolerans ANSP101.

Deoxynivalenol (DON), primarily generated by Fusarium species, often exists in agricultural products. It can be transformed to 3-epi-deoxynivalenol (3-epi-DON), with a relatively low toxicity, via two steps. DDH in Pelagibacterium halotolerans ANSP101 was proved to convert DON to 3-keto-deoxynivalenol (3-keto-DON). In the present research, AKR4, a NADPH-dependent aldo/keto reductase from P. halotolerans ANSP101, was identified to be capable of converting 3-keto-DON into 3-epi-DON. Our results demonstrated that AKR4 is clearly a NADPH-dependent enzyme, for its utilization of NADPH is higher than that of NADH. AKR4 functions at a range of pH 5-10 and temperatures of 20-60 °C. AKR4 is able to degrade 89% of 3-keto-DON in 90 min at pH 7 and 50 °C with NADPH as the cofactor. The discovery of AKR4, serving as an enzyme involved in the final step in DON degradation, might provide an option for the final detoxification of DON in food and feed.

Pelagibacterium flavum sp. nov., Isolated from Soil Sample.

A Gram-stain-negative, yellow, moist and circular, aerobic, motile, and rod-shaped bacterium, designated YIM 151497T, was isolated from soil sample collected from Blue-Bridge, Weizhou Island, Guangxi province, China. Classification using a polyphasic approach suggested that strain YIM 151497T belonged to the genus Pelagibacterium, and was closely relevant to Pelagibacterium nitratireducens JLT2005T (98.8%), Pelagibacterium halotolerans CGMCC 1.7692T (98.7%), Pelagibacterium lixinzhangensis H64T (98.1%), and Pelagibacterium luteolum CGMCC 1.10267T (97.1%). The growth ranges of temperature, pH, and NaCl were 4-40℃, pH 4.0-10.0, and 0-7% NaCl, respectively. It was positive for catalase and oxidase. The primary respiratory quinone was Q-10. The elemental fatty acids were Summed Feature 8 (constituting C18:1ω7c and/or C18:1ω6c), C19:0 cyclo ω8c, C16:0, and C18:1ω7c 11-methyl. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and three unidentified glycolipids. The DNA G+C content based on the complete genome sequence was 60.7mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between strain YIM 151497T and species of Pelagibacterium were in the ranges of 73.9-86.3% and 19.7-31.3%, respectively. The Average Amino Acid Identity (AAI) between strain YIM 151497T and species of Pelagibacterium were in the ranges of 68.8-88.8%. On the basis of these data, strain YIM 151497T is considered to represent a novel species of the genus Pelagibacterium with the name of Pelagibacterium flavum sp. nov. Type strain is strain YIM 151497T (= KCTC 49826T = CGMCC 1.61521T = MCCC 1K08053T).

A quinoprotein dehydrogenase from Pelagibacterium halotolerans ANSP101 oxidizes deoxynivalenol to 3-keto-deoxynivalenol

Deoxynivalenol (DON), a notorious mycotoxin commonly present in cereal grains, poses severe health risks to human and livestock. A quinoprotein from Pelagibacterium halotolerans ANSP101, named deoxynivalenol dehydrogenase (DDH), was identified by comparative genome analysis of P. halotolerans ANSP101, Devosia mutan 17-2-E−8 and Devosia strain IFO13580. DDH was responsible for oxidation of DON into 3-keto-DON with hydrogen acceptor phenazine methosulfate (PMS) or dichlorophenolindophenol (DCPIP). DDH had two catalytic amino acid residues (Ser478, Glu480), and 57.55% sequence identity with quinoprotein dehydrogenases DepA from Devosia sp. 17-2-E−8 and QDDH from Devosia sp. D6-9 by sequence alignment. The Met516 of DDH was changed to Glu516 by site-specific mutagenesis, and the new mutant was designated as TDDH. The mutant TDDH possessing catalytic triplet SEE (Ser478, Glu480, Glu516) could degrade DON to 3-keto-DON with pyrroloquinoline quinone (PQQ) as hydrogen acceptor besides PMS and DCPIP. Importantly, the mutant TDDH exhibited stronger degradation ability for DON in the presence of the same hydrogen acceptor compared with wild DDH. The mutant TDDH had an effect on degrading DON with PQQ at a wide range of pH (6.0–11.0) and temperatures (20–45 °C). The results provide potential use of TDDH as a detoxification agent to mitigate DON hazard on human and animals applied in food and feed.

Enzymatic degradation of deoxynivalenol by a novel bacterium, Pelagibacterium halotolerans ANSP101

Deoxynivalenol (DON), a toxic secondary metabolite produced by Fusarium species that mainly infests cereals such as wheat and corn, threatens human and livestock health. The present study describes the characterization of a novel bacterial strain, Pelagibacterium halotolerans ANSP101 which is capable of transforming DON to less-toxic product 3-keto-deoxynivalenol by the oxidation of the C3 hydroxyl group. Strain ANSP101 was isolated from a seawater sample from a depth of 55 m in Chinese Bohai sea. The strain was identified as Pelagibacterium halotolerans by morphology characterization and 16S rDNA gene sequencing. The DON degrading activity of strain ANSP101 was predominantly attributed to the bacterial cell lysate. Besides, the cell lysate was sensitive to sodium dodecyl sulfate, heat, and proteinase K treatment, indicating that the intracellular proteins or enzymes are responsible for the DON degradation. The optimal temperature and pH for the maximal degradation of DON were 40 °C and pH 8.0 by the cell lysate. These results provide the potential use of P. halotolerans ANSP101 as a detoxification agent for DON decontamination in cereals and feed.

Pelagibacterium montanilacus sp. nov., an alkaliphilic bacterium isolated from Lake Cuochuolong on the Tibetan Plateau.

A Gram-stain negative, alkaliphilic and halotolerant bacterium, designated CCL18T, was isolated from Lake Cuochuolong on the Tibetan Plateau. The strain was aerobic, short rod-shaped, catalase- and oxidase-positive, and motile by means of several polar flagella. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain CCL18T belongs to the genus Pelagibacterium, with its two closest neighbours being Pelagibacterium halotolerans B2T (96.6 %, 16S rRNA gene sequence similarity) and Pelagibacterium luteolum 1_C16_27T (96.1 %). The predominant respiratory quinone of strain CCL18T was Q-10, with Q-9 as a minor component. The major fatty acids were C18 : 1ω6c/C18 : 1ω7c (60.4 %), C19 : 0cyclo ω8c (8.1 %) and C18 : 0 (6.8 %). The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, seven kinds of unidentified lipids and three kinds of glycolipids. The DNA G+C content was 60.1 mol%. DNA-DNA hybridization showed 35.2 % relatedness between strain CCL18T and P. halotolerans B2T and 24.6 % relatedness to P. luteolum 1_C16_27T. Based on phylogenetic analysis, DNA-DNA hybridization and a range of physiological and biochemical characteristics, strain CCL18T was clearly distinguishable from the other strains of the genus Pelagibacterium. It was evident that strain CCL18T could be classified as a novel species of the genus Pelagibacterium, for which the name Pelagibacterium montanilacus sp. nov. is proposed. The type strain is CCL18T (=CGMCC 1.16231T=KCTC 62030T).

Pelagibacterium lentulum sp. nov., a marine bacterium from the culture broth of Picochlorum sp. 122.

A Gram-stain-negative, motile, non-spore-forming, rod-shaped bacterium, designated strain B2T, was isolated from the culture broth of a marine microalga, Picochlorum sp. 122. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain B2T forms a robust cluster with members of the genus Pelagibacterium, and shares the highest sequence similarity of 96.80 % with Pelagibacterium halotolerans CGMCC 1.7692T. Optimal growth of strain B2T was observed at 33 °C, at pH 8 and in the presence of 1 % (w/v) NaCl. The predominant ubiquinone of strain B2T was Q-10, and the G+C content of the genomic DNA was 58.6 mol%. The major fatty acid profile comprised C18 : 1ω7c/ω6c, C19 : 0 cyclo ω8c and C16 : 0. The major polar lipids of strain B2T were diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids and seven unidentified lipids. Comprehensive analyses based on a polyphasic characterization of strain B2T indicated that it represents a novel species of the genus Pelagibacterium, for which the name Pelagibacterium lentulum sp. nov. is proposed. The type strain is B2T (=MCCC 1K03218T=CGMCC 1.15896T=KCTC 52551T).

Crystal structure of Pelagibacterium halotolerans PE8: New insight into its substrate-binding pattern

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.

Genetic diversity of nitrate reducing bacteria in marine and brackish water nitrifying bacterial consortia generated for activating nitrifying bioreactors in recirculating aquaculture systems

Nitrate reducing potency of 88 bacterial isolates segregated from marine and brackish water nitrifying bacterial consortia (NBC), generated for activation of nitrifying bioreactors, was confirmed by determining the nitrate reducing capability under aerobic conditions as maintained in nitrifying bioreactors. All the isolates had the potential to be used as bio-augmentors for activating nitrate dissimilation in recirculating aquaculture system. The existence of nitrate reducers with nitrifiers in NBC and in the reactor configuration negates the requirement of integrating anoxic denitrifying system for effective removal of NO3−-N. Phylogenetic analyses of representative isolates from each cluster of the dendrograms generated based on phenotypic characterization and amplified ribosomal DNA restriction analysis revealed profound diversity of nitrate reducing bacterial flora in the NBC. They were composed of Streptomyces enissocaesilis, Marinobacter sp., Pseudomonas sp., Microbacterium oxydans, Pelagibacterium halotolerans and Alcanivorax dieselolei from marine NBC and Streptomyces tendae, Nesterenkonia sp., Bacillus cereus, Microbacterium oxydans and Brevibacterium sp. from brackish water NBC. The diversity indices of the consortia were calculated using Mega 5.0, primer 7 and VITCOMIC softwares. Marine NBC exhibited higher Shannon wiener diversity and mean population diversity than brackish water NBC. The study delineated higher species richness and diversity in marine NBC than in its brackish water counterpart, a possible reflection of the higher biodiversity of marine systems, and hence, the former is more promising to be used as start-up cultures for the activation of nitrifying bioreactors after appropriate acclimatization to the desired salinity.

Pelagibacterium lixinzhangensis sp. nov., a Novel Member of the Genus Pelagibacterium.

A Gram-negative, rod shaped and non-spore-forming bacterium, strain H642(T), was isolated from a desert sample collected from Xinjiang province of China, and subjected to a polyphasic taxonomic investigation. Strain H642(T) grew between 20 and 40 °C (optimal 28-37 °C), pH 6.0-10.0 (optimal 7.0-9.0) and at NaCl concentrations between 1.0 and 20.0 % (optimal 2.0-8.0 %). It was positive for catalase, but negative for oxidase. Ubiquinone-10 was present as the major respiratory ubiquinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, four unknown glycolipids and one unknown lipid. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain H642(T) belongs to the genus Pelagibacterium. Strain H642(T) was most closely related to Pelagibacterium halotolerans (with 98.4 % similarity), Pelagibacterium nitratireducens (97.3 %) and Pelagibacterium luteolum (96.2 %). DNA-DNA reassociation values between strain H642(T), P. halotolerans B2(T) and P. nitratireducens JLT2005(T) were 38.0 and 25.3 %, respectively. The genomic DNA G + C content of strain H642(T) was determined to be 44.4 mol%. Based on genotypic, chemotaxonomic and phenotypic data, strain H642(T) represents a novel species of the genus Pelagibacterium, for which the name Pelagibacterium lixinzhangensis sp. nov is proposed. The type strain is H642(T) (=CGMCC 1.10230(T)=JCM 16597(T)).

Characterization of a novel metallo-β-lactamases fold hydrolase from Pelagibacterium halotolerans, a marine halotolerant bacterium isolated from East China Sea.

In this study, a novel metallo-β-lactamases fold hydrolase PH-1 was identified from Pelagibacterium halotolerans B2(T). This novel member of the family Hyphomicrobiaceae was isolated from the East China Sea. In silico analysis demonstrated that PH-1 and its relative homologues cluster in a unique branch and constitute a new subgroup among MBLs. PH-1 was cloned and overexpressed in Escherichia coli BL21 in a soluble form. SDS-PAGE, MALDI-TOF/TOF-MS, and size-exclusion chromatography analysis demonstrated that the PH-1 was a monomer with molecular weight of about 29kDa. Substrate specificity study showed PH-1 preferred penicillin type β-lactams and exhibited maximum activity toward penicillin-G. Additionally, our experiments also revealed that PH-1 was a halotolerant enzyme since it is active under 4M NaCl. The enzyme activity of PH-1 was negatively affected by 1mM Mn(2+) and EDTA. These observations lay a foundation for further study of MBLs from marine bacterium.

Cloning, expression and characterization of a new enantioselective esterase from a marine bacterium Pelagibacterium halotolerans B2T

• An esterase was cloned from a marine bacterium and overexpressed in Escherichia coli . • The enzyme was alkalitolerance and halotolerance. • The enzyme preferred short chain p -nitrophenyl esters, and was not a metalloenzyme. • The enzyme was useful in the synthesis of methyl ( R )-3-(4-fluorophenyl)glutarate. • ( R )-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction. An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium halotolerans B2 T and overexpressed in Escherichia coli Rosetta, resulting an active, soluble protein which constituted 23.1% of the total cell protein content. Phylogenetic analysis of the protein showed it was a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8 preferred short chain p -nitrophenyl esters (C2–C6), exhibited maximum activity toward p -nitrophenyl acetate, and was not a metalloenzyme. PE8 was an alkaline esterase with an optimal pH of 9.5 and an optimal temperature of 45 °C toward p -nitrophenyl acetate. Furthermore, it was found that PE8 exhibited activity and enantioselectivity in the synthesis of methyl ( R )-3-(4-fluorophenyl)glutarate (( R )-3-MFG) from the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG). ( R )-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction under optimized conditions (0.6 M phosphate buffer (pH 8.0) containing 17.5% 1,4-dioxane under 30 °C). In addition, PE8 was tolerant to extremely strong basic and high ionic strength solutions as it exhibited high activity even at pH 11.0 in 1 M phosphate buffer. Given its highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8 could be a promising candidate for the production of ( R )-3-MFG in industry. The results also demonstrate the potential of the marine environment as a source of useful biocatalysts.

Pelagibacterium nitratireducens sp.nov., A Marine Alphaproteobacterium Isolated from the East China Sea

A Gram-negative, rod-shaped, non-spore-forming aerobic bacterium, motile with a single polar flagellum, strain JLT2005(T), was isolated from surface seawater collected from the East China Sea and formed ivory white colonies on a rich organic medium. The strain was positive for catalase, oxidase, and urease. It grew in the presence of 0-12 % (w/v) NaCl (optimum 5 %), at 20-35 °C (optimum 25 °C), or at pH 6-10 (optimum pH 9). The major fatty acids (>10 %) were C(18:1)ω7c, C(19:0)ω8c cyclo, C(16:0), and C(18:0). The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and five unidentified glycolipids. Ubiquinone-10 and Ubiquinone-11 were present as the major quinones. The DNA G+C content was 74.3 mol%. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain JLT2005(T) belongs to the genus Pelagibacterium in the family Hyphomicrobiaceae, class Alphaproteobacteria. The closest neighbors were Pelagibacterium halotolerans B2(T) (98.7 % similarity) and Pelagibacterium luteolum 1_C16_27(T) (97.1 % similarity). DNA-DNA relatedness values of strain JLT2005(T) with P. halotolerans B2(T) and with P. luteolum 1_C16_27(T) were 31.6 and 25 %. Evidence from genotypic, chemotaxonomic, and phenotypic data shows that strain JLT2005(T) represents a novel species of the genus Pelagibacterium, for which the name Pelagibacterium nitratireducens sp. nov is proposed. The type strain is JLT2005(T) (=CGMCC 1.10829(T) =JCM 17767(T)).

Cloning, expression and characterization of a halotolerant esterase from a marine bacterium Pelagibacterium halotolerans B2T

An esterase PE10 (279 aa) from Pelagibacterium halotolerans B2(T) was cloned and overexpressed in Escherichia coli Rosetta in a soluble form. The deduced protein was 29.91kDa and the phylogenetic analysis of the deduced amino acids sequence showed it represented a new family of lipolytic enzymes. The recombinant protein was purified by Ni-NTA affinity chromatography column and the characterization showed its optimal temperature and pH were 45°C and pH 7.5, respectively. Substrate specificity study showed PE10 preferred short chain p-nitrophenyl esters and exhibited maximum activity toward p-nitrophenyl acetate. In addition, PE10 was a halotolerant esterase as it was still active under 4M NaCl. Three-dimensional modeling of PE10 suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment. With this halotolerance property, PE10 could be a candidate for industrial use.

Complete Genome Sequence of Pelagibacterium halotolerans B2T

Pelagibacterium halotolerans B2(T) is a marine halotolerant bacterium that was isolated from a seawater sample collected from the East China Sea. Here, we present the complete genome sequence of the type strain P. halotolerans B2(T), which consists of one chromosome (3,944,837 bp; 61.4% G+C content) and one plasmid (4,050 bp; 56.1% G+C content). This is the first complete genome of a member of the Pelagibacterium genus.

Pelagibacterium halotolerans gen. nov., sp. nov. and Pelagibacterium luteolum sp. nov., novel members of the family Hyphomicrobiaceae

Two Gram-negative, motile, aerobic bacterial strains, designated B2(T) and 1_C16_27(T), were respectively isolated from a seawater sample collected from the East China Sea and a semi-coke sample from north-eastern Estonia. Their genetic, phenotypic and chemotaxonomic properties were studied. The isolates were short rods with polar flagella and were positive for catalase and oxidase activities. Q-10 was the predominant respiratory ubiquinone. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids. The major fatty acids were nonadecanoic (C(19 : 0) cyclo), octadecanoic (C(18 : 0) and C(18 : 0) 3-OH), octadecenoic (C(18 : 1)) and hexadecanoic (C(16 : 0)) acids. The G+C content of the genomic DNA was 58.1-59.3 mol%. 16S rRNA gene sequence analysis revealed that the two isolates represent a distinct lineage within the family Hyphomicrobiaceae. The phylogenetically closest relatives were Cucumibacter (92.7-93.7 % 16S rRNA gene sequence similarity), Devosia (92.9-94.4 %) and Zhangella (91.7-92.1 %). Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strains B2(T) and 1_C16_27(T) could be differentiated from each other and from members of the genera Cucumibacter, Devosia and Zhangella. Therefore, it is proposed that strains B2(T) and 1_C16_27(T) represent two novel species in a new genus, for which the names Pelagibacterium halotolerans gen. nov., sp. nov. (the type species; type strain B2(T) = CGMCC 1.7692(T) = JCM 15775(T)) and Pelagibacterium luteolum sp. nov. (type strain 1_C16_27(T) = CGMCC 1.10267(T) = JCM 16552(T) = CELMS EEUT 1C1627(T)) are proposed.