Gramella sediminis sp. nov., isolated from a tidal flat of the Yellow Sea.

A novel species of the genus Gramella, designated ASW11-100T, was isolated from a tidal flat sediment in the Yellow Sea, PR China. Phylogenetic analysis based on 16S rRNA gene sequences and single-copy orthologous clusters revealed that strain ASW11-100T belonged to the genus Gramella, and exhibited 16S rRNA gene sequence similarities of 98.9, 98.8 and 98.7 % to Gramella sabulilitoris HSMS-1T, Gramella sediminilitoris GHTF-27T and Gramella forsetii KT0803T, respectively. The genome of strain ASW11-100T harbours 2950 protein-coding genes and 105 carbohydrate-active enzymes including 38 glycoside hydrolases. Seventeen of the glycoside hydrolases are organized in five distinct polysaccharide utilization loci, which are predicted to involve in the degradation of starch, glucans, arabinoxylans, arabinomannan, arabinans and arabinogalactans. The genomic DNA G+C content was 37.3 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain ASW11-100T and its closely related relatives were in ranges of 19.8-23.9% and 76.6-80.9 %, respectively. Cells of the isolate were Gram-negative, aerobic, non-flagellated and short rod-shaped. Carotenoid pigments were produced, but flexirubin-type pigments were absent. The major fatty acids (>10 %) were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c). The sole respiratory quinone was menaquinone-6 and the major polar lipid was phosphatidylethanolamine. Based on the above polyphasic evidence, strain ASW11-100T should be considered to represent a novel Gramella species, for which the name Gramella sediminis sp. nov. is proposed. The type strain is ASW11-100T (=KCTC 82502T=MCCC 1K05580T).

Influence of genetic polymorphisms in P2Y12 receptor signaling pathway on antiplatelet response to clopidogrel in coronary heart disease.

BACKGROUNDS: Remarkable interindividual variability in clopidogrel response is observed, genetic polymorphisms in P2RY12 and its signal pathway is supposed to affect clopidogrel response in CHD patients. METHODS: 539 CHD patients treated with clopidogrel were recruited. The platelet reaction index (PRI) indicated by VASP-P level were detected in 12-24 h after clopidogrel loading dose or within 5-7 days after initiation of maintain dose clopidogrel. A total of 13 SNPs in relevant genes were genotyped in sample A (239 CHD patients). The SNPs which have significant differences in PRI will be validated in another sample (sample B, 300 CHD patients). RESULTS: CYP2C19*2 increased the risk of clopidogrel resistance significantly. When CYP2C19*2 and CYP2C19*3 were considered, CYP2C19 loss of function (LOF) alleles were associated with more obviously increased the risk of clopidogrel resistance; P2RY12 rs6809699C > A polymorphism was also associated with increased risk of clopidogrel resistance (AA vs CC: P = 0.0398). This difference still existed after stratification by CYP2C19 genotypes. It was also validated in sample B. The association was also still significant even in the case of stratification by CYP2C19 genotypes in all patients (sample A + B). CONCLUSION: Our data suggest that P2RY12 rs6809699 is associated with clopidogrel resistance in CHD patients. Meanwhile, the rs6809699 AA genotype can increase on-treatment platelet activity independent of CYP2C19 LOF polymorphisms.

Salinimicrobium sediminilitoris sp. nov., Isolated from a Tidal Flat.

A Gram-negative, facultatively anaerobic, motile, and rod-shaped bacterium, designated ASW11-47 T, was isolated from a tidal flat sediment taken from the coast of Qingdao, PR China. Phylogenetic analysis of 16S rRNA gene sequence showed that strain ASW11-47 T belongs to the genus Salinimicrobium and is most closely related to Salinimicrobium terrae YIM-C338T (98.68% similarity). The length of draft genome is 3,594,457 bp, and DNA G + C content is 40.8 mol%. The values of average nucleotide identity and digital DNA-DNA hybridization between strain ASW11-47 T and closely related strains were in ranges of 75.9-85.9 and 19.7-31.5%, respectively. The major fatty acids (> 10%) were iso-C15:0 and iso-C17:0 3-OH. The predominant respiratory quinone was menaquinone-6 and the major polar lipid was phosphatidylethanolamine. On the basis of genotypic, phenotypic, and chemotaxonomic analysis, strain ASW11-47 T represents a novel species within the genus Salinimicrobium, for which the name Salinimicrobium sediminilitoris sp. nov. is proposed. The type strain is ASW11-47 T (= KCTC 82501 T = MCCC 1K05586T).

Pontixanthobacter rizhaonensis sp. nov., a marine bacterium isolated from surface seawater of the Yellow Sea, and proposal of Pseudopontixanthobacter gen. nov., Pseudopontixanthobacter confluentis comb. nov. and Pseudopontixanthobacter sediminis comb. nov.

A bacterial strain designated RZ02T was isolated from surface seawater collected from the Yellow Sea in PR China and characterized by polyphasic taxonomy. Cells of strain RZ02T were Gram-stain-negative, aerobic, non-motile, catalase- and oxidase-positive rods forming ochre-pigmented colonies. Growth occurred at 7-36 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0) and with 1-5 % (optimum, 2 %) NaCl. The major cellular fatty acids (>10 %) of strain RZ02T were summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and C16 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol and sphingoglycolipid. The genome size of strain RZ02T was 2.79 Mbp with a G+C content of 55.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RZ02T was mostly related to Pontixanthobacter luteolus SW-109T and Pontixanthobacter aestiaquae HDW-31T (97.3 and 97.1% sequence similarity, respectively), and formed a phyletic lineage with members of the genus Pontixanthobacter. The phylogenetic analysis based on the up-to-date bacterial core gene sequences confirmed that strain RZ02T clustered within the genus Pontixanthobacter. The average nucleotide identity and in silico DNA-DNA hybridization values between strain RZ02T and P. luteolus SW-109T and P. aestiaquae HDW-31T were 72.8 and 72.9 % and 18.7 and 18.5%, respectively. Based on these evidences, strain RZ02T is proposed to represent a novel species of the genus Pontixanthobacter under the name Pontixanthobacter rizhaonensis sp. nov. The type strain is RZ02T (=KCTC 62828T=MCCC 1K04521T). In addition, based on the results of whole genome analyses, proposals of Pseudopontixanthobacter gen. nov., Pseudopontixanthobacter confluentis comb. nov. and Pseudopontixanthobacter sediminis comb. nov. are also included.

Pseudopuniceibacterium antarcticum sp. nov., isolated from an Antarctic marine sponge.

A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated strain HQ09T, was isolated from a marine sponge off the coast of Fields Peninsula, West Antarctica. Strain HQ09T grew at 4-35 °C (optimum, 25 °C), pH 5-9 (optimum, pH 7.0), and with 1-10% NaCl (optimum, 2 %). Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain HQ09T was affiliated with the genus Pseudopuniceibacterium in the family Rhodobacteraceae, sharing 99.64 % identity with the type strain of Pseudopuniceibacterium sediminis, the only known species in the genus. However, the low digital DNA-DNA hybridization (dDDH) (27.2 %) and average nucleotide identity (ANI) (83.63 %) values between strain HQ09T and the type strain of Pseudopuniceibacterium sediminis indicated that they did not belong to the same species. Strain HQ09T could also be differentiated from Pseudopuniceibacterium sediminis by many phenotypic characteristics. The major fatty acids (>5 %) of strain HQ09T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), 11-methyl C18 : 1 ω7c, C16 : 0 and C19 : 0 cyclo ω8c. The polar lipids included phosphatidylglycerol, phosphatidylcholine, two unidentified aminolipids and one unidentified phospholipid. The predominant respiratory quinone was ubiquinone 10 (Q-10). The genomic DNA G+C content was 62.63 mol%. Four secondary metabolite biosynthetic gene clusters were detected in the genome, potentially producing ectoine and three types of unknown compounds. On the basis of the polyphasic evidences obtained in this study, strain HQ09T represents a novel species of the genus Pseudopuniceibacterium, for which the name Pseudopuniceibacterium antarcticum sp. nov. is proposed, with the type strain being HQ09T (=KCTC 52229T=CGMCC 1.15538T).

Zongyanglinia huanghaiensis gen. nov., sp. nov., a novel denitrifying bacterium isolated from the yellow sea, and transfer of Pelagicola marinus to Zongyanglinia gen. nov. as Zongyanglinia marinus comb. nov.

Two Gram-stain-negative, oxidase- and catalase-positive, aerobic strains (CY05T and H18S-6) were isolated from sediment samples of the Yellow Sea, China. The strains were positive for denitrification. Optimum growth was observed at 20 °C, pH 7.5-8.0 and with 2.0%-3.0% NaCl. The predominant cellular fatty acids (> 10%) were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), major respiratory quinone was ubiquinone-10 and main polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and one unidentified aminolipid. The approximate genome size of strains CY05T and H18S-6 were 4.86 and 5.04 Mbp, the genomic G + C content of them were 54.2 and 54.5%, respectively. Both of the phylogenetic analysis based on 16S rRNA gene sequences and the up-to-date bacterial core gene (UBCG) sequences revealed that strains CY05T, H18S-6 and Pelagicola marinus DSW4-44T formed a distinct monophyletic clade within the family Rhodobacteraceae. The ANI and isDDH values between strains CY05T and H18S-6 were 94.0% and 56.5%, between CY05T and Pelagicola marinus DSW4-44T were 94.1% and 59.8%, respectively, all below the accepted threshold value for species delineation. But the ANI and isDDH values between strains H18S-6 and Pelagicola marinus DSW4-44T were 96.8% and 76.7% respectively, indicating that strains H18S-6 and Pelagicola marinus DSW4-44T belong to the same species. Based on the distinctive polyphasic evidence, CY05T represent a novel species of a novel genus of the family Rhodobacteraceae, for which the name Zongyanglinia huanghaiensis gen. nov., sp. nov. is proposed. The type strain is CY05T (= MCCC 1K04409T = KCTC 62200T). Moreover, the reclassification of Pelagicola marinus Choi et al. 2019 as Zongyanglinia marinus comb. nov. (type strain DSW4-44T = KCTC 62762T = KCCM 43261T = JCM 33637T) is proposed based on the polyphasic taxonomic data obtained in this study.

Phylogenomic Analyses of a Clade Within the Family Flavobacteriaceae Suggest Taxonomic Reassignments of Species of the Genera Algibacter, Hyunsoonleella, Jejuia, and Flavivirga, and the Proposal of Pseudalgibacter gen. nov. and Pseudalgibacter alginicilyticus comb. nov.

The family Flavobacteriaceae forms a major branch within the phylum Bacteroidetes. Whole-genome sequence-based analysis could significantly improve the accuracy of taxonomic assignments. In this study, phylogenomic analyses were carried out to revisit the taxonomic status of a clade of the family Flavobacteriaceae. Taking genome-based phylogeny as the primary guideline and average amino acid identity and phenotypic information as supplements, the following taxonomic proposals were put forward: Arenitalea lutea should be reclassified into the genus Algibacter; Algibacter aquaticus should be reclassified into the genus Flavivirga; Jejuia pallidilutea and Algibacter aestuarii should be reclassified into the genus Hyunsoonleella; Algibacter alginicilyticus should be reclassified into the novel genus Pseudalgibacter gen. nov. This study builds up a solid framework for taxonomic decisions of a clade of the family Flavobacteriaceae and will contribute to further insights into the evolution of this family.

First report of Colletotrichum gloeosporioides causing leaf spot on Cyclobalanopsis glauca in China.

Cyclobalanopsis glauca (Thunb.) Oerst. is one of the most widely distributed species of evergreen broad-leaved tree in subtropical areas of China. It is also grown in Korea, Japan, and India. Because of its beautiful shape, C. glauca is commonly used for greening gardens and walkways. In July 2018, leaf spots on C. glauca were observed in Zhejiang province (Lishui, N: 28°26' 6.75";E: 119°54'11.22), China. About 70% of the trees were found to be diseased, with approximately 50% of leaves showing symptoms. The symptoms on C. glauca leaves initially appeared as small brown-yellow spots which gradually expanded, developing a light brown central and dark brown to black margin. The spots ranged from 4 to 15 mm in diameter. Ten symptomatic fragments measuring approximately 5×5 mm from each leaf were surface disinfested with 70% ethanol for 30 s, and then they were rinsed in sterile distilled water and placed on potato dextrose agar (PDA) medium at 25 °C in the dark for five days. Segments of colony perimeters were then transferred to new plates. The colonies initially produced white mycelia that later turned gray-white with pink and occasionally black dots scattered on the surface of the mycelium. Spores were aseptate, cylindrical, 8 to 15 µm in length, and 3 to 5 µm wide, most with rounded ends, a few with one apex round and the other fusiform, as described for Colletotrichum gloeosporioides (Penz.) Sacc. (Agostini et al. 1993). The internal transcribed spacer rDNA (ITS: MK758005) and two nuclear protein-coding genes (CHS: MK784770, ACT: MK784769) were amplified with ITS1/ITS4, CHS-79F/CHS-345, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequence had 99.61% identity to GQ485605 for ITS, 99.56% to GQ856782 for ACT, and 100% to GQ856733 for CHS of C. gloeosporioides CBS 953.97 in GenBank, respectively. To fulfill Koch's postulates, spores (1×108) of the isolate were sprayed onto leaves of twelve 2-year-old C. glauca plants (at least six leaves per plant). The fungus was inoculated on one side of each leaf, and distilled water was used as a mock inoculated control on the other side. The plants were cultivated in the greenhouse to maintain high humidity and a temperature near 25 °C. After 9 days, 100% of the leaf halves that had been inoculated had symptoms identical to those observed on affected C. glauca leaves in the field, while no symptoms were observed on the mock inoculated half of each leaf. The fungus was reisolated from the symptoms and identified as C. gloeosporioides using techniques previously described. To our knowledge, this is the first report of C. gloeosporioides infecting C. glauca in China. This study will establish a foundation for the further study of C. gloeosporioides to address the disease effectively. References: Agostini, J. P., et al. 1993. Phytopathology. 82:1177. Weir, B. S., et al. 2012. Stud. Mycol. 73:115. This work was supported by the National Science Foundation for Young Scientists of China (31800035).

Maribacter luteus sp. nov., a marine bacterium isolated from intertidal sand of the Yellow Sea.

A novel rod-shaped and Gram-stain-negative bacterium, designated strain RZ05T, was isolated from a sand sample collected from the intertidal zone of the Yellow Sea, PR China. Results of phylogenetic analysis based on 16S rRNA gene sequences revealed that strain RZ05T clusters within the genus Maribacter, a member of the family Flavobacteriaceae, and has the highest sequence similarity to Maribacter polysiphoniae KCTC 22021T (97.8 %), followed by Maribacter arenosus KCTC 52191T (97.2 %). Cells of this strain were observed to be aerobic, oxidase- and catalase-positive, motile by gliding and formed yellow colonies. Growth occurred at 7-40 °C (optimum, 30 °C), at pH 6.5-9.5 (optimum, pH 7.0) and with 0.5-6 % (optimum, 2 %) NaCl. Its polar lipid profile included phosphatidylethanolamine, two unidentified glycolipids, one unidentified aminolipid and four unidentified lipids. The major cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, iso-C16 : 0 3-OH, iso-C15 : 0 3-OH, summed feature 9 (10-methyl C16 : 0/iso-C17 : 1 ω9c) and summed feature 3 (iso-C15 : 0 2-OH/C16 : 1 ω7c/C16 : 1 ω6c). The only respiratory quinone was menaquinone 6 (MK-6). The genome of strain RZ05T was 4.65 Mbp with a G+C content of 38.9 mol%. The average nucleotide identity and in silico DNA-DNA hybridization values between strain RZ05T and its most closely related type strain M. polysiphoniae KCTC 22021T were 80.3 and 26.3  %, respectively. The results of phylogenetic, phenotypic and chemotaxonomic analyses indicated that strain RZ05T represents a novel species of the genus Maribacter, for which the name Maribacter luteus sp. nov. is proposed. The type strain is RZ05T (=KCTC 62834T=MCCC 1K03617T).

Whole genome analysis calls for a taxonomic rearrangement of the genus Colwellia.

Microbial taxonomy is the foundation of microbiology and rapid advancements in DNA sequencing technologies are providing new approaches to address prevailing questions in this field. The family Colwelliaceae, which currently comprises four genera, is a diverse and globally abundant group of Gamaproteobacteria. Based on 14 publically available genomes of bacteria strains labeled as members of the family Colwelliaceae, phylogenomic analyses were conducted to revisiting the taxonomic status of this family both in the genus and species level. Using genome-based phylogeny as a primary guideline and genome-based similarity indexes including average amino acid identity, percentage of conserved proteins, average nucleotide identity, and the digital DNA-DNA hybridization as supplements, the following taxonomic proposals were proposed: Colwellia polaris, Colwellia beringensis, Colwellia sediminilitoris, Colwellia aestuarii, Colwellia chukchiensis and Colwellia mytili should be reclassified into the novel genus Cognaticolwellia; Colwellia agarivorans should be reclassified into the novel genus Pseudocolwellia. Our results constitute a solid framework for current and future taxonomic decisions within this family, which will be helpful for avoiding confusion with ecological and evolutionary interpretations in subsequent studies.

Litorilituus lipolyticus sp. nov., isolated from intertidal sand of the Yellow Sea in China, and emended description of Colwellia asteriadis.

A Gram-stain negative, rod-shaped, aerobic, oxidase-positive and catalase-weakly positive bacterial strain with polar or subpolar flagellum, designated RZ04T, was isolated from an intertidal sand sample collected from a coastal area of the Yellow Sea, China. The organism was observed to grow optimally at 25 °C and pH 6.5-7.0 with 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain RZ04T was closely related to Colwellia asteriadis (similarity 96.9%) and Litorilituus sediminis (similarity 96.8%), and 94.4-96.4% sequence similarities to other type strains of species of the genera belonged to the family Colwelliaceae. The dominant fatty acids of strain RZ04T were determined to be C17:1ω8c, C15:1ω8c, C16:0 and summed feature 3 (C16:1ω6c and/or C16:1ω7c), and the predominant isoprenoid quinone was determined to be quinone 8 (Q-8). Phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid and four unidentified lipids were determined to be the major constituents of the polar lipids. The genome of strain RZ04T is 4.14 Mbp with a G + C content of 37.4 mol%. A total of 3631 genes are predicted, with 3531 protein-coding genes, 75 RNA genes and 25 pseudogenes. Based on phenotypic, genotypic and phylogenetic analysis, strain RZ04T is considered to represent a novel species in the genus Litorilituus, for which the name Litorilituus lipolyticus is proposed. The type strain is RZ04T (= MCCC 1K03616T = KCTC 62835T). An emended description of Colwellia asteriadis is also provided.

Pseudopuniceibacterium sediminis gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from sediment.

A novel Gram-stain-negative bacterium, strain CY03T, was isolated from sediment of the Yellow Sea, PR China. Cells of strain CY03T were rods, aerobic and non-flagellated. Growth occurred at 5-40 °C (optimum, 30 °C), pH 5.5-9.5 (pH 7.5) and with 0.5-9.0 % NaCl (1.5-2.0 %). The 16S rRNA gene sequence comparison showed affiliation to the family Rhodobacteraceae with Puniceibacterium confluentis (97.0 %) as the most closely related species, followed by members of the genus Pseudooceanicola, Pseudooceanicola antarcticus (96.8 %) and Pseudooceanicola nitratireducens (96.7 %). The major cellular fatty acids were cyclo-C19 : 0 ω8c, C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and 11-methyl C18 : 1 ω7c. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, one unidentified phospholipid, one unidentified aminolipid and five unidentified lipids. The predominant respiratory quinone was Q-10. The DNA G+C content of the type strain was 62.8 mol%. Based on the results of the polyphasic characterization for strain CY03T, it represents a novel species of a novel genus of the family Rhodobacteraceae, for which the name Pseudopuniceibacterium sediminis gen. nov., sp. nov. is proposed. The type strain is CY03T (=CCTCC AB 2017195T=KCTC 62198T).

Flavobacterium phocarum sp. nov., isolated from soils of a seal habitat in Antarctica.

A Gram-stain-negative, yellow-pigmented, non-flagellated, gliding, rod-shaped, oxidase-negative and catalase-positive bacterium, designated SE14T, was isolated from soil on King George Island, South Shetland Islands, Antarctica. Strain SE14T grew at 4-25 °C (optimum, 20 °C), at pH 6.0-9.0 (optimum, pH 7.0-7.5) and with 0-3.0 % NaCl (optimum, 1.0-1.5 %), and could not produce flexirubin-type pigments. 16S rRNA gene sequence analysis showed the the isolate belonged to the genus Flavobacterium. Strain SE14T had the highest 16S rRNA gene sequence similarity to Flavobacterium antarcticum, F. tegetincola and F. degerlachei with 95.8, 95.5 and 95.2 %, respectively. The strain SE14T consisted of a clade with Flavobacteriumnoncentrifugens (16S rRNA gene sequence similarity 94.9 %) and F. qiangtangense (16S rRNA gene sequence similarity 94.2 %) and simultaneously formed a distinct phyletic lineage in the neighbour-joining phylogenetic tree. Polar lipids of the strain included phosphatidylethanolamine and four unidentified aminolipids. Strain SE14T contained anteiso-C15 : 0, iso-C15 : 0 and a mixture of iso-C15 : 0 2-OH and/or C16 : 1ω7c as the main fatty acids, and the only respiratory quinone was menaquinone-6. The genomic DNA G+C content was 42.3 mol%. The polyphasic taxonomic study revealed that strain SE14T belongs to a novel species within the genus Flavobacterium , and the name Flavobacterium phocarum sp. nov. is proposed. The type strain is SE14T (=CCTCC AB 2017225T=KCTC 52612T).

Neptunicoccus sediminis gen. nov., sp. nov., a member of the family Rhodobacteraceae isolated from the Yellow Sea.

A novel Gram-stain-negative bacterium, designated strain CY02T, was isolated from sediment of the Yellow Sea. Cells of CY02T were aerobic, coccus or short rods. Growth occurred at 5-42 °C (optimum, 35 °C), pH 6-10 (optimum, 8.0) and 0.5-9.0 % NaCl (optimum, 1.5-3.0 %). Phylogenetic analysis of 16S rRNA gene sequences revealed that CY02T was a member of the family Rhodobacteraceae and exhibited less than 95 % sequence similarities with the closely related type strains of the family Rhodobacteraceae. The genomic DNA G+C content of CY02T was 57.5 mol%. The major respiratory quinone was ubiquinone-10 (Q-10). The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, three unidentified lipids, one unidentified phospholipid and one unidentified aminolipid. The predominant cellular fatty acids were C18 : 1ω7c (57.6 %), 11-methyl C18 : 1ω7c (22.8 %) and C16 : 0 (10.6 %). Based on the results of morphological, physiological, biochemical, chemotaxonomic, genotypic and phylogenetic analyses, strain CY02T represents a novel species of a novel genus of the family Rhodobacteraceae, for which the name Neptunicoccus sediminis gen. nov., sp. nov. is proposed. The type strain of Neptunicoccus sediminis is CY02T (=CCTCC AB 2015430T=KCTC 42985T=NBRC 111872T=MCCC 1K03518).

Subcellular localisation of lipoproteins of Vibrio vulnificus by the identification of outer membrane vesicles components.

Vibrio vulnificus, a Gram-negative halophilic bacterium, is an opportunistic human pathogen that is responsible for the majority of seafood-associated deaths worldwide. Lipoproteins are important components of the bacterial cell envelope and have been shown to be involved in a wide variety of cellular processes. Little is known about the localisation or transport mechanism of lipoproteins in V. vulnificus. To assess the localisation of lipoproteins in V. vulnificus, we tested two established techniques for the rapid separation of membrane-associated proteins: detergent extraction with Sarkosyl and outer membrane vesicles (OMVs) preparation. The results showed that Sarkosyl extraction was not useful for the separation of lipoproteins from the different membranes of V. vulnificus. On the other hand, we confirmed that OMVs produced by V. vulnificus contained lipoproteins from the outer but not the inner membrane. Analysis of the OMVs components confirmed the localisation of several well-known lipoproteins to membranes that were different from expected, based on their predicted functions. Using this technique, we found that Asp at position +2 of mature lipoproteins can function as an inner membrane retention signal in V. vulnificus. Interestingly, the Escherichia coli "+2 rule" does not apply to the V. vulnificus lipoprotein IlpA (G2D) mutant, as a Ser to Asp mutation at position +2 of IlpA did not affect its outer membrane localisation. Furthermore, an IlpA tether-mRFP chimeric lipoprotein and its G2D mutant also behaved like IlpA. Together, these results suggest that the sorting rule of lipoproteins in V. vulnificus might be different from that in E. coli.

High Expression of AHSP, EPB42, GYPC and HEMGN Predicts Favorable Prognosis in FLT3-ITD-Negative Acute Myeloid Leukemia.

BACKGROUND/AIMS: Acute myeloid leukemia (AML) is a heterogeneous clonal disease and patients with AML who harbor an FMS-like tyrosine kinase 3 (FLT3) mutation present several dilemmas for the clinician. This study aims to identify novel targets for explaining the dilemmas. METHODS: We analyzed four microarray gene expression profiles to investigate changes in whole genome expression associated with FLT3-ITD mutation. RESULTS: We identified 22 differentially expressed genes which are commonly expressed among all four profiles. Kaplan-Meier analysis of the dataset GSE12417 revealed that low expression of AHSP, EPB42, GYPC and HEMGN predicted poor prognosis (AHSP: P=0.0317, HR=1.894; EPB42: P=0.0382, HR=1.859; GYPC: P=0.0015, HR=2.051; HEMGN: P=0.0418, HR=1.838 in GSE12417 test cohort; AHSP: P=0.0279, HR=1.548; EPB42: P=0.0398, HR=1.505; GYPC: P=0.0408, HR=1.501; HEMGN: P=0.0143, HR=1.630 in GSE12417 validation cohort). When patients were FLT3-ITD positive, the expression of FLT3 was significantly increased (all P<0.05 in four profiles), and correleation analysis of four profiles revealed that the expression of the four candidate genes negatively correlated with FLT3 expression. CONCLUSIONS: Our findings suggest that AHSP, EPB42, GYPC and HEMGN may be suitable biomarkers for diagnostic or therapeutic strategies for FLT3-ITD-positive AML patients.

Flavobacterium ardleyense sp. nov., isolated from Antarctic soil.

A Gram-stain-negative, aerobic, yellow-pigmented, non-flagellated, non-gliding, rod-like, oxidase- and catalase-positive bacterium, designated A2-1T, was isolated from soil on Ardley Island, South Shetland Islands, Antarctica. Strain A2-1T grew at 4-22 °C (optimum, 10 °C), at pH 6.0-8.0 (optimum, pH 6.5) and with 0-1.5 % NaCl (optimum, 0.5 %), but could not produce flexirubin-type pigments. 16S rRNA gene sequence analysis showed that the isolates belonged to the genus Flavobacterium. Strain A2-1T had the highest 16S rRNA gene sequence similarity to Flavobacterium cucumis, F. ahnfeltiae and F. cheniae with 95.7, 95.6 and 95.4 %, respectively. The strain A2-1T consisted of a clade with F. cucumis and F. cheniae and simultaneously formed a distinct phyletic lineage in the neighbour-joining phylogenetic tree. Polar lipids of the strain included phosphatidylethanolamine (PE), four unidentified aminolipids and one unidentified lipid. The strain A2-1T contained anteiso-C15 : 0 (20.2 %), iso-C15 : 0 (16.2 %) and C15 : 1 G (11.0 %) as the main fatty acids and the only respiratory quinone was menaquinone MK-6. The genomic DNA G+C content was 34.0 mol%. The polyphasic taxonomic study revealed that the strain A2-1T belongs to a novel species within the genus Flavobacterium and the name Flavobacterium ardleyense sp. nov. is proposed. The type strain is A2-1T (=CCTCC AB 2017157T=KCTC 52644T).

Euzebyella marina sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, yellow-pigmented, non-flagellated, non-gliding, oxidase- and catalase-positive bacterium, designated CY01T, was isolated from seawater of the Yellow Sea. CY01T grew at 15-37 °C (optimum, 30 °C), pH 5-8 (optimum, 6.5-7.5) and with 0.5-12 % (w/v) NaCl (optimum, 0.5-3.5 %). It could not produce flexirubin-type pigment or reduce nitrate to nitrite. CY01T showed the highest 16S rRNA gene sequence similarity to the type strain of Euzebyella saccharophila (97.0 %) and clustered tightly with the species of the genus Euzebyella in the phylogenetic trees based on the 16S rRNA gene sequences. The major cellular fatty acids of CY01T were iso-C15 : 0, iso-C15 : 1G and iso-C17 : 0 3-OH and the major respiratory quinone was menaquinone MK-6. Polar lipids included phosphatidylethanolamine (PE), four unidentified lipids and one unidentified aminolipid. The genomic DNA G+C content was 38.2 mol%. Based on the results of the polyphasic characterization of CY01T, it represents a novel species of the genus Euzebyella, for which the name Euzebyella marina sp. nov. is proposed. The type strain is CY01T (=CCTCC AB 2014348T=KCTC 42440T).

Development of a regulatable expression system for the functional study of Vibrio vulnificus essential genes.

We developed a regulatable gene expression system for Vibrio vulnificus, which contains a lacIq-pTrc cassette. Monomeric red fluorescence protein (mRFP) was used as a reporter to test this system. The results showed that this system tightly controlled the expression of mRFP without leaky expression and was suitable for the controlled expression of genes encoding recombinant proteins in V. vulnificus. To demonstrate the utility of this system, a dominant negative form of V. vulnificus VVMO6_RS04990, a homolog of Escherichia coli LolD that is essential in lipoprotein transport and membrane biogenesis, was inducibly expressed. Expression of the dominant negative LolD homolog, which has a mutation in the ATPase domain, resulted in a growth defect in V. vulnificus cells and impaired cell envelope stability. This result suggests that the V. vulnificus LolD homolog plays a role in cell envelope biogenesis. This tight and titratable expression system will therefore be a valuable tool for the study of essential genes in V. vulnificus.