Godanti bhasma (anhydrous CaSO4) induces massive cytoplasmic vacuolation in mammalian cells: A model for phagocytosis assay.

Phagocytosis is an essential physiological mechanism; its impairment is associated with many diseases. A highly smart particle is required for understanding detailed sequential cellular events in phagocytosis. Recently, we identified an Indian traditional medicine named Godanti Bhasma (GB), a bioactive calcium sulfate particle prepared by thermo-transformation ofgypsum. Thermal processing of the gypsum transforms its native physicochemical properties by removing water molecules into the anhydrous GB, which was confirmed by Raman and FT-IR spectroscopy. GB particle showed a 0.5-5 µm size range and a neutral surface charge. Exposure of mammalian cells to GB particles showed a rapid cellular uptake through phagocytosis and induced massive cytoplasmic vacuolation in cells. Interestingly, no cellular uptake and cytoplasmic vacuolation were observed with the parent gypsum particle. The presence of the GB particles in intra-vacuolar space was confirmed using FESEM coupled with EDX. Flow cytometry analysis and live tracking of GB-treated cells showed particle internalization, vacuole formation, particle dissolution, and later vacuolar turnover. Quantification of GB-induced vacuolation was done using neutral red uptake assay in cells. Treatment of lysosomal inhibitors (BFA1 or CQ) with GB could not induce vacuolation, suggesting the requirement of an acidic environment for the vacuolation. In the mimicking experiment, GB particle dissolution in acidic cell-free solution suggested that degradation of GB occurs by acidic pH inside the cell vacuole. Vacuole formation generally accompanies with cell death, whereas GB-induced massive vacuolation does not cause cell death. Moreover, the cell divides and proliferates with the vacuolar process, intra-vacuolar cargo degradation, and eventually vacuolar turnover. Taken together, the sequential cellular events in this study suggest that GB can be used as a smart particle for phagocytosis assay development in animal cells.

Phylogeny and structural insights of lipase from Halopseudomonas maritima sp. nov., isolated from sea sand.

A Gram-negative, aerobic bacterial strain RR6T was isolated from the sea sand to produce lipase and proposed as a novel species of Halopseudomonas. The optimum growth occurred at 28-37 °C, and the pH was 6.0-8.0. The optimum growth occurred at 3.0 -6.5% (w/v) NaCl. The major cellular fatty acids were C10:0 3OH, C12:0, C16:1 ω7c/16:1 ω6c, 18:1 ω7c and/or 18:1 ω6c, and C16:0. The predominant polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, unidentified phospholipid, and unidentified lipids. The genome is 3.93 Mb, and the G + C content is 61.3%. The 16S rRNA gene sequences shared 99.73-99.87% sequence similarity with the closely related type strains of Halopseudomonas. The average nucleotide identity and average amino acid identity of strain RR6T with reference type strains were below 95-96%, and the corresponding in-silico DNA-DNA hybridization values were below 70%. Strain RR6T clustered with Halopseudomonas gallaeciensis V113T and Halopseudomonas pachastrellae CCUG 46540 T in the phylogenetic tree. Further, lipase produced by this bacterium belongs to α/ß hydrolase lipase family and exhibits structural similarity to the lactonizing lipase. Based on the polyphasic analysis, the new isolates RR6T represent a novel species of Halopseudomonas for which Halopseudomonas maritima sp. nov. is proposed. The type strain is RR6T (= NBRC 115418 T = TBRC 15628 T).

Genomic evidence and virulence properties decipher the extra-host origin of Bordetella bronchiseptica.

Until recently, members of the classical Bordetella species comprised only pathogenic bacteria that were thought to live exclusively in warm-blooded animals. The close phylogenetic relationship of Bordetella with Achromobacter and Alcaligenes, which include primarily environmental bacteria, suggests that the ancestral Bordetellae were probably free-living. Eventually, the Bordetella species evolved to infect and live within warm-blooded animals. The modern history of pathogens related to the genus Bordetella started towards the end of the 19th century when it was discovered in the infected respiratory epithelium of mammals, including humans. The first identified member was Bordetella pertussis, which causes whooping cough, a fatal disease in young children. In due course, B. bronchiseptica was recovered from the trachea and bronchi of dogs with distemper. Later, a second closely related human pathogen, B. parapertussis, was described as causing milder whooping cough. The classical Bordetellae are strictly host-associated pathogens transmitted via the host-to-host aerosol route. Recently, the B. bronchiseptica strain HT200 has been reported from a thermal spring exhibiting unique genomic features that were not previously observed in clinical strains. Therefore, it advocates that members of classical Bordetella species have evolved from environmental sources. This organism can be transmitted via environmental reservoirs as it can survive nutrient-limiting conditions and possesses a motile flagellum. This study aims to review the molecular basis of origin and virulence properties of obligate host-restricted and environmental strains of classical Bordetella.

Moraxella tetraodonis sp. nov., isolated from freshwater pufferfish (Tetraodon cutcutia) skin.

A novel aerobic bacterium, strain PS-22 of the genus Moraxella, was isolated from the skin of freshwater pufferfish (Tetraodon cutcutia). Cells were Gram stain negative, aerobic, non-motile, and coccoid. Optimum growth occurred at 28-30 °C and pH 6.5-7.5. The major cellular fatty acids were C18:1 ω9c, C10:0, C16:0, and C12:0 anteiso. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipid, amino lipid, and seven unknown lipids. The genome size is 2.68 Mbp, and the DNA G + C content was 43.3%. A gene ontology study revealed that the major fraction of genes were associated with biological processes (46.81%), followed by molecular function (34.27%) and cellular components (18.8%). Comparisons of 16S rRNA gene sequences revealed 99.11-90% sequence similarity with the closely related type strains of the genus Moraxella. The average nucleotide identity (ANI) and average amino acid identity (AAI) of strain PS-22 with reference type strains of the genus Moraxella were below 95-96%, and the corresponding in silico DNA-DNA hybridization (DDH) values were below 70%. A phylogenetic tree based on genome-wide core genes and 16S rRNA gene sequences revealed that strain PS-22 clustered with Moraxella osloensis CCUG350T in both the phylogenetic trees. Genotypic and phenotypic characteristics of strain PS-22 represent a novel species for which Moraxella tetraodonis sp. nov. is proposed. The type strain is PS-22T (= TBRC 15232T = NBRC 115236T).

Pseudomonas phenolilytica sp. nov., a novel phenol-degrading bacterium.

This study describes a bacterium strain RBPA9 isolated from a municipality waste dumping area capable of degrading phenol, proposed as a novel species of Pseudomonas. Cells are Gram-negative, rod shaped, aerobic and motile. The genome is 3.92 Mb, and the G + C content is 64.64%. The overall genome relatedness indices such as in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) values were below 70% and 95-96%, respectively. Phylogenetic analysis based on genome-wide core genes and 16S rRNA gene sequences revealed that strain RBPA9 clustered with Pseudomonas stutzeri ATCC 17588 T in both the phylogenetic trees. Maximum growth was recorded at 200 mg /L concentration of phenol which was consumed within 24 h. A gene cluster of phenol degradation pathway was detected. The quantitative real-time PCR (RT-PCR) demonstrated the expression of all the genes required in the meta-cleavage pathway of phenol in RBPA9. Our results reveal that strain RBPA9 represents a novel species for which Pseudomonas phenolilytica sp. nov. is proposed. The type strain is RBPA9T (= TBRC 15231 T = NBRC 115284 T).

Genome analysis of Pseudomonas species reveals that Pseudomonas panacis Park et al. 2005 is a later heterotypic synonym of Pseudomonas marginalis (Brown 1918) Stevens 1925 (Approved Lists 1980).

We described the comparative genomic analysis of Pseudomonas panacis DSM 18529T and Pseudomonas marginalis DSM 13124T of the genus Pseudomonas to define the taxonomic assignment. When conducting this analysis, genomic information for 203 type strains was available in the NCBI genome database. The ANI, AAI and isDDH data were higher than the threshold values between Pseudomonas panacis DSM 18529T and Pseudomonas marginalis DSM 13124T. Whole-genome comparisons show 97 % average nucleotide identity, 98 % average amino acid identity and 75 % in silico DNA-DNA hybridization values. Pseudomonas marginalis (Brown 1918) Stevens 1925 (Approved Lists 1980) have priority over the name Pseudomonas panacis Park et al. 2005, therefore nomenclatural authorities propose that Pseudomonas panacis Park et al. 2005 is a later heterotypic synonym of Pseudomonas marginalis (Brown 1918) Stevens 1925 (Approved Lists 1980). The type strain is ATCC 10844T (=DSM 13124T=NCPPB 667T).

Influence of Geochemistry in the Tropical Hot Springs on Microbial Community Structure and Function.

Thermophiles inhabiting high temperatures are considered primitive microorganisms on early Earth. In this regard, several works have demonstrated microbial community composition in geothermal environments. Despite that, studies on hot springs located in the Indian subcontinent viz., Surajkund in the district Hazaribag, Jharkhand; Bakreshwar in the district Birbhum, West Bengal; Tantloi in the district Dumka, and Sidpur in the district Pakur, Jharkhand are scanty. Nonetheless, the metagenomic analysis of these hot springs showed significant differences in the predominant phyla corresponding to geochemical properties. The Chloroflexi, Proteobacteria, Actinobacteria, Deinococcus-Thermus, and Firmicutes were dominant phyla in all the samples. In contrast, Meiothermus was more in comparatively low-temperature hot springs. In addition, archaeal phyla, Euryarchaeota, Candidatus Bathyarchaeota, and Crenarchaeota were predominant in all samples. The canonical correspondence analysis (CCA) showed the abundance of Deinococcus, Thermus, Pyrobaculum, Kocuria, and Geodermatophilus positively correlated with the aqueous concentration of sulfate, fluoride, and argon in relatively high-temperature (≥ 72 °C) hot springs. However, at a lower temperature (≤ 63 °C), Thermodesulfovibrio, Caldilinea, Chloroflexus, Meiothermus, and Tepidimonas are positively correlated with the concentration of zinc, iron, and dissolved oxygen. Further, hierarchical clustering exhibits variations in its functional attributes depending on the temperature gradients. Metagenome analysis predicted carbon, methane, sulfur, and nitrogen metabolism genes, indicating a wide range of bacteria and archaea habitation in these hot springs. In addition, identified several genes encode polyketide biosynthesis pathways. The present study described the microbial community composition and function in the tropical hot springs and their relationship with the environmental variables.

Description of Acinetobacter kanungonis sp. nov., based on phylogenomic analysis.

A novel strain of a member of the genus Acinetobacter, strain PS-1T, was isolated from the skin of fresh water pufferfish (Tetraodon cutcutia) collected from Mahanadi River, India. Cells were Gram-stain-negative, aerobic, coccoid and non-motile. The predominant polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phospholipid (PL) and the cell wall sugars were glucose, galactose and ribose. The major cellular fatty acids of PS-1T were C18 : 1ω9c (30.67 %), C16 : 1ω7c (19.54 %), C16 : 0 (15.87 %), C12 : 0 (7.35 %) and C12 : 0 3-OH (6.77 %). The genome size was 3.5 Mbp and the DNA G+C content was 41.97 %. Gene ontology study revealed that the major fraction of genes were associated with biological processes (53.99 %) followed by molecular function (30.42 %) and cellular components (15.58 %). Comparisons of 16S rRNA gene sequences revealed 97.94-97.05 % sequence similarity with the closely related type strains of species of the genus Acinetobacter. The average nucleotide identity (ANI) and average amino acid identity (AAI) of PS-1T with reference strains of species of the genus Acinetobacter with validly published names were bellow 95-96 and the corresponding in-silico DNA-DNA hybridization (DDH) values were below 70 %. A phylogenomic tree based on core genome analysis supported these results. Genotypic and phenotypic characteristics of PS-1T indicate that the strain represents a novel species of the genus Acinetobacter and the name Acinetobacter kanungonis sp. nov. is proposed. The type strain is PS-1T (=JCM 34131T=NCIMB 15260T).

Glutamicibacter mishrai sp. nov., isolated from the coral Favia veroni from Andaman Sea.

A novel aerobic marine actinobacterium (strain S5-52T) belonging to the genus Glutamicibacter was isolated from the coral Favia veroni sampled from the Andaman Sea, India. Cells are Gram stain positive and rod shaped. The DNA G+C content was 58.7 mol%. The major quinones were MK-8 and MK-9. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid, trimannosyldiacylglycerol, phospholipid and dimannosylglyceride. The peptidoglycan type was A4α. Strain S5-52T showed a maximum 16S rRNA similarity of 99.36% with Glutamicibacter halophytocola DSM 101718T. The genome of strain S5-52T was 3.57 Mb that contains 3274 protein coding sequences (CDS). DNA-DNA similarity and ANI values between S5-52T and the reference strains were below 70% and 95-96%, respectively. Analysis of genomic reduction events in the evolutionary path from the LUCA (last universal common ancestor) to G. mishrai LMG 29155T and G. halophytocola DSM 101718T exhibit a number of genes involved in amino acid metabolism, cell wall biogenesis and replication, recombination and repair mechanism that reduced in both the species. Based on phenotypic, chemotaxonomic properties and comparative genomic studies, the strain S5-52T is considered a novel species of the genus Glutamicibacter, for which the name Glutamicibacter mishrai sp. nov. is proposed. The type strain is S5-52T (= KCTC 39846T = LMG 29155T).

Composition and functional characterization of the gut microbiome of freshwater pufferfish (Tetraodon cutcutia).

This study describes the community composition and functions of the gut microbiome of the freshwater omnivorous pufferfish based on metagenomic approach. Metagenome sequence data showed a dominance of the class Gammaproteobacteria followed by Fusobacteria, Actinobacteria, Anerolineae, Betaproteobacteria, Deinococci, Clostridia and Deltaproteobacteria. At the order level, the most abundant groups were Aeromonadales, Fusobacteriales, Enterobacterales, Synechococcales. The genus Aeromonas was the most predominant followed by Plesiomonas and Cetobacterium. Additionally, within the domain Archaea, class Methanomicrobia was most abundant followed by Hadesarchaea, Thermoplasmata, Candidatus Altiarchaeales, Candidatus Bathyarchaeota and Thermoprotei. The metabolic profile of the bacterial community exhibited a high prevalence of genes associated with core housekeeping functions, such as synthesis of cofactors, vitamins, prosthetic groups, pigments, amino acids and its derivatives, carbohydrate and protein metabolism. Comparative analysis with other fish gut microbiome showing similarity in protein metabolism with carnivorous Salmon and carbohydrate metabolism with herbivorous grass carp respectively. This study describes the bacterial community compositions are influenced by the trophic level.

Phylogenomic Analysis Reveals That Arthrobacter mysorens Nand and Rao 1972 (Approved Lists 1980) and Glutamicibacter mysorens Busse 2016 are Later Heterotypic Synonyms of Arthrobacter nicotianae Giovannozzi-Sermanni 1959 (Approved Lists 1980) and Glutamicibacter nicotianae Busse 2016.

We described the comparative genomic analysis to delineate the taxonomic relationship between the two Glutamicibacter species Glutamicibacter mysorens NBRC 103060T and Glutamicibacter nicotianae NBRC 14234T. Phylogenetic tree based on concatenated ribosomal protein marker genes showed both species clade together. The average nucleotide identity (ANI) values between G. mysorens NBRC 103060T and G. nicotianae NBRC 14234T ranged from 97.23 to 97.97%. Further, the average amino acid identity (AAI) between two strains were more than 97.61%. The ANI, AAI and in silico DNA-DNA hybridization (isDDH) data were higher than the threshold value for bacterial species delineation. The two strains have identical profile of fatty acids, sugars and lipid composition; and overall similar phenotypic characteristics. It therefore becomes evident that the two species actually belong to the same species. Arthrobacter nicotianae (Giovannozzi-Sermanni 1959) (Approved Lists 1980) and Glutamicibacter nicotianae Busse 2016 have priority over the names Arthrobacter mysorens (Nand and Rao 1972) (Approved Lists 1980) and Glutamicibacter mysorens Busse 2016, therefore we proposed that Glutamicibacter mysorens Busse 2016 is a later heterotypic synonym of Glutamicibacter nicotianae Busse 2016. The type strain is ATCC 15236T = CCM 1648T = BCRC (formerly CCRC) 11219T = CCUG 23842T = CDA 883T = CIP 82.107T = DSM 20123T = HAMBI 1859T = IAM 12342T = IFO (now NBRC) 14234T = IMET 10353T = JCM 1333T = LMG 16305T = NCIMB 9458T = NRIC 0153T.

Colchicine: Isolation, LC-MS QTof Screening, and Anticancer Activity Study of Gloriosa superba Seeds.

Colchicine was extracted from Gloriosa superba seeds using the Super Critical Fluid (CO2) Extraction (SCFE) technology. The seeds were purified upto 99.82% using column chromatography. Colchicine affinity was further investigated for anticancer activity in six human cancer cell lines, i.e., A549, MCF-7, MDA-MB231, PANC-1, HCT116, and SiHa. Purified colchicine showed the least cell cytotoxicity and antiproliferation and caused no G2/M arrest at clinically acceptable concentrations. Mitotic arrest was observed in only A549 and MDA-MB231 cell lines at 60nM concentration. Our finding indicated the possible use of colchicine at a clinically acceptable dose and provided insight into the science behind microtubule destabilization. However, more studies need to be conducted beforethese findings could be established.

Microbiological studies of hot springs in India: a review.

The earliest microbiological studies on hot springs in India date from 2003, a much later date compared to global attention in this striking field of study. As of today, 28 out of 400 geothermal springs have been explored following both culturable and non-culturable approaches. The temperatures and pH of the springs are 37-99 °C and 6.8-10, respectively. Several studies have been performed on the description of novel genera and species, characterization of different bio-resources, metagenomics of hot spring microbiome and whole genome analysis of few isolates. 17 strains representing novel species and many thermostable enzymes, including lipase, protease, chitinase, amylase, etc. with potential biotechnological applications have been reported by several authors. Influence of physico-chemical conditions, especially that of temperature, on shaping the hot spring microbiome has been established by metagenomic investigations. Bacteria are the predominant life forms in all the springs with an abundance of phyla Firmicutes, Proteobacteria, Actinobacteria, Thermi, Bacteroidetes, Deinococcus-Thermus and Chloroflexi. In this review, we have discussed the findings on all microbiological studies that have been carried out to date, on the 28 hot springs. Further, the possibilities of extrapolating these studies for practical applications and environmental impact assessment towards protection of natural ecosystem of hot springs have also been discussed.

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T).

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa 3 ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

Idiomarina andamanensis sp. nov., an alkalitolerant bacterium isolated from Andaman Sea water.

Two closely related aerobic, Gram-negative rod shaped bacteria (strain W5T and W3) were isolated from Andaman Sea. Heterotrophic growth on marine agar was observed at 15-45 °C and pH 6-10. Strain W5T showed maximum 16S rRNA sequence similarity of 99.58% with Idiomarina marina JCM 15083T. DNA fingerprinting analysis by ERIC-REP PCR, PFGE and MLSA revealed differences in banding patterns, also DNA-DNA hybridization values were well below 70% confirming W5T to be a new species. DNA G+C content was 46.7 mol%. Major fatty acids were iso-C15:0, iso-C17:0, iso-C17:1 ω9c, iso-C13:0 3OH, iso-C11:0 3OH and C16:0. Polar lipids included phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) diphosphatidylglycerol (DPG) phospholipid (PL) two aminolipids (AL) and two unidentified lipids (L1-2). Q-8 is the predominant ubiquinone. On the basis of polyphasic taxonomic study, strain W5T is considered to be representative of a new species of the genus Idiomarina, for which the name Idiomarina andamanensis sp. nov. is being proposed. The type strain W5T (= LMG 29773T = JCM 31645T) was isolated from Andaman Sea.

Sulfitobacter pontiacus subsp. fungiae subsp. nov., Isolated from Coral Fungia seychellensis from Andaman Sea, and Description of Sulfitobacter pontiacus subsp. pontiacus subsp. nov.

Two closely related aerobic, Gram reaction-negative rod-shaped bacteria (S7-75T and S7-80) were isolated from mucus of coral Fungia seychellensis from Andaman Sea, India. Heterotrophic growth on marine agar was observed at 4-35 °C and pH 6.5-10.5; optimum growth occurred at 25-30 °C and pH 7-8. 16 S rRNA sequence analysis confirmed the strains belonged to the genus Sulfitobacter and the two isolates shared more than 99.28% pairwise sequence similarity. DNA-DNA similarity between two isolates S7-75T and S7-80 was above 96%. Strain S7-75T showed maximum 16S rRNA similarity of 99.64% with Sulfitobacter pontiacus LMG 19752T. However, DNA-DNA relatedness between strain S7-75T and S. pontiacus LMG 19752T confirmed the placement of strain S7-75T as subspecies under the species S. pontiacus. Further, pulsed-field gel electrophoresis (PFGE), REP-PCR, ERIC-PCR fingerprint patterns and lipid profiles also differentiated strain S7-75T from the reference strain of S. pontiacus LMG 19752T. The DNA G+C content was 59.8 mol%. Q10 was the major respiratory quinone. Based on polyphasic analysis, the isolate S7-75T represents a subspecies of S. pontiacus for which the name S. pontiacus subsp. fungiae subsp. nov. is proposed with S7-75T (=JCM 31094T = LMG 29158T) as type strain.

Subretinal AAV2.COMP-Ang1 suppresses choroidal neovascularization and vascular endothelial growth factor in a murine model of age-related macular degeneration.

To assess whether Tie2-mediated vascular stabilization ameliorates neovascular age-related macular degeneration (AMD), we investigated the impact of adeno-associated virus-mediated gene therapy with cartilage oligomeric matrix protein angiopoietin-1 (AAV2.COMP-Ang1) on choroidal neovascularization (CNV), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF) in a mouse model of the disease. We treated mice with subretinal injections of AAV2.COMP-Ang1 or control (AAV2.AcGFP, AAV2.LacZ, and phosphate-buffered saline). Subretinal AAV2 localization and plasmid protein expression was verified in the retinal pigment epithelium (RPE)/choroid of mice treated with all AAV2 constructs. Laser-assisted simulation of neovascular AMD was performed and followed by quantification of HIF, VEGF, and CNV in each experimental group. We found that AAV2.COMP-Ang1 was associated with a significant reduction in VEGF levels (29-33%, p < 0.01) and CNV volume (60-70%, p < 0.01), without a concomitant decrease in HIF1-α, compared to all controls. We concluded that a) AAV2 is a viable vector for delivering COMP-Ang1 to subretinal tissues, b) subretinal COMP-Ang1 holds promise as a prospective treatment for neovascular AMD, and c) although VEGF suppression in the RPE/choroid may be one mechanism by which AAV2.COMP-Ang1 reduces CNV, this therapeutic effect may be hypoxia-independent. Taken together, these findings suggest that AAV2.COMP-Ang1 has potential to serve as an alternative or complementary option to anti-VEGF agents for the long-term amelioration of neovascular AMD.

Psychrobacter pocilloporae sp. nov., isolated from a coral, Pocillopora eydouxi.

Two closely related aerobic, Gram-stain-negative, rod-shaped bacteria (S6-60T and S6-67) were isolated from the mucus of the coral, Pocilloporaeydouxi, from the Andaman Sea, India. Heterotrophic growth on marine agar was observed at 4-37 °C and at pH 6.5-10.0; optimum growth occurred at 25-30 °C and at pH 7-9. 16S rRNA gene sequence analysis confirmed that the isolates belong to the genus Psychrobacter; the two isolates shared more than 99.5 % pairwise sequence similarity. Strain S6-60T showed a maximum 16S rRNA similarity of 98.92 % with Psychrobacter pacificensis DSM 23406T. DNA-DNA homology between the two isolates, S6-60T and S6-67, was above 90 %, whereas strain S6-60T showed less than 70 % homology with closely related type species. The DNA G+C content was 47.7 mol%. It contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipid as the major polar lipids. C10 : 0, C12 : 0 3OH, C16 : 0, C18 : 1ω9c, C17 : 1ω8c and C16 : 1ω7c were found to be the predominant fatty acids. Based on a polyphasic analysis, the isolates (S6-60T and S6-67) represent a novel species of the genus Psychrobacter for which the name Psychrobacter pocilloporae sp. nov. is proposed with S6-60T(=JCM 31058T=LMG 29157T) as the type strain.

Sulfitobacter faviae sp. nov., isolated from the coral Faviaveroni.

Three closely related, non-sporulating, aerobic, Gram-stain-negative, motile, rod-shaped isolates (S5-53T, S6-62 and S6-64) were obtained from mucus of corals Favia veroni from the Andaman Sea, India. Colonies grown on marine agar were small, circular and cream-coloured. Heterotrophic growth was observed at 10-40 °C and pH 6-10; optimum growth occurred at 25-30 °C and pH 7-8. 16S rRNA gene sequence analysis confirmed the isolates belonged to the genus Sulfitobacter and the three isolates shared more than 99 % pairwise sequence similarity. Strain S5-53T shared highest 16S rRNA gene sequence similarity of 98.43 % with Sulfitobacter dubius KMM 3554T. DNA-DNA relatedness among the three isolates was above 70 % whereas strain S5-53T showed less than 70 % relatedness with the type strains of closely related species. The DNA G+C content of strain S5-53T was 61 mol%. It contained phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and diphosphatidylglycerol as major polar lipids. Predominant fatty acids included C18 : 1ω7c, C18 : 1ω7c 11-methyl, C16 : 0 and C10 : 0 3-OH. Q10 was the major respiratory quinone. Based on this polyphasic analysis, the new isolates (S5-53T, S6-62 and S6-64) are considered to represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter faviae sp. nov. is proposed. The type strain is S5-53T(=JCM 31093T=LMG 29156T).

Functional analysis of ars gene cluster of Pannonibacter indicus strain HT23(T) (DSM 23407(T)) and identification of a proline residue essential for arsenate reductase activity.

Arsenic is a naturally occurring ubiquitous highly toxic metalloid. In this study, we have identified ars gene cluster in Pannonibacter indicus strain HT23(T) (DSM 23407(T)), responsible for reduction of toxic pentavalent arsenate. The ars gene cluster is comprised of four non-overlapping open reading frames (ORFs) encoding a transcriptional regulator (ArsR), a low molecular weight protein tyrosine phosphatases (LMW-PTPase) with hypothetical function, an arsenite efflux pump (Acr3), and an arsenate reductase (ArsC). Heterologous expression of arsenic inducible ars gene cluster conferred arsenic resistance to Escherichia coli ∆ars mutant strain AW3110. The recombinant ArsC was purified and assayed. Site-directed mutagenesis was employed to ascertain the role of specific amino acids in ArsC catalysis. Pro94X (X = Ala, Arg, Cys, and His) amino acid substitutions led to enzyme inactivation. Circular dichroism spectra analysis suggested Pro94 as an essential amino acid for enzyme catalytic activity as it is indispensable for optimum protein folding in P. indicus Grx-coupled ArsC.