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.