Metabolomic and genomic insights into TMA degradation by a novel halotolerant strain - Paracoccus sp. PS1.

Trimethylamine N-oxide (TMAO) is a gut metabolite that acts as a biomarker for chronic diseases, and is generated by the oxidation of trimethylamine (TMA) produced by gut microflora. Since, microbial degradation of TMA is predicted to be used to restrict the production of TMAO, we aimed to isolate bacterial strains that could effectively degrade TMA before being oxidized to TMAO. As marine fish is considered to have a rich content of TMAO, we have isolated TMA degrading isolates from fish skin. Out of the fourteen isolates, depending on their rapid TMA utilization capability in mineral salt medium supplemented with TMA as a sole carbon and nitrogen source, isolate PS1 was selected as our desired isolate. Its TMA degrading capacity was further confirmed through spectrophotometric, Electrospray Ionization Time-of-Flight Mass Spectrometry (ESI TOF-MS) and High performance liquid chromatography (HPLC) analysis and in silico analysis of whole genome (WG) gave further insights of protein into its TMA degradation pathways. PS1 was taxonomically identified as Paracoccus sp. based on its 16S rRNA and whole genome sequence analysis. As PS1 possesses the enzymes required for degradation of TMA, clinical use of this isolate has the potential to reduce TMAO generation in the human gut.

Biocontrol of biofilm forming Burkholderia cepacia using a quorum quenching crude lactonase enzyme extract from a marine Chromohalobacter sp. strain D23.

Biofilm plays advantageous role in Burkholderia cepacia by exerting multi-drug resistance. As quorum sensing (QS) system regulates biofilm formation and pathogenicity in B. cepacia strains, quorum quenching (QQ) may be a novel strategy to control persistent B. cepacia infections. In these regards, 120 halophilic bacteria were isolated from marine sample and tested using Chromobacterium violaceum and C. violaceum CV026-based bioassays initially, showing reduced violacein synthesis by QQ enzyme by 6 isolates. Among them, Chromohalobacter sp. D23 significantly degraded both C6-homoserine lactone (C6-HSL) and C8-HSL due to potent lactonase activity, which was detected by C. violaceum CV026 biosensor. Further high-performance liquid chromatography (HPLC) study confirmed degradation of N-acyl homoserine lactones (N-AHLs) particularly C6-HSL and C8-HSL by crude lactonase enzyme. Chromohalobacter sp. D23 reduced biofilm formation in terms of decreased total biomass and viability in biofilm-embedded cells in B. cepacia significantly which was also evidenced by fluorescence microscopic images. An increase in antibiotic susceptibility of B. cepacia biofilm was achieved when crude lactonase enzyme of Chromohalobacter sp. strain D23 was combined with chloramphenicol (1-5 × MIC). Chromohalobacter sp. D23 also showed prominent decrease in QS-mediated synthesis of virulence factors such as extracellular polymeric substances (EPS), extracellular protease, and hemolysin in B. cepacia. Again crude lactonase enzyme of Chromohalobacter sp. strain D23 inhibited B. cepacia biofilm formation inside nasal oxygen catheters in vitro. Finally, antibiotic susceptibility test and virulence tests revealed sensitivity of Chromohalobacter sp. strain D23 against a wide range of conventional antibiotics as well as absence of gelatinolytic, hemolytic, and serum coagulating activities. Therefore, the current study shows potential quorum quenching as well as anti-biofilm activity of Chromohalobacter sp. D23 against B. cepacia.

Metaproteomic Discovery and Characterization of a Novel Lipolytic Enzyme From an Indian Hot Spring.

Lipolytic enzymes are produced by animals, plants and microorganisms. With their chemo-, regio-, and enantio-specific characteristics, lipolytic enzymes are important biocatalysts useful in several industrial applications. They are widely used in the processing of fats and oils, detergents, food processing, paper and cosmetics production. In this work, we used a new functional metaproteomics approach to screen sediment samples of the Indian Bakreshwar hot spring for novel thermo- and solvent-stable lipolytic enzymes. We were able to identify an enzyme showing favorable characteristics. DS-007 showed high hydrolytic activity with substrates with shorter chain length (C10, significantly less hydrolytic activity was observed. A preference for short chain acyl groups is characteristic for esterases, suggesting that DS-007 is an esterase. Consistent with the high temperature at its site of isolation, DS-007 showed a temperature optimum at 55°C and retained 80% activity even after prolonged exposure to temperatures as high as 60°C. The enzyme showed optimum activity at pH 9.5, with more than 50% of its optimum activity between pH 8.0 and pH 9.5. DS-007 also exhibited tolerance toward organic solvents at a concentration of 1% (v/v). One percent of methanol increased the activity of DS-007 by 40% in comparison to the optimum conditions without solvent. In the presence of 10% methanol, DMSO or isopropanol DS-007 still showed around 50% activity. This data indicates that DS-007 is a temperature- and solvent-stable thermophilic enzyme with reasonable activity even at lower temperatures as well as a catalyst that can be used at a broad range of pH values with an optimum in the alkaline range, showing the adaptation to the habitat's temperature and alkaline pH.

Plant Growth-Promoting Traits of a Thermophilic Strain of the Klebsiella Group with its Effect on Rice Plant Growth.

In agriculture, instead of synthetic fertilizers, natural bio-inoculants can be used to increase growth and yield of crops. For this purpose, we report a thermophilic bacteria Klebsiella sp. strain PMnew, isolated from Paniphala hot spring. The strain was characterized and assessed for plant growth-promoting traits. Oryza sativa L. var Swarna (rice) seeds were inoculated with the strain to study the bacterization effect on vegetative and reproductive growth of rice plants. The results indicate that PMnew produces organic acids to solubilize phosphate (550.16 ± 0.04 µg/ml), fixes nitrogen, produces indole compounds, siderophore, and ACC deaminase, and shows heavy metal resistance to chromium, cobalt, arsenic, cadmium, and mercury. It also possesses the ability to utilize several monomeric and polymeric sugars as sole carbon source including starch, agar, xylan, gelatin, and pectin, and can grow under both nutrient-rich and deficient conditions. Inoculated rice plants grew twice the length of control plants and surpassed the total grain mass yield of control plants by almost 18 times. Thus, this study brings forth a broad spectrum and easy to cultivate bio-inoculant, which can be used to increase rice production.

Microbiome and ecology of a hot spring-microbialite system on the Trans-Himalayan Plateau.

Little is known about life in the boron-rich hot springs of Trans-Himalayas. Here, we explore the geomicrobiology of a 4438-m-high spring which emanates ~70 °C-water from a boratic microbialite called Shivlinga. Due to low atmospheric pressure, the vent-water is close to boiling point so can entropically destabilize biomacromolecular systems. Starting from the vent, Shivlinga's geomicrobiology was revealed along the thermal gradients of an outflow-channel and a progressively-drying mineral matrix that has no running water; ecosystem constraints were then considered in relation to those of entropically comparable environments. The spring-water chemistry and sinter mineralogy were dominated by borates, sodium, thiosulfate, sulfate, sulfite, sulfide, bicarbonate, and other macromolecule-stabilizing (kosmotropic) substances. Microbial diversity was high along both of the hydrothermal gradients. Bacteria, Eukarya and Archaea constituted >98%, ~1% and <1% of Shivlinga's microbiome, respectively. Temperature constrained the biodiversity at ~50 °C and ~60 °C, but not below 46 °C. Along each thermal gradient, in the vent-to-apron trajectory, communities were dominated by Aquificae/Deinococcus-Thermus, then Chlorobi/Chloroflexi/Cyanobacteria, and finally Bacteroidetes/Proteobacteria/Firmicutes. Interestingly, sites of >45 °C were inhabited by phylogenetic relatives of taxa for which laboratory growth is not known at >45 °C. Shivlinga's geomicrobiology highlights the possibility that the system's kosmotrope-dominated chemistry mitigates against the biomacromolecule-disordering effects of its thermal water.

CdS quantum dots embedded in PVP: Inorganic phosphate ion sensing in real sample and its antimicrobial activity.

Polyvinyl-pyrrolidone capped spherical cadmium sulphide quantum dots (CdS-PVP QDs), 2-6 nm in size, were developed as a selective turn-on fluorescence nanosensor for monohydrogen phosphate ion (HPO42-) in aqueous medium. Fluorescence intensity of CdS-PVP QDs significantly increased with addition of HPO42- ions, whereas the other common inorganic ions had very little effect on the fluorescence intensity. The proposed sensor may be efficiently used for the detection of HPO42- ions at a low level of concentration up to 213 nM in real urine sample. Cell imaging study indicates that the CdS-PVP QDs are cell permeable and can detect the intracellular distribution of HPO42- ions under fluorescence microscope. The CdS-PVP QDs showed considerable activity against Staphylococcus aureus also.

A simple urea-based multianalyte and multichannel chemosensor for the selective detection of F-, Hg2+ and Cu2+ in solution and cells and the extraction of Hg2+ and Cu2+ from real water sources: a logic gate mimic ensemble.

Herein, a hydrazine-based chromogenic, fluorogenic and electrochemical chemosensor BCC [1,5-bis(4-cyanophenyl) carbonohydrazide] was premeditated and synthesized through a simple one-step synthetic procedure for the selective detection of toxic anions, such as F-, in a DMSO-ACN medium and cations, such as Hg2+ and Cu2+, in a MeOH-water medium. The detection limit for F- was reckoned to be 0.5 ppm, and for Hg2+ and Cu2+, it was 0.8 ppm and 50 nM, respectively. The chemosensor exhibited a distinct change in colour from colourless to dark blue in the presence of F-, and upon the addition of Hg2+ and Cu2+, the BCC turned from colourless to light blue and purple accordingly. Moreover, turn-on fluorescence response transpired by the attenuation of PET signified the selective sensing of analytes with a zero-order rate constant. Sophisticated analytical experiments, such as ESI-MS, UV-Vis, photoluminescence, cyclic voltammetry, FTIR, and 1H-NMR, along with the theoretical calculations corroborated the probable sensing pathways. The reversible colorimetric response of BCC towards F- and H+ can be advantageous in the design of electronic circuits derived from Boolean algebra. The complexation ability of the sensor with toxic Hg2+- and Cu2+-like ions made it an efficient material to remove these metal ions from real water sources polluted with these toxic elemental ions. Furthermore, the in vitro studies were accomplished using the Bauhinia acuminate pollen cell to check the cell penetrability of the sensor molecule.

Application of rice (Oryza sativa L.) root endophytic diazotrophic Azotobacter sp. strain Avi2 (MCC 3432) can increase rice yield under green house and field condition.

Use of plant-associated beneficial microbes, especially endophytes are getting popular day by day as they occupy a relatively privileged niche inside different plant tissues with lesser competition for food and shelter than rhizosphere. The effects of different physical factors like temperature, rainfall, and seasonal variation and UV radiation on plant growth promoting endophytic communities are less pronounced than those on the rhizospheric and phylloplane microbes. This present work has been compromised with further utilization of an indigenous rice (Oryza sativa L.) root endophytic Azotobacter sp. strain Avi2 (MCC 3432) (AzA) as a bio-formulation for sustainable rice production based on several physiological parameters (plant height, root length/weight, leaf area, yield, chlorophyll contain), in-vitro comparative plant growth promoting assays, greenhouse and field experiments (dry and wet season). Treatments with AzA exhibited higher yield as well as maximal chlorophyll fluorescence (Fm) of flag leaves in flowering and grain filling stages indicating higher photosynthetic rates. Scanning electron microscopic image of rice roots demonstrated accumulation of bacterial biofilm at the junction of primary and lateral root confirming the root-colonizing ability of the bacterium. The results of the study were quite encouraging as AzA exhibited better vegetative and reproductive growth of rice in pot and field experiment compared to formulated rhizospheric Azotobacter sp. (commercial product). Apart from that plants treated with AzA (supplemented 50% nitrogenous fertilizer of recommended dose) exhibited similar yield parameters when it was compared with the recommended dose of fertilizer (RDF; 120:60:60 mg N:P:K kg-1 soil/ without any bacterial). Therefore, it can be concluded that application of this plant growth promoting endophyte can reduce a substantial amount of N-fertilizer for field application.

Nanomolar-level selective dual channel sensing of Cu2+ and CN- from an aqueous medium by an opto-electronic chemoreceptor.

A dual-channel chromogenic and fluorogenic fused-aromatic-system-based chemoreceptor (2-(benzo[d]thiazol-2-yl)-1-((pyren-8-yl)methylene)hydrazine) (TyM) was designed for ditopic sensitivity towards heavy and transition metal ions (HTMs), such as Cu2+, in an aqueous medium and lethal CN- in a semi-aqueous medium. The chemoreceptor displayed proclivity towards the targeted analytes with a distinct optical response (yellow to colourless in the case of Cu2+ and yellow to bright red for CN-). TyM formed a 2 : 1 adduct with Cu2+ with a detection limit of 40 nM. A 1 : 1 binding stoichiometry was confirmed with the chemoreceptor TyM with CN- in sub-nano molar limit of detection. In addition to sophisticated spectroscopic analysis, such as UV-vis, fluorescence, FTIR, 1H-NMR, 13C-NMR, ESI-MS, and HRMS, the plausible mechanistic course of sensing was also established from a theoretical perspective. The reversible UV-vis response of the chemoreceptor TyM towards CN- and H+ can mimic different molecular logic functions and therefore can be exploited for designing several complex electronic circuits principally based on Boolean Algebra. In vitro fluorescence imaging in male microspores of seed plants (Bohonia Nigalandra) and Monilia Albicans (diploid fungus) with TyM and Cu2+ confirmed the permeability of the chemoreceptor TyM at the cellular level as well as its ability to investigate transition metals, such as Cu2+, in biological samples.

Trace Level Recognition of Zn2+ and Cd2+ by Biocompatible Chemosensor inside Androecium, Diagnosis of Pick's Disease from Urine and Biomimetic ß-Cell Exocytosis.

Two chemosensors with varying substitution have been synthesized for selective detection of d10 metal ion analyte Zn2+ and Cd2+ by fluorometric method from aqueous medium at very low limit of detection. Density functional theory (DFT)-based Loewdin spin population analysis reveals that methoxy-substituted chemosensor is much stronger donor than bromo-substituted chemosensor. Eventually, bromo and methoxy substituted chemosensors are moderate and strong donor, respectively, toward selective detection of Cd2+ and Zn2+ by luminescence induced phenomenon (blue for Cd2+ and cyan for Zn2+). The mechanism of sensing could be explained by PET-CHEF-C = N isomerization-ILCT pathway. 1H NMR, ESI-MS and FT-IR has been carried out in order to explore the selective ion sensing mechanism. Intracellular detection of Zn2+ and Cd2+ has been carried out inside androecium (filament and pollens) of Tecoma Stans. Extracellular detection of Zn2+ for yeast cells represents the bio mimetic model experiments toward ß-cells exocytosis as a marker of diabetes mellitus. The unprecedented and novel feature of the present biocompatible chemosensor is its application as biosensor to detect in vivo Zn2+ from human urine specimen which could be a next generation diagnostic tool for Pick's disease.

Antioxidant properties of the carotenoid extracts of three Deinococcus­Thermus phylum bacteria, Meiothermus sp. strains RP and TP and Thermus sp. strain YY from Paniphala hot spring, India

BACKGROUND:The purpose of the study was to extract carotenoids from thermophilic bacteria which show efficient antioxidant and protein oxidation inhibition properties, characterize and identify those isolates, extract the carotenoids in different solvents, quantify the carotenoids and perform concentration-dependent and solvent-dependent quantitative assays validated and analysed by appropriate statistical tests. METHODS: Three pigment-forming thermophilic strains were isolated from water sample of Paniphala hot spring, India, and tentatively identified by 16S ribosomal DNA (rDNA) homology. Different concentrations of the carotenoid extracts (100, 80, 40 and 20µg) in three solvents, methanol, DMSO and water, were used to determine the antioxidant activity through five methods: the DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, the ABTS (2,2-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid)) assay, the hydrogen peroxide assay, TOC (total antioxidant capacity) assay and inhibition of protein oxidation assay. Statistical analysis of mean, standard deviation, ANOVA and Pearson correlationcoefficient was performed in Microsoft Excel statistical package.Results:The isolates were tentatively identified as Meiothermussp. strain RP, Meiothermussp. strain TP and Thermusstrain YY.Meiothermussp. formed red coloured pigment, where as Thermussp. formed yellow coloured pigment. Allof the extracts showed positive results in DPPH assay, ABTS assay and hydrogen peroxide radical scavenging assaywith best results obtained when the extracts were dissolved in water. Total antioxidant capacity assay was also highin all the extracts. Protein oxidation inhibition activity was only seen in extracts of strain YY. One-way ANOVA(analysis of variance) clearly showed that choice of solvent influenced the antioxidant capacity of all of the extracts. CONCLUSIONS: Newer and efficient antioxidative compounds are constantly being searched for, and the carotenoid extracts of RP, TP and YY have been shown to catalyze various types of antioxidative reactions, including proteinoxidation inhibition by YY. Thus, all these extracts have huge potential to be industrially and pharmaceutically useful.

Tuning of azine derivatives for selective recognition of Ag+ with the in vitro tracking of endophytic bacteria in rice root tissue.

Several azine derivatives have been prepared and structurally characterized by spectroscopic and single-crystal X-ray diffraction analysis. Two of them, viz. naphthalene based (A10) and anthracene based (A11) show fluorescence enhancement in the presence of Ag+ in aqueous-methanol. Moreover, A11 efficiently tracks Ag+in vitro endophytic bacteria infected rice root tissue. Experimental results have been substantiated by theoretical DFT calculations.

Genome Sequence of the Multiple-Protease-Producing Strain Geobacillus thermoleovorans N7, a Thermophilic Bacterium Isolated from Paniphala Hot Spring, West Bengal, India.

Here, we present the draft genome sequence of Geobacillus thermoleovorans strain N7 (MCC 3175), isolated from Paniphala Hot Spring, West Bengal, India, which contains genes that encode several industrially and medically important thermostable enzymes like neutral protease, xylose isomerase, rhamnogalacturonan acetylesterase, nitrate and nitrite reductase, l-asparaginase, glutaminase, and RNase P.

Genome Sequence of the Arsenic-Resistant Haladaptatus sp. Strain R4 Isolated from Ramnagar, West Bengal, India.

Here, we present the draft genome of Haladaptatus sp. strain R4, a halophilic archaea that produces an orange-pink pigment and is capable of growing in a wide salinity range. The genome assembly shows genes for arsenic resistance, siderophore production, trehalose and glycine betaine biosynthesis, uptake and transporters of sodium, potassium, and chloride ions.

Genome Sequence of the Red Pigment-Forming Meiothermus taiwanensis Strain RP Isolated from Paniphala Hot Spring, India.

Here we report the draft genome sequence of Meiothermus taiwanensis strain RP (MCC 2966), isolated from the Paniphala hot spring of India, which contains genes encoding for enzymes of the methyl erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis and carotenoid backbone synthesis.

Dual mode ratiometric recognition of zinc acetate: nanomolar detection with in vitro tracking of endophytic bacteria in rice root tissue.

Several naphthalene-based aldazine derivatives were developed as efficient colorimetric and fluorescence probes for selective ratiometric recognition of traces of zinc acetate. The derivative structures were characterized by single-crystal X-ray diffraction. The probes were used for in vitro tracking of zinc acetate in endophytic bacteria within rice root tissue and to image zinc acetate in human breast cancer cells (MCF7) by normal and fluorescence microscopy. Density functional theoretical studies were in close agreement with the experimental findings.

Colorimetric and fluorimetric response of Schiff base molecules towards fluoride anion, solution test kit fabrication, logical interpretations and DFT-D3 study.

Two newly synthesized Schiff base molecules are herein reported as anion sensors. -NO2 substituted receptor (P1) is comparatively more acidic and can sense F(-), OAc(-) and H2PO4(-), whereas -CN substituted receptor (P2) is less acidic and is selective for F(-) only. Reversible UV-Vis response for both receptors with F(-) can mimic multiple logic gate functions, and several complex electronic circuits based on XNOR, XOR, OR, AND, NOT and NOR logic operations with 'Write-Read-Erase-Read' options have been executed. Interesting 'turn on and off' fluorescence responses were noticed for the receptors with F(-). Intracellular F(-) detection as a diagnosis of non-skeletal fluorosis was successful using a fluorescence microscope with Candida albicans (prokaryotic cell, a diploid fungus) and pollen grains of Tecoma stans (eukaryotic cell) incubated in 10(-6) M fluoride-contaminated hand-pump water collected from Bankura, West Bengal, India. Furthermore, a solution test kit was fabricated for easy and selective detection of F(-) in an aqueous solvent.

Pyridine Based Fluorescence Probe: Simultaneous Detection and Removal of Arsenate from Real Samples with Living Cell Imaging Properties.

Pyridine based fluorescence probe, DFPPIC and its functionalized Merrifield polymer has been synthesized, characterized and used as an arsenate selective fluorescence sensor. Arsenate induced fluorescence enhancement is attributed to inter-molecular H-bonding assisted CHEF process. The detection limit for arsenate is 0.001 µM, much below the WHO recommended tolerance level in drinking water. DFPPIC can detect intracellular arsenate in drinking water of Purbasthali, West Bengal, India efficiently. Graphical Abstract DFPPIC and its Merrifield conjugate polymer are used for selective determination and removal of arsenate from real drinking water samples of Purbasthali, a highly arsenic contaminated region of West Bengal, India. DFPPIC is very promising to imaging arsenate in living cells.

Femtomolar level sensing of inorganic arsenic(III) in water and in living-systems using a non-toxic fluorescent probe.

A highly selective femtomolar level sensing of inorganic arsenic(III) as arsenious acid has been accomplished in water medium and in living-systems (on pollen grains of Tecoma stans; Candida albicans cells (IMTECH No. 3018) and Peperomia pellucida stem section) using a non-toxic fluorescent probe of a Cu(II)-complex.

Functional molecular lumino-materials to probe serum albumins: solid phase selective staining through noncovalent fluorescent labeling.

Selective staining of human serum albumin protein in gel electrophoresis over wide range of other protein(s) is extremely important because it contains more than 60% volume of serum fluid in human body. Given the nonexistence of suitable dye materials for selective staining of serum albumins in gel electrophoresis, we report a new class of easy synthesizable and low molecular weight staining agents based on 3-amino-N-alkyl-carbazole scaffold for selective staining of serum albumins in solid phase. A detailed structure-efficiency relationship (SER) study enabled us to develop two such potent functional molecular probes which stain both human and bovine serum albumin selectively in gel electrophoresis in the presence of other proteins and enzymes. The present gel staining process was found to be very simple and less time-consuming as compared to the conventional coomassie blue staining which in turn makes these probes a new class of serum albumin-specific staining materials in proteome research. Moreover, these molecular lumino-materials can detect serum albumins at subnanomolar level in the presence of broad spectrum of other proteins/enzymes in aqueous buffer (99.9% water, pH = 7.3) keeping the protein secondary structure intact. Our experimental and the docking simulation results show that these probes bind preferentially at 'binding site I' of both the serum proteins.