Avinash Mishra

Isolation and characterization of extracellular polymeric substances from micro-algae Dunaliella salina under salt stress

Extracellular polymeric substances (EPSs), produced by Dunaliella salina strain, increase concomitantly with salt concentration and maximum (944 mg/l) were obtained at 5M NaCl, whereas minimum (56 mg/l) at 0.5M salinity. Emulsifying activity was measured in terms of strength to retain the emulsion and comparatively 85.76% retention was observed at 0.5M salinity thereafter it intends to decline. The FT-IR-spectra reveal characteristic functional groups NH stretching, asymmetrical CH stretching vibration of aliphatic CH(2)-group, CC stretching of aromatic, CN stretch of aliphatic amine, NH wag of primary amine and CX stretch of alkyl-halides with a stretching of COC, CO corresponding to the presence of carbohydrates. The FT-IR-spectra substantiated the presence of primary amine-group, aromatic-compound, halide-group, aliphatic alkyl-group and polysaccharides. Four monosaccharides (glucose, galactose, fructose and xylose) were also detected by HPLC analysis. Production of EPSs may allow further exploration of D. salina as potential EPSs producer and make it as a promising candidate for biotechnological and industrial exploitation.

Expression of SbGSTU (tau class glutathione S-transferase) gene isolated from Salicornia brachiata in tobacco for salt tolerance

Tau class glutathione transferases (GSTU) genes are plant specific, induced by different abiotic stress, and important for protecting plants against oxidative damage. GST gene was isolated using 5′ RACE from an extreme halophyte Salicorniabrachiata, cloned, sequenced and its protein structure was predicted. Transcript profiling of SbGST gene expression was studied under different abiotic stress conditions and plant growth regulator treatments, viz. salt, cold, drought, ABA and salicylic acid, with time period point and concentration point. The expression of SbGST gene was up-regulated in all stress conditions, except SA treatment. Seed germination percentage, GST enzyme assay, fresh weight and other growth parameters (root length, shoot length and leaf area) were studied and results indicate that over-expression of SbGST gene in transgenic tobacco leads to enhanced seed germination and growth under salt stress. Transgenic lines were evaluated for their performance under salt stress and tobacco plants over-expressing SbGST showed higher seed germination and survival compared to wild type. These results confirm that expression of SbGST gene is up-regulated by different stresses and over-expression of tau class SbGST gene in transgenic tobacco plays a vital role in abiotic stress tolerance. SbGST gene expressed conspicuously under salt stress leading to enhance seed germination and better growth. Furthermore, GST is a potential candidate gene to be used in genetic engineering for enhancing abiotic stress tolerance.

Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli.

Salicornia brachiata is an extreme halophyte growing luxuriantly in the coastal marshes and frequently exposed to various abiotic stresses including heavy metals. A full length type 2 metallothionein (SbMT-2) gene was isolated using RACE and its copy number was confirmed by southern blot analysis. Transcript expression of SbMT-2 gene was analyzed by semi-quantitative Rt-PCR and real time quantitative (qRT) PCR. Expression of SbMT-2 gene was up-regulated concurrently with zinc, copper, salt, heat and drought stress, down regulated by cold stress while unaffected under cadmium stress. Heterologous expression of SbMT-2 gene enhances metal accumulation and tolerance in E. coli. Metal-binding characteristics of SbMT-2 protein show its possible role in homeostasis and/or detoxification of heavy metals. Significant tolerance was observed by E. coli cells expressing recombinant SbMT-2 for Zn(++), Cu(++) and Cd(++) compared to cells expressing GST only. Sequestration of zinc was 4-fold higher compared to copper and in contrast SbMT-2 inhibits the relative accumulation of cadmium by 1.23-fold compared to GST protein. Fusion protein SbMT-2 showed utmost affinity to zinc (approx. 2.5 fold to Cu(++) and Cd(++)) followed by copper and cadmium ions with same affinity. Halophyte S. brachiata has inherent resilience of varying abiotic tolerance therefore SbMT-2 gene could be a potential candidate to be used for enhanced metal tolerance and heavy metal phytoremediation.

Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan

A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11°E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0–1.16 ± 0.05 μg chl. a and 0.2–0.29 ± 0.01 μg chl. b per 106 cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 ± 0.12 μg chl. a and 0.84 ± 0.4 μg chl. b per 106 cells) was observed which declined again at 5.5 M NaCl concentration (2.0 ± 0.1 μg chl. a and 0.52 ± 0.03 μg chl. b per 106 cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 ± 0.07 μg), glycine betaine (5.7 ± 0.28 μg), glycerol (3.7 ± 0.18 ml) and total sugar (250 ± 12.5 μg) per 105 cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 ± 0.01 μg per 105 cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na+ remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K+ and Mg++ was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliellasalina can be used to explore its biotechnological and industrial potential.

Developing transgenic Jatropha using the SbNHX1 gene from an extreme halophyte for cultivation in saline wasteland.

Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na+/H+ antiporter gene that compartmentalises excess Na+ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.

Over-expression of the Peroxisomal Ascorbate Peroxidase (SbpAPX) Gene Cloned from Halophyte Salicornia brachiata Confers Salt and Drought Stress Tolerance in Transgenic Tobacco

Salicornia brachiata Roxb., an extreme halophyte, is a naturally adapted higher plant model for additional gene resources to engineer salt tolerance in plants. Ascorbate peroxidase (APX) plays a key role in protecting plants against oxidative stress and thus confers abiotic stress tolerance. A full-length SbpAPX cDNA, encoding peroxisomal ascorbate peroxidase, was cloned from S. brachiata. The open reading frame encodes for a polypeptide of 287 amino acid residues (31.3-kDa protein). The deduced amino acid sequence of the SbpAPX gene showed characteristic peroxisomal targeting sequences (RKRAI) and a C-terminal hydrophobic region of 39 amino acid residues containing a transmembrane domain (TMD) of 23 amino acid residues. Northern blot analysis showed elevated SbpAPX transcript in response to salt, cold, abscisic acid and salicylic acid stress treatments. The SbpAPX gene was transformed to tobacco for their functional validation under stresses. Transgenic plants over-expressing SbpAPX gene showed enhanced salt and drought stress tolerance compared to wild-type plants. Transgenic plants showed enhanced vegetative growth and germination rate both under normal and stressed conditions. Present study revealed that the SbpAPX gene is a potential candidate, which not only confers abiotic stress tolerance to plants but also seems to be involved in plant growth.

Microbial population index and community structure in saline–alkaline soil using gene targeted metagenomics

Population indices of bacteria and archaea were investigated from saline-alkaline soil and a possible microbe-environment pattern was established using gene targeted metagenomics. Clone libraries were constructed using 16S rRNA and functional gene(s) involved in carbon fixation (cbbL), nitrogen fixation (nifH), ammonia oxidation (amoA) and sulfur metabolism (apsA). Molecular phylogeny revealed the dominance of Actinobacteria, Firmicutes and Proteobacteria along with archaeal members of Halobacteraceae. The library consisted of novel bacterial (20%) and archaeal (38%) genera showing ≤95% similarity to previously retrieved sequences. Phylogenetic analysis indicated ability of inhabitant to survive in stress condition. The 16S rRNA gene libraries contained novel gene sequences and were distantly homologous with cultured bacteria. Functional gene libraries were found unique and most of the clones were distantly related to Proteobacteria, while clones of nifH gene library also showed homology with Cyanobacteria and Firmicutes. Quantitative real-time PCR exhibited that bacterial abundance was two orders of magnitude higher than archaeal. The gene(s) quantification indicated the size of the functional guilds harboring relevant key genes. The study provides insights on microbial ecology and different metabolic interactions occurring in saline-alkaline soil, possessing phylogenetically diverse groups of bacteria and archaea, which may be explored further for gene cataloging and metabolic profiling.

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO(2) by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO(2). Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed <95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real-time PCR (qRT PCR), was 9.38 ± 0.75 × 10(7) and 5.43 ± 0.79 × 10(8) (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO(2)-fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.

Physicochemical characterization of biosurfactant and its potential to remove oil from soil and cotton cloth

An alkaliphilic bacterium, Klebsiella sp. strain RJ-03, produced a biosurfactant, which showed low viscosity with pseudoplastic rheological behavior and exhibited emulsification activity with oils and hydrocarbons. The biosurfactant has excellent oil removing efficiency as compared to chemical surfactants. The isolated biosurfactant has compatibility with detergents and enhanced oil removing efficiency from soil and cotton cloths. It comprised of sugar, uronic acid, protein and sulfate. GC-MS analysis confirmed the presence of six monosaccharides (w/w), glucose (6.65%), galactose (23.98%), rhamnose (14.94%), mannose (17.54%), fucose (9.47%) and 6-O-Me-galactose (1.4%). It is a high molecular weight, thermostable biopolymer showing degradation above 300 °C. Positive ion reflector mode of MALDI TOF-TOF MS analysis revealed series of low and mid range mass peaks (m/z) corresponding to mono-, di-, tri- and oligo-saccharides content. The NMR, FT-IR, EDX-SEM, AFM and PSD analysis confirmed the presence of functional groups, bonds, elements and particle size respectively.

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus.

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. 1HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CIxrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.