Isolation of a Halophilic,Bacteriorhodopsin-producing Archaeon,Haloferax larsenii RG3D.1 from the Rocky Beach of Malvan,West Coast of India

Haloarchaea live in saline environments and require salt for their growth. The present study reports on the isolation of a haloarchaeal strain RG3D.1 identified as Haloferax larsenii using Sehgal and Gibbons medium with 3.42 M NaCl, from Rock Garden at Malvan in the West Coast of India. The rock type of Malvan Coast is correlated based on geological setting with the basal member of the Bababudan group placed at 2728 ± 102 million years ago (Ma). The haloarchaeal strain produced bacteriorhodopsin (BR) (0.137 g l^?1) and generated electric potential of 49.2 mV when exposed to sunlight. Thus, this is the first report on the isolation of a haloarchaeon, Haloferax larsenii, from red rocky substrate and production of BR.     

Salt dependent stability and unfolding of [Fe2-S2] ferredoxin of Halobacterium salinarum: spectroscopic investigations

Ferredoxin from the haloarchaeon Halobacterium salinarum is a 14. 6-kDa protein with a [Fe2-S2] center and is involved in the oxidative decarboxylation of 2-oxoacids. It possesses a high molar excess of acidic amino acid residues and is stable at high salt concentration. We have purified the protein from this extreme haloarchaeon and investigated its salt-dependent stability by circular dichroism, fluorescence, and absorption techniques. The predominantly beta-sheeted protein is stable in salt concentrations of >/=1.5 M NaCl. At lower concentrations a time-dependent increase in fluorescence intensity ratio (I(360):I(330)), a decrease in the absorption at 420 nm, and a decrease in ellipticity values are observed. The rate of fluorescence intensity change at any low salt concentration is the highest, followed by absorption and ellipticity. This suggests that at low salt the unfolding of ferredoxin starts with the loss of tertiary structure, which leads to the disruption of the [Fe2-S2] center, resulting in the loss of secondary structural elements.     

Delineating Kocuria turfanensis 2M4 as a credible PGPR: a novel IAA-producing bacteria isolated from saline desert

Indole-3-acetic acid (IAA)-producing bacteria Kocuria turfanensis strain 2M4 was isolated from the rhizospheric soil of halotolerant plant Suaeda fruticosa from a unique saline desert of Little Rann of Kutch, Gujarat, India. Rhizobacteria was bright orange pigmented, gram-positive, coccoid, non-endospore forming, and aerobic in nature. 16S rRNA gene sequence analysis showed that 2M4 isolate matched best with type strain of K. turfanensis HO-9042^T. Isolate optimally produced 38 µg ml^?1 IAA when growth medium was supplemented with 600 µg ml^?1 of L-tryptophan. Thin layer chromatography and Fourier transform infrared spectroscopy analysis were performed to corroborate IAA production. To characterize rhizobacterial isolate as a plant growth-promoting bacteria, it was tested for phosphate solubilization where it solubilized maximum 12 µg ml^?1 phosphate in presence of fructose, produced 53% siderophore units under iron-free minimal MM9 medium and produced 1.8 µmol ml^?1 ammonia in peptone water broth. Plant growth promotion by test isolate was studied on groundnut (Arachis hypogaea L.) under non-saline and saline soil. There was increase by 18% in total plant length and 30% in fresh biomass observed under non-saline control soil. Under saline soil, test isolate showed 17% increase in total length of the plant and 13% increase in fresh biomass.     

Draft Genome Sequence of the Extremely Halophilic Archaeon Halogranum salarium B-1T

Halogranum salarium is an extremely halophilic archaeon isolated from evaporitic salt crystals and belongs to the family Halobacteriaceae. Here, we present the 4.5-Mb draft genome sequence of the type strain (B-1^T) of H. salarium. This is the first report of the draft genome sequence of a haloarchaeon in the genus Halogranum.     

MAP KINASE PHOSPHATASE1 promotes osmotolerance by suppressing PHYTOALEXIN DEFICIENT4-independent immunity

Initial exposure of plants to osmotic stress caused by drought, cold, or salinity leads to acclimation, termed acquired tolerance, to subsequent severe stresses. Acquired osmotolerance induced by salt stress is widespread across Arabidopsis (Arabidopsis thaliana) accessions and is conferred by disruption of a nucleotide-binding leucine-rich repeat gene, designated ACQUIRED OSMOTOLERANCE. De-repression of this gene under osmotic stress causes detrimental autoimmunity via ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 (PAD4). However, the mechanism underlying acquired osmotolerance remains poorly understood. Here, we isolated an acquired osmotolerance-defective mutant (aod13) by screening 30,000 seedlings of an ion beam-mutagenized M2 population of Bu-5, an accession with acquired osmotolerance. We found that AOD13 encodes the dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1), which negatively regulates MITOGEN-ACTIVATED PROTEIN KINASE3/6 (MPK3/6). Consistently, MPK3/6 activation was greater in aod13 than in the Bu-5 wild-type (WT). The aod13 mutant was sensitive to osmotic stress but tolerant to salt stress. Under osmotic stress, pathogenesis-related genes were strongly induced in aod13 but not in the Bu-5 WT. Loss of PAD4 in pad4 aod13 plants did not restore acquired osmotolerance, implying that activation of immunity independent of PAD4 renders aod13 sensitive to osmotic stress. These findings suggest that AOD13 (i.e. MKP1) promotes osmotolerance by suppressing the PAD4-independent immune response activated by MPK3/6.

Under osmotic stress, MAP KINASE PHOSPHATASE1 represses the MITOGEN-ACTIVATED PROTEIN KINASE3/6-dependent immune response that impairs osmotolerance of Arabidopsis thaliana.     

A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning,heterologous expression and biochemical characterization

A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (k _cat/K _m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg^?1 s^?1 (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl₂. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.     

Geodermatophilus chilensis sp. nov., from soil of the Yungay core-region of the Atacama Desert,Chile

A polyphasic study was undertaken to establish the taxonomic status of three representative Geodermatophilus strains isolated from an extreme hyper-arid Atacama Desert soil. The strains, isolates B12^T, B20 and B25, were found to have chemotaxonomic and morphological properties characteristic of the genus Geodermatophilus. The isolates shared a broad range of chemotaxonomic, cultural and physiological features, formed a well-supported branch in the Geodermatophilus 16S rRNA gene tree in which they were most closely associated with the type strain of Geodermatophilus obscurus. They were distinguished from the latter by BOX-PCR fingerprint patterns and by chemotaxonomic and other phenotypic properties. Average nucleotide identity, average amino acid identity and digital DNA–DNA hybridization values between the whole genome sequences of isolate B12^T and G. obscurus DSM 43160^T were 89.28%, 87.27% and 37.4%, respectively, metrics consistent with its classification as a separate species. On the basis of these data, it is proposed that the isolates be assigned to the genus Geodermatophilus as Geodermatophilus chilensis sp. nov. with isolate B12^T (CECT 9483^T = NCIMB 15089^T) as the type strain. Analysis of the whole genome sequence of G. chilensis B12^T with 5341 open reading frames and a genome size of 5.5 Mb highlighted genes and gene clusters that encode for properties relevant to its adaptation to extreme environmental conditions prevalent in extreme hyper-arid Atacama Desert soils.     

Hyperthermostable <Emphasis Type="Italic">Thermotoga maritima</Emphasis> xylanase XYN10B shows high activity at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids

The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15–35 % hydrophilic ILs, even at 75–90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15–25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower V _max. However, only minor changes in kinetic parameter K _m showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential.     

A highly thermoactive and salt-tolerant α-amylase isolated from a pilot-plant biogas reactor

Aiming at the isolation of novel enzymes from previously uncultured thermophilic microorganisms, a metagenome library was constructed from DNA isolated from a pilot-plant biogas reactor operating at 55 °C. The library was screened for starch-degrading enzymes, and one active clone was found. An open reading frame of 1,461 bp encoding an α-amylase from an uncultured organism was identified. The amy13A gene was cloned in Escherichia coli, resulting in high-level expression of the recombinant amylase. The novel enzyme Amy13A showed the highest sequence identity (75 %) to α-amylases from Petrotoga mobilis and Halothermothrix orenii. Amy13A is highly thermoactive, exhibiting optimal activity at 80 °C, and it is also highly salt-tolerant, being active in 25 % (w/v) NaCl. Amy13A is one of the few enzymes that tolerate high concentrations of salt and elevated temperatures, making it a potential candidate for starch processing under extreme conditions.     

A meta-analysis of the association of glutathione S-transferase P1 gene polymorphism with the susceptibility of breast cancer

Glutathione S-transferase P1 (GSTP1) is one of the important mutant sites for the cancer risk at present. The conclusions of the published reports on the relationship between GSTP1 A/G gene polymorphism and the risk of breast cancer are still debated. This meta-analysis was performed to evaluate the association between GSTP1 and the risk of breast cancer. The association reports were identified from PubMed and Cochrane Library, and eligible studies were included and synthesized using meta-analysis method. 35 investigations were included into this meta-analysis for the association of GSTP1 A/G gene polymorphism and breast cancer susceptibility, consisting of 40,347 subjects (18,665 patients with breast cancer and 21,682 controls). The association between GSTP1 A/G gene polymorphism and breast cancer risk was not found for overall population, Caucasians and Africans. Interestingly, the GSTP1 A/G gene polymorphism was associated with the susceptibility of breast cancer in Asians (G allele: OR = 1.10, 95 % CI: 1.04–1.17, P = 0.001; GG genotype: OR = 1.36, 95 % CI: 1.14–1.62, P = 0.0008; AA genotype: OR = 0.92, 95 % CI: 0.85–0.98, P = 0.02). Furthermore, the GSTP1 A/G gene polymorphism was associated with the susceptibility of breast cancer for the analysis of the controls from hospital. In conclusion, GSTP1 A/G gene polymorphism is associated with the breast cancer susceptibility in Asians. However, more studies on the relationship between GSTP1 A/G gene polymorphism and the risk of breast cancer should be performed in further.     

Indigenous salt-tolerant rhizobacterium <Emphasis Type="Italic">Pantoea dispersa</Emphasis> (PSB3) reduces sodium uptake and mitigates the effects of salt stress on growth and yield of chickpea

Salt stress has multiple damaging effects on plants including physiological damage, reduced growth, and productivity. Plant growth-promoting rhizobacteria (PGPR) are one of the valuable options to mitigate the negative effects of this stress. In the present study, native bacteria from chickpea’s rhizosphere were isolated, and checked for their salt tolerance and plant growth-promoting attributes (phosphate (P) solubilization, siderophores, indole-3-acetic acid (IAA) production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production). One isolate, subsequently identified as Pantoea dispersa, showed appreciable production of IAA (218.3 µg/ml) and siderophores (60.33% SU), P-solubilization (3.64 µg/ml) and ACC deaminase activity (207.45 nmol/mg/h) in the presence of 150 mM NaCl, under laboratory conditions. Salt stress in uninoculated chickpea (GPF2 cultivar) plants induced high accumulation of Na⁺ ions (3.86 mg g^?1 dw) in the leaves, along with significant reduction in K⁺ uptake, membrane integrity, chlorophyll concentration, and leaf water content, thus resulting in impaired growth of the plant and yield (pods and seeds) in a salt concentration-dependent manner. The damage due to salt stress was restored significantly in plants inoculated with P. dispersa. A significant improvement in biomass (32–34%), pods number (31–34.5%), seeds number (32–35.7%), pods weight (30–32.6%), and seeds weight (27–35%) per plant occurred in salt stress-affected plants, which was associated with significant reduction in Na⁺ uptake, reduced membrane damage, significantly improved leaf water content, chlorophyll content, and K⁺ uptake. This study suggests for the first time that native P. dispersa strain PSB3 can be used to alleviate the negative effects of salt stress on chickpea plants and holds the potential to be used as a biofertilizer.     

Cloning and characterization of GPAT gene from Lepidium latifolium L.: a step towards translational research in agri-genomics for food and fuel

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes first and the rate limiting step in glycerolipid synthesis pathway, which in turn contribute to stabilization of plasma membrane structure and oil lipid synthesis in plant cells. Here, we report cloning and characterization of GPAT gene from Lepidium latifolium (LlaGPAT). The cDNA sequence (1,615 bp) of LlaGPAT gene consisted of 1,113 bp ORF encoding a protein of 370 aa residues, with deduced mass of 41.2 kDa and four acyltransferase (AT) motifs having role in catalysis and in glycerol-3-phosphate binding. Southern blot analysis suggested presence of a single copy of the gene in the genome. Tissue specific expression of the gene was seen more abundantly in aerial parts, compared to the roots. Quantitative real-time PCR indicated down-regulation of the gene by cold (4 °C), drought (PEG6000), salt (300 mM NaCl) and ABA (100 μM) treatments. Considering the vitality of the function of encoded enzyme, LlaGPAT can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.     

Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage (Brassica rapa L.)

The extreme resistance to Turnip mosaic virus observed in the Chinese cabbage (Brassica rapa) line, BP8407, is monogenic and recessive. Bulked segregant analysis was carried out to identify simple sequence repeat and Indel markers linked to this recessive resistance gene, termed recessive Turnip mosaic virus resistance 02 (retr02). Mapping of PCR-specific Indel markers on 239 individuals of a BP8407 × Ji Zao Chun F₂ population, located this resistance gene to a 0.9-cM interval between two Indel markers (BrID10694 and BrID101309) and in scaffold000060 or scaffold000104 on chromosome A04 of the B. rapa genome. Eleven eukaryotic initiation factor 4E (eIF4E) and 14 eukaryotic initiation factor 4G (eIF4G) genes are predicted in the B. rapa genome. A candidate gene, Bra035393 on scaffold000104, was predicted within the mapped resistance locus. The gene encodes the eIF(iso)4E protein. Bra035393 was sequenced in BP8407 and Ji Zao Chun. A polymorphism (A/G) was found in exon 3 between BP8407 and Ji Zao Chun. This gene was analysed in four resistant and three susceptible lines. A correlation was observed between the amino acid substitution (Gly/Asp) in the eIF(iso)4E protein and resistance/susceptibility. eIF(iso)4E has been shown previously to interact with the TuMV genome-linked protein, VPg.     

Equilibrium and Kinetics of Adsorption of Mn2+ by Haloarchaeon Halobacterium sp. GUSF (MTCC3265)

The coastal waters of countries bordering on an ocean show increases in manganese pollution due to runoff from mining activity and from industries dealing with production of ferroalloys, steel, iron, petrochemicals, and fertilizers. One gram of dried cells of haloarchaeon Halobacterium sp. GUSF (MTCC3265) adsorbed 99% Mn²⁺ in 60 min at pH 6.8 and 30ºC on contact with 109.54 mg Mn²⁺ per liter in saline solution. Adsorbed Mn²⁺ was quantified by atomic absorption spectrometry and demonstrated on the cell surface by SEM-EDX. Mn²⁺ adsorbed to functional groups of the adsorbent was studied by FTIR. The adsorption process of Mn²⁺ showed saturation and followed pseudo–second-order kinetics; was consistent with the homogeneity of the Langmuir model (R² of 0.99); exhibited a Q_max of 62.5 mg g^?1 and a binding energy of 0.018 L mg^?1. The Mn²⁺adsorption was also consistent with the heterogeneity of the Freundlich model by exhibiting a K_f of 1.0 mg g^?1 with an n value of 1.1. Adsorption efficiency of 99% was retained even after a third adsorption-desorption cycle. This is the first report on metal ion adsorption, using Mn²⁺ as an example, by the haloarchaeon Halobacterium sp. GUSF (MTCC3265) in the domain Archaea.