Ensifer meliloti overexpressing Escherichia coli phytase gene (appA) improves phosphorus (P) acquisition in maize plants.

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 (lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti {E. m (pVA1)} increased its phosphatase and phytase activity by ∼9- and ∼50-fold, respectively, compared to the transformants containing empty plasmid as control {E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein {E. m (pVA1)} and {E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both {E. m (pVA1)} and {E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with {E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Microbial desalination cell for enhanced biodegradation of waste engine oil using a novel bacterial strain Bacillus subtilis moh3.

Microbial desalination cell (MDC) is a bioelectrochemical system developed recently from microbial fuel cells (MFCs), for producing green energy from organic wastes along with desalination of saltwater. MDC is proved to be a better performer than MFC in terms of power output and chemical oxygen demand removal, with desalination as an additional feature. This study investigates the application potential of MDC for integrated biodegradation of waste engine oil. This study showed, for the first time, that waste engine oil could be used as an organic substrate in MDC, achieving biodegradation of engine oil along with considerable desalination and power production. Utilization of these wastes in MDC can protect the environment from waste engine oil contamination. Indigenous oil-degrading bacteria were isolated and identified from engine oil contaminated sludge. Degradation of waste engine oil by these novel isolates was studied in batch cultures and optimized the growth conditions. The same cultures when used in MDC, gave enhanced biodegradation (70.1 +/- 0.5%) along with desalination (68.3 +/- 0.6%) and power production (3.1 +/- 0.3 mW/m2). Fourier transform-infrared spectroscopy and gas chromatography-mass spectrometry analyses were performed to characterize the degradation metabolites in the anolyte of MDC which clearly indicated the biodegradation of long chain, branched and cyclic hydrocarbons present in waste engine oil.

Pseudomonas fluorescens ATCC 13525 containing an artificial oxalate operon and Vitreoscilla hemoglobin secretes oxalic acid and solubilizes rock phosphate in acidic alfisols.

Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 µM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 µM and 351 µM P, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil.

Antimicrobial and antioxidant effects of spice extracts on the shelf life extension of raw chicken meat.

The antimicrobial and antioxidant effects of different spice extracts in raw chicken meat during storage for 15 days at 4 °C were studied. Raw chicken meat was treated with BHT (positive control), Syzygium aromaticum (SA), Cinnmomum cassia (CC), Origanum vulgare (OV), and Brassica nigra (BN) extracts and the different combinations as well as the results were compared to raw chicken meat without any additive (negative control). The antioxidant and antimicrobial activities of spice extracts were determined. Total phenolic contents and flavonoid contents were ranged from 14.09 ± 0.78 to 24.65 ± 0.83 mg of GAE/g and 7.07 ± 0.15 to 12.13 ± 0.24 mg of quercetin/g, respectively. The pH, instrumental color (CIE L*, a*, b*), total viable counts (TVC), Lactic Acid Bacteria (LAB) counts, Enterobacteriaceae counts, Pseudomonas spp. counts and 2-thiobarbituric acid reactive substances (TBARS) were determined at a gap of 3 days interval for a period of 15 days. The bacterial counts of T-W-SA+T-W-CC+T-W-OV samples were lower than control samples during storage. T-W-SA+T-W-CC+T-W-OV samples maintained significantly (P<0.05) higher L*, a* and b* values while storing. The TBARS values of T-W-SA+T-W-CC+T-W-OV samples were lowest among the samples. These results show that spice extracts are very effective against microbial growth, lipid oxidation and has potential as a natural antioxidant in raw chicken meats.

Preliminary pharmacognostic and phytochemical standardization of Dhataki [Woodfordia fruticosa (L.) Kurz.] leaves.

BACKGROUND: Woodfordia fruticosa (L.) Kurz., known as Dhataki, is an important medicinal plant used in Ayurveda. Recent studies on leaf showed that it contains important chemical constituents responsible for biological activities. The ethnic folk from India and Nepal are using the leaf to treat ulcers, rheumatism, fever, hemoptysis and as a disinfectant. It is also reported to be used in perfume, leather and textile industries. AIM: To investigate preliminary pharmacognostical and phytochemical parameters of leaf to standardize the drug. MATERIALS AND METHODS: Identification of plant was done as per the standard guidelines given in the floras. Macro and microscopic evaluation performed as per the routine laboratory procedures. Phytochemical, physico-chemical, florescence analysis, behavior of powdered drug have been conducted as per the WHO guidelines. RESULTS: Unique arrangement of the vascular bundle in mid rib region is observed. Aqueous and alcoholic extracts showed the presence of alkaloids, phenols, tannins, and flavonoids. CONCLUSION: The findings of this study will be helpful in the identification of Dhataki leaf.

In vitro propagation of a rare medicinal fern of Western Ghats--Diplazium esculentum (Reytz.).

Present study aimed for in vitro culture of circinate part of young leaves of D. esculentum which is amongst the leafy vegetables consumed as vegetable by Paniya and Chetti tribes of Western Ghats. The circinate part of young leaves (crosiers), excised before the beginning of foliar expansion, was inoculated on half strength Murashige and Skoog (MS) medium supplemented with auxins indole-3-butyric acid (IBA) or alpha-napthalene acetic acid (NAA) or 2,4-Dichlorophenoxyacetic acid (2,4-D) and cytokinin 6- benzylaminopurine (BA) in a range 0.5 to 2.5 mg L(-1). Combinations of different concentrations of 2,4 D + BA, IBA + BA as well as of NAA+ BA were also tested in half strength MS medium with 3% sucrose and with pH 5.8. The best morphogenic response was obtained with half strength MS medium supplemented with 2,4-D 0.5 mg L(-1) and BA 2.5 mg L(-1), 3% sucrose, at pH 5.8. For rooting of the microshoots, half strength MS medium supplemented with 2,4-D ( 2 and 1 mg L(-1)) exhibited best results. Present study reports the successful in vitro culturing of D. esculentum.

Plasmid load adversely affects growth and gluconic acid secretion ability of mineral phosphate-solubilizing rhizospheric bacterium Enterobacter asburiae PSI3 under P limited conditions.

Effect of the metabolic load caused by the presence of plasmids on mineral phosphate-solubilizing (MPS) Enterobacter asburiae PSI3, was monitored with four plasmid cloning vectors and one native plasmid, varying in size, nature of the replicon, copy number and antibiotic resistance genes. Except for one plasmid, the presence of all other plasmids in E. asburiae PSI3 resulted in the loss of the MPS phenotype as reflected by the failure to bring about a drop in pH and release soluble P when grown in media containing rock phosphate (RP) as the sole P source. When 100 µM soluble P was supplemented along with RP, the adverse effects of plasmids on MPS phenotype and on growth parameters was reduced for some plasmid bearing derivatives, as monitored in terms of specific growth rates, glucose consumed, gluconic acids yields and P released. When 10 mM of soluble P as the only P source, was added to the medium all transformants showed growth and pH drop comparable with native strain. It may be concluded that different plasmids impose, to varying extents, a metabolic load in the phosphate-solubilizing bacterium E. asburiae PSI3 and results in diminishing its growth and P-solubilizing ability in P deficient conditions.

Effect of transgenic rhizobacteria overexpressing Citrobacter braakii appA on phytate-P availability to mung bean plants.

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-P available to plants. In the present study, Citrobacter braakii phytase gene (appA) was over-expressed in rhizobacteria possessing plant growth promoting (PGP) traits for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broad host-range expression vector pBBR1MCS2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains) and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying level of phytase activity, ranging from 10 folds to 538 folds higher than the corresponding control strains. Transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed highest expression of phytase (~8 U/ mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having ability to secrete organic acid, showed significantly higher release of P from Ca-phytate in buffered minimal media. AppA over-expressing rhizobacteria showed increased P content, dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semi solid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of over-expression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

Metabolic channeling of glucose towards gluconate in phosphate-solubilizing Pseudomonas aeruginosa P4 under phosphorus deficiency.

Most phosphate-solubilizing bacteria (PSB), including the Pseudomonas species, release P from sparingly soluble mineral phosphates by producing high levels of gluconic acid from extracellular glucose, in a reaction catalyzed by periplasmic glucose dehydrogenase, which is an integral component of glucose catabolism of pseudomonads. To investigate the differences in the glucose metabolism of gluconic acid-producing PSB pseudomonads and low gluconic acid-producing/non-PSB strains, several parameters pertaining to growth and glucose utilization under P-sufficient and P-deficient conditions were monitored for the PSB isolate Pseudomonas aeruginosa P4 (producing approximately 46 mM gluconic acid releasing 437 microM P) and non-PSB P. fluorescens 13525. Our results show interesting differences in the channeling of glucose towards gluconate and other catabolic end-products like pyruvate and acetate with respect to P status for both strains. However, PSB strain P. aeruginosa P4, apart from exhibiting better growth under both low and high Pi conditions, differed from P. fluorescens 13525 in its ability to accumulate gluconate under P-solubilizing conditions. These alterations in growth, glucose utilization and acid secretion are correlated with glucose dehydrogenase, glucose-6-phosphate dehydrogenase and pyruvate carboxylase activities. The ability to shift glucose towards a direct oxidative pathway under P deficiency is speculated to underlie the differential gluconic acid-mediated P-solubilizing ability observed amongst pseudomonads.

Characterization of carbamoyl phosphate synthetase of Streptomyces spp.

Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc).