StAR-Related Lipid Transfer (START) Domains Across the Rice Pangenome Reveal How Ontogeny Recapitulated Selection Pressures During Rice Domestication.

The StAR-related lipid transfer (START) domain containing proteins or START proteins, encoded by a plant amplified family of evolutionary conserved genes, play important roles in lipid binding, transport, signaling, and modulation of transcriptional activity in the plant kingdom, but there is limited information on their evolution, duplication, and associated sub- or neo-functionalization. Here we perform a comprehensive investigation of this family across the rice pangenome, using 10 wild and cultivated varieties. Conservation of START domains across all 10 rice genomes suggests low dispensability and critical functional roles for this family, further supported by chromosomal mapping, duplication and domain structure patterns. Analysis of synteny highlights a preponderance of segmental and dispersed duplication among STARTs, while transcriptomic investigation of the main cultivated variety Oryza sativa var. japonica reveals sub-functionalization amongst genes family members in terms of preferential expression across various developmental stages and anatomical parts, such as flowering. Ka/Ks ratios confirmed strong negative/purifying selection on START family evolution, implying that ontogeny recapitulated selection pressures during rice domestication. Our findings provide evidence for high conservation of START genes across rice varieties in numbers, as well as in their stringent regulation of Ka/Ks ratio, and showed strong functional dependency of plants on START proteins for their growth and reproductive development. We believe that our findings advance the limited knowledge about plant START domain diversity and evolution, and pave the way for more detailed assessment of individual structural classes of START proteins among plants and their domain specific substrate preferences, to complement existing studies in animals and yeast.

Identification and Downstream Analyses of Domains Amplified in Plant Genomes: The Case of StAR-Related Lipid Transfer (START) Domains in Rice.

Plant genomes can withstand small- and large-scale duplications, at a far greater success than any other kingdom in the tree of life, resulting in the existence and evolution of gene families, often with over a hundred members! The gene families, in turn, go through subfunctionalization or neofunctionalization, to form protein domains performing unique or grouped functions in context of the original activity. Due to the large number of such cases in the plant kingdom, it has become a routine task for plant biologists to investigate their specific gene family of interest. In this chapter, we provide a simple and standard pipeline for this effort, taking the example of steroidogenic acute regulatory protein (StAR) related lipid transfer (START) domains in rice, as reference. We describe the extraction, processing, and downstream analysis of Oryza sativa var. japonica proteome towards identification and comparative exploration of START domains. This was done by training profile Hidden Markov Models (HMM) of 35 reported START domains in Arabidopsis, which were then used to search potential homologs in rice. Downstream investigations included domain structure analysis, visualization of exon-intron patterns, chromosomal localization of START genes, and phylogenetic studies, followed by identification of cis-regulatory elements and gene regulatory network construction. Additionally, we have also highlighted various alternative tools and techniques that can be used to perform similar analyses, along with salient features.

Evaluation of Probiotic Properties and Prebiotic Utilization Potential of Weissella paramesenteroides Isolated From Fruits.

Weissella paramesenteroides has gained a considerable attention as bacteriocin and exopolysaccharide producers. However, potential of W. paramesenteroides to utilize different prebiotics is unexplored area of research. Fruits being vectors of various probiotics, five W. paramesenteroides strains, namely, FX1, FX2, FX5, FX9, and FX12, were isolated from different fruits. They were screened and selected based on their ability to survive at pH 2.5 and in 1.0% sodium taurocholate, high cell surface hydrophobicity, mucin adhesion, bile-induced biofilm formation, antimicrobial activity (AMA) against selected enteropathogens, and prebiotic utilization ability, implicating the functional properties of these strains. In vitro safety evaluation showed that strains were susceptible to antibiotics except vancomycin and did not harbor any virulent traits such as biogenic amine production, hemolysis, and DNase production. Based on their functionality, two strains FX5 and FX9 were selected for prebiotic utilization studies by thin layer chromatography (TLC) and short-chain fatty acids (SCFAs) production by high performance liquid chromatography. TLC profile evinced the ability of these two strains to utilize low molecular weight galactooligosaccharides (GOS) and fructooligosaccharides (FOS), as only the upper low molecular weight fractions were disappeared from cell-free-supernatants (CFS). Enhanced ß-galactosidase activity correlated with galactose accumulation in residual CFS of GOS displayed GOS utilization ability. Both the strains exhibited AMA against E. coli and Staph. aureus and high SCFAs production in the presence of prebiotic, suggesting their synbiotic potential. Thus, W. paramesenteroides strains FX5 and FX9 exhibit potential probiotic properties with prebiotic utilization and can be taken forward to evaluate synergistic synbiotic potential in detail.

Probiotics, prebiotics and colorectal cancer prevention.

Colorectal cancer (CRC), the third major cause of mortality among various cancer types in United States, has been increasing in developing countries due to varying diet and dietary habits and occupational hazards. Recent evidences showed that composition of gut microbiota could be associated with the development of CRC and other gut dysbiosis. Modulation of gut microbiota by probiotics and prebiotics, either alone or in combination could positively influence the cross-talk between immune system and microbiota, would be beneficial in preventing inflammation and CRC. In this review, role of probiotics and prebiotics in the prevention of CRC has been discussed. Various epidemiological and experimental studies, specifically gut microbiome research has effectively improved the understanding about the role of probiotics and microbial treatment as anticarcinogenic agents. A few human studies support the beneficial effect of probiotics and prebiotics; hence, comprehensive understanding is urgent to realize the clinical applications of probiotics and prebiotics in CRC prevention.

Enhancement of stress tolerance in the polyhydroxyalkanoate producers without mobilization of the accumulated granules.

Poly(3-hydroxybutyrate) [P(3HB)], a polymer belonging to the polyhydroxyalkanoate (PHA) family, is accumulated by numerous bacteria as carbon and energy storage material. The mobilization of accumulated P(3HB) is associated with increased stress and starvation tolerance. However, the potential function of accumulated copolymer such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] remained unknown. In this study, Delftia acidovorans DS 17 was used to evaluate the contributions of P(3HB) and P(3HB-co-3HV) granules during simulated exogenous carbon deprivation on cell survival by transferring cells with PHAs to carbon-free mineral salt medium supplemented with 1% (w/v) nitrogen source. By mobilizing the intracellular P(3HB) and P(3HB-co-3HV) at 11 and 40 mol% 3HV compositions, the cells survived starvation. Surprisingly, D. acidovorans containing P(3HB-co-94 mol% 3HV) also survived although the mobilization was not as effective. Similarly, recombinant Escherichia coli pGEM-T::phbCAB(Cn) (harboring the PHA biosynthesis genes of Cupriavidus necator) containing P(3HB) granules had a higher viable cell counts compared to those without P(3HB) granules but without any P(3HB) mobilization when exposed to oxidative stress by photoactivated titanium dioxide. This study provided strong evidence that enhancement of stress tolerance in PHA producers can be achieved without mobilization of the previously accumulated granules. Instead, PHA biosynthesis may improve bacterial survival via multiple mechanisms.

Potential of probiotics, prebiotics and synbiotics for management of colorectal cancer.

Colorectal Cancer (CRC) is the second leading cause of cancer-related mortality and is the fourth most common malignant neoplasm in USA. Escaping apoptosis and cell mutation are the prime hallmarks of cancer. It is apparent that balancing the network between DNA damage and DNA repair is critical in preventing carcinogenesis. One-third of cancers might be prevented by nutritious healthy diet, maintaining healthy weight and physical activity. In this review, an attempt is made to abridge the role of carcinogen in colorectal cancer establishment and prognosis, where special attention has been paid to food-borne mutagens and functional role of beneficial human gut microbiome in evading cancer. Further the significance of tailor-made prebiotics, probiotics and synbiotics in cancer management by bio-antimutagenic and desmutagenic activity has been elaborated. Probiotic bacteria are live microorganisms that, when administered in adequate amounts, confer a healthy benefit on the host. Prebiotics are a selectively fermentable non-digestible oligosaccharide or ingredient that brings specific changes, both in the composition and/or activity of the gastrointestinal microflora, conferring health benefits. Synbiotics are a combination of probiotic bacteria and the growth promoting prebiotic ingredients that purport "synergism."

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-640.

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-640 with various stabilizers at different temperatures was studied. Dextransucrase was stable at lower temperatures (10-30°C) and lost the activity at above 30°C. The salts such as CaCl(2), CoCl(2) and MgCl(2) enhanced the dextransucrase activity. A 22% higher dextransucrase activity was obtained by 4 mM CoCl(2). The dextransucrase activity was lost by 50% at 1 mM EDTA. Urea denatured the enzyme and caused 45%, 90% and 98% loss of activity in 30 min when treated with 1 M, 3 M, and 5 M urea concentrations, respectively. Amongst the stabilizers Tween 80, glycerol, PEG-8000, dextran (500 kDa) and glutaraldehyde, Tween 80 provided the maximum stability at 30°C. In the presence of Tween 80 the enzyme lost only 8% activity at 30°C in 20 h but, it lost 65% of activity with out any stabilizer. The enzyme lost 92% of activity with in 4 days at 30°C and lost only 25% of activity at -20°C after 14 days.

Structural and biochemical properties of lichenase from Clostridium thermocellum.

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and ß-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or ß-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu(2+) ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu(2+) (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni(2+), Co(2+) and Zn(2+) did not affect the activity of the enzyme at low concentrations (0-10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (ß/α)(8)-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.

Application of response surface methodology for maximizing dextransucrase production from Leuconostoc mesenteroides NRRL B-640 in a bioreactor.

The production of dextransucrase from Leuconostoc mesenteroides NRRL B-640 was investigated using statistical approaches. Plackett-Burman design with six variables, viz. sucrose, yeast extract, K(2)HPO(4), peptone, beef extract, and Tween 80, was used to screen the nutrients that significantly affected the dextransucrase production. 2(4)-Central composite design with four selected variables (sucrose, K(2)HPO(4), yeast extract, and beef extract) was used for response surface methodology (RSM) for optimizing the enzyme production. The culture was grown under flask culture with 100 ml optimized medium containing 30 g/l sucrose, 18.5 g/l yeast extract, 15.3 g/l K(2)HPO(4), and 5 g/l beef extract at 25 degrees C and shaking at 200 rpm gave dextransucrase with specific activity of 0.68 U/mg. Whereas the same optimized medium in a 3.0-l bioreactor (1.4 l working volume) gave an experimentally determined value of specific activity of 0.70 U/mg, which was in perfect agreement with the predicted value of 0.65 U/mg by the statistical model.

Screening and optimization of nutritional factors for higher dextransucrase production by Leuconostocmesenteroides NRRL B-640 using statistical approach.

To improve dextransucrase production from Leuconostocmesenteroides NRRL B-640 culture medium was screened and optimized using the statistical design techniques of Plackett-Burman and response surface methodology (RSM). Plackett-Burman design with six variables viz. sucrose, yeast extract, K2HPO4, peptone, beef extract and Tween 80 was performed to screen the nutrients that were significantly affecting dextransucrase production. The variables sucrose, K2HPO4, yeast extract and beef extract showed above 90% confidence levels for dextransucrase production and were considered as significant factors for optimization using response surface methodology. 2(4)-central composite design was used for RSM optimization. The experimental results were fitted to a second-order polynomial model which gave a coefficient of determination R2=0.95. The optimized composition of 30g/l sucrose, 18.9g/l yeast extract, 19.4g/l K2HPO4 and 15g/l beef extract gave an experimental value of dextransucrase activity of 10.7U/ml which corresponded well with the predicted value of 10.9U/ml by the model.

Identification, effective purification and functional characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640.

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol fractionation (PEG) and gel-filtration. The cell free extract was subjected to fractionation by PEG-200, 400 and 1500. The 10% (w/v) PEG-1500 gave dextransucrase with maximum specific activity of 23 with 40 fold purification in a single step. The purified enzyme showed multiple molecular forms on SDS-PAGE, however the same sample showed a single band on non-denaturing native-PAGE. The purified dextransucrase fractions obtained from PEG-1500, confirmed the presence of dextran, when run on SDS-PAGE under non-denaturing gels for in situ activity detection by Periodic Acid Schiff's staining. The activity bands corresponded to the native and active form of the purified dextransucrase of approximately, 180kDa molecular size, that appeared on the denaturing gels stained with Coomassie Brilliant Blue. No bands appeared after staining the activity of dextransucrase on non denaturing SDS-PAGE gels with raffinose, which excluded the presence of fructosyltransferases. Further purification of 10% PEG-1500 purified dextransucrase by gel-filtration gave dextransucrase with specific activity of 35 with 61 fold purification.

An overview of purification methods of glycoside hydrolase family 70 dextransucrase.

The enzyme dextransucrase (sucrose:1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2.4.1.5) catalyses the synthesis of exopolysaccharide, dextran from sucrose. This class of polysaccharide has been extensively exploited in pharmaceutical industry as blood volume expander, as stabiliser in food industry and as a chromatographic medium in fine chemical industry because of their nonionic nature and stability. Majority of the dextrans are synthesized from sucrose by dextransucrase secreted mainly by bacteria belonging to genera Leuconostoc, Streptococcus and Lactobacillus. Bulk of the information on purification of extracellular dextransucrase has been generated from Leuconostoc species. Various methods such as precipitation by ammonium sulphate, ethanol or polyethylene glycol, phase partitioning, ultrafiltration and chromatography have been used to purify the enzyme. Purification of dextransucrase is rendered difficult by the presence of viscous dextran in the medium. However, processes like ultra-filtration, salt and PEG precipitation, chromatography and phase partitioning have been standardized and successfully used for higher scale purification of the enzyme. A recombinant dextransucrase from Leuconostoc mesenteroides B-512F with a histidine tag has been expressed in E. coli cells and purifi ed by immobilized metal ion chromatography. This review reports the available information on purifi cation methods of dextransucrase from Leuconostoc mesenteroides strains.