Genetically Engineered Escherichia coli Nissle 1917 Synbiotics Reduce Metabolic Effects Induced by Chronic Consumption of Dietary Fructose.

AIMS: To assess protective efficacy of genetically modified Escherichia coli Nissle 1917 (EcN) on metabolic effects induced by chronic consumption of dietary fructose. MATERIALS AND METHODS: EcN was genetically modified with fructose dehydrogenase (fdh) gene for conversion of fructose to 5-keto-D-fructose and mannitol-2-dehydrogenase (mtlK) gene for conversion to mannitol, a prebiotic. Charles foster rats weighing 150-200 g were fed with 20% fructose in drinking water for two months. Probiotic treatment of EcN (pqq), EcN (pqq-glf-mtlK), EcN (pqq-fdh) was given once per week 109 cells for two months. Furthermore, blood and liver parameters for oxidative stress, dyslipidemia and hyperglycemia were estimated. Fecal samples were collected to determine the production of short chain fatty acids and pyrroloquinoline quinone (PQQ) production. RESULTS: EcN (pqq-glf-mtlK), EcN (pqq-fdh) transformants were confirmed by restriction digestion and functionality was checked by PQQ estimation and HPLC analysis. There was significant increase in body weight, serum glucose, liver injury markers, lipid profile in serum and liver, and decrease in antioxidant enzyme activity in high-fructose-fed rats. However the rats treated with EcN (pqq-glf-mtlK) and EcN (pqq-fdh) showed significant reduction in lipid peroxidation along with increase in serum and hepatic antioxidant enzyme activities. Restoration of liver injury marker enzymes was also seen. Increase in short chain fatty acids (SCFA) demonstrated the prebiotic effects of mannitol and gluconic acid. CONCLUSIONS: Our study demonstrated the effectiveness of probiotic EcN producing PQQ and fructose metabolizing enzymes against the fructose induced hepatic steatosis suggesting that its potential for use in treating fructose induced metabolic syndrome.

Probiotic Escherichia coli CFR 16 producing pyrroloquinoline quinone (PQQ) ameliorates 1,2-dimethylhydrazine-induced oxidative damage in colon and liver of rats.

Inflammation of the gastrointestinal tract is associated with reactive oxygen species (ROS) genesis. Alleviation of oxidative stress is achieved by using antioxidants and probiotics. Present study investigates a synergistic effect of the probiotic Escherichia coli CFR 16 containing Vitreoscilla haemoglobin gene (vgb), green fluorescent protein (gfp) gene and pyrroloquinoline quinone (pqq) gene cluster on oxidative stress induced by 1,2-dimethylhydrazine (DMH). Adult virgin Charles foster male rats (3-4 months) weighing 200-250 g were administered with DMH (25 mg/kg body weight, s.c.) twice a week for eight consecutive weeks. Rats receiving only DMH dose showed increased lipid peroxidation in liver and intestinal tissues with reduced activity of antioxidant enzymes, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Oral dose of E. coli CFR 16::vgb-gfp harbouring pqq gene cluster increased rat faecal PQQ concentration by twofold, reduced lipid peroxidation and retained SOD, CAT and GPx activities close to normal levels in liver and colonic tissues following DMH treatment. In addition, significant protection was found in colonic histological sections of these rat groups. This study demonstrates a protective efficacy in the following order: E. coli CFR 16 < E. coli CFR 16::vgb-gfp < vitamin C = PQQ < E. coli CFR 16::vgb-gfp (pqq).

Impact of targeted specific antibiotic delivery for gut microbiota modulation on high-fructose-fed rats.

The objective of present investigation was to study the effect of gut microbiota alteration by oral administration of targeted delivery of pH sensitive cefdinir microspheres to high-fructose-fed (HFD) rats. Rats were fed with a high-fructose diet with or without cefdinir microsphere administration for 30 days. The fecal microbiota community, oral glucose tolerance, the markers of liver injury, plasma and hepatic lipids profile, and histological evaluation were investigated. The levels of blood glucose, liver injury markers, lipid profile in plasma and liver, and fat tissue were significantly increased in high-fructose-fed rats. However, after pH-sensitive cefdinir microsphere administration, the elevation of these parameters was significantly suppressed. Cef EL significantly lowered the increased AST (p < 0.05) and ALT (p < 0.001) levels in HFD group. There is a significant lower (p < 0.01) AUCglucose level in Cef EL group than HFD group The histological changes in the liver and the small and large intestines were more profound in HFD group as compared to cefdinir-treated HFD and control groups. Feeding of cefdinir microsphere sustained lactobacilli and bifidobacteria and significantly decreased (p < 0.05) the number of Enterobacteriaceae induced by HFD. Experimental evidences demonstrated that the effectiveness of pH-specific cefdinir microsphere on reducing insulin resistance and development of metabolic changes in high-fructose-fed rats and suggested that it may be a promising therapeutic agent in treating type 2 diabetes. Intestinal-targeted antibiotic delivery needs to be further explored for its therapeutic applications.

Mechanism of phosphate solubilization and antifungal activity of Streptomyces spp. isolated from wheat roots and rhizosphere and their application in improving plant growth.

The application of plant-growth-promoting rhizobacteria (PGPR) at field scale has been hindered by an inadequate understanding of the mechanisms that enhance plant growth, rhizosphere incompetence and the inability of bacterial strains to thrive in different soil types and environmental conditions. Actinobacteria with their sporulation, nutrient cycling, root colonization, bio-control and other plant-growth-promoting activities could be potential field bio-inoculants. We report the isolation of five rhizospheric and two root endophytic actinobacteria from Triticum aestivum (wheat) plants. The cultures exhibited plant-growth-promoting activities, namely phosphate solubilization (1916 mg l(-1)), phytase (0.68 U ml(-1)), chitinase (6.2 U ml(-1)), indole-3-acetic acid (136.5 mg l(-1)) and siderophore (47.4 mg l(-1)) production, as well as utilizing all the rhizospheric sugars under test. Malate (50-55 mmol l(-1)) was estimated in the culture supernatant of the highest phosphate solublizer, Streptomyces mhcr0816. The mechanism of malate overproduction was studied by gene expression and assays of key glyoxalate cycle enzymes - isocitrate dehydrogenase (IDH), isocitrate lyase (ICL) and malate synthase (MS). The significant increase in gene expression (ICL fourfold, MS sixfold) and enzyme activity (ICL fourfold, MS tenfold) of ICL and MS during stationary phase resulted in malate production as indicated by lowered pH (2.9) and HPLC analysis (retention time 13.1 min). Similarly, the secondary metabolites for chitinase-independent biocontrol activity of Streptomyces mhcr0817, as identified by GC-MS and (1)H-NMR spectra, were isoforms of pyrrole derivatives. The inoculation of actinobacterial isolate mhce0811 in T. aestivum (wheat) significantly improved plant growth, biomass (33%) and mineral (Fe, Mn, P) content in non-axenic conditions. Thus the actinobacterial isolates reported here were efficient PGPR possessing significant antifungal activity and may have potential field applications.

Plant growth promoting potential and soil enzyme production of the most abundant Streptomyces spp. from wheat rhizosphere.

AIM: To evaluate the plant growth promotion (PGP) potential and soil enzyme production under solid state fermentation (SSF) by most abundant Streptomyces spp. isolated from the wheat rhizosphere and to evaluate their effect on plant growth parameters. METHODS AND RESULTS: Actinomycetes were isolated from wheat rhizosphere and screened for PGP activities. Three actinomycete isolates having significantly higher PGP activities (Streptomyces rochei IDWR19, Streptomyces carpinensis IDWR53, Streptomyces thermolilacinus IDWR81) were selected. The soil enzymes production potential of these isolates using soil extract and wheat straw under ssf was assessed. Utilization of soil extract as a fermentation medium for soil enzyme production by Actinomycetes has been reported first time in this study. Maximum chitinase (S. rochei IDWR19 12·2 U mg(-1) protein) and phytase activity (S. carpinensis IDWR53 5·2 U mg(-1) protein) was produced on 7th day of incubation, whereas maximum alkaline protease (S. rochei IDWR19 3·2 U mg(-1) protein) was produced on 6th day of incubation. For cellulase (S. rochei IDWR19 7·4 U mg(-1) protein) and invertase (S. carpinensis IDWR53 451 U mg(-1) protein) maximum activity was observed on 4th as well as 5th day of incubation. On the basis of PGP activity and enzyme production, two actinomycete isolates (S. rochei IDWR19 and S. thermolilacinus IDRWR81) were selected for plant growth experiment. An increase of 12·2 and 24·5% in shoot length of plants inoculated with S. rochei IDWR19 and S. thermolilacinus IDWR81 was observed, respectively. A similar increase in biomass of 1·8- and 2·3-fold was also recorded for the two isolates, respectively. CONCLUSIONS: It could be concluded that Streptomyces sp. with high PGP activities and soil enzyme production capability significantly improved growth and development of wheat cv. SIGNIFICANCE AND IMPACT OF THE STUDY: The abundant Actinomycetes obtained in this study (S. rochei IDWR19 and S. thermolilacinus IDWR81) are rhizosphere competent and effective strains.

Excision of Anabaena PCC 7120 nifD element in Escherichia coli: Growth kinetics and RecA regulated xisA expression and DNA rearrangement.

Anabaena PCC 7120 nifHDK operon is interrupted by an 11 kb DNA element which is excised during the development of heterocysts by Excisase A, encoded by the xisA gene residing on the element. The excision is a site-specific recombination event that occurs at the 11 base pair direct repeats flanking the element. Earlier work showed the excision of the 11 kb element in Escherichia coli at a frequency 0.3%. We report here the excision of this element at 1.1% and 1.98% in E. coli DH5alpha, and 1.9% and 10.9% in E. coli JM 101 when grown on Luria broth and minimal media, respectively. Excision of nifD element in isogenic recA(-) (RK1) and recA+ (RK2) E. coli JM101 P1 transductants, showed similar results to that of E. coli JM101 and DH5alpha, respectively. A plasmid pMX32, carrying a xisA defective 11kb element, showed no excision in E. coli RK2 strain. In contrast to Anabaena PCC 7120, excision of nifD element did not increase in E. coli DH5alpha grown in iron-deficient conditions. A PxisA::lacZ transcriptional fusion, used to detect the expression of elusive xisA gene, showed maximal beta-galactosidase activity in the stationary phase. The results suggest that the excision event in E. coli may involve additional factors, such as RecA and that the physiological status can influence the excision of nifD element.

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).