Effect of fiber source on cecal fermentation and nitrogen recycled through cecotrophy in rabbits.
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The influence of fiber source on fiber digestion in rabbits was investigated. Six fibrous feedstuffs with wide differences in chemical composition and particle size were selected: paprika meal, olive leaves, alfalfa hay, soybean hulls, sodium hydroxide-treated barley straw, and sunflower hulls. Six diets were formulated to contain one of these ingredients as the sole source of fiber. To avoid nutrient imbalances, fiber sources were supplemented with different proportions of a fiber-free concentrate, based on soy protein isolate, wheat flour, lard, and a vitamin and mineral mix, to obtain diets containing at least 3% nitrogen and 5% starch. Daily soft feces excretion, and its NDF, and total and microbial nitrogen content were determined in 60 fattening rabbits (10 per diet). Seven days after the last cecotrophy control, the same animals were used to determine weight of stomach, cecum and their contents, and cecal fermentation traits (pH, VFA and ammonia concentrations, and buffer properties of cecal contents). Stepwise regression analysis showed a positive effect (P < .001) on soft feces excretion, total and microbial nitrogen concentrations in soft feces, cecal acidity, and total VFA in the cecum of dietary pectic constituents (2.9, 3.5, 2.5, .9, and 6.6%) and proportion of fine particles (< .315 mm) (1.8, .9, 1.3, .15, and .9%) per each increment of one percentage unit of the independent variables. Proportion of fine particles also increased weight of cecal contents (P < .001). Soft feces excretion and weight of stomach and of its contents increased (P < .001) by 5.2, 2.8, and 10.2% per each percentage unit increment of proportion of large particles (> 1.25 mm). Degree of lignification of NDF decreased total nitrogen concentration in soft feces and cecal VFA concentration (P < .001). Source of fiber affected cecal pH not only by its influence on the cecal concentrations of the final products of fermentation, but also through its effect on the pH of dry cecal contents (P < .001). The latter was negatively correlated with dietary proportion of fine particles, degree of lignification of NDF, and base-buffering capacity of dry cecal contents (r = -.52, -.37, and -.49, respectively). From these results, we conclude that pectic constituent concentration, degree of lignification of NDF, and particle size are the variables that best characterize the influence of the source of fiber on soft feces excretion and cecal fermentation traits in rabbits. (+info)
Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics.
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Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N-terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N-terminal extensions could be pro-enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases. (+info)
Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti.
(51/896)
Genes coding for components of the pyruvate dehydrogenase (PDH) multienzyme complex (PDHc) from Sinorhizobium meliloti, the alfalfa symbiont, have been isolated on the basis of their high expression in symbiotic bacteria. The Elp component, PDH, is encoded by two genes, pdhAalpha (1,047 bp) and pdhAbeta (1,383 bp), a situation encountered in the alpha-proteobacteria Rickettsia prowazekii and Zymomonas mobilis as well as in some gram-positive bacteria and in mitochondria. pdhAalpha and pdhAbeta precede pdhB (1,344 bp), which encodes the E2p component, dihydrolipoamide acetyltransferase, of the PDHc. No gene encoding the E3 component, lipoamide dehydrogenase, was found in the immediate vicinity of pdhA and pdhB genes. pdhAalpha, pdhAbeta and pdhB likely constitute an operon. Here, we provide evidence that pdhA expression is induced in the symbiotic stage, compared with free-living conditions. We demonstrate that symbiotic expression of pdhA genes does not depend on the fix LJ regulatory cascade that regulates nitrogen fixation and respiration gene expression in symbiotic S. meliloti cells. Induction of pdhA expression could be obtained under free-living conditions upon the addition of pyruvate to the culture medium. Induction by pyruvate and symbiotic activation of pdh gene expression take place at the same promoter. (+info)
Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis.
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Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-beta-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 microg per g fresh weight) and oldest stem internode segments (>10 microg per g fresh weight) while roots contained only trace amounts (<0.2 microg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malonyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field. (+info)
Sinorhizobium meliloti nfe (nodulation formation efficiency) genes exhibit temporal and spatial expression patterns similar to those of genes involved in symbiotic nitrogen fixation.
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The nfe genes (nfeA, nfeB, and nfeD) are involved in the nodulation efficiency and competitiveness of the Sinorhizobium meliloti strain GR4 on alfalfa roots. The nfeA and nfeB genes are preceded by functional nif consensus sequences and NifA binding motifs. Here, we determined the temporal and spatial expression patterns of the nfe genes in symbiosis with alfalfa. Translational fusions of the nfe promoters with the gusA gene and reverse transcription-polymerase chain reaction analyses indicate that they are expressed and translated within mature nitrogen-fixing nodules and not during early steps of nodule development. Within the nodules the three nfe genes exhibit a spatial expression pattern similar to that of genes involved in symbiotic nitrogen fixation. We show that nfeB and nfeD genes are expressed not only from their own promoters but also from the upstream nfe promoter sequences. Furthermore, with the use of specific antibodies the NfeB and NfeD proteins were detected within the root nodule bacteroid fraction. Finally, NfeB was inmunolocalized in the bacteroid cell membrane whereas NfeD was detected in the bacteroid cytoplasm. (+info)
Disruption of a gene essential for sulfoquinovosyldiacylglycerol biosynthesis in Sinorhizobium meliloti has no detectable effect on root nodule symbiosis.
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The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3' of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sulfolipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered. (+info)
Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization.
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Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed. (+info)
Body weight and tissue gain in lambs fed an all-concentrate diet and implanted with trenbolone acetate or grazed on alfalfa.
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Targhee x Hampshire lambs (average BW 23 +/- 1 kg) were used in two experiments to determine the effects of finishing on concentrate with an anabolic implant or forage grazing after concentrate feeding on growth, organ and viscera weights, and carcass tissue accretion. In Exp. 1 and 2 lambs were penned by sex and assigned for slaughter at initial (23 kg), intermediate (37 kg), or end BW (ewes, 47.7; wethers 50.4 kg). From 23 to 37 kg BW, lambs were fed all-concentrate diets in drylot (DL) or grazed on alfalfa (ALF). Experiment 1 was a 2 x 2 factorial with 28 lambs; factors were wether vs ewe lambs and unimplanted vs DL implanted with trenbolone acetate-estradiol benzoate. There were no differences in organ and viscera weights due to implant status. However, ADG (P < .03) and lean gain (P < .02) were greater for implanted than for unimplanted wethers (507 vs 357 g and 1,314 vs 656 g, respectively). Ewes did not respond to the implant. Fat accretion was not affected by implantation. Experiment 2 was a 2 x 3 factorial with 42 lambs; factors were wether vs ewe lambs and drylot during growing and finishing phases (DL-DL) vs drylot during growing and alfalfa grazing during finishing (DL-ALF) vs alfalfa grazing during growing and finishing phases (ALF-ALF). In Exp. 2, ADG of DL-DL lambs was greater (P < .01) than ADG of DL-ALF or ALF-ALF lambs. Lambs on ALF-ALF had smaller (P < .05) livers and rumen/reticulum weights but heavier (P < .04) kidney, omasum, small and large intestine, and cecum weights than those on DL. In Exp. 2, DL-ALF and ALF-ALF lambs had overall hindsaddle lean gain equal to those on DL-DL with less mesenteric fat and 100 g less separable fat. Finishing lambs on alfalfa reduced fat accretion without decreasing lean accretion, whereas trenbolone acetate implants for lambs fed concentrate increased BW gain and lean accretion without affecting fat accretion. (+info)