Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™.

The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig™, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3' to the other copies with a 6 kbp deletion in the 5' promoter region.

Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization.

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes.

Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders, and conventional and genetically enhanced lines.

Porcine liver proteome iTRAQ analysis enabled the confident identification of 880 proteins with a rate of false positive identifications of less than 5%. Proteins involved in energy metabolism, catabolism, protein biosynthesis, electron transport, and other oxidoreductase reactions were highly enriched confirming the central role of liver as the major chemical and energy factory. Comparative analysis with human and mouse liver proteomes demonstrated that 80% of proteins were common to all three liver proteomes.

Cel9D, an atypical 1,4-beta-D-glucan glucohydrolase from Fibrobacter succinogenes: characteristics, catalytic residues, and synergistic interactions with other cellulases.

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified.

Chemical structures of manure from conventional and phytase transgenic pigs investigated by advanced solid-state NMR spectroscopy.

Nonpoint phosphorus (P) pollution from animal manure is becoming a serious global problem. The current solution for the swine industry includes the enzyme phytase as a component in oil meal and cereal grain-based swine diets. A long-term approach is the production of transgenic phytase pigs that express phytase in the salivary glands and secrete it in the saliva.

Characterization and synergistic interactions of Fibrobacter succinogenes glycoside hydrolases.

The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme.

Outer membrane proteins of Fibrobacter succinogenes with potential roles in adhesion to cellulose and in cellulose digestion.

Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4.

Transgene and mitochondrial DNA are indicators of efficient composting of transgenic pig carcasses.

Composting is an environmentally sound method for the disposal of on-farm livestock mortalities that generates material suitable for use as fertilizer; however, this method is not generally permitted for disposal of transgenic livestock mortalities during the research and development phase. This study has explored the application of the polymerase chain reaction (PCR) as a method for assessing the persistence of transgene and mitochondrial DNA markers during the composting of euthanized transgenic pig. There was at least a 10(7) fold reduction of genetic material to a level that not either transgene or mitochondrion markers were detectable.

Characteristics of adjacent family 6 acetylxylan esterases from Fibrobacter succinogenes and the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan.

Acetylxylan esterase genes axe6A and axe6B located adjacent to one another on a Fibrobacter succinogenes chromosome have been separately cloned and their properties characterized. The corresponding esterases contained an N-terminal carbohydrate esterase family 6 catalytic domain (CD) and a C-terminal family 6 carbohydrate-binding module (CBM). The amino acid sequences of the CDs and CBMs were found to exhibit 52% and 40% amino acid similarity, respectively.

Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization.

Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium Fibrobacter intestinalis DR7 not shared by Fibrobacter succinogenes S85. Subtractive clones from F. intestinalis were sequenced and assembled to form 712 nonredundant contigs with an average length of 525 bp.

Characteristics of a cluster of xylanase genes in Fibrobacter succinogenes S85.

Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized. Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences. The amino acid sequences exhibited similarities of between 53 and 60%.

Functional insights revealed by the crystal structures of Escherichia coli glucose-1-phosphatase.

The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell.

Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase.

When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.

Properties of the major non-specific endonuclease from the strict anaerobe Fibrobacter succinogenes and evidence for disulfide bond formation in vivo.

DNase A is a non-specific endonuclease of Fibrobacter succinogenes. The enzyme was purified to homogeneity and its properties studied both in vitro and in vivo. Magnesium but not calcium was essential for nucleolytic activity.