scholarly journals Adaptation of Gut Microbiotas to Transgenic Pigs Secreting β-Glucanase, Xylanase and Phytase

2020 ◽  
Author(s):  
Jianxin Mo ◽  
Guoling Li ◽  
Guangyan Huang ◽  
Haoqiang Wang ◽  
Junsong Shi ◽  
...  
Keyword(s):  
Feed Efficiency ◽  
Digestive Enzymes ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Transgenic Pigs ◽  
Intestinal Microbes ◽  
Type D ◽  
Aspartate Carbamoyltransferase ◽  
Fractional Abundance ◽  
Marker Free

Abstract BackgroundThe gut microbiotas play an important role in digestive function and feed efficiency in pigs. However, the effect of exogenous digestive enzymes on the composition and functional contributions of swine intestinal microbes is unclear. The objective of this study was to investigate the change of gut microbiotas in the transgenic pigs secreting microbial digestive enzymes in their salivary glands.MethodsEGFP marker-free transgenic (MF-TG) pigs were generated by deleted the EGFP coding genes in the transgenic pigs we previously generated. Samples of chyme from the ileum, caecum and colon of five MF-TG and five wild-type (WT) sows were collected for investigating the gut microbiomes via metagenomics analyses.ResultsThe levels of probiotics were abundant in the caecum of MF-TG pigs and higher than those of WT pigs. By contrast, the levels of some harmful microorganisms were higher in the caecum of WT pigs than those of MF-TG pigs. In addition, the microorganisms in the colon of MF-TG pigs had high fractional abundance in DNA (cytosine-5)-methyltransferase 1 and serine-type D-Ala-D-Ala carboxypeptidase, whereas the aspartate carbamoyltransferase regulatory subunit and outer membrane protein pathways were enriched in WT pigs. Moreover, the levels of numerous carbohydrases in the caecum of MF-TG pigs were higher than those of WT pigs. ConclusionsThe results indicated that intestinal microbes can change adaptively to the secretion of transgenic enzymes, thereby forming a benign cooperation with their host.

scholarly journals Adaptation of Gut Microbiome to Transgenic Pigs Secreting β-Glucanase, Xylanase, and Phytase

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianxin Mo ◽  
Guoling Li ◽  
Guangyan Huang ◽  
Haoqiang Wang ◽  
Junsong Shi ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Glycoside Hydrolase ◽  
Lactobacillus Reuteri ◽  
Nutrient Loss ◽  
Regulatory Subunit ◽  
Glycoside Hydrolase Family ◽  
Transgenic Pigs ◽  
Intestinal Microbes ◽  
Type D ◽  
Hydrolase Family

We previously generated transgenic pigs with enhanced growth rate and reduced nutrient loss. However, the composition of their gut microbiome is unknown. In this study, we successfully generated EGFP marker-free transgenic (MF-TG) pigs with high expression levels of microbial β-glucanase, xylanase, and phytase in the parotid gland. We collected intestinal contents from the ileum, cecum and colon of five MF-TG and five wild-type (WT) sows and investigated the gut microbiome of the transgenic pigs via metagenomic analysis. Results showed that the levels of probiotics, such as Lactobacillus reuteri and Streptococcus, were more abundant in the cecum of the MF-TG pigs and higher than those of WT pigs. By contrast, the levels of harmful microorganisms, such as Campylobacter, Chlamydia trachomatis, and Campylobacter fetus, and various unidentified viruses, were higher in the cecum of the WT pigs than those of the MF-TG pigs. By comparing unigenes and the eggNOG database, we found that the microorganisms in the colon of the MF-TG pigs had high fractional abundance in DNA (cytosine-5)-methyltransferase 1 and serine-type D-Ala-D-Ala carboxypeptidase, whereas the aspartate carbamoyltransferase regulatory subunit and outer membrane protein pathways were enriched in the WT pigs. Moreover, the microorganisms in the cecum of the MF-TG pigs were active in GlycosylTransferase Family 8 (GT8), Glycoside Hydrolase Family 13 (GH13), and Glycoside Hydrolase Family 32 (GH32). Furthermore, the levels of numerous carbohydrases, such as glucan 1,3-beta-glucosidase, xylan 1,4-beta-xylosidase and exo-1,3-1,4-glucanase, were higher in the cecum of the MF-TG pigs than those of the WT pigs. The results indicated that intestinal microbes can change adaptively to the secretion of transgenic enzymes, thereby forming a benign cooperation with their host. This cooperation could be beneficial for improving feed efficiency.

scholarly journals Generation of Multi-Transgenic Pigs Using PiggyBac Transposons Co-expressing Pectinase, Xylanase, Cellulase, β-1.3-1.4-Glucanase and Phytase

2020 ◽  
Vol 11 ◽  
Author(s):  
Haoqiang Wang ◽  
Guoling Li ◽  
Cuili Zhong ◽  
Jianxin Mo ◽  
Yue Sun ◽  
...  
Keyword(s):  
Environmental Impact ◽  
Feed Efficiency ◽  
Cell Walls ◽  
Digestive Enzymes ◽  
Feed Utilization ◽  
Cell Cloning ◽  
Transgenic Pigs ◽  
Enzymatic Assays ◽  
Pork Industry ◽  
Low Levels

The current challenges facing the pork industry are to maximize feed efficiency and minimize fecal emissions. Unlike ruminants, pigs lack several digestive enzymes such as pectinase, xylanase, cellulase, β-1.3-1.4-glucanase, and phytase which are essential to hydrolyze the cell walls of grains to release endocellular nutrients into their digestive tracts. Herein, we synthesized multiple cellulase and pectinase genes derived from lower organisms and then codon-optimized these genes to be expressed in pigs. These genes were then cloned into our previously optimized XynB (xylanase)- EsAPPA (phytase) bicistronic construct. We then successfully generated transgenic pigs that expressed the four enzymes [Pg7fn (pectinase), XynB (xylanase), EsAPPA (phytase), and TeEGI (cellulase and β-glucanase)] using somatic cell cloning. The expression of these genes was parotid gland specific. Enzymatic assays using the saliva of these founders demonstrated high levels of phytase (2.0∼3.4 U/mL) and xylanase (0.25∼0.42 U/mL) activities, but low levels of pectinase (0.06∼0.08 U/mL) activity. These multi-transgenic pigs are expected to contribute to enhance feed utilization and reduce environmental impact.

scholarly journals Generation of a novel growth-enhanced and reduced environmental impact transgenic pig strain

2018 ◽  
Author(s):  
Xianwei Zhang ◽  
Zicong Li ◽  
Huaqiang Yang ◽  
Dewu Liu ◽  
Gengyuan Cai ◽  
...  
Keyword(s):  
Feed Efficiency ◽  
Single Copy ◽  
Transgenic Pig ◽  
Wild Type ◽  
Feed Conversion ◽  
Transgenic Pigs ◽  
Nutrient Emissions ◽  
Environmental Emissions ◽  
Excessive Nutrients ◽  
Feed Conversion Rate

ABSTRACTIn pig production, insufficient feed digestion causes excessive nutrients such as phosphorus and nitrogen, which are then released to the environment. To address the issue of environmental emissions, we have established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, β-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphate outputs were reduced by 23%–46%, and growth rate improved by 23.4% (gilts) and 24.4% (boars) when the pigs were fed on a corn and soybean-based diet and high-NSP diet. The transgenic pigs showed a 11.5%– 14.5% improvement in feed conversion rate compared to the age-matched wild-type littermates. These findings indicate that transgenic pigs are promising resources for improving feed efficiency and reducing nutrient emissions to the environment.

scholarly journals Generation of multi-transgenic pigs using PiggyBac transposons co-expressing pectinase, xylanase, cellulase, β-1.3-1.4-glucanase and phytase

2020 ◽  
Author(s):  
Haoqiang Wang ◽  
Guoling Li ◽  
Cuili Zhong ◽  
Jianxin Mo ◽  
Yue Sun ◽  
...  
Keyword(s):  
Environmental Impact ◽  
Feed Efficiency ◽  
Cell Walls ◽  
Digestive Enzymes ◽  
Feed Utilization ◽  
Cell Cloning ◽  
Transgenic Pigs ◽  
Enzymatic Assays ◽  
Pork Industry ◽  
Low Levels

AbstractThe current challenges facing the pork industry are to maximize feed efficiency and minimize fecal emissions. Unlike ruminants, pigs lack a number of digestive enzymes like pectinase, xylanase, cellulase, β-1.3-1.4-glucanase and phytase to hydrolyze the cell walls of grains to release endocellular nutrients into their digestive tracts. Herein, we synthesized multiple cellulase and pectinase genes derived from lower organisms and then codon optimized these genes to be expressed in pigs. These genes were then cloned into our previously optimized XynB (xylanase)- EsAPPA (phytase) bicistronic construct. We then successfully generated transgenic pigs that expressed four enzymes (Pg7fn (pectinase), XynB (xylanase), EsAPPA (phytase) and TeEGI (cellulase and β-glucanase)) using somatic cell cloning. Expression of these genes was parotid gland specific. Enzymatic assays using the saliva of these founders demonstrated high levels of phytase (2.0~3.4 U/mL) and xylanase (0.25~0.42 U/mL) activity, but low levels of pectinase (0.06~0.08 U/mL) activity. These multi-transgenic pigs are expected to contribute to enhance feed utilization and reduce environmental impact.

scholarly journals CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Jihong Li ◽  
Menglin Ma ◽  
Mahfuzur R. Sarker ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Null Mutant ◽  
Intestinal Infection ◽  
Wild Type ◽  
Global Regulator ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Type D ◽  
Epsilon Toxin ◽  
Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

scholarly journals Comparative Proteomics Reveals the Potential Targets of BcNoxR, a Putative Regulatory Subunit of NADPH Oxidase of Botrytis cinerea

2016 ◽  
Vol 29 (12) ◽  
pp. 990-1003 ◽  
Author(s):  
Hua Li ◽  
Zhanquan Zhang ◽  
Chang He ◽  
Guozheng Qin ◽  
Shiping Tian
Keyword(s):  
Nadph Oxidase ◽  
Botrytis Cinerea ◽  
Proteomic Analysis ◽  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Tomato Fruit ◽  
Polymerase Chain Reaction Analysis ◽  
Regulatory Subunit ◽  
Wild Type

The NADPH oxidase (NOX) complex has been shown to play a crucial role in stress response and in the virulence of various fungal pathogens. The underlying molecular mechanisms of NOX, however, remain largely unknown. In the present study, a comparative proteomic analysis compared changes in protein abundance in wild-type Botrytis cinerea and ΔbcnoxR mutants in which the regulatory subunit of NOX was deleted. The ΔbcnoxR mutants exhibited reduced growth, sporulation, and impaired virulence. A total of 60 proteins, representing 49 individual genes, were identified in ΔbcnoxR mutants that exhibited significant differences in abundance relative to wild-type. Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that the differences in transcript levels for 36 of the genes encoding the identified proteins were in agreement with the proteomic analysis, while the remainder exhibited reverse levels. Functional analysis of four proteins that decreased abundance in the ΔbcnoxR mutants indicated that 6-phosphogluconate dehydrogenase (BcPGD) played a role in the growth and sporulation of B. cinerea. The Δbcpgd mutants also displayed impaired virulence on various hosts, such as apple, strawberry, and tomato fruit. These results suggest that NOX can influence the expression of BcPGD, which has an impact on growth, sporulation, and virulence of B. cinerea.

The morphology and development of Drosophila eye

Development ◽  
1967 ◽  
Vol 17 (3) ◽  
pp. 491-499
Author(s):  
E. W. Hanly ◽  
C. William Fuller ◽  
M. S. Millam Stanley
Keyword(s):  
Drosophila Melanogaster ◽  
Metabolic Pathways ◽  
Biosynthetic Pathways ◽  
Wild Type ◽  
Specific Enzyme ◽  
Type D ◽  
Red Color ◽  
Drosophila Eye ◽  
The Subject

The development of pigment in the eye of Drosophila melanogaster and other insects has been the subject of many studies and much controversy. It has been established that the red color of eyes of wild-type D. melanogaster is due to the presence of two classes of pigments, ommochromes and pteridines (Ziegler, 1961). The relationships among the various members of each class are still obscure; the biosynthetic pathways are yet to be elucidated. No specific enzyme involved in the synthesis of any member of either group has been isolated or characterized. It has been suggested, however (Hadorn, 1955), that these metabolic pathways may involve several organs, including the eye, but that the final deposition and conversion occur only in the eye. The recent development of a satisfactory technique for the culture of Drosophila organs (Schneider, 1964) has made possible the study of pigment development in the isolated eye and in eyes associated with selected organs.

Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase

1982 ◽  
Vol 2 (10) ◽  
pp. 1229-1237
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Regulatory Subunit ◽  
Mutational Event ◽  
Wild Type ◽  
Cell Mutant ◽  
Dependent Protein Kinase ◽  
Cell Extracts ◽  
Counter Selection ◽  
Dependent Protein ◽  
Gene Lesion

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

scholarly journals The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85

2019 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
Francesca Troilo ◽  
Francesca Malagrinò ◽  
Lorenzo Visconti ◽  
Angelo Toto ◽  
Stefano Gianni
Keyword(s):  
Protein Interactions ◽  
Specific Interaction ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Sh2 Domains ◽  
Trans Isomerization ◽  
A Site ◽  
Kinetics Of

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

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