scholarly journals Determination of Butyrate Synthesis Capacity in Gut Microbiota: Quantification of but Gene Abundance by qPCR in Fecal Samples

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1303
Author(s):  
Nikola Daskova ◽  
Marie Heczkova ◽  
Istvan Modos ◽  
Petra Videnska ◽  
Petra Splichalova ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Functional Capacity ◽  
Sequence Similarity ◽  
Gene Copy ◽  
Fecal Dna ◽  
Degenerate Primers ◽  
Bacterial Gene ◽  
Bacterial Fermentation ◽  
Gene Abundance ◽  
Coa Transferase

Butyrate is formed in the gut during bacterial fermentation of dietary fiber and is attributed numerous beneficial effects on the host metabolism. We aimed to develop a method for the assessment of functional capacity of gut microbiota butyrate synthesis based on the qPCR quantification of bacterial gene coding butyryl-CoA:acetate CoA-transferase, the key enzyme of butyrate synthesis. In silico, we identified bacteria possessing but gene among human gut microbiota by searching but coding sequences in available databases. We designed and validated six sets of degenerate primers covering all selected bacteria, based on their phylogenetic nearness and sequence similarity, and developed a method for gene abundance normalization in human fecal DNA. We determined but gene abundance in fecal DNA of subjects with opposing dietary patterns and metabolic phenotypes—lean vegans (VG) and healthy obese omnivores (OB) with known fecal microbiota and metabolome composition. We found higher but gene copy number in VG compared with OB, in line with higher fecal butyrate content in VG group. We further found a positive correlation between the relative abundance of target bacterial genera identified by next-generation sequencing and groups of but genecontaining bacteria determined by specific primers. In conclusion, this approach represents a simple and feasible tool for estimation of microbial functional capacity.

scholarly journals Impact of Body Mass Index (BMI) on the Effect of a Lactobacillus Rhamnosus GG (LGG)/Bifidobacterium Animalis Subspecies Lactis BB-12 (BB-12) Combination on Gut Microbiota (P20-023-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Debra Poutsiaka ◽  
Lori Stern ◽  
Virginia Riquelme ◽  
Emily Hollister ◽  
Julia Cope ◽  
...  
Keyword(s):  
Community Structure ◽  
Gut Microbiota ◽  
Lactobacillus Rhamnosus ◽  
Detailed Examination ◽  
Global Changes ◽  
Shotgun Metagenomics ◽  
Gene Abundance ◽  
Funding Sources ◽  
And Function ◽  
Relative Gene

Abstract Objectives This exploratory study builds upon an earlier study of probiotic supplementation1 to assess the effects of a probiotic combination (P) of LGG and BB-12 on human gut microbiota composition and function, and to uncover an association with BMI. Methods Healthy subjects ingested P for 21 days (n = 18, P group) or did not (n = 7, C group). Fecal samples obtained at baseline (D_0) and after 21 days of supplementation (D_21) underwent 16S ribosomal RNA gene and shotgun metagenomics sequencing to characterize the bacterial and archaeal communities to the genus/species level and identify functional community genes. Results Following P ingestion, no global differences in microbiota community structure or relative gene abundance were detected. In targeted analyses, the abundances of LGG and BB-12 in the P group at D_21 increased in a statistically significant manner as the BMI decreased (Spearman correlation, P = 0.04 and P = 0.01, respectively). The relative abundance of LGG but not BB-12 appeared increased in P subjects at D_21 with BMI < 25 compared to BMI > 25 (P = 0.09). P group subjects with BMI < 25 demonstrated trends toward or statistically significant increases in the relative abundances of 5 genes involved with flagellar structure (KEGG orthologs K02422, P = 0.04; K03406, P = 0.06; K02407, P = 0.08; K02397, P = 0.08; K02396, P = 0.09) at D_21 compared to those with BMI > 25. No such differences were observed for the C group nor were there differences in relative gene abundance at D_0 in the P group with BMI < 25 vs BMI > 25. Conclusions We observed no global changes in the fecal microbial community structure or function with P ingestion in this sample of healthy persons. However, we did observe patterns suggestive of a potential link between BMI and the response of the gut microbiota to P. Although our results are based on a small number of subjects, they are in line with previous findings related to LGG supplementation and the expression of flagellar genes2. We agree with other recent reports that future studies would benefit from a detailed examination of the transcriptome, proteome and/or metabolome to better understand the potential impact of probiotics on the gut microbiota, and the mechanism of the effect of BMI. Funding Sources Pfizer Inc.

scholarly journals Bias in Template-to-Product Ratios in Multitemplate PCR

1998 ◽  
Vol 64 (10) ◽  
pp. 3724-3730 ◽  
Author(s):  
Martin F. Polz ◽  
Colleen M. Cavanaugh
Keyword(s):  
Bacterial Species ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Binding Energies ◽  
Site Directed Mutagenesis ◽  
Universal Primers ◽  
Gene Copy ◽  
Degenerate Primers ◽  
Relative Product ◽  
Genomic Dnas

ABSTRACT Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

scholarly journals Distribution and Diversity of Geminiviruses in Trinidad and Tobago

1998 ◽  
Vol 88 (12) ◽  
pp. 1262-1268 ◽  
Author(s):  
Pathmanathan Umaharan ◽  
Malla Padidam ◽  
Ralph H. Phelps ◽  
Roger N. Beachy ◽  
Claude M. Fauquet
Keyword(s):  
Trinidad And Tobago ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Sweet Pepper ◽  
Yellow Mosaic Virus ◽  
Restriction Enzyme Digestion ◽  
Weed Species ◽  
Dot Blot ◽  
Degenerate Primers

Seven crop and eight weed species from 12 agricultural locations in Trinidad and Tobago were assayed for the presence of whitefly-transmitted geminiviruses (WTGs) by using dot blot hybridization and polymerase chain reaction (PCR) amplification of the N-terminal coat protein sequence with degenerate primers. The amplified fragments were cloned and analyzed by restriction enzyme digestion to determine fragment length polymorphism among the cloned fragments. Representative clones were then sequenced and subjected to phylogenetic analysis to determine the sequence similarity to known WTGs. WTGs were found in every location sampled and in 10 of the 15 species investigated: Lycopersicon esculentum(tomato), Capsicum annuum (pepper), Capsicum frutescens (sweet pepper), Abelmoschus esculentus (okra), Phaseolus vulgaris (beans), Alternanthera tenella, Desmodium frutescens, Euphorbia heterophylla, Malva alceifolia, and Sida acuta. The geminiviruses infecting these plants were closely related to potato yellow mosaic virus from Venezuela (PYMV-VE) and tomato leaf curl virus from Panama (ToLCV-PA). However, in pepper, sweet pepper, okra, Alternanthera tenella, Euphorbia heterophylla, Des-modium frutescens, and in one sample of tomato, a PYMV-VE-related virus was found in mixed infections with a virus related to pepper huasteco virus. Full-length infectious DNA-A and DNA-B of a tomato-infecting geminivirus from Trinidad and Tobago were cloned and sequenced. DNA-A appears to be a recombinant derived from PYMV-VE or ToLCV-PA, and Sida golden mosaic from Honduras. The implications of these findings in the control of WTGs are discussed.

scholarly journals MECHANISMS IN ENDOCRINOLOGY: Gut microbiota in patients with type 2 diabetes mellitus

2015 ◽  
Vol 172 (4) ◽  
pp. R167-R177 ◽  
Author(s):  
Kristine H Allin ◽  
Trine Nielsen ◽  
Oluf Pedersen
Keyword(s):  
Type 2 Diabetes ◽  
Gut Microbiota ◽  
Metabolic Diseases ◽  
Short Chain Fatty Acids ◽  
Intestinal Content ◽  
Low Grade ◽  
Bacterial Fermentation ◽  
Underlying Mechanisms

Perturbations of the composition and function of the gut microbiota have been associated with metabolic disorders including obesity, insulin resistance and type 2 diabetes. Studies on mice have demonstrated several underlying mechanisms including host signalling through bacterial lipopolysaccharides derived from the outer membranes of Gram-negative bacteria, bacterial fermentation of dietary fibres to short-chain fatty acids and bacterial modulation of bile acids. On top of this, an increased permeability of the intestinal epithelium may lead to increased absorption of macromolecules from the intestinal content resulting in systemic immune responses, low-grade inflammation and altered signalling pathways influencing lipid and glucose metabolism. While mechanistic studies on mice collectively support a causal role of the gut microbiota in metabolic diseases, the majority of studies in humans are correlative of nature and thus hinder causal inferences. Importantly, several factors known to influence the risk of type 2 diabetes, e.g. diet and age, have also been linked to alterations in the gut microbiota complicating the interpretation of correlative studies. However, based upon the available evidence, it is hypothesised that the gut microbiota may mediate or modulate the influence of lifestyle factors triggering development of type 2 diabetes. Thus, the aim of this review is to critically discuss the potential role of the gut microbiota in the pathophysiology and pathogenesis of type 2 diabetes.

scholarly journals Indole-Diterpene Gene Cluster from Aspergillus flavus

2004 ◽  
Vol 70 (11) ◽  
pp. 6875-6883 ◽  
Author(s):  
Shuguang Zhang ◽  
Brendon J. Monahan ◽  
Jan S. Tkacz ◽  
Barry Scott
Keyword(s):  
Aspergillus Flavus ◽  
Biosynthetic Pathway ◽  
Sequence Similarity ◽  
Soil Fungus ◽  
Amino Acid Sequence Similarity ◽  
Degenerate Primers ◽  
Functional Homolog ◽  
Penicillium Paxilli ◽  
Diterpene Biosynthesis ◽  
Tremorgenic Mycotoxin

ABSTRACT Aflatrem is a potent tremorgenic mycotoxin produced by the soil fungus Aspergillus flavus and is a member of a large structurally diverse group of secondary metabolites known as indole-diterpenes. By using degenerate primers for conserved domains of fungal geranylgeranyl diphosphate synthases, we cloned two genes, atmG and ggsA (an apparent pseudogene), from A. flavus. Adjacent to atmG are two other genes, atmC and atmM. These three genes have 64 to 70% amino acid sequence similarity and conserved synteny with a cluster of orthologous genes, paxG, paxC, and paxM, from Penicillium paxilli which are required for indole-diterpene biosynthesis. atmG, atmC, and atmM are coordinately expressed, with transcript levels dramatically increasing at the onset of aflatrem biosynthesis. A genomic copy of atmM can complement a paxM deletion mutant of P. paxilli, demonstrating that atmM is a functional homolog of paxM. Thus, atmG, atmC, and atmM are necessary, but not sufficient, for aflatrem biosynthesis by A. flavus. This provides the first genetic evidence for the biosynthetic pathway of aflatrem in A. flavus.

scholarly journals Genomic Microdiversity of Bifidobacterium pseudocatenulatum Underlying Differential Strain-Level Responses to Dietary Carbohydrate Intervention

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Guojun Wu ◽  
Chenhong Zhang ◽  
Huan Wu ◽  
Ruirui Wang ◽  
Jian Shen ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Human Health ◽  
Dietary Intervention ◽  
Genomic Diversity ◽  
Strain Level ◽  
Transport Systems ◽  
Gene Copy ◽  
Dietary Carbohydrate ◽  
A Genome ◽  
Differential Responses

ABSTRACT The genomic basis of the response to dietary intervention of human gut beneficial bacteria remains elusive, which hinders precise manipulation of the microbiota for human health. After receiving a dietary intervention enriched with nondigestible carbohydrates for 105 days, a genetically obese child with Prader-Willi syndrome lost 18.4% of his body weight and showed significant improvement in his bioclinical parameters. We obtained five isolates (C1, C15, C55, C62, and C95) of one of the most abundantly promoted beneficial species, Bifidobacterium pseudocatenulatum , from a postintervention fecal sample. Intriguingly, these five B. pseudocatenulatum strains showed differential responses during the dietary intervention. Two strains were largely unaffected, while the other three were promoted to different extents by the changes in dietary carbohydrate resources. The differential responses of these strains were consistent with their functional clustering based on the COGs (Clusters of Orthologous Groups), including those involved with the ABC-type sugar transport systems, suggesting that the strain-specific genomic variations may have contributed to the niche adaption. Particularly, B. pseudocatenulatum C15, which had the most diverse types and highest gene copy numbers of carbohydrate-active enzymes targeting plant polysaccharides, had the highest abundance after the dietary intervention. These studies show the importance of understanding genomic diversity of specific members of the gut microbiota if precise nutrition approaches are to be realized. IMPORTANCE The manipulation of the gut microbiota via dietary approaches is a promising option for improving human health. Our findings showed differential responses of multiple B. pseudocatenulatum strains isolated from the same habitat to the dietary intervention, as well as strain-specific correlations with bioclinical parameters of the host. The comparative genomics revealed a genome-level microdiversity of related functional genes, which may have contributed to these differences. These results highlight the necessity of understanding strain-level differences if precise manipulation of gut microbiota through dietary approaches is to be realized.

Sequence and phylogenetic analysis of the SNF4/AMPK gamma subunit gene from Drosophila melanogaster

Genome ◽  
1999 ◽  
Vol 42 (6) ◽  
pp. 1077-1087 ◽  
Author(s):  
Erin N Yoshida ◽  
Bernhard F Benkel ◽  
Ying Fong ◽  
Donal A Hickey
Keyword(s):  
Phylogenetic Analysis ◽  
Drosophila Melanogaster ◽  
Gene Family ◽  
Genomic Sequence ◽  
Sequence Similarity ◽  
Energy Levels ◽  
Metabolic Stress ◽  
Gene Copy ◽  
Gamma Subunit ◽  
Snf1 Kinase

To optimize gene expression under different environmental conditions, many organisms have evolved systems which can quickly up- and down-regulate the activity of other genes. Recently, the SNF1 kinase complex from yeast and the AMP-activated protein kinase complex from mammals have been shown to represent homologous metabolic sensors that are key to regulating energy levels under times of metabolic stress. Using heterologous probing, we have cloned the Drosophila melanogaster homologue of SNF4, the noncatalytic effector subunit from this kinase complex. A sequence corresponding to the partial genomic sequence as well as the full-length cDNA was obtained, and shows that the D. melanogaster SNF4 is encoded in a 1944-bp cDNA representing a protein of 648 amino acids (aa). Southern analysis of Drosophila genomic DNA in concert with a survey of mammalian SNF4 ESTs indicates that in metazoans, SNF4 is a duplicated gene, and possibly even a larger gene family. We propose that one gene copy codes for a short (330 aa) protein, whereas the second locus codes for a longer version (<410 aa) that is extended at the carboxy terminus, as typified by the Drosophila homologue presented here. Phylogenetic analysis of yeast, invertebrate, and multiple mammalian isoforms of SNF4 shows that the gene duplication likely occurred early in the metazoan lineage, as the protein products of the different loci are relatively divergent. When the phylogeny was extended beyond the SNF4 gene family, SNF4 shares sequence similarity with other cystathionine-β-synthase domain-containing proteins, including IMP dehydrogenase and a variety of uncharacterized Methanococcus proteins.Key words: SNF4, AMPK gamma subunit, derepression, gene family, phylogeny.

scholarly journals Microbial community composition and abundance after millennia of submarine permafrost warming

2019 ◽  
Author(s):  
Julia Mitzscherling ◽  
Fabian Horn ◽  
Maria Winterfeld ◽  
Linda Mahler ◽  
Jens Kallmeyer ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Pore Water ◽  
Negative Correlation ◽  
Microbial Community Composition ◽  
Microbial Abundance ◽  
Bacterial Gene ◽  
Gene Abundance ◽  
Permafrost Warming

Abstract. Warming of the Arctic led to an increase of permafrost temperatures by about 0.3 °C during the last decade. Permafrost warming is associated with increasing sediment water content, permeability and diffusivity and could on the long-term alter microbial community composition and abundance even before permafrost thaws. We studied the long-term effect (up to 2500 years) of submarine permafrost warming on microbial communities along an onshore-offshore transect on the Siberian Arctic Shelf displaying a natural temperature gradient of more than 10 °C. We analysed the in-situ development of bacterial abundance and community composition through total cell counts (TCC), quantitative PCR of bacterial gene abundance and amplicon sequencing, and correlated the microbial community data with temperature, pore water chemistry and sediment physicochemical parameters. On time-scales of centuries, permafrost warming coincided with an overall decreasing microbial abundance while millennia after warming microbial abundance was similar to cold onshore permafrost and DOC content was least. Based on correlation analysis TCC unlike bacterial gene abundance showed a significant rank-based negative correlation with increasing temperature while both TCC and bacterial gene copy numbers showed a negative correlation with salinity. Bacterial community composition correlated only weakly with temperature but strongly with pore-water stable isotope signatures and depth, while it showed no correlation with salinity. Microbial community composition showed substantial spatial variation and an overall dominance of Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes and Proteobacteria which are amongst the microbial taxa that were found to be active in other frozen permafrost environments as well. We suggest that, millennia after permafrost warming by over 10 °C, microbial community composition and abundance show some indications for proliferation but mainly reflect the sedimentation history and paleo-environment and not a direct effect through warming.

scholarly journals Red Clover Hydroxycinnamoyl-CoA:L-DOPA Hydroxycinnamoyl Transferase, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides

2021 ◽  
Vol 12 ◽  
Author(s):  
Michael L. Sullivan ◽  
Benjamin J. Knollenberg
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Aromatic Amino Acids ◽  
Red Clover ◽  
In Planta ◽  
Degenerate Primers ◽  
Coa Transferase ◽  
Rapid Amplification ◽  
Coa Thioesters ◽  
High Degree

Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play roles in protecting the plant from biotic and abiotic stresses but can also help preserve protein during harvest and storage of the forage via oxidation by an endogenous polyphenol oxidase. We previously identified and characterized, a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. A combination of PCR with degenerate primers based on BAHD hydroxycinnamoyl-CoA transferase sequences and 5′ and 3′ rapid amplification of cDNA ends was used to clone two nearly identical cDNAs from red clover. When expressed in Escherichia coli, the encoded proteins were capable of transferring hydroxycinnamic acids (p-coumaric, caffeic, or ferulic) from the corresponding CoA thioesters to the aromatic amino acids L-Phe, L-Tyr, L-DOPA, or L-Trp. Kinetic parameters for these substrates were determined. Stable expression of HDT in transgenic alfalfa resulted in foliar accumulation of p-coumaroyl- and feruloyl-L-Tyr that are not normally present in alfalfa, but not derivatives containing caffeoyl or L-DOPA moieties. Transient expression of HDT in Nicotiana benthamiana resulted in the production of caffeoyl-L-Tyr, but not clovamide. Coexpression of HDT with a tyrosine hydroxylase resulted in clovamide accumulation, indicating the host species’ pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.

scholarly journals Arid Ecosystem Vegetation Canopy-Gap Dichotomy: Influence on Soil Microbial Composition and Nutrient Cycling Functional Potential

2020 ◽  
Vol 87 (5) ◽  
Author(s):  
Priyanka Kushwaha ◽  
Julia W. Neilson ◽  
Albert Barberán ◽  
Yongjian Chen ◽  
Catherine G. Fontana ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Community Composition ◽  
Functional Capacity ◽  
N Mineralization ◽  
Organic N ◽  
Arid Ecosystem ◽  
Gene Abundance ◽  
Soil Microbial ◽  
Functional Potential ◽  
The Impact

ABSTRACT Increasing temperatures and drought in desert ecosystems are predicted to cause decreased vegetation density combined with barren ground expansion. It remains unclear how nutrient availability, microbial diversity, and the associated functional capacity vary between the vegetated canopy and gap soils. The specific aim of this study was to characterize canopy versus gap microsite effect on soil microbial diversity, the capacity of gap soils to serve as a canopy soil microbial reservoir, nitrogen (N)-mineralization genetic potential (ureC gene abundance) and urease enzyme activity, and microbial-nutrient pool associations in four arid-hyperarid geolocations of the western Sonoran Desert, Arizona, United States. Microsite combined with geolocation explained 57% and 45.8% of the observed variation in bacterial/archaeal and fungal community composition, respectively. A core microbiome of amplicon sequence variants was shared between the canopy and gap soil communities; however, canopy soils included abundant taxa that were not present in associated gap communities, thereby suggesting that these taxa cannot be sourced from the associated gap soils. Linear mixed-effects models showed that canopy soils have significantly higher microbial richness, nutrient content, and organic N-mineralization genetic and functional capacity. Furthermore, ureC gene abundance was detected in all samples, suggesting that ureC is a relevant indicator of N mineralization in deserts. Additionally, novel phylogenetic associations were observed for ureC, with the majority belonging to Actinobacteria and uncharacterized bacteria. Thus, key N-mineralization functional capacity is associated with a dominant desert phylum. Overall, these results suggest that lower microbial diversity and functional capacity in gap soils may impact ecosystem sustainability as aridity drives open-space expansion in deserts. IMPORTANCE Increasing aridity will drive a shift in desert vegetation and interspace gap (microsite) structure toward gap expansion. To evaluate the impact of gap expansion, we assess microsite effects on soil nutrients, microbiome community composition and functional capacity, and the potential of gap soils to serve as microbial reservoirs for plant root-associated microbiomes in an arid ecosystem. Results indicate that gap soils have significantly lower bioavailable nutrients, microbial richness, and N-mineralization functional capacity. Further, abundance of the bacterial urease gene (ureC) correlates strongly with N availability, and its major phylogenetic association is with Actinobacteria, the dominant phylum found in deserts. This finding is relevant because it identifies an important N-mineralization capacity indicator in the arid soil microbiome. Such indicators are needed to understand the relationships between interplant gap expansion and microbial diversity and functional potential associated with plant sustainability. This will be a critical step in recovery of land degraded by aridity stress.

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