The Gastrointestinal Tract of the White-Throated Woodrat (Neotoma albigula) Harbors Distinct Consortia of Oxalate-Degrading Bacteria

ABSTRACTThe microbiota inhabiting the mammalian gut is a functional organ that provides a number of services for the host. One factor that may regulate the composition and function of gut microbial communities is dietary toxins. Oxalate is a toxic plant secondary compound (PSC) produced in all major taxa of vascular plants and is consumed by a variety of animals. The mammalian herbivoreNeotoma albigulais capable of consuming and degrading large quantities of dietary oxalate. We isolated and characterized oxalate-degrading bacteria from the gut contents of wild-caught animals and used high-throughput sequencing to determine the distribution of potential oxalate-degrading taxa along the gastrointestinal tract. Isolates spanned three genera:Lactobacillus,Clostridium, andEnterococcus. Over half of the isolates exhibited significant oxalate degradationin vitro, and allLactobacillusisolates contained theoxcgene, one of the genes responsible for oxalate degradation. Although diverse potential oxalate-degrading genera were distributed throughout the gastrointestinal tract, they were most concentrated in the foregut, where dietary oxalate first enters the gastrointestinal tract. We hypothesize that unique environmental conditions present in each gut region provide diverse niches that select for particular functional taxa and communities.

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Effect of Dietary Oxalate on the Gut Microbiota of the Mammalian Herbivore Neotoma albigula

Applied and Environmental Microbiology ◽

10.1128/aem.00216-16 ◽

2016 ◽

Vol 82 (9) ◽

pp. 2669-2675 ◽

Cited By ~ 26

Author(s):

Aaron W. Miller ◽

Kelly F. Oakeson ◽

Colin Dale ◽

M. Denise Dearing

Keyword(s):

Gut Microbiota ◽

High Throughput Sequencing ◽

Fecal Microbiota ◽

Content Type ◽

Form And Function ◽

Plant Secondary Compound ◽

Mammalian Herbivore ◽

And Function ◽

Dietary Oxalate

ABSTRACTDiet is one of the primary drivers that sculpts the form and function of the mammalian gut microbiota. However, the enormous taxonomic and metabolic diversity held within the gut microbiota makes it difficult to isolate specific diet-microbe interactions. The objective of the current study was to elucidate interactions between the gut microbiota of the mammalian herbivoreNeotoma albigulaand dietary oxalate, a plant secondary compound (PSC) degraded exclusively by the gut microbiota. We quantified oxalate degradation inN. albigulafed increasing amounts of oxalate over time and tracked the response of the fecal microbiota using high-throughput sequencing. The amount of oxalate degradedin vivowas linearly correlated with the amount of oxalate consumed. The addition of dietary oxalate was found to impact microbial species diversity by increasing the representation of certain taxa, some of which are known to be capable of degrading oxalate (e.g.,Oxalobacterspp.). Furthermore, the relative abundances of 117 operational taxonomic units (OTU) exhibited a significant correlation with oxalate consumption. The results of this study indicate that dietary oxalate induces complex interactions within the gut microbiota that include an increase in the relative abundance of a community of bacteria that may contribute either directly or indirectly to oxalate degradation in mammalian herbivores.

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Advances in aptamer evolution and engineering

10.32469/10355/60401 ◽

2016 ◽

Author(s):

◽

Khalid Kamal Alam

Keyword(s):

High Throughput Sequencing ◽

Target Selection ◽

Three Dimensional ◽

Rna Aptamers ◽

Hiv Reverse Transcriptase ◽

University Of Missouri ◽

Selection Approach ◽

And Function ◽

The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Aptamers are single-stranded nucleic acids that fold into unique three-dimensional shapes that allow them to bind with high affinity and specificity to targets of interest. They are selected through the process of in vitro evolution, wherein large libraries of randomized sequence are iteratively partitioned and amplified to enrich for high-fitness, functional molecules. Selected libraries are sequenced and individual aptamers are characterized for their structure and function. Aptamers have found use as research tools, diagnostics, and therapeutics and in the control of biological systems. The work described herein presents several advancements to the selection and application of aptamers. I first describe an aptamer bioinformatics platform, FASTAptamer, which performs the primary sequence tasks common to all combinatorial selection techniques. I then describe a poly-target selection approach that leverages high-throughput sequencing, the aptamer bioinformatics platform, and parallel selections against a family of related targets to identify the first RNA aptamers capable of potent broad-spectrum inhibition of HIV reverse transcriptase. Finally, this work describes the engineering and in vitro validation of a bifurcated aptamer, Split-Broccoli, for direct visualization of RNA:RNA processes.

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How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions

mBio ◽

10.1128/mbio.01559-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Nicolas Kint ◽

Carolina Alves Feliciano ◽

Maria C. Martins ◽

Claire Morvan ◽

Susana F. Fernandes ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Anaerobic Bacteria ◽

Growth Defect ◽

Strictly Anaerobic ◽

Low Oxygen ◽

Air Exposure ◽

Vegetative Cells ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

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Intestinal drug transporters in pathological states: an overview

Pharmacological Reports ◽

10.1007/s43440-020-00139-6 ◽

2020 ◽

Vol 72 (5) ◽

pp. 1173-1194 ◽

Cited By ~ 1

Author(s):

Marek Drozdzik ◽

Izabela Czekawy ◽

Stefan Oswald ◽

Agnieszka Drozdzik

Keyword(s):

Gastrointestinal Tract ◽

Kidney Failure ◽

Drug Transporters ◽

Drug Bioavailability ◽

Gastrointestinal Pathology ◽

Endogenous Compounds ◽

Clinical Consequences ◽

Drug Actions ◽

And Function

Abstract Emerging information suggests that gastrointestinal and systemic pathology states may affect expression and function of membrane transporters in the gastrointestinal tract. Altered status of the transporters could affect drug as well as endogenous compounds handling with subsequent clinical consequences. It seems that in some pathologies, e.g., liver or kidney failure, changes in the intestinal transporter function provide compensatory functions, eliminating substrates excreted by dysfunctional organs. A literature search was conducted on Ovid and Pubmed databases to select relevant in vitro, animal and human studies that have reported expression, protein abundance and function of intestinal drug transporters. The accumulated data suggest that gastrointestinal pathology (inflammatory bowel disease, celiac disease, cholestasis) as well as systemic pathologies (kidney failure, liver failure, hyperthyroidism, hyperparathyroidism, obesity, diabetes mellitus, systemic inflammation and Alzheimer disease) may affect drug transporter expression and function in the gastrointestinal tract. The altered status of drug transporters may provide compensatory activity in handling endogenous compounds, affect local drug actions in the gastrointestinal tract as well as impact drug bioavailability. Graphic abstract

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Elucidation of Insertion Elements Carried on Plasmids andIn VitroConstruction of Shuttle Vectors from the Toxic Cyanobacterium Planktothrix

Applied and Environmental Microbiology ◽

10.1128/aem.01188-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4887-4897 ◽

Cited By ~ 11

Author(s):

Guntram Christiansen ◽

Alexander Goesmann ◽

Rainer Kurmayer

Keyword(s):

Homologous Recombination ◽

High Throughput ◽

High Throughput Sequencing ◽

Gene Clusters ◽

Content Type ◽

Shuttle Vectors ◽

Is Element ◽

Toxic Cyanobacterium ◽

Is Elements

ABSTRACTSeveral gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacteriumPlanktothrix agardhiiNIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively)in vitroby PCR amplification and the subsequent transposition of the Tn5 cattransposon containing the R6Kγ origin of replication ofEscherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cattransposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing andin vitroproduction of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.

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Extrahepatic Drug Transporters in Liver Failure: Focus on Kidney and Gastrointestinal Tract

International Journal of Molecular Sciences ◽

10.3390/ijms21165737 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5737 ◽

Cited By ~ 1

Author(s):

Marek Droździk ◽

Stefan Oswald ◽

Agnieszka Droździk

Keyword(s):

Gastrointestinal Tract ◽

Liver Failure ◽

Liver Dysfunction ◽

Drug Transporters ◽

Endogenous Compounds ◽

Clinical Consequences ◽

Drug Actions ◽

And Function ◽

Drug Pharmacokinetics

Emerging information suggests that liver pathological states may affect the expression and function of membrane transporters in the gastrointestinal tract and the kidney. Altered status of the transporters could affect drug as well as endogenous compounds handling with subsequent clinical consequences. It seems that changes in intestinal and kidney transporter functions provide the compensatory activity of eliminating endogenous compounds (e.g., bile acids) generated and accumulated due to liver dysfunction. A literature search was conducted on the Ovid and PubMed databases to select relevant in vitro, animal and human studies that have reported expression, protein abundance and function of the gastrointestinal and kidney operating ABC (ATP-binding cassette) transporters and SLC (solute carriers) carriers. The accumulated data suggest that liver failure-associated transporter alterations in the gastrointestinal tract and kidney may affect drug pharmacokinetics. The altered status of drug transporters in those organs in liver dysfunction conditions may provide compensatory activity in handling endogenous compounds, affecting local drug actions as well as drug pharmacokinetics.

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The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

mBio ◽

10.1128/mbio.00448-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Rogério F. Lourenço ◽

Saumya Saurabh ◽

Jonathan Herrmann ◽

Soichi Wakatsuki ◽

Lucy Shapiro

Keyword(s):

Dna Binding ◽

Caulobacter Crescentus ◽

Genetic Material ◽

Bacterial Cells ◽

Structural Elements ◽

Content Type ◽

Time Dynamic ◽

Associated Proteins ◽

And Function

ABSTRACT Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA. IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

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Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology

International Journal of Molecular Sciences ◽

10.3390/ijms21228774 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8774

Author(s):

Natalia Komarova ◽

Daria Barkova ◽

Alexander Kuznetsov

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

High Throughput Sequencing ◽

Combinatorial Libraries ◽

Structure And Function ◽

Huge Number ◽

Systematic Evolution ◽

And Function ◽

Exponential Enrichment

Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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Characterization of Intracellular Growth RegulatoricgRby Utilizing Transcriptomics To Identify Mediators of Pathogenesis in Shigella flexneri

Infection and Immunity ◽

10.1128/iai.00537-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3068-3076 ◽

Cited By ~ 8

Author(s):

Carolyn R. Morris ◽

Christen L. Grassel ◽

Julia C. Redman ◽

Jason W. Sahl ◽

Eileen M. Barry ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Diarrheal Disease ◽

Intracellular Growth ◽

Bioinformatics Analyses ◽

Content Type ◽

Regulatory Pathways

ABSTRACTShigellaspecies Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods within vitroandin vivoassays to provide new insights into pathogenesis. Comparisons ofin vivoandin vitrogene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present inS. flexneriisolates but not in the three otherShigellaspecies. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific toS. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designatedicgR, forintracellulargrowthregulator. Deletion oficgRcaused no difference in growthin vitrobut resulted in increased intracellular replication in HCT-8 cells. Furtherin vitroandin vivostudies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ΔicgRmutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown inShigellapathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

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