Mixed-Species Genomic Microarray Analysis of Fecal Samples Reveals Differential Transcriptional Responses of Bifidobacteria in Breast- and Formula-Fed Infants

ABSTRACT Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto- and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions.

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Simulation of P2X-mediated calcium signaling in microglia

10.1101/354142 ◽

2018 ◽

Author(s):

Ben Chun ◽

Bradley D. Stewart ◽

Darin Vaughan ◽

Adam D. Bachstetter ◽

Peter M. Kekenes-Huskey

Keyword(s):

Computational Model ◽

Purinergic Receptor ◽

Receptor Activation ◽

Quantitative Model ◽

Published Data ◽

Transcriptional Responses ◽

Extracellular Adenosine ◽

Wide Range ◽

Intracellular Ca

AbstractMicroglia function is orchestrated through highly-coupled signaling pathways that depend on calcium (Ca2+). In response to extracellular adenosine triphosphate (ATP), transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis and potentially their release. While steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic puriner-gic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumor necrosis factor α (TNFα) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of NFAT transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our key findings are that TNFα production via P2X4 is maximized at low ATP when subject to high frequency ATP stimulation, whereas P2X7 contributes most significantly at millimolar ATPranges. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

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Amino Acid Substitutions in Yeast TFIIF Confer Upstream Shifts in Transcription Initiation and Altered Interaction with RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10975-10985.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10975-10985 ◽

Cited By ~ 45

Author(s):

Mohamed A. Ghazy ◽

Seth A. Brodie ◽

Michelle L. Ammerman ◽

Lynn M. Ziegler ◽

Alfred S. Ponticelli

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Primer Extension ◽

Site Directed Mutagenesis ◽

Protein Encoding ◽

Transcription Factor Iif ◽

Growth Properties ◽

Encoding Genes

ABSTRACT Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.

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Genome-Wide Effects of Selenium and Translational Uncoupling on Transcription in the Termite Gut Symbiont Treponema primitia

mBio ◽

10.1128/mbio.00869-13 ◽

2013 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Eric G. Matson ◽

Adam Z. Rosenthal ◽

Xinning Zhang ◽

Jared R. Leadbetter

Keyword(s):

Protein Synthesis ◽

Large Body ◽

Transcriptional Responses ◽

Translational Coupling ◽

Genome Wide ◽

A Genome ◽

Protein Encoding ◽

Termite Gut ◽

Inhibiting Protein ◽

Encoding Genes

ABSTRACTWhen prokaryotic cells acquire mutations, encounter translation-inhibiting substances, or experience adverse environmental conditions that limit their ability to synthesize proteins, transcription can become uncoupled from translation. Such uncoupling is known to suppress transcription of protein-encoding genes in bacteria. Here we show that the trace element selenium controls transcription of the gene for the selenocysteine-utilizing enzyme formate dehydrogenase (fdhFSec) through a translation-coupled mechanism in the termite gut symbiontTreponema primitia, a member of the bacterial phylumSpirochaetes. We also evaluated changes in genome-wide transcriptional patterns caused by selenium limitation and by generally uncoupling translation from transcription via antibiotic-mediated inhibition of protein synthesis. We observed that inhibiting protein synthesis inT. primitiainfluences transcriptional patterns in unexpected ways. In addition to suppressing transcription of certain genes, the expected consequence of inhibiting protein synthesis, we found numerous examples in which transcription of genes and operons is truncated far downstream from putative promoters, is unchanged, or is even stimulated overall. These results indicate that gene regulation in bacteria allows for specific post-initiation transcriptional responses during periods of limited protein synthesis, which may depend both on translational coupling and on unclassified intrinsic elements of protein-encoding genes.IMPORTANCEA large body of literature demonstrates that the coupling of transcription and translation is a general and essential method by which bacteria regulate gene expression levels. However, the potential role of noncanonical amino acids in regulating transcriptional output via translational control remains, for the most part, undefined. Furthermore, the genome-wide transcriptional state in response to translational decoupling is not well quantified. The results presented here suggest that the noncanonical amino acid selenocysteine is able to tune transcription of an important metabolic gene via translational coupling. Furthermore, a genome-wide analysis reveals that transcriptional decoupling produces a wide-ranging effect and that this effect is not uniform. These results exemplify how growth conditions that impact translational processivity can rapidly feed back on transcriptional productivity of prespecified groups of genes, providing bacteria with an efficient response to environmental changes.

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Comparative Sequence Analysis of Mycobacterium leprae and the New Leprosy-Causing Mycobacterium lepromatosis

Journal of Bacteriology ◽

10.1128/jb.00762-09 ◽

2009 ◽

Vol 191 (19) ◽

pp. 6067-6074 ◽

Cited By ~ 59

Author(s):

Xiang Y. Han ◽

Kurt C. Sizer ◽

Erika J. Thompson ◽

Juma Kabanja ◽

Jun Li ◽

...

Keyword(s):

16S Rrna ◽

Phylogenetic Trees ◽

Mycobacterium Leprae ◽

16S Rrna Genes ◽

Rrna Genes ◽

Neutral Evolution ◽

Rrna Gene ◽

Protein Encoding ◽

Mycobacterium Lepromatosis ◽

Encoding Genes

ABSTRACT Mycobacterium lepromatosis is a newly discovered leprosy-causing organism. Preliminary phylogenetic analysis of its 16S rRNA gene and a few other gene segments revealed significant divergence from Mycobacterium leprae, a well-known cause of leprosy, that justifies the status of M. lepromatosis as a new species. In this study we analyzed the sequences of 20 genes and pseudogenes (22,814 nucleotides). Overall, the level of matching of these sequences with M. leprae sequences was 90.9%, which substantiated the species-level difference; the levels of matching for the 16S rRNA genes and 14 protein-encoding genes were 98.0% and 93.1%, respectively, but the level of matching for five pseudogenes was only 79.1%. Five conserved protein-encoding genes were selected to construct phylogenetic trees and to calculate the numbers of synonymous substitutions (dS values) and nonsynonymous substitutions (dN values) in the two species. Robust phylogenetic trees constructed using concatenated alignment of these genes placed M. lepromatosis and M. leprae in a tight cluster with long terminal branches, implying that the divergence occurred long ago. The dS and dN values were also much higher than those for other closest pairs of mycobacteria. The dS values were 14 to 28% of the dS values for M. leprae and Mycobacterium tuberculosis, a more divergent pair of species. These results thus indicate that M. lepromatosis and M. leprae diverged ∼10 million years ago. The M. lepromatosis pseudogenes analyzed that were also pseudogenes in M. leprae showed nearly neutral evolution, and their relative ages were similar to those of M. leprae pseudogenes, suggesting that they were pseudogenes before divergence. Taken together, the results described above indicate that M. lepromatosis and M. leprae diverged from a common ancestor after the massive gene inactivation event described previously for M. leprae.

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What Is the Best Lens? Comparing the Resolution Power of Genome-Derived Markers and Standard Barcodes

Microorganisms ◽

10.3390/microorganisms9020299 ◽

2021 ◽

Vol 9 (2) ◽

pp. 299

Author(s):

Angela Conti ◽

Laura Corte ◽

Debora Casagrande Pierantoni ◽

Vincent Robert ◽

Gianluigi Cardinali

Keyword(s):

Single Copy ◽

Sensu Stricto ◽

Fungal Species ◽

Rrna Genes ◽

Taxonomic Resolution ◽

Protein Encoding ◽

The Many ◽

Next Generation Sequencing Ngs ◽

Encoding Genes

Fungal species delimitation was traditionally carried out with multicopy ribosomal RNA (rRNA) genes, principally for their ease of amplification. Since the efficacy of these markers has been questioned, single-copy protein-encoding genes have been proposed alone or in combination for Multi-Locus Sequence Typing (MLST). In this context, the role of the many sequences obtained with Next-Generation Sequencing (NGS) techniques, in both genomics and metagenomics, further pushes toward an analysis of the efficacy of NGS-derived markers and of the metrics to evaluate the marker efficacy in discriminating fungal species. This paper aims at proposing MeTRe (Mean Taxonomic Resolution), a novel index that could be used both for measuring marker efficacy and for assessing the actual resolution (i.e., the level of separation) between species obtained with different markers or their combinations. In this paper, we described and then employed this index to compare the efficacy of two rRNAs and four single-copy markers obtained from public databases as both an amplicon-based approach and genome-derived sequences. Two different groups of species were used, one with a pathogenic species of Candida that was characterized by relatively well-separated taxa, whereas the other, comprising some relevant species of the sensu stricto group of the genus Saccharomyces, included close species and interspecific hybrids. The results showed the ability of MeTRe to evaluate marker efficacy in general and genome-derived markers specifically.

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Characterization of Phosphopantetheinyl Hydrolase from Mycobacterium tuberculosis

Microbiology Spectrum ◽

10.1128/spectrum.00928-21 ◽

2021 ◽

Author(s):

Shilpika Pandey ◽

Amrita Singh ◽

Guangli Yang ◽

Felipe B. d’Andrea ◽

Xiuju Jiang ◽

...

Keyword(s):

Infectious Disease ◽

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Posttranslational Modification ◽

Content Type ◽

Protein Encoding ◽

And Function ◽

Encoding Genes

Tuberculosis (TB), caused by Mycobacterium tuberculosis , was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis ’s phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium’s cell wall and virulence.

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Bio-electrospraying the nematode Caenorhabditis elegans : studying whole-genome transcriptional responses and key life cycle parameters

Journal of The Royal Society Interface ◽

10.1098/rsif.2009.0364 ◽

2009 ◽

Vol 7 (45) ◽

pp. 595-601 ◽

Cited By ~ 22

Author(s):

Napachanok Mongkoldhumrongkul ◽

Suresh C. Swain ◽

Suwan N. Jayasinghe ◽

Stephen Stürzenbaum

Keyword(s):

Caenorhabditis Elegans ◽

Heat Shock ◽

Model Organism ◽

Laboratory Culture ◽

Whole Genome ◽

Transcriptional Responses ◽

Wide Range ◽

Shock Proteins ◽

Cellular Components

Bio-electrospray, the direct jet-based cell handling approach, is able to handle a wide range of cells (spanning immortalized, primary to stem cells). Studies at the genomic, genetic and the physiological levels have shown that, post-treatment, cellular integrity is unperturbed and a high percentage (more than 70%, compared with control) of cells remain viable. Although, these results are impressive, it may be argued that cell-based systems are oversimplistic. Therefore, it is important to evaluate the bio-electrospray technology using sensitive and dynamically developing multi-cellular organisms that share, at least some, similarities with multi-cell microenviorments encountered with tissues and organs. This study addressed this issue by using a well-characterized model organism, the non-parasitic nematode Caenorhabditis elegans . Nematode cultures were subjected to bio-electrospraying and compared with positive (heat shock) and negative controls (appropriate laboratory culture controls). Overall, bio-electrospraying did not modulate the reproductive output or induce significant changes in in vivo stress-responsive biomarkers (heat shock proteins). Likewise, whole-genome transcriptomics could not identify any biological processes, cellular components or molecular functions (gene ontology terms) that were significantly enriched in response to bio-electrospraying. This demonstrates that bio-electrosprays can be safely applied directly to nematodes and underlines its potential future use in the creation of multi-cellular environments within clinical applications.

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Complete Genome Sequence of the plant pathogenic fungus Colletotrichum lupini.

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-21-0173-a ◽

2021 ◽

Author(s):

Riccardo Baroncelli ◽

Flora Pensec ◽

Daniele Da Lio ◽

Thaís Regina Boufleur ◽

Isabel Vicente ◽

...

Keyword(s):

Plant Pathogens ◽

Pathogenic Fungus ◽

Lupinus Albus ◽

Genome Structure ◽

White Lupin ◽

Sequencing Technologies ◽

Wide Range ◽

Protein Encoding ◽

Colletotrichum Lupini ◽

Encoding Genes

Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into eleven nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41Mb and 36.55 Kb, respectively. A total of 18324 protein encoding genes have been predicted, of which only 39 are specific to C. lupini. To the best of our knowledge this is the first genome of this species to be fully sequenced and to get publicly released. This resource will provide insight into pathogenicity factors and will help to get a better understanding of the evolution and genome structure of this important plant pathogen.

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Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo.

Genetics ◽

10.1093/genetics/135.3.665 ◽

1993 ◽

Vol 135 (3) ◽

pp. 665-676 ◽

Cited By ~ 9

Author(s):

G Prelich ◽

F Winston

Keyword(s):

Protein Kinase ◽

Histone H3 ◽

Wild Type ◽

Protein Encoding ◽

Mutant Strains ◽

Upstream Activating Sequence ◽

Suc2 Gene ◽

Encoding Genes ◽

Type Strains

Abstract Regulated transcription of most protein-encoding genes in Saccharomyces cerevisiae requires an upstream activating sequence (UAS); in the absence of UAS elements, little or no transcription occurs. In certain mutant strains, however, promoters that have been deleted for their UAS can direct significant levels of transcription, indicating that the remaining promoter elements (the basal promoter) are capable of directing higher levels of transcription, but they are normally represented in wild-type strains. To analyze this repression, we have selected for mutations that cause increased transcription of the SUC2 gene in the absence of its UAS. In addition to some previously studied genes, this selection has identified five genes that we have designated BUR1, BUR2, BUR3, BUR5 and BUR6 (for Bypass UAS Requirement). The bur mutations cause pleiotropic phenotypes, indicating that they affect transcription of many genes. Furthermore, some bur mutations suppress the requirement for the SNF5 trans-activator at both SUC2 and Ty. Additional analysis has demonstrated that BUR1 is identical to SGV1, which encodes a CDC28-related protein kinase. This result indicates that protein phosphorylation is important for repression of the SUC2 basal promoter as well as other aspects of transcription in vivo. Finally, BUR5 is identical to HHT1, encoding histone H3, further implicating chromatin structure as important for expression of SUC2.

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Identification of Lactobacillus plantarum Genes That Are Induced in the Gastrointestinal Tract of Mice

Journal of Bacteriology ◽

10.1128/jb.186.17.5721-5729.2004 ◽

2004 ◽

Vol 186 (17) ◽

pp. 5721-5729 ◽

Cited By ~ 175

Author(s):

Peter A. Bron ◽

Corinne Grangette ◽

Annick Mercenier ◽

Willem M. de Vos ◽

Michiel Kleerebezem

Keyword(s):

Lactobacillus Plantarum ◽

Active Form ◽

Model System ◽

Hypothetical Proteins ◽

Gi Tract ◽

Protein Encoding ◽

Specific Factors ◽

Encoding Genes ◽

Harsh Conditions

ABSTRACT Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of environmental niches, including the human gastrointestinal (GI) tract. Moreover, this lactic acid bacterium can survive passage through the human or mouse stomach in an active form. To investigate the genetic background of this persistence, resolvase-based in vivo expression technology (R-IVET) was performed in L. plantarum WCFS1 by using the mouse GI tract as a model system. This approach identified 72 L. plantarum genes whose expression was induced during passage through the GI tract as compared to laboratory media. Nine of these genes encode sugar-related functions, including ribose, cellobiose, sucrose, and sorbitol transporter genes. Another nine genes encode functions involved in acquisition and synthesis of amino acids, nucleotides, cofactors, and vitamins, indicating their limited availability in the GI tract. Four genes involved in stress-related functions were identified, reflecting the harsh conditions that L. plantarum encounters in the GI tract. The four extracellular protein encoding genes identified could potentially be involved in interaction with host specific factors. The rest of the genes are part of several functionally unrelated pathways or encode (conserved) hypothetical proteins. Remarkably, a large number of the functions or pathways identified here have previously been identified in pathogens as being important in vivo during infection, strongly suggesting that survival rather than virulence is the explanation for the importance of these genes during host residence.

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