AtxA-Controlled Small RNAs of Bacillus anthracis Virulence Plasmid pXO1 Regulate Gene Expression in trans

Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.

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atxA Controls Bacillus anthracis Capsule Synthesis via acpA and a Newly Discovered Regulator, acpB

Journal of Bacteriology ◽

10.1128/jb.186.2.307-315.2004 ◽

2004 ◽

Vol 186 (2) ◽

pp. 307-315 ◽

Cited By ~ 67

Author(s):

Melissa Drysdale ◽

Agathe Bourgogne ◽

Susan G. Hilsenbeck ◽

Theresa M. Koehler

Keyword(s):

Gene Expression ◽

Bacillus Anthracis ◽

Gene Transcription ◽

Double Mutant ◽

Regulatory Proteins ◽

Toxin Gene ◽

Virulence Plasmid ◽

Predicted Amino Acid Sequence ◽

Null Mutant ◽

Virulence Plasmids

ABSTRACT Two regulatory genes, acpA and atxA, have been reported to control expression of the Bacillus anthracis capsule biosynthesis operon capBCAD. The atxA gene is located on the virulence plasmid pXO1, while pXO2 carries acpA and the cap genes. acpA has been viewed as the major regulator of the cap operon because it is essential for capsule gene expression in a pXO1− pXO2+ strain. atxA is essential for toxin gene transcription but has also been implicated in control of the cap genes. The molecular functions of the regulatory proteins are unknown. We examined cap gene expression in a genetically complete pXO1+ pXO2+ strain. Our results indicate that another pXO2 gene, acpB (previously called pXO2-53; accession no. NC002146.1 :49418-50866), has a role in cap expression. The predicted amino acid sequence of AcpB is 62% similar to that of AcpA and 50% similar to that of AtxA. Assessment of cap gene transcription revealed that cap expression was not affected in a pXO1+ pXO2+ acpB-null mutant and was slightly reduced in an isogenic acpA mutant. However, cap gene expression was abolished in an acpA acpB double mutant. Microscopic examination of capsule synthesis by the mutants corroborated these findings. acpA and acpB expression is controlled by atxA; capsule synthesis and transcription of acpA and acpB were markedly reduced in an atxA mutant. The data suggest that, in a strain containing both virulence plasmids, atxA is the major regulator of capsule synthesis and controls capBCAD expression indirectly, via positive regulation of acpA and acpB.

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The Global Regulator CodY Regulates Toxin Gene Expression in Bacillus anthracis and Is Required for Full Virulence

Infection and Immunity ◽

10.1128/iai.00716-09 ◽

2009 ◽

Vol 77 (10) ◽

pp. 4437-4445 ◽

Cited By ~ 60

Author(s):

Willem van Schaik ◽

Alice Château ◽

Marie-Agnès Dillies ◽

Jean-Yves Coppée ◽

Abraham L. Sonenshein ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Anthracis ◽

Mutant Strain ◽

Virulence Gene ◽

Etiologic Agent ◽

Toxin Gene ◽

Promoter Regions ◽

Virulence Gene Expression ◽

Toxin Gene Expression ◽

Virulence Regulator

ABSTRACT In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis, the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis. Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA, as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally.

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Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

Vanderbilt Undergraduate Research Journal ◽

10.15695/vurj.v9i0.3772 ◽

2013 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Tara A Shrout

Keyword(s):

Gene Expression ◽

Transcriptional Control ◽

Striated Muscle ◽

Binding Capacity ◽

Structural Features ◽

Neonatal Rat ◽

Regulatory Control ◽

Untranslated Regions ◽

Luciferase Reporter ◽

Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

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Establishing Standard Protocols for Bacterial Culture in Biological Research in Canisters (BRIC) Hardware

Gravitational and Space Research ◽

10.2478/gsr-2016-0013 ◽

2020 ◽

Vol 4 (2) ◽

pp. 58-69 ◽

Cited By ~ 4

Author(s):

Patricia Fajardo-Cavazos ◽

Wayne L. Nicholson

Keyword(s):

Gene Expression ◽

Bacterial Culture ◽

Culture Conditions ◽

Media Culture ◽

Ground Control ◽

Biological Research ◽

Confounding Factors ◽

Cross Experiment ◽

Spaceflight Experiments ◽

Control Samples

AbstractThe NASA GeneLab Data System (GLDS) was recently developed to facilitate cross-experiment comparisons in order to understand the response of microorganisms to the human spaceflight environment. However, prior spaceflight experiments have been conducted using a wide variety of different hardware, media, culture conditions, and procedures. Such confounding factors could potentially mask true differences in gene expression between spaceflight and ground control samples. In an attempt to mitigate such confounding factors, we describe here the development of a standardized set of hardware, media, and protocols for liquid cultivation of microbes in Biological Research in Canisters (BRIC) spaceflight hardware, using the model bacteria Bacillus subtilis strain 168 and Staphylococcus aureus strain UAMS-1 as examples.

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Mechanical culture conditions effect gene expression

10.2514/6.2001-5016 ◽

2001 ◽

Author(s):

J. Love ◽

T. Hammond ◽

P. Allen ◽

L. Cubano ◽

T. Baker ◽

...

Keyword(s):

Gene Expression ◽

Culture Conditions ◽

Effect Gene

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Study of Dosage Compensation in Drosophila

Genetics ◽

10.1093/genetics/165.3.1167 ◽

2003 ◽

Vol 165 (3) ◽

pp. 1167-1181

Author(s):

Pei-Wen Chiang ◽

David M Kurnit

Keyword(s):

Gene Expression ◽

X Chromosome ◽

Dosage Compensation ◽

Qpcr Assay ◽

Male And Female ◽

Multiple Genes ◽

Chromosomal Changes ◽

Regulatory Effects

Abstract Using a sensitive RT-QPCR assay, we analyzed the regulatory effects of sex and different dosage compensation mutations in Drosophila. To validate the assay, we showed that regulation for several genes indeed varied with the number of functional copies of that gene. We then confirmed that dosage compensation occurred for most genes we examined in male and female flies. Finally, we examined the effects on regulation of several genes in the MSL pathway, presumed to be involved in sex-dependent determination of regulation. Rather than seeing global alterations of either X chromosomal or autosomal genes, regulation of genes on either the X chromosome or the autosomes could be elevated, depressed, or unaltered between sexes in unpredictable ways for the various MSL mutations. Relative dosage for a given gene between the sexes could vary at different developmental times. Autosomal genes often showed deranged regulatory levels, indicating they were in pathways perturbed by X chromosomal changes. As exemplified by the BR-C locus and its dependent Sgs genes, multiple genes in a given pathway could exhibit coordinate regulatory modulation. The variegated pattern shown for expression of both X chromosomal and autosomal loci underscores the complexity of gene expression so that the phenotype of MSL mutations does not reflect only simple perturbations of genes on the X chromosome.

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Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

Scientific Reports ◽

10.1038/s41598-021-82853-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kornphimol Kulthong ◽

Guido J. E. J. Hooiveld ◽

Loes Duivenvoorde ◽

Ignacio Miro Estruch ◽

Victor Marin ◽

...

Keyword(s):

Gene Expression ◽

Culture Conditions ◽

Gene Set Enrichment Analysis ◽

Shear Stresses ◽

Static Culture ◽

Dynamic Culture ◽

Intestinal Segments ◽

On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

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Comparison of the Essential Cellular Functions of the Two murA Genes of Bacillus anthracis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01594-07 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2009-2013 ◽

Cited By ~ 9

Author(s):

G. C. Kedar ◽

Vickie Brown-Driver ◽

Daniel R. Reyes ◽

Mark T. Hilgers ◽

Mark A. Stidham ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Anthracis ◽

High Resistance ◽

Gene Replacement ◽

Cellular Growth ◽

Cellular Functions

ABSTRACT Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug.

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Polycombs and microRNA-223 regulate human granulopoiesis by transcriptional control of target gene expression

Blood ◽

10.1182/blood-2011-08-371344 ◽

2012 ◽

Vol 119 (17) ◽

pp. 4034-4046 ◽

Cited By ~ 101

Author(s):

Giuseppe Zardo ◽

Alberto Ciolfi ◽

Laura Vian ◽

Linda M. Starnes ◽

Monia Billi ◽

...

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Transcriptional Control ◽

Transcriptional Level ◽

Seed Region ◽

Mrna Targets ◽

Epigenetic Events ◽

Evolutionarily Conserved ◽

Lineage Decision ◽

Chromatin Remodeling Complexes

Abstract Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin “bivalent domains,” hypermethylation, recruitment of polycomb (PcG)–RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.

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