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  • 1
    Language: English
    In: Nucleic acids research, 2007, Vol.35(4), pp.1279-88
    Description: Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative sigma factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase sigma(S). We identified stress conditions that induce high MicA levels even during exponential growth-a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor sigma(E) is activated. The strict dependence of micA transcription on sigma(E) is supported by three observations. Induced overexpression of sigma(E) increases micA transcription, an DeltarpoE mutant displays undetectable MicA levels and the micA promoter has the consensus sigma(E) signature. Thus, MicA is part of the sigma(E) regulon and downregulates its target gene, ompA, probably to alleviate membrane stress.
    Keywords: Gene Expression Regulation, Bacterial ; Escherichia Coli -- Genetics ; RNA, Antisense -- Biosynthesis ; RNA, Bacterial -- Biosynthesis ; Sigma Factor -- Metabolism ; Transcription Factors -- Metabolism
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 2
    Language: English
    In: Nucleic Acids Research, 2018, Vol. 46(8), pp. 4188-4199
    Description: Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5-tails-as standby sites-with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats-and to a lesser extent, AU-repeats-dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16-18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem-loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails.
    Keywords: Natural Sciences ; Biological Sciences ; Biochemistry And Molecular Biology ; Naturvetenskap ; Biologiska Vetenskaper ; Biokemi Och Molekylärbiologi
    ISSN: 0305-1048
    E-ISSN: 13624962
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  • 3
    Language: English
    In: Nucleic Acids Research, 2013, Vol. 41(12), p. e122
    Description: We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5'-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.
    Keywords: Natural Sciences ; Naturvetenskap ; Medical And Health Sciences ; Medicin Och Hälsovetenskap
    ISSN: 0305-1048
    E-ISSN: 13624962
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  • 4
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2019, Vol. 116(32), pp. 15901-15906
    Description: In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30 Delta S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (similar to 100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.
    Keywords: Translation Initiation ; Ribosome Standby Site ; Rna Secondary Structure ; Ribosomal Protein S1 ; Fluorescence Anisotropy ; Natural Sciences ; Biological Sciences ; Microbiology ; Naturvetenskap ; Biologiska Vetenskaper ; Mikrobiologi ; Natural Sciences ; Biological Sciences ; Biochemistry And Molecular Biology ; Naturvetenskap ; Biologiska Vetenskaper ; Biokemi Och Molekylärbiologi
    ISSN: 0027-8424
    E-ISSN: 10916490
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  • 5
    In: Applied and Environmental Microbiology, 2005, Vol. 71(11), p.7292
    Description: Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of this organism as a biocontrol agent has not been very efficient. In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting bacterium Paenibacillus polymyxa, 2003). Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a plasmid-borne gfp gene. Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly the root tip, where they formed biofilms. Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder. Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues. Studies were performed in both a gnotobiotic system and a soil system. The fact that similar observations were made in both systems suggests that colonization by this bacterium can be studied in a more defined system. Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant growth-promoting bacteria are discussed. ; Includes references ; p. 7292-7300.
    Keywords: Root Tips ; Green Fluorescent Protein ; Bacterial Colonization ; Plant Growth-Promoting Rhizobacteria ; Intercellular Spaces ; Arabidopsis Thaliana ; Biofilm ; Roots ; Reporter Genes ; Gene Expression ; Paenibacillus Polymyxa;
    ISSN: 0099-2240
    ISSN: 00992240
    E-ISSN: 10985336
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  • 6
    In: Molecular Microbiology, March 2017, Vol.103(6), pp.1020-1033
    Description: Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR‐1 toxin‐antitoxin system of to appreciate the role of the RNA antitoxin IstR‐1 in TisB‐dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug‐tolerant by arresting growth. The RNA antitoxin IstR‐1 sets a threshold for TisB‐dependent depolarization under DNA‐damaging conditions, resulting in two sub‐populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5′ UTR structure in the mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub‐population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity. In Escherichia coli, two regulatory RNA elements set a threshold for production of toxin TisB and, as a consequence, depolarization of the inner membrane upon DNA damage. Downstream generation of persister cells is considered as “primed” by DNA damage as environmental trigger. Deletion of both RNA elements renders persister formation “stochastic”, since no environmental trigger is needed for TisB production in the double deletion strain.
    Keywords: Antitoxins ; Decision Making ; Translation ; DNA Damage ; Inner Membranes ; Survival ; Stochasticity ; Toxins ; Escherichia Coli ; DNA Metabolism & Structure;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 7
    Language: English
    In: Scientific Reports, 2017, Vol. 7
    Description: CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.
    Keywords: Natural Sciences ; Biological Sciences ; Naturvetenskap ; Biologiska Vetenskaper
    ISSN: 2045-2322
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  • 8
    In: Molecular Microbiology, May 2012, Vol.84(3), pp.414-427
    Description: Roughly 10% of all genes in are controlled by the global transcription factor Lrp, which responds to nutrient availability. Bioinformatically, we identified as one of several putative targets for the sRNA MicF, which is transcriptionally downregulated by Lrp. Deleting results in higher Lrp levels, while overexpression of MicF inhibits Lrp synthesis. This effect is by antisense; mutations in the predicted interaction region relieve MicF‐dependent repression of Lrp synthesis, and regulation is restored by compensatory mutations. , MicF sterically interferes with initiation complex formation and inhibits mRNA translation. , MicF indirectly activates genes in the Lrp regulon by repressing Lrp, and causes severely impaired growth in minimal medium, a phenotype characteristic of deletion strains. The double negative feedback between MicF and Lrp may promote a switch for adequate Lrp‐dependent adaptation to nutrient availability. Lrp adds to the growing list of transcription factors that are targeted by sRNAs, thus indicating that perhaps the majority of all bacterial genes may be directly or indirectly controlled by sRNAs.
    Keywords: Biology;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 9
    Language: English
    In: The Journal of biological chemistry, 12 September 2003, Vol.278(37), pp.35558-63
    Description: The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems. This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B'. Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or pi-turn) in the loop of CopT might determine the directionality of helix progression. Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics. Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended. In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn.
    Keywords: RNA, Antisense -- Chemistry
    ISSN: 0021-9258
    E-ISSN: 1083351X
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  • 10
    Language: English
    In: mBio, 2016, Vol. 7(6)
    Description: VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF(30) (30 kDa), and the shorter VirF(21) (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF(30) and VirF(21) and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF(21) is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF(21). The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF(30) is responsible for activation of the virulence system, VirF(21) negatively autoregulates virF expression itself. Since VirF(21) modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. IMPORTANCE Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF(30) and VirF(21), that are functionally distinct. The major form, VirF(30), activates the genes necessary for virulence, whereas the minor VirF(21), which shares the C-terminal two-thirds of VirF(30), negatively autoregulates virF expression itself. VirF(21) is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.
    Keywords: Natural Sciences ; Biological Sciences ; Microbiology ; Naturvetenskap ; Biologiska Vetenskaper ; Mikrobiologi
    ISSN: 2161-2129
    ISSN: 21507511
    E-ISSN: 21507511
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