111 research outputs found

    H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during Their Integration in Host Chromosomes

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    <div><p>Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, <i>Escherichia coli</i> expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to <i>E</i>. <i>coli</i> survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three <i>E</i>. <i>coli</i> strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in <i>E</i>. <i>coli</i> strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the <i>ybdO</i> promoter and coding regions have diversified the potential for H-NS-independent negative regulation among <i>E</i>. <i>coli</i> strains. The <i>ybdO</i> expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during <i>E</i>. <i>coli</i> evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated <i>E</i>. <i>coli</i> adaptation to new ecological niches.</p></div

    Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli.

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    The horizontally transferred chromosomal segments, which are the main source of genetic diversity among bacterial pathogens, are bound by the nucleoid protein H-NS, resulting in the formation of a nucleoprotein complex and the silencing of gene expression. The de-silencing or activation of virulence genes necessary for the colonization of enterohemorrhagic Escherichia coli is achieved mainly by the action of two regulators, Pch and Ler, which are encoded by horizontally transferred elements. Although Ler has been shown to activate transcription by counteracting H-NS silencing, the mechanism for Pch is poorly understood. We show here that Pch activates the LEE1 promoter and also enhances the Ler-mediated activation of other LEE promoters. Transcriptional activation was completely dependent on repression by the H-NS/StpA/Hha/YdgT complex, indicating that Pch-derived activation was achieved by alleviating H-NS-mediated silencing. Expression of pch reduced the binding of H-NS at LEE1 promoter and altered the nucleoprotein complex. Furthermore, in vitro reconstruction of the protein-DNA complex on LEE1 promoter DNA confirmed the exclusive effect of Pch on H-NS binding. These results demonstrated that Pch is another anti-silencing regulator and a modulator of H-NS-containing nucleoprotein complexes. Thus, the anti-silencing mechanism plays a key role in the coordinated regulation of virulence genes in EHEC

    The dynamic balance of import and export of zinc in Escherichia coli suggests a heterogeneous population response to stress

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    Zinc is essential for life, but toxic in excess. Thus all cells must control their internal zinc concentration. We used a systems approach, alternating rounds of experiments and models, to further elucidate the zinc control systems in Escherichia coli. We measured the response to zinc of the main specific zinc import and export systems in the wild-type, and a series of deletion mutant strains. We interpreted these data with a detailed mathematical model and Bayesian model fitting routines. There are three key findings: first, that alternate, non-inducible importers and exporters are important. Second, that an internal zinc reservoir is essential for maintaining the internal zinc concentration. Third, our data fitting led us to propose that the cells mount a heterogeneous response to zinc: some respond effectively, while others die or stop growing. In a further round of experiments, we demonstrated lower viable cell counts in the mutant strain tested exposed to excess zinc, consistent with this hypothesis. A stochastic model simulation demonstrated considerable fluctuations in the cellular levels of the ZntA exporter protein, reinforcing this proposal. We hypothesize that maintaining population heterogeneity could be a bet-hedging response allowing a population of cells to survive in varied and fluctuating environments

    Antisense transcription regulates the expression of the enterohemorrhagic Escherichia coli virulence regulatory gene ler in response to the intracellular iron concentration.

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    Enteric pathogens, such as enterohemorrhagic E. coli (EHEC) O157:H7, encounter varying concentrations of iron during their life cycle. In the gastrointestinal tract, the amount of available free iron is limited because of absorption by host factors. EHEC and other enteric pathogens have developed sophisticated iron-responsive systems to utilize limited iron resources, and these systems are primarily regulated by the Fur repressor protein. The iron concentration could be a signal that controls gene expression in the intestines. In this study, we explored the role of iron in LEE (locus for enterocyte effacement) virulence gene expression in EHEC. In contrast to the expression of Fur-regulated genes, the expression of LEE genes was greatly reduced in fur mutants irrespective of the iron concentration. The expression of the ler gene, the LEE-encoded master regulator, was affected at a post-transcription step by fur mutation. Further analysis showed that the loss of Fur affected the translation of the ler gene by increasing the intracellular concentration of free iron, and the transcription of the antisense strand was necessary for regulation. The results indicate that LEE gene expression is closely linked to the control of intracellular free iron homeostasis

    High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq

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    Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences

    High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by <it>Bacillus subtilis</it>

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    Abstract Background Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system. Results Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1) of AmyK38 was obtained using strain MGB874. Conclusions We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.</p

    Complete Genome Sequence of Bacillus cereus NC7401, Which Produces High Levels of the Emetic Toxin Cereulide

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    We report the complete and annotated genome sequence of Bacillus cereus NC7401, a representative of a specific group of emetic strains. The emetic toxin, cereulide, is produced by a non-ribosomal protein synthesis (NRPS) system that is encoded by a gene cluster on a large resident plasmid, pNCcld.We report the complete and annotated genome sequence of Bacillus cereus NC7401, a representative of the strain group that causes emetic-type food poisoning. The emetic toxin, cereulide, is produced by a nonribosomal protein synthesis (NRPS) system that is encoded by a gene cluster on a large resident plasmid, pNCcld

    AMDORAP: Non-targeted metabolic profiling based on high-resolution LC-MS

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    <p>Abstract</p> <p>Background</p> <p>Liquid chromatography-mass spectrometry (LC-MS) utilizing the high-resolution power of an orbitrap is an important analytical technique for both metabolomics and proteomics. Most important feature of the orbitrap is excellent mass accuracy. Thus, it is necessary to convert raw data to accurate and reliable <it>m/z </it>values for metabolic fingerprinting by high-resolution LC-MS.</p> <p>Results</p> <p>In the present study, we developed a novel, easy-to-use and straightforward <it>m/z </it>detection method, AMDORAP. For assessing the performance, we used real biological samples, <it>Bacillus subtilis </it>strains 168 and MGB874, in the positive mode by LC-orbitrap. For 14 identified compounds by measuring the authentic compounds, we compared obtained <it>m/z </it>values with other LC-MS processing tools. The errors by AMDORAP were distributed within ±3 ppm and showed the best performance in <it>m/z </it>value accuracy.</p> <p>Conclusions</p> <p>Our method can detect <it>m/z </it>values of biological samples much more accurately than other LC-MS analysis tools. AMDORAP allows us to address the relationships between biological effects and cellular metabolites based on accurate <it>m/z </it>values. Obtaining the accurate <it>m/z </it>values from raw data should be indispensable as a starting point for comparative LC-orbitrap analysis. AMDORAP is freely available under an open-source license at <url>http://amdorap.sourceforge.net/</url>.</p
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