Deep sequencing reveals as-yet-undiscovered small RNAs in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>In <it>Escherichia coli</it>, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of <it>E. coli </it>with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length.</p> <p>Results</p> <p>We discovered 229 novel candidate sRNAs (≥ 50 nt) with computational or experimental evidence of transcription initiation. Among them, the expression of seven intergenic sRNAs and three <it>cis</it>-antisense sRNAs was detected by northern blot analysis. Interestingly, five novel sRNAs are expressed from prophage regions and we note that these sRNAs have several specific characteristics. Furthermore, we conducted an evolutionary conservation analysis of the candidate sRNAs and summarised the data among closely related bacterial strains.</p> <p>Conclusions</p> <p>This comprehensive screen for <it>E. coli </it>sRNAs using a deep sequencing approach has shown that many as-yet-undiscovered sRNAs are potentially encoded in the <it>E. coli </it>genome. We constructed the <it>Escherichia coli </it>Small RNA Browser (ECSBrowser; <url>http://rna.iab.keio.ac.jp/</url>), which integrates the data for previously identified sRNAs and the novel sRNAs found in this study.</p

Towards improving the safety and diagnostic yield of stereotactic biopsy in a single centre

Background: Previously, we reported on our single centre results regarding the diagnostic yield of stereotactic needle biopsies of brain lesions. The yield then (1996-2006) was 89.4%. In the present study, we review and evaluate our experience with intraoperative frozen-section histopathologic diagnosis on-demand in order to improve the diagnostic yield. Methods:

Escherichia coli genome-wide promoter analysis: Identification of additional AtoC binding target elements

<p>Abstract</p> <p>Background</p> <p>Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of <it>E. coli </it>activates the expression of <it>atoDAEB </it>operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that <it>atoSC </it>is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the <it>atoDAEB </it>promoter as the single, <it>cis</it>-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the <it>E. coli </it>genome.</p> <p>Results</p> <p>Through the implementation of a computational <it>de novo </it>motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the <it>E. coli </it>genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the <it>in vivo </it>binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences.</p> <p>Conclusions</p> <p>This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.</p

Feedback Inhibition in the PhoQ/PhoP Signaling System by a Membrane Peptide

The PhoQ/PhoP signaling system responds to low magnesium and the presence of certain cationic antimicrobial peptides. It regulates genes important for growth under these conditions, as well as additional genes important for virulence in many gram-negative pathogens. PhoQ is a sensor kinase that phosphorylates and activates the transcription factor PhoP. Since feedback inhibition is a common theme in stress-response circuits, we hypothesized that some members of the PhoP regulon may play such a role in the PhoQ/PhoP pathway. We therefore screened for PhoP-regulated genes that mediate feedback in this system. We found that deletion of mgrB (yobG), which encodes a 47 amino acid peptide, results in a potent increase in PhoP-regulated transcription. In addition, over-expression of mgrB decreased transcription at both high and low concentrations of magnesium. Localization and bacterial two-hybrid studies suggest that MgrB resides in the inner-membrane and interacts directly with PhoQ. We further show that MgrB homologs from Salmonella typhimurium and Yersinia pestis also repress PhoP-regulated transcription in these organisms. In cell regulatory circuits, feedback has been associated with modulating the induction kinetics and/or the cell-to-cell variability in response to stimulus. Interestingly, we found that elimination of MgrB-mediated feedback did not have a significant effect on the kinetics of reporter protein production and did not decrease the variability in expression among cells. Our results indicate MgrB is a broadly conserved membrane peptide that is a critical mediator of negative feedback in the PhoQ/PhoP circuit. This new regulator may function as a point of control that integrates additional input signals to modulate the activity of this important signaling system

Leaf colour as a signal of chemical defence to insect herbivores in wild cabbage (Brassica Oleracea)

Leaf colour has been proposed to signal levels of host defence to insect herbivores, but we lack data on herbivory, leaf colour and levels of defence for wild host populations necessary to test this hypothesis. Such a test requires measurements of leaf spectra as they would be sensed by herbivore visual systems, as well as simultaneous measurements of chemical defences and herbivore responses to leaf colour in natural host-herbivore populations. In a large-scale field survey of wild cabbage (Brassica oleracea) populations, we show that variation in leaf colour and brightness, measured according to herbivore spectral sensitivities, predicts both levels of chemical defences (glucosinolates) and abundance of specialist lepidopteran (Pieris rapae) and hemipteran (Brevicoryne brassicae) herbivores. In subsequent experiments, P. rapae larvae achieved faster growth and greater pupal mass when feeding on plants with bluer leaves, which contained lower levels of aliphatic glucosinolates. Glucosinolate-mediated effects on larval performance may thus contribute to the association between P. rapae herbivory and leaf colour observed in the field. However, preference tests found no evidence that adult butterflies selected host plants based on leaf coloration. In the field, B. brassicae abundance varied with leaf brightness but greenhouse experiments were unable to identify any effects of brightness on aphid preference or performance. Our findings suggest that although leaf colour reflects both levels of host defences and herbivore abundance in the field, the ability of herbivores to respond to colour signals may be limited, even in species where performance is correlated with leaf colour

Hemotin, a regulator of phagocytosis encoded by a small ORF and xonserved across metazoans

Translation of hundreds of small ORFs (smORFs) of less than 100 amino acids has recently been revealed in vertebrates and Drosophila. Some of these peptides have essential and conserved cellular functions. In Drosophila, we have predicted a particular smORF class encoding ~80 aa hydrophobic peptides, which may function in membranes and cell organelles. Here, we characterise hemotin, a gene encoding an 88aa transmembrane smORF peptide localised to early endosomes in Drosophila macrophages. hemotin regulates endosomal maturation during phagocytosis by repressing the cooperation of 14-3-3ζ with specific phosphatidylinositol (PI) enzymes. hemotin mutants accumulate undigested phagocytic material inside enlarged endo-lysosomes and as a result, hemotin mutants have reduced ability to fight bacteria, and hence, have severely reduced life span and resistance to infections. We identify Stannin, a peptide involved in organometallic toxicity, as the Hemotin functional homologue in vertebrates, showing that this novel regulator of phagocytic processing is widely conserved, emphasizing the significance of smORF peptides in cell biology and disease