Small RNA (sRNA) regulates extreme Temperature adaptation of Geobacillus thermoleovorans isolated from a Malaysian hot spring.

RNA kecil (sRNA) telah ditunjukkan memainkan peranan penting dalam pengawalan gen dalam eukariot (juga dikenali sebagai microRNA) dan prokariot. Dalam bakteria, sRNA mengawalatur pembentukan biofilem, penderian korum, kevirulenan dan gerak balas stres persekitaran. Small RNA (sRNA) has been shown to play important gene regulatory roles in both eukaryotes (commonly known as microRNA) and prokaryotes. In bacteria, sRNAs regulate biofilm formation, quorum sensing, virulence, and environmental stress response

Finding sRNA generative locales from high-throughput sequencing data with NiBLS.

Journal ArticleCopyright © 2010 MacLean et al; licensee BioMed Central Ltd.BACKGROUND: Next-generation sequencing technologies allow researchers to obtain millions of sequence reads in a single experiment. One important use of the technology is the sequencing of small non-coding regulatory RNAs and the identification of the genomic locales from which they originate. Currently, there is a paucity of methods for finding small RNA generative locales. RESULTS: We describe and implement an algorithm that can determine small RNA generative locales from high-throughput sequencing data. The algorithm creates a network, or graph, of the small RNAs by creating links between them depending on their proximity on the target genome. For each of the sub-networks in the resulting graph the clustering coefficient, a measure of the interconnectedness of the subnetwork, is used to identify the generative locales. We test the algorithm over a wide range of parameters using RFAM sequences as positive controls and demonstrate that the algorithm has good sensitivity and specificity in a range of Arabidopsis and mouse small RNA sequence sets and that the locales it generates are robust to differences in the choice of parameters. CONCLUSIONS: NiBLS is a fast, reliable and sensitive method for determining small RNA locales in high-throughput sequence data that is generally applicable to all classes of small RNA.Gatsby Charitable Foundatio

Light-induced transcriptional responses associated with proteorhodopsin-enhanced growth in a marine flavobacterium

Proteorhodopsin (PR) is a photoprotein that functions as a light-driven proton pump in diverse marine Bacteria and Archaea. Recent studies have suggested that PR may enhance both growth rate and yield in some flavobacteria when grown under nutrient-limiting conditions in the light. The direct involvement of PR, and the metabolic details enabling light-stimulated growth, however, remain uncertain. Here, we surveyed transcriptional and growth responses of a PR-containing marine flavobacterium during carbon-limited growth in the light and the dark. As previously reported (Gómez-Consarnau et al., 2007), Dokdonia strain MED134 exhibited light-enhanced growth rates and cell yields under low carbon growth conditions. Inhibition of retinal biosynthesis abolished the light-stimulated growth response, supporting a direct role for retinal-bound PR in light-enhanced growth. Among protein-coding transcripts, both PR and retinal biosynthetic enzymes showed significant upregulation in the light. Other light-associated proteins, including bacterial cryptochrome and DNA photolyase, were also expressed at significantly higher levels in the light. Membrane transporters for Na+/phosphate and Na+/alanine symporters, and the Na+-translocating NADH-quinone oxidoreductase (NQR) linked electron transport chain, were also significantly upregulated in the light. Culture experiments using a specific inhibitor of Na+-translocating NQR indicated that sodium pumping via NQR is a critical metabolic process in the light-stimulated growth of MED134. In total, the results suggested the importance of both the PR-enabled, light-driven proton gradient, as well as the generation of a Na+ ion gradient, as essential components for light-enhanced growth in these flavobacteria.Gordon and Betty Moore FoundationNational Science Foundation (U.S.) (NSF Science and Technology Center Award EF0424599.)Japan Society for the Promotion of Science (Postdoctoral Fellowships for Research Abroad

Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq

Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria

Identification of microRNAs Actively Involved in Fatty Acid Biosynthesis in Developing Brassica napus Seeds Using High-Throughput Sequencing

Seed development has a critical role during the spermatophyte life cycle. In Brassica napus, a major oil crop, fatty acids are synthesized and stored in specific tissues during embryogenesis, and understanding the molecular mechanism underlying fatty acid biosynthesis during seed development is an important research goal. In this study, we constructed three small RNA libraries from early seeds at 14, 21 and 28 days after flowering (DAF) and used high-throughput sequencing to examine microRNA (miRNA) expression. A total of 85 known miRNAs from 30 families and 1,160 novel miRNAs were identified, of which 24, including 5 known and 19 novel miRNAs, were found to be involved in fatty acid biosynthesis. bna-miR156b, bna-miR156c, bna-miR156g, novel_mir_1706, novel_mir_1407, novel_mir_173, and novel_mir_104 were significantly down-regulated at 21 DAF and 28 DAF, whereas bna-miR159, novel_mir_1081, novel_mir_19 and novel_mir_555 were significantly up-regulated. In addition, we found that some miRNAs regulate functional genes that are directly involved in fatty acid biosynthesis and that other miRNAs regulate the process of fatty acid biosynthesis by acting on a large number of transcription factors. The miRNAs and their corresponding predicted targets were partially validated by quantitative RT-PCR. Our data suggest that diverse and complex miRNAs are involved in the seed development process and that miRNAs play important roles in fatty acid biosynthesis during seed development

An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukemia cases using multiple polymerases

Chronic lymphocytic leukaemia (CLL) is the most common clonal B-cell disorder characterized by clonal diversity, a relapsing and remitting course, and in its aggressive forms remains largely incurable. Current front-line regimes include agents such as fludarabine, which act primarily via the DNA damage response pathway. Key to this is the transcription factor p53. Mutations in the TP53 gene, altering p53 functionality, are associated with genetic instability, and are present in aggressive CLL. Furthermore, the emergence of clonal TP53 mutations in relapsed CLL, refractory to DNA-damaging therapy, suggests that accurate detection of sub-clonal TP53 mutations prior to and during treatment may be indicative of early relapse. In this study, we describe a novel deep sequencing workflow using multiple polymerases to generate sequencing libraries (MuPol-Seq), facilitating accurate detection of TP53 mutations at a frequency as low as 0.3%, in presentation CLL cases tested. As these mutations were mostly clustered within the regions of TP53 encoding DNA-binding domains, essential for DNA contact and structural architecture, they are likely to be of prognostic relevance in disease progression. The workflow described here has the potential to be implemented routinely to identify rare mutations across a range of diseases

Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.This work has been funded by Projects PI12/00552 and PI15/00860 to GB, PI11/01034 to MP, CP13/00226 to AB, and P14/000059 to MP and AB, all integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the ISCIII - General Subdirection of Assessment and Promotion of the Research – European Regional Development Fund (FEDER) 'A way of making Europe'. Miami University Research Funds from the department of Microbiology supported this work. The authors also want to thank the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to GB), co-financed by the European Development Regional Fund (EDRF) 'A Way to Achieve Europe', Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad as well as Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). A.P. was financially supported by the Galician Plan for Research, Innovation and Growth (I2C Plan 2012-2016). J.A. Vallejo was financially supported by the Sara Borrell Programme (ISCIII, Spain CD13/00373). S. Rumbo-Feal was financially supported by the Agustí Pumarola Grant (Societat Catalana de Malalties Infeccioses i Microbiologia Clínica, SCMIMC) and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC)

The role of the RNA-binding protein Hfq in the model pathogen Salmonella Typhimurium

Hfq is a RNA-binding protein which exists in homohexamers in vivo. Based on its folding, containing the highly conserved Sm1 and Sm2 motifs, it belongs to the growing family of Sm and Sm-like (Lsm) proteins. It has been shown, that Hfq is a pleiotropic regulator in bacteria which is involved in a broad variety of functions. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium Even though hfq has turned out to have no severe influence on the growth or the viability of the pathogenic bacterium Salmonella Typhimurium under laboratory conditions, we could show that it is strongly involved in the regulation of pathogenicity. A Δhfq mutant leads to loss of effector protein expression and secretion and thereby to reduced invasion of non-phagocytic cells and to reduced ability of intracellular replication in macrophages. Based on these observations, loss of infectivity in a mouse-model of infection could be proven. Further studies revealed not only lack of secreted proteins in the Δhfq mutant, but showed severe changes in the overall protein pattern when compared to its isogenic wild type strain, with an overrepresentation of membrane and membrane-associated proteins. Concerning the virulence phenotype, we have been able to restore effector protein expression (even if not their secretion) by overexpression of one of the major transcription factors involved in expression of virulence genes encoded in Salmonella pathogenicity island 1 (SPI1), namely HilA. Additionally, we could show that alteration in mRNA stability is causing for example the increase of the major outer membrane protein, OmpD or the decrease in the flagellar protein, FliC. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq Our analysis represents a demonstration for usage of high throughput pyrosequencing (HTPS) in bacteria to determine the large regulon of the pleiotropic regulator, Hfq. The combination of transcriptomics with co-immunoprecipitation (coIP) of direct binding partners of Hfq and subsequent cDNA library synthesis and its sequencing allowed the dissection of genes directly influenced by Hfq and downstream effects based on deregulation of transcription factors. By analysis of RNA co-immunoprecipitated with Hfq compared to control coIPs in Salmonella Typhimurium lysates we were able to determine specific enrichment factors for a large set of mRNAs as well as sRNAs. Comparison with the transcriptomic data showed that Hfq regulates multiple major transcription factors, like a transcription factor of SPI1, HilD, and the major transcription factor, FlhD2C2, regulating the large class of flagellar genes in Salmonella and other bacterial species. By overexpression of these transcription factors we could restore phenotypes of a Δhfq mutant, e.g. loss of effector protein expression and secretion and reduced expression of the class III flagellar gene, FliC. Concerning sRNA expression in Salmonella, we found 10 new sRNAs in this pathogen and were able to verify the expression of a large set of sRNAs that have been known to be conserved in the model organism, Escherichia coli. Aside noncoding RNAs also two mRNAs encoding for small open reading frames (ORFs) in E. coli could be detected in the coIP RNA sample from Salmonella Typhimurium

Next Generation Sequencing Technologies for Insect Virus Discovery

Insects are commonly infected with multiple viruses including those that cause sublethal, asymptomatic, and latent infections. Traditional methods for virus isolation typically lack the sensitivity required for detection of such viruses that are present at low abundance. In this respect, next generation sequencing technologies have revolutionized methods for the discovery and identification of new viruses from insects. Here we review both traditional and modern methods for virus discovery, and outline analysis of transcriptome and small RNA data for identification of viral sequences. We will introduce methods for de novo assembly of viral sequences, identification of potential viral sequences from BLAST data, and bioinformatics for generating full-length or near full-length viral genome sequences. We will also discuss implications of the ubiquity of viruses in insects and in insect cell lines. All of the methods described in this article can also apply to the discovery of viruses in other organisms