Specific and Global RNA Regulators in Pseudomonas Aeruginosa
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Quorum Sensing in Pseudomonas Aeruginosa Mediated by Rhlr Is
www.nature.com/scientificreports OPEN Quorum sensing in Pseudomonas aeruginosa mediated by RhlR is regulated by a small RNA PhrD Received: 21 May 2018 Anuja Malgaonkar & Mrinalini Nair Accepted: 15 November 2018 Pseudomonas aeruginosa is a highly invasive human pathogen in spite of the absence of classical host Published: xx xx xxxx specifc virulence factors. Virulence factors regulated by quorum sensing (QS) in P. aeruginosa cause acute infections to shift to chronic diseases. Several small regulatory RNAs (sRNAs) mediate fne- tuning of bacterial responses to environmental signals and regulate quorum sensing. In this study, we show that the quorum sensing regulator RhlR is positively infuenced upon over expression of the Hfq dependent small RNA PhrD in Pseudomonas. RhlR transcripts starting from two of the four diferent promoters have same sequence predicted to base pair with PhrD. Over expression of PhrD increased RhlR transcript levels and production of the biosurfactant rhamnolipid and the redox active pyocyanin pigment. A rhlR::lacZ translational fusion from one of the four promoters showed 2.5-fold higher expression and, a 9-fold increase in overall rhlR transcription was seen in the wild type when compared to the isogenic phrD disruption mutant. Expression, in an E. coli host background, of a rhlR::lacZ fusion in comparison to a construct that harboured a scrambled interaction region resulted in a 10-fold increase under phrD over expression. The interaction of RhlR-5′UTR with PhrD in E. coli indicated that this regulation could function without the involvement of any Pseudomonas specifc proteins. Overall, this study demonstrates that PhrD has a positive efect on RhlR and its associated physiology in P. -
Genome-Wide Analysis Reveals a Rhla-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1
Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1 Michele Castro Federal University of Rio de Janeiro Graciela Maria Dias Federal University of Rio de Janeiro Tiago Salles Federal University of Rio de Janeiro Núbia Cabral Federal University of Rio de Janeiro Danielly Mariano Federal University of Rio de Janeiro Hadassa Oliveira Federal University of Rio de Janeiro Eliana Abdelhay Instituto Nacional do Câncer Renata Gomes Instituto Nacional do Câncer Bianca Neves ( [email protected] ) Federal University of Rio de Janeiro Research Article Keywords: Pseudomonas aeruginosa, Rhamnolipids, Swarming Motility, Flagella, Transcriptomic Prole Posted Date: February 8th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-154442/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Genome-wide analysis reveals a RhlA-dependent modulation of flagellar genes in Pseudomonas aeruginosa PAO1 Michele R. Castro,1,2 Graciela M. Dias,1 Tiago S. Salles,1 Núbia M. Cabral,1 Danielly C. O. Mariano,1 Hadassa L. Oliveira,1 Eliana S. F. W. Abdelhay 3, Renata B. Gomes,3 and Bianca C. Neves1* 1 Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 2 Department of Biology, Federal Institute of Rio de Janeiro (IFRJ), Rio de Janeiro, Brazil 3 National Cancer Institute (INCA), Rio de Janeiro, Brazil *Corresponding author Bianca C. Neves Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ) Avenida Athos da Silveira Ramos, 149, Lab A537 Rio de Janeiro, RJ, 21941-919, Brazil Telephone: +55 21 3938-7355 FAX: +55 21 3938-7266 e-mail: [email protected] Abstract Background: Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. -
Redundancy in Ribonucleotide Excision Repair: Competition, Compensation, and Cooperation
DNA Repair 29 (2015) 74–82 Contents lists available at ScienceDirect DNA Repair j ournal homepage: www.elsevier.com/locate/dnarepair Redundancy in ribonucleotide excision repair: Competition, compensation, and cooperation ∗ Alexandra Vaisman, Roger Woodgate Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA a r t i c l e i n f o a b s t r a c t Article history: The survival of all living organisms is determined by their ability to reproduce, which in turn depends Received 1 November 2014 on accurate duplication of chromosomal DNA. In order to ensure the integrity of genome duplication, Received in revised form 7 February 2015 DNA polymerases are equipped with stringent mechanisms by which they select and insert correctly Accepted 9 February 2015 paired nucleotides with a deoxyribose sugar ring. However, this process is never 100% accurate. To fix Available online 16 February 2015 occasional mistakes, cells have evolved highly sophisticated and often redundant mechanisms. A good example is mismatch repair (MMR), which corrects the majority of mispaired bases and which has been Keywords: extensively studied for many years. On the contrary, pathways leading to the replacement of nucleotides Ribonucleotide excision repair with an incorrect sugar that is embedded in chromosomal DNA have only recently attracted significant Nucleotide excision repair attention. This review describes progress made during the last few years in understanding such path- Mismatch repair Ribonuclease H ways in both prokaryotes and eukaryotes. Genetic studies in Escherichia coli and Saccharomyces cerevisiae Flap endonuclease demonstrated that MMR has the capacity to replace errant ribonucleotides, but only when the base is DNA polymerase I mispaired. -
Supporting Information
Supporting Information Figure S1. The functionality of the tagged Arp6 and Swr1 was confirmed by monitoring cell growth and sensitivity to hydeoxyurea (HU). Five-fold serial dilutions of each strain were plated on YPD with or without 50 mM HU and incubated at 30°C or 37°C for 3 days. Figure S2. Localization of Arp6 and Swr1 on chromosome 3. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 3L, Tel 3R, CEN3, and the RP gene are shown under the panels. Figure S3. Localization of Arp6 and Swr1 on chromosome 4. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) in the whole chromosome region are compared. The position of Tel 4L, Tel 4R, CEN4, SWR1, and RP genes are shown under the panels. Figure S4. Localization of Arp6 and Swr1 on the region including the SWR1 gene of chromosome 4. The binding of Arp6- FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position and orientation of the SWR1 gene is shown. Figure S5. Localization of Arp6 and Swr1 on chromosome 5. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 5L, Tel 5R, CEN5, and the RP genes are shown under the panels. Figure S6. Preferential localization of Arp6 and Swr1 in the 5′ end of genes. Vertical bars represent the binding ratio of proteins in each locus. -
Potent Inhibition of Human Telomerase by U-73122
Journal of Biomedical Science (2006) 13:667–674 667 DOI 10.1007/s11373-006-9100-z Potent inhibition of human telomerase by U-73122 Yi-Jui Chen, Wei-Yun Sheng, Pei-Rong Huang & Tzu-Chien V. Wang* Department of Molecular and Cellular Biology, Chang Gung University, Kwei-San, Tao-Yuan, 333, Taiwan Received 28 April 2006; accepted 14 June 2006 Ó 2006 National Science Council, Taipei Key words: alkylating agents, cancer therapyzU-73122, N-ethylmaleimide, telomerase inhibitor Summary Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC50 value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 lM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC50 values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hemato- poietic cancer cells. -
Diverse Effects of Natural and Synthetic Surfactants on the Inhibition of Staphylococcus Aureus Biofilm
pharmaceutics Article Diverse Effects of Natural and Synthetic Surfactants on the Inhibition of Staphylococcus aureus Biofilm Gianna Allegrone †, Chiara Ceresa *,†, Maurizio Rinaldi and Letizia Fracchia Department of Pharmaceutical Sciences, Università del Piemonte Orientale “A. Avogadro”, 28100 Novara, Italy; [email protected] (G.A.); [email protected] (M.R.); [email protected] (L.F.) * Correspondence: [email protected]; Tel.: +39-0321-375-837 † These authors contributed equally to this work. Abstract: A major challenge in the biomedical field is the creation of materials and coating strategies that effectively limit the onset of biofilm-associated infections on medical devices. Biosurfactants are well known and appreciated for their antimicrobial/anti-adhesive/anti-biofilm properties, low toxicity, and biocompatibility. In this study, the rhamnolipid produced by Pseudomonas aeruginosa 89 (R89BS) was characterized by HPLC-MS/MS and its ability to modify cell surface hydrophobicity and membrane permeability as well as its antimicrobial, anti-adhesive, and anti-biofilm activity against Staphylococcus aureus were compared to two commonly used surfactants of synthetic origin: Tween® 80 and TritonTM X-100. The R89BS crude extract showed a grade of purity of 91.4% and was composed by 70.6% of mono-rhamnolipids and 20.8% of di-rhamnolipids. The biological activities of R89BS towards S. aureus were higher than those of the two synthetic surfactants. In particular, the anti-adhesive and anti-biofilm properties of R89BS and of its purified mono- and di-congeners were similar. R89BS inhibition of S. aureus adhesion and biofilm formation was ~97% and 85%, respectively, Citation: Allegrone, G.; Ceresa, C.; and resulted in an increased inhibition of about 33% after 6 h and of about 39% after 72 h when Rinaldi, M.; Fracchia, L. -
E. Coli Ribonuclease H (Rnase H), Recombinant # 02-060 1,000 Units, # 02-060-5 5 X 1,000 Units
Manufacturer E. coli Ribonuclease H (RNase H), Recombinant # 02-060 1,000 units, # 02-060-5 5 x 1,000 units Ribonuclease H (RNase H) is an endoribonuclease which specifically degrades the RNA strand of an RNA/DNA hybrid, leaving the DNA strand and unhybridized RNA intact. E. coli RNase H (RNaseHI) was over-expressed in E. coli as a recombinant protein and the protein then purified. MW is 17.6 kDa. Applications 1. Removal of mRNA in DNA/RNA hybrid prior to the synthesis of the second strand of cDNA (1, 2) 2. Removal of poly (A) tails from mRNA after hybridization with oligo (dT) (3) 3. Oligodeoxyribonucleotide-directed site-specific cleavage of RNA (4) Specification Form: 50 units/ul in 20mM Tris-HCl (pH 7.5), 100mM KCl, 1mM DTT, 50% Glycerol Specific Activity: 100,000 units/mg protein Unit Definition: 1 unit is defined as the amount of the enzyme that hydrolyzes 1 nmol of the RNA in 3H labeled M13 DNA/RNA hybrid to acid-soluble ribonucleotides in 20 min at 37°C. Storage: -20°C Quality: Greater than 95% protein determined by SDS-PAGE (Fig. 1, CBB staining). Endo- and exo-DNase activities and RNase activity were not detected with 100 U/ml RNaseH in 50 ul reaction at 37°C. Reagents Supplied with Enzyme: 10X RNaseH Reaction Buffer: 100 mM Tris-HCl (pH 8.0), 100 mM MgCl2, 500 mM NaCl, 10 mM DTT, 500 ug/ml BSA (Bovine Serum Albumin) Caution: To avoid contamination of trace amounts of nucleic acids in BSA, use reaction buffer that does not contain BSA and use RNaseH at higher concentrations. -
The Characterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry
Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1999 The hC aracterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry. Lenore Marie Polo Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Polo, Lenore Marie, "The hC aracterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry." (1999). LSU Historical Dissertations and Theses. 6923. https://digitalcommons.lsu.edu/gradschool_disstheses/6923 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter free, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. -
Hunting RNA Motifs
Hunting RNA motifs Paul Gardner July 23, 2012 Paul Gardner Hunting RNA motifs Classification problems I What is this? I The second most abundant M. tuberculosis transcript I No hits to Rfam, no homologs have been identified, no known function Mycobacteria tuberculosis genome: RNA−seq and annotations 7e+05 +ve strand R ank R eads Gene −ve strand 6e+05 1 5,741K SSU rR N A 2 689K sRNA/RUF? 5e+05 3 236K R N aseP R N A 4e+05 4 197K LSU rR N A 5 119K AT P synthase 3e+05 Read counts 6 119K HSP X 2e+05 7 115K tmR N A CDSs ncRNAs 8 103K Secreted protein 1e+05 Pfam domain terminators 0e+00 Arnvig et al. Rv3661 miscrna00343 (2011) PLoS HAD Rv3662c Pathog. 4099500 4100000 4100500 4101000 4101500 4102000 genome coordinate Paul Gardner Hunting RNA motifs Classification problems I What about this? I The seventh most abundant N. gonorrhoeae transcript I No hits to Rfam, no homologs have been identified, no known function Neisseria gonorrhoeae genome: RNA−seq and annotations R ank R eads Gene 80000 +ve strand −ve strand 1 206K R N aseP R N A 2 178K tR N A -Ser 60000 3 130K ZapA 3‘ U T R ? 4 120K P hage protein 40000 5‘ U T R ? 5 97K tmR N A Read counts 6 93K BRO protein 20000 CDSs ncRNAs 7 81K sR N A ? Pfam domain terminators 8 81K Hsp70 protein 0 9 76K tRNA-Asp NGO2161 terminator12061 terminator12063 Inositol_P terminator12062 NGO2162 terminator12064 Isabella & Clark SEC−C (2011) BMC Genomics. -
Clusters of Bacterial RNA Polymerase Are Biomolecular Condensates That Assemble Through Liquid–Liquid Phase Separation
Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation Anne-Marie Ladouceura,1, Baljyot Singh Parmarb,1, Stefan Biedzinskia, James Walla, S. Graydon Topea, David Cohna, Albright Kima, Nicolas Soubrya, Rodrigo Reyes-Lamothea, and Stephanie C. Webera,b,2 aDepartment of Biology, McGill University, Montreal, QC H3A 1B1, Canada; and bDepartment of Physics, McGill University, Montreal, QC H3A 2T8, Canada Edited by Richard A. Young, Massachusetts Institute of Technology, Cambridge, MA, and approved June 23, 2020 (received for review March 17, 2020) Once described as mere “bags of enzymes,” bacterial cells are in possibility that prokaryotes also use LLPS to compartmentalize fact highly organized, with many macromolecules exhibiting non- their cells. uniform localization patterns. Yet the physical and biochemical In eukaryotes, LLPS is mediated by proteins containing mul- mechanisms that govern this spatial heterogeneity remain largely tivalent domains and disordered regions (22–24) that bring unknown. Here, we identify liquid–liquid phase separation (LLPS) as molecules together into dynamic condensates through transient a mechanism for organizing clusters of RNA polymerase (RNAP) in interactions. Sequence analysis predicts that bacterial proteomes Escherichia coli . Using fluorescence imaging, we show that RNAP have a much lower frequency of disordered regions than quickly transitions from a dispersed to clustered localization pattern eukaryotic proteomes: Only 4.2% compared to 33%, respectively as cells enter log phase in nutrient-rich media. RNAP clusters are (25). This paucity of disorder raises doubts about the prevalence sensitive to hexanediol, a chemical that dissolves liquid-like com- of LLPS in this domain of life. -
The Landscape of Nucleotide Polymorphism Among 13,500 Genes of the Conifer Picea Glauca, Relationships with Functions, and Comparison with Medicago Truncatula
GBE The Landscape of Nucleotide Polymorphism among 13,500 Genes of the Conifer Picea glauca, Relationships with Functions, and Comparison with Medicago truncatula Nathalie Pavy1,*,y,AstridDescheˆnes1,y, Sylvie Blais1, Patricia Lavigne2, Jean Beaulieu1,3, Nathalie Isabel1,2, John Mackay1, and Jean Bousquet1 1Canada Research Chair in Forest and Environmental Genomics, Centre for Forest Research and Institute for Systems and Integrative Biology, Universite´ Laval, Que´bec, Canada 2Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Que´bec, Canada 3Natural Resources Canada, Canadian Wood Fibre Centre, Laurentian Forestry Centre, Que´bec, Canada *Corresponding author: E-mail: [email protected]. yThese authors contributed equally to this work. Accepted: September 15, 2013 Data deposition: This project has been deposited in dbSNP at: http://www.ncbi.nlm.nih.gov/SNP/snp_viewBatch.cgi?sbid¼1058878. Abstract Gene families differ in composition, expression, and chromosomal organization between conifers and angiosperms, but little is known regarding nucleotide polymorphism. Using various sequencing strategies, an atlas of 212k high-confidence single nucleo- tide polymorphisms (SNPs) with a validation rate of more than 92% was developed for the conifer white spruce (Picea glauca). Nonsynonymous and synonymous SNPs were annotated over the corresponding 13,498 white spruce genes representative of 2,457 known gene families. Patterns of nucleotide polymorphisms were analyzed by estimating the ratio of nonsynonymous to synonymous numbers of substitutions per site (A/S). A general excess of synonymous SNPs was expected and observed. However, the analysis from several perspectives enabled to identify groups of genes harboring an excess of nonsynonymous SNPs, thus potentially under positive selection. -
DNA Polymerase I
INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type o f computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 The Role of Magnesium in Hydrolytic Nuclease Enzymes. The Use of Inert Chromium and Cobalt Probes in Understanding Magnesium Activation. Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University by Christopher B.