DOCSLIB.ORG

The Metabolic Potential of Escherichia Coli BL21 in Defined and Rich Medium Zhaopeng Li1,2, Manfred Nimtz1 and Ursula Rinas1,2*

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Metabolic Potential of Escherichia Coli BL21 in Defined and Rich Medium Zhaopeng Li1,2, Manfred Nimtz1 and Ursula Rinas1,2* Li et al. Microbial Cell Factories 2014, 13:45 http://www.microbialcellfactories.com/content/13/1/45 RESEARCH Open Access The metabolic potential of Escherichia coli BL21 in defined and rich medium Zhaopeng Li1,2, Manfred Nimtz1 and Ursula Rinas1,2* Abstract Background: The proteome reflects the available cellular machinery to deal with nutrients and environmental challenges. The most common E. coli strain BL21 growing in different, commonly employed media was evaluated using a detailed quantitative proteome analysis. Results: The presence of preformed biomass precursor molecules in rich media such as Luria Bertani supported rapid growth concomitant to acetate formation and apparently unbalanced abundances of central metabolic pathway enzymes, e.g. high levels of lower glycolytic pathway enzymes as well as pyruvate dehydrogenase, and low levels of TCA cycle and high levels of the acetate forming enzymes Pta and AckA. The proteome of cells growing exponentially in glucose-supplemented mineral salt medium was dominated by enzymes of amino acid synthesis pathways, contained more balanced abundances of central metabolic pathway enzymes, and a lower portion of ribosomal and other translational proteins. Entry into stationary phase led to a reconstruction of the bacterial proteome by increasing e.g. the portion of proteins required for scavenging rare nutrients and general cell protection. This proteomic reconstruction during entry into stationary phase was more noticeable in cells growing in rich medium as they have a greater reservoir of recyclable proteins from the translational machinery. Conclusions: The proteomic comparison of cells growing exponentially in different media reflected the antagonistic and competitive regulation of central metabolic pathways through the global transcriptional regulators Cra, Crp, and ArcA. For example, the proteome of cells growing exponentially in rich medium was consistent with a dominating role of phosphorylated ArcA most likely a result from limitations in reoxidizing reduced quinones in the respiratory chain under these growth conditions. The proteomic alterations of exponentially growing cells into stationary phase cells were consistent with stringent-like and stationary phase responses and a dominating control through DksA-ppGpp and RpoS. Keywords: Escherichia coli, Growth rate control, Metabolic balance, Overflow metabolism, Proteome, Stationary phase response, Transcriptional control, Two-dimensional gel electrophoresis Background conditions, negatively effects the production of proteins Escherichia coli is still the most utilized bacterial work- and other biomolecules. horse for the production of biomolecules, in particular For rapid and convenient lab-scale production of recom- the strain BL21 is the most employed host for the pro- binant proteins, cells are usually grown in rich medium duction of recombinant proteins. E. coli BL21 is consid- such as Luria Bertani or Terrific broth. However, for large- ered a very robust strain compared to E. coli K12 strains scale recombinant protein production, defined mineral salt as it produces less acetate [1,2]. Acetate, produced as media are generally preferred as they allow the implemen- major by-product during fast growth in carbon excess tation of fed-batch based high-cell density cultivations [3]. E. coli isnotonlyusedashostforrecombinantprotein production but is also gaining increasing importance in * Correspondence: [email protected] synthetic biology for the production of heterologous low 1Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 molecular weight compounds [4-6] and also for overpro- Braunschweig, Germany duction of homologous metabolites such as amino acids 2Leibniz University of Hannover, Technical Chemistry–Life Science, Callinstraße 5, D-30167 Hannover, Germany [7] or for the utilization as a whole cell biocatalyst © 2014 Li et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Microbial Cell Factories 2014, 13:45 Page 2 of 17 http://www.microbialcellfactories.com/content/13/1/45 employed in biotransformation processes [8]. Thus, a more profound comprehension of the metabolic poten- 14 A tial of exponentially growing or stationary phase and 12 resting cells is of considerable importance to under- stand and improve production processes but also for 10 the pure knowledge gain to better understand bacterial 8 600 ] A1 physiology. - OD600 6 Based on a proteomic study we present here for the 400 first time a detailed description of the metabolic poten- 4 200 tial of E. coli BL21 during growth in defined and rich 2 Acetate [mg L 0 medium. The quantitative proteome analysis was not 02468 only done for exponentially growing cells but also for 0 Time [h] stationary phase cells to analyze the metabolic capabil- 100 B B1 ] 80 ities as well as the adaptation potential to changing en- -1 1.6 vironmental conditions. 60 40 1.2 Results 20 Dissolved oxygen [%] 0 During growth in rich medium such as the commonly 02468 0.8 employed media Luria Bertani or Terrific Broth cells do Time [h] not need to synthesize most of the precursor molecules (e.g. amino acids) as they are already present in the 0.4 medium. Thus, cells can spend more resources to pro- Specific growth rate [h duce the macromolecules required for their own prolif- 0.0 eration and can grow more rapidly compared to cells 024681012141618 growing in defined mineral salt medium (Figure 1). Re- Time [h] markably, E. coli BL21 also secretes low but detectable Figure 1 Growth properties of E. coli BL21 in defined and rich levels of acetate during rapid growth in rich medium medium. Cells were grown in glucose supplemented mineral salt even in the absence of oxygen limiting conditions (Figure 1). medium (DNB, gray symbols, gray solid line and arrows) and rich For a better understanding of the bacterial physiology medium (LB, open symbols, black solid line and arrows) at 37°C. of E. coli BL21 under these different conditions of nu- Samples for proteome analysis were taken at the time-points indicated: exponential phase (open arrow) and stationary phase (stealth arrow). trient availability a detailed comparative analysis of the Cell growth (OD600), specific growth rate, acetate and dissolved bacterial proteome was carried out. A first brief visual oxygen concentrations are given. examination revealed a less complex nature of the proteome during rapid growth in rich medium com- pared to the slower growth in the glucose supple- Glycolysis, pyruvate dehydrogenase, and pentose mented mineral salt medium (Figure 2). Most notably, phosphate pathway enzymes required for amino acid biosynthesis are virtually Enzymes of the upper glycolytic pathway are present in absent during rapid growth in rich medium (Figure 2). A slightly higher amounts in cells growing in defined lumped quantitative analysis of the abundance levels of all medium with glucose as carbon substrate compared to identified proteins in cells from exponential and stationary the cells growing in rich medium (Figures 3 and 4). How- phase in defined and rich media is given in Figure 3 and ever, the opposite is observed for the lower glycolytic in more detail visualized in Additional file 1: Figures S1-S9 pathway; enzymes belonging to this part of the glycolysis with corresponding values shown in the Additional file 1: and also the subunits of the pyruvate dehydrogenase com- Table S1 (values with standard deviation also in Additional plex, the enzyme connecting the glycolytic pathway and file 2: Table S5). the tricarbocyclic acid cycle, are present in significantly lower amounts in cells growing in defined medium Central (carbon) metabolism (Figures 3 and 4). Also, most identified enzymes be- The comparative proteome analysis of cells growing ex- longing to the pentose phosphate pathway (e.g. Gnd, ponentially in defined and rich medium with special at- TktA, TalB, and Eda) are found in lower amounts in tention to enzymes of the central metabolic pathways cells growing in defined medium (Figures 3 and 4). revealed a complex picture corroborating a “suboptimal” Thus, the higher amounts of enzymes of the lower coupling of catabolic and anabolic pathways especially glycolytic and pentose phosphate pathways as well as during rapid growth in rich medium also for E. coli of pyruvate dehydrogenase during rapid growth in rich BL21 (Figure 4). medium point to an increased capacity for carbon flux Li et al. Microbial Cell Factories 2014, 13:45 Page 3 of 17 http://www.microbialcellfactories.com/content/13/1/45 pH 4 pH 7 pH 4 pH 7 pH 4 pH 7 13 160 kDa 13 13 14 14 14 3 8 3 3 8 10 10 8 10 9 9 9 12 12 12 7 7 7 16 16 16 4 4 4 18 18 18 11 11 11 1 1 1 15 6 17 15 6 17 15 6 17 5 5 5 A 2 B 2 C 2 25 kDa Figure 2 Proteomic fingerprint of cells growing in defined and rich medium. Cells growing at exponential phase in glucose supplemented mineral salt medium (A) and in LB medium at exponential (B) and stationary phases (C) were analyzed by 2D gel electrophoresis. Images of gel sections indicating representative metabolic enzymes are shown: upper glycolysis, FbaB 1; lower glycolysis, GpmA 2, PpsA 3; TCA cycle: GltA 4, SucD 5, Mdh 6; anaplerotic reactions, PckA 7, MaeB 8; by-product metabolism, Acs 9, Pta 10, AckA 11, PoxB 12, AdhE 13; amino acid biosynthesis, MetE 14, IlvE 15, IlvC 16, CysK 17, and amino acid degradation, TnaA 18. Sampling time-points are indicated in Figure 1. The entire gel images representing the proteome of cells grown in defined and rich (LB and TB) media at exponential and stationary phases indicating all identified proteins are given in the Additional file 1: Figures S1-S6.
Load more

Recommended publications
  • Inactivation Mechanisms of Alternative Food Processes on Escherichia Coli O157:H7
    INACTIVATION MECHANISMS OF ALTERNATIVE FOOD PROCESSES ON ESCHERICHIA COLI O157:H7 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Aaron S. Malone, M.S. ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor Ahmed E. Yousef, Adviser Professor Polly D. Courtney ___________________________________ Professor Tina M. Henkin Adviser Food Science and Nutrition Professor Robert Munson ABSTRACT Application of high pressure (HP) in food processing results in a high quality and safe product with minimal impact on its nutritional and organoleptic attributes. This novel technology is currently being utilized within the food industry and much research is being conducted to optimize the technology while confirming its efficacy. Escherichia coli O157:H7 is a well studied foodborne pathogen capable of causing diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The importance of eliminating E. coli O157:H7 from food systems, especially considering its high degree of virulence and resistance to environmental stresses, substantiates the need to understand the physiological resistance of this foodborne pathogen to emerging food preservation methods. The purpose of this study is to elucidate the physiological mechanisms of processing resistance of E. coli O157:H7. Therefore, resistance of E. coli to HP and other alternative food processing technologies, such as pulsed electric field, gamma radiation, ultraviolet radiation, antibiotics, and combination treatments involving food- grade additives, were studied. Inactivation mechanisms were investigated using molecular biology techniques including DNA microarrays and knockout mutants, and quantitative viability assessment methods. The results of this research highlighted the importance of one of the most speculated concepts in microbial inactivation mechanisms, the disruption of intracellular ii redox homeostasis.
    [Show full text]
  • Subcellular Localization in Bacteria: from Envz/Ompr to Transertion
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2011 Subcellular Localization in Bacteria: From EnvZ/OmpR to Transertion Elizabeth Libby University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biological and Chemical Physics Commons, and the Microbiology Commons Recommended Citation Libby, Elizabeth, "Subcellular Localization in Bacteria: From EnvZ/OmpR to Transertion" (2011). Publicly Accessible Penn Dissertations. 974. https://repository.upenn.edu/edissertations/974 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/974 For more information, please contact [email protected]. Subcellular Localization in Bacteria: From EnvZ/OmpR to Transertion Abstract The internal structures of the bacterial cell and the associated dynamic changes as a function of physiological state have only recently begun to be characterized. Here we explore two aspects of subcellular localization in E. coli cells: the cytoplasmic distribution of the response regulator OmpR and its regulated chromosomal genes, and the subcellular repositioning of chromosomal loci encoding membrane proteins upon induction. To address these questions by quantitative fluorescence microscopy, we developed a simple system to tag virtually any chromosomal location with arrays of lacO or tetO by extending and modifying existing tools. An unexplained subcellular localization was reported for a functional fluorescent protein fusion to the response regulator OmpR in Escherichia coli. The pronounced regions of increased fluorescence, or foci, are dependent on OmpR phosphorylation, and do not occupy fixed, easily identifiable positions, such as the poles or midcell. Here we show that the foci are due to OmpR-YFP binding specific sites in the chromosome.
    [Show full text]
  • Biological Circuits with Small RNA Switches
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Stealth regulation: biological circuits with small RNA switches Susan Gottesman Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA So you thinkyou finally understand the regulation of temporal RNAs (stRNAs) or microRNAs, and that these your favorite gene? The transcriptional regulators have RNAs are processed by some of the same protein cofac- been identified; the signaling cascades that regulate syn- tors as is RNAi, have put regulatory RNAs in the spot- thesis and activity of the regulators have been found. light in eukaryotes as well. Recent searches have con- Possibly you have found that the regulator is itself un- firmed that flies, worms, plants, and humans all harbor stable, and that instability is necessary for proper regu- significant numbers of small RNAs likely to play regu- lation. Time to lookfor a new project, or retire and rest latory roles. on your laurels? Not so fast—there’s more. It is rapidly Along with the rapid expansion in RNAs doing inter- becoming apparent that another whole level of regula- esting things, has come a proliferation of nomenclature. tion lurks, unsuspected, in both prokaryotic and eukary- Noncoding RNAs (ncRNA) has been used recently, as otic cells, hidden from our notice in part by the tran- the most general term (Storz 2002). Among the noncod- scription-based approaches that we usually use to study ing RNAs, the subclass of relatively small RNAs that gene regulation, and in part because these regulators are frequently act as regulators have been called stRNAs very small targets for mutagenesis and are not easily (small temporal RNAs, eukaryotes) and sRNAs (small found from genome sequences alone.
    [Show full text]
  • Fur Is the Master Regulator of the Extraintestinal Pathogenic
    Downloaded from RESEARCH ARTICLE crossmark Fur Is the Master Regulator of the Extraintestinal Pathogenic mbio.asm.org Escherichia coli Response to Serum on August 3, 2015 - Published by Sagi Huja,a Yaara Oren,a Dvora Biran,a Susann Meyer,b Ulrich Dobrindt,c Joerg Bernhard,b Doerte Becher,b Michael Hecker,b Rotem Sorek,d Eliora Z. Rona,e Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israela; Institute for Microbiology, Ernst-Moritz-Arndt-Universität, Greifswald, Germanyb; Institute of Hygiene, Westfälische Wilhelms-Universität, Münster, Germanyc; Department of Molecular Genetics Weizmann Institute of Science, Rehovot, Israeld; MIGAL, Galilee Research Center, Kiriat Shmona, Israele ABSTRACT Drug-resistant extraintestinal pathogenic Escherichia coli (ExPEC) strains are the major cause of colisepticemia (coli- bacillosis), a condition that has become an increasing public health problem in recent years. ExPEC strains are characterized by high resistance to serum, which is otherwise highly toxic to most bacteria. To understand how these bacteria survive and grow in serum, we performed system-wide analyses of their response to serum, making a clear distinction between the responses to nu- tritional immunity and innate immunity. Thus, mild heat inactivation of serum destroys the immune complement and abolishes mbio.asm.org the bactericidal effect of serum (inactive serum), making it possible to examine nutritional immunity. We used a combination of deep RNA sequencing and proteomics in order to characterize ExPEC genes whose expression is affected by the nutritional stress of serum and by the immune complement. The major change in gene expression induced by serum—active and inactive—in- volved metabolic genes.
    [Show full text]
  • Journal of Microbiological Methods a Proteome-Wide Screen to Identify Transcription Factors Interacting with the Vibrio Cholerae
    Journal of Microbiological Methods 165 (2019) 105702 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth Note A proteome-wide screen to identify transcription factors interacting with the Vibrio cholerae rpoS promoter T ⁎ ⁎ Julio C. Ayala1, Jorge A. Benitez , Anisia J. Silva Morehouse School of Medicine, Department of Microbiology, Biochemistry and Immunology, 720 Westview Dr., SW, Atlanta, GA 30310, USA ABSTRACT We describe a proteomic approach to identify transcription factors binding to a target promoter. The method's usefulness was tested by identifying proteins binding to the Vibrio cholerae rpoS promoter in response to cell density. Proteins identified in this screen included the nucleoid-associated protein Fis and the quorum sensing regulator HapR. Isolation of regulatory mutants has played a major role in increasing with agitation at 37 °C to stationary phase. Culture media were sup- our understanding of the regulation of gene expression (Shuman and plemented with ampicillin (Amp, 100 μg/mL), kanamycin (Km, 25 μg/ Silhavy, 2003). There are cases, however, in which the above approach mL), polymyxin B (PolB, 100 units/mL) or L-arabinose (0.2%) as re- can encounter significant obstacles. For instance, the methods used to quired. Chromosomal deletions of fis and/or hapR were created in strain genetically manipulate model organisms are not always equally effec- C7258ΔlacZ by allelic exchange as described previously (Wang et al., tive in less studied microorganisms; some regulatory mutations can be 2011; Wang et al., 2012; Wang et al., 2014). Briefly, genomic DNA highly pleiotropic, often be deleterious, unstable and difficult to select.
    [Show full text]
  • Genome-Wide Transcriptional Response to Varying Rpos Levels
    RESEARCH ARTICLE crossm Genome-Wide Transcriptional Response Downloaded from to Varying RpoS Levels in Escherichia coli K-12 Garrett T. Wong,a* Richard P. Bonocora,b Alicia N. Schep,a* Suzannah M. Beeler,a* Anna J. Lee Fong,a* Lauren M. Shull,a* Lakshmi E. Batachari,a Moira Dillon,a Ciaran Evans,c* Carla J. Becker,a Eliot C. Bush,a Johanna Hardin,c http://jb.asm.org/ Joseph T. Wade,b,d Daniel M. Stoebela Department of Biology, Harvey Mudd College, Claremont, California, USAa; Wadsworth Center, New York State Department of Health, Albany, New York, USAb; Department of Mathematics, Pomona College, Claremont, California, USAc; Department of Biomedical Sciences, School of Public Health, University at Albany, SUNY, Albany, New York, USAd ABSTRACT The alternative sigma factor RpoS is a central regulator of many stress on April 17, 2017 by CALIFORNIA INSTITUTE OF TECHNOLOGY responses in Escherichia coli. The level of functional RpoS differs depending on the Received 21 October 2016 Accepted 12 stress. The effect of these differing concentrations of RpoS on global transcriptional January 2017 Accepted manuscript posted online 23 responses remains unclear. We investigated the effect of RpoS concentration on the January 2017 transcriptome during stationary phase in rich media. We found that 23% of genes in Citation Wong GT, Bonocora RP, Schep AN, the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed Beeler SM, Lee Fong AJ, Shull LM, Batachari LE, genes and promoters. We observed three distinct classes of response to RpoS by Dillon M, Evans C, Becker CJ, Bush EC, Hardin J, Wade JT, Stoebel DM.
    [Show full text]
  • Regulatory Interplay Between Small Rnas and Transcription Termination Factor Rho Lionello Bossi, Nara Figueroa-Bossi, Philippe Bouloc, Marc Boudvillain
    Regulatory interplay between small RNAs and transcription termination factor Rho Lionello Bossi, Nara Figueroa-Bossi, Philippe Bouloc, Marc Boudvillain To cite this version: Lionello Bossi, Nara Figueroa-Bossi, Philippe Bouloc, Marc Boudvillain. Regulatory interplay be- tween small RNAs and transcription termination factor Rho. Biochimica et Biophysica Acta - Gene Regulatory Mechanisms , Elsevier, 2020, pp.194546. 10.1016/j.bbagrm.2020.194546. hal-02533337 HAL Id: hal-02533337 https://hal.archives-ouvertes.fr/hal-02533337 Submitted on 6 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Regulatory interplay between small RNAs and transcription termination factor Rho Lionello Bossia*, Nara Figueroa-Bossia, Philippe Bouloca and Marc Boudvillainb a Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France b Centre de Biophysique Moléculaire, CNRS UPR4301, rue Charles Sadron, 45071 Orléans cedex 2, France * Corresponding author: [email protected] Highlights Repression
    [Show full text]
  • Osmosensing by the Bacterial Phoq/Phop Two-Component System
    Osmosensing by the bacterial PhoQ/PhoP two-component system Jing Yuana,b,1, Fan Jina,b, Timo Glattera, and Victor Sourjika,b,1 aMax Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany; and bLOEWE Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved November 6, 2017 (received for review October 5, 2017) The PhoQ/PhoP two-component system plays an essential role in domain and the cytoplasmic domains (10, 11), with the latter the response of enterobacteria to the environment of their mam- detecting the cytoplasmic pH change. The PhoQ TM domain is a malian hosts. It is known to sense several stimuli that are poten- dimer of two TM helices that form a four-helix bundle (12). tially associated with the host, including extracellular magnesium Upon activation of the sensory domain, the periplasmic ends of limitation, low pH, and the presence of cationic antimicrobial the two TM1 helices move closer to each other and push the two peptides. Here, we show that the PhoQ/PhoP two-component TM2 helices farther apart, accompanied by a TM2 rotation, systems of Escherichia coli and Salmonella can also perceive an which requires the solvation of a semichannel in the four-helix osmotic upshift, another key stimulus to which bacteria become bundle (12). Asn202, located near the center of the TM2 helix exposed within the host. In contrast to most previously estab- and proximal to this semichannel, affects the solvation of the lished stimuli of PhoQ, the detection of osmotic upshift does not cavity, and mutations of Asn202 to a nonpolar residue lock PhoQ require its periplasmic sensor domain.
    [Show full text]
  • Supplementary Material
    Lacoux et al. Dynamics of transcription initiation and RNA processing Supplementary Material Dynamic insights on transcription initiation and RNA processing during bacterial adaptation Caroline Lacoux1,#, Aymeric Fouquier d’Hérouël2, Françoise Wessner- Le Bohec1, Nicolas Innocenti1,3,#, Chantal Bohn4, Sean P. Kennedy5,#, Tatiana Rochat6, Rémy A. Bonnin4,#, Pascale Serror1, Erik Aurell3,7, Philippe Bouloc4 and Francis Repoila1,* Email adresses: CL: [email protected], AFD: [email protected], FW: [email protected], NI: [email protected], CB: [email protected], SPK: [email protected], TR: [email protected], RB: [email protected], PS: [email protected], EA: [email protected], PB: [email protected], FR: [email protected] Corresponding author : [email protected] Running head: Dynamics of transcription initiation and RNA processing This file contains: Section S1: Dynamics of RNA levels in response to σE induction (Contains Figures S1 and S2). Section S2: Assignment of 5’ RNA ends. Section S3: Figure S3. Patterns of changes in TIF at σE-TSSs assigned as UNDs or PSSs in this study. Figure S4. Examples of changes in TIF at σE-independent TSSs Section S4: RNA dynamics at the bepA-yfgD operon (Contains Figures S5) References 1 Lacoux et al. Dynamics of transcription initiation and RNA processing Section S1: Dynamics of RNA levels in response to σE induction Global view of the evolving transcriptome Our experimental system consisted of a plasmid (pZE21-rpoE) that harbor the σE encoding sequence under control of the inducible promoter PLtetO-1 in E. coli K12 strain MG1655 (Fig.
    [Show full text]
  • Influence of Glucose Availability and CRP Acetylation on The
    ORIGINAL RESEARCH published: 23 May 2018 doi: 10.3389/fmicb.2018.00941 Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis Daniel V. Guebel 1 and Néstor V. Torres 2* 1 Biotechnology Counselling Services, Buenos Aires, Argentina, 2 Systems Biology and Mathematical Modelling Group, Department of Biochemistry, Microbiology, Cellular Biology and Genetics, Institute of Biomedical Technologies, Center for Biomedical Research of the Canary Islands, University of La Laguna, San Cristóbal de La Laguna, Spain Background: While in eukaryotes acetylation/deacetylation regulation exerts multiple Edited by: pleiotropic effects, in Escherichia coli it seems to be more limited and less known. Hence, Marie-Joelle Virolle, we aimed to progress in the characterization of this regulation by dealing with three Centre National de la Recherche convergent aspects: the effector enzymes involved, the master regulator CRP, and the Scientifique (CNRS), France dependence on glucose availability. Reviewed by: Abhijit Shukla, Methods: The transcriptional response of E. coli BW25113 was analyzed across 14 Memorial Sloan Kettering Cancer Center, United States relevant scenarios. These conditions arise when the wild type and four isogenic mutants Feng Jiang, (defective in deacetylase CobB, defective in N(ε)-lysine acetyl transferase PatZ, Q- and Chinese Academy of Medical R-type mutants of protein CRP) are studied under three levels of glucose availability Sciences, China (glucose-limited chemostat and glucose-excess or glucose-exhausted in batch culture). *Correspondence: acetylated Néstor V. Torres The Q-type emulates a permanent stage of CRP , whereas the R-type emulates [email protected] a permanent stage of CRPdeacetylated.
    [Show full text]
  • Sral Srna Interaction Regulates the Terminator by Preventing Premature Transcription Termination of Rho Mrna
    SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA Inês Jesus Silvaa,1, Susana Barahonaa,2, Alex Eyraudb,2, David Lalaounab, Nara Figueroa-Bossic, Eric Masséb, and Cecília Maria Arraianoa,1 aInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal; bRNA Group, Department of Biochemistry, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada; and cInstitute for Integrative Biology of the Cell (I2BC), Commissariat à l’énergie atomique, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France Edited by Tina M. Henkin, The Ohio State University, Columbus, OH, and approved December 28, 2018 (received for review July 5, 2018) Transcription termination is a critical step in the control of gene sequence, leading to changes in translation and/or mRNA degra- expression. One of the major termination mechanisms is mediated dation (9–11). However, distinct mechanisms of action have been by Rho factor that dissociates the complex mRNA-DNA-RNA poly- increasingly reported in the literature (11). For instance, the Sal- merase upon binding with RNA polymerase. Rho promotes termina- monella sRNA ChiX was shown to induce premature transcription tion at the end of operons, but it can also terminate transcription termination within the coding sequence of chiP as a result of its within leader regions, performing regulatory functions and avoiding interaction with 5′-UTR of the operon chiPQ, thus affecting the pervasive transcription. Transcription of rho is autoregulated through expression of both genes of the operon (12). Conversely, it was a Rho-dependent attenuation in the leader region of the transcript.
    [Show full text]
  • Identification and Characterization of Molecular Targets for Disease Management in Tree Fruit Pathogens
    IDENTIFICATION AND CHARACTERIZATION OF MOLECULAR TARGETS FOR DISEASE MANAGEMENT IN TREE FRUIT PATHOGENS By Jingyu Peng A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Pathology – Doctor of Philosophy 2020 ABSTRACT IDENTIFICATION AND CHARACTERIZATION OF MOLECULAR TARGETS FOR DISEASE MANAGEMENT IN TREE FRUIT PATHOGENS By Jingyu Peng Fire blight, caused by Erwinia amylovora, is a devastating bacterial disease threatening the worldwide production of pome fruit trees, including apple and pear. Within host xylem vessels, E. amylovora cells restrict water flow and cause wilting symptoms through formation of biofilms, that are matrix-enmeshed surface-attached microcolonies of bacterial cells. Biofilm matrix of E. amylovora is primarily composed of several exopolysaccharides (EPSs), including amylovora, levan, and, cellulose. The final step of biofilm development is dispersal, which allows dissemination of a subpopulation of biofilm cells to resume the planktonic mode of growth and consequentially cause systemic infection. In this work, we demonstrate that identified the Hfq-dependent small RNA (sRNA) RprA positively regulates amylovoran production, T3SS, and flagellar-dependent motility, and negatively affects levansucrase activity and cellulose production. We also identified the in vitro and in vivo conditions that activate RprA, and demonstrated that RprA activation leads to decreased formation of biofilms and promotes the dispersal movement of biofilm cells. This work supports the involvement of RprA in the systemic infection of E. amylovora during its pathogenesis. Bacterial toxin-antitoxin (TA) systems are small genetic loci composed of a proteinaceous toxin and a counteracting antitoxin. In this work, we identified and characterized a chromosomally encoded hok/sok-like type I TA system in Erwinia amylovora Ea1189.
    [Show full text]