DOCSLIB.ORG

Positive Factors

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Positive Factors Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8958-8962, October 1991 Biochemistry Functional map of the a subunit of Escherichia coli RNA polymerase: Two modes of transcription activation by positive factors (positive control/subunit assembly/transcription factor/protein-protein contact/deletion mutant) KAZUHIKO IGARASHIt, AKEMI HANAMURAt§, Kozo MAKINO¶, HIROFUMI AIBAII, HIROJI AIBA*§, TAKESHI MIZUNOII, ATSUO NAKATA¶, AND AKIRA ISHIHAMAt** tNational Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411, Japan; *Tsukuba University, Department of Chemistry, Tsukuba, Ibaraki 305, Japan; 1Osaka University, Institute of Microbial Diseases, Suita, Osaka 565, Japan; and 1Nagoya University, School of Agriculture, Chikusa, Nagoya 464, Japan Communicated by Motoo Kimura, June 24, 1991 ABSTRACT The role of the a subunit of Escherichia coli subunit is involved in positive control by certain transcription RNA polymerase in transcription activation by positive factors activators (10-14). was investigated using two reconstituted mutant RNA poly- In the present study, we extended this line of experiments merases (containing C-terminally truncated a subunits) and to type II CRP-dependent promoters. Most CRP-dependent three positive factors [the cAMP receptor protein (CRP), promoters can be classified into two types depending on the OmpR, and PhoB]. The mutant RNA polymerases did not location of the CRP site relative to the transcription start site respond to transcription activation by activator proteins that (15, 16). Type I promoters are associated with a CRP site that bind upstream of the respective promoters. Transcription by is separated from the basic promoter (-10 and -35 signals) these mutant enzymes was, however, activated in the cases and include the lac P1 and uxuAB promoters. On type II where activators bind to target sites that overlap the promoter promoters, the CRP site is centered around -41/-42 and therefore partially overlaps the basic promoter at the -35 -35 region. Two different mechanisms are proposed for the region. In addition, we examined the responsiveness of the positive control of transcription by activator proteins, one mutant RNA polymerases to transcription stimulation by two requiring the C-terminal domain of the a subunit, and the other activators, PhoB, an activator of the phosphate regu- other not requiring it. lon, and OmpR, which is involved in osmoregulation of the ompF and ompC genes (reviewed in ref. 17). The results Positive control of transcription is one of the common suggest the presence oftwo different mechanisms for positive mechanisms of regulated gene expression in both prokary- control of transcription by activator proteins: one requires otes and eukaryotes. Two mechanisms have been proposed the C-terminal domain of the a subunit, and the other does for this activation, (i) activator-induced conformational not. change of DNA structure around the promoter and (ii) direct contact between activator and RNA polymerase on the DNA MATERIALS AND METHODS (for review see ref. 1). Several mutants ofA repressor protein or cAMP receptor protein (CRP) that still bind to their RNA Polymerases. Wild-type and mutant RNA polymer- respective target sites but are unable to activate transcription ases were assembled in vitro from individually overproduced have been isolated, indicating direct interaction between and purified (3, (3', and (770 subunits and one of the wild-type these activator proteins and RNA polymerase (2-5). or mutant a subunits, as described by Igarashi and Ishihama RNA polymerase of Escherichia coli is composed of four (9). different subunits, a, (3, (3', and one of a variety ofao subunits Transcription Factors. CRP was purified as described (18). (reviewed in ref. 6). One major function of the a subunit is to Purification of OmpR protein and the truncated form of a mediate the assembly ofthe two large subunits, (3and (3', into mutant EnvZll protein, EnvZll*, was described previously the core enzyme structure (7, 8). A set of mutant RNA (19, 20). EnvZll* lacks the N-terminal hydrophobic region. polymerases were constructed that were assembled in vitro Overproduction and purification of PhoB protein and from wild-type ,(, (3', and a,70 subunits and a mutant a subunit PhoR1084, a truncated form of PhoR lacking the N-terminal lacking the C-terminal 73 or 94 amino acid residues (9). The hydrophobic region, were described previously (21, 22). mutant RNA polymerase core enzymes containing C-termi- Template DNAs. All the truncated DNA templates used are nally truncated a subunits exhibited essentially the same illustrated in Fig. 1A. The 240-bp Hpa II fragment containing specific activity of RNA synthesis as the native enzyme. the gal promoter region was isolated from plasmid pBdCl o70 (23). The 344-bp Hinfl fragment containing the pBR-P4 After addition of subunit, these mutant holoenzymes were promoter was isolated from plasmid pBR322 (24). The 516-bp able to initiate transcription accurately from certain promot- HindII1 fragment containing the ompC promoter region was ers. However, they did not respond to transcription activa- obtained from plasmid pUCI-OL (25). The 115-bp BamHI- tion by cAMP/CRP at two type I CRP-dependent promoters HindIII fragment containing the pstS promoter was obtained on the lac and uxuAB genes (9). These results led us to from plasmid pOS1 (26), and its ends were filled-in using the conclude that the C-terminal domain (residues 257-329) ofthe Klenow fragment of DNA polymerase I. The 205-bp EcoRI a subunit is involved in the response of RNA polymerase to fragment containing the lacUV5 promoter was obtained from transcriptional activation by cAMP/CRP. Phenotypes result- plasmid pKB252 (27). ing from known rpoA mutations also suggest that the a Abbreviation: CRP, cAMP receptor protein. The publication costs of this article were defrayed in part by page charge §Present address: Nagoya University, Faculty of Science, Chikusa, payment. This article must therefore be hereby marked "advertisement" Nagoya 464, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. **To whom reprint requests should be addressed. 8958 Biochemistry: Igarashi et al. Proc. Natl. Acad. Sci. USA 88 (1991) 8959 A pBR P4 I.: 344 bp S-----lAl-l 241 b gal I- 240 b~p -d 45 b (P1) 6 50 b (P2) ompC 516 bp - 98 b pstS .Z1J 15 bp 41 b B3 ux uAB GAGTGAAATTGTTGTGATGTGGTTAACCCAATTAGAATTCGGGATTGACATGTCTTACCAAAAGGTAGAACTTATACGC CRP site -35 -10 lac P1 AATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA CRP site 35 -10 pBR P4 CGGTATTTTCTCCTTACGCATCTGTiG-CGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTfG CRP site -10 gal P1 TCTTGTGTAAACGATTCCACTAATTTATTTCCATGTCACACTTTTCGCATCTTTGTTATGCTATGGTTATTTCA ompC CRP site "10 TTGCATTTANCAT T TTGAAACATCTATAGCGATAAATGAAACATCTTAAAAGTTTTAGTATCATATTCGTGTTGGATTATTCTGCAT TTTTGGCGAGAAGGACT OrnpR site OmpR site OmpR site .735 - 1 0M pstS TCICTCTGTCATAAAACTGTCATATTCCTTACATATAACTGTCACCTGTTTGTCCTATTTTGCTTCTCG PhoB site PhoB site 0 FIG. 1. Activator-dependent E. coli promoters. (A) Truncated DNA templates used for in vitro transcription. DNA fragments, each carrying a promoter(s) (black areas) and an activator binding site(s) (gray areas), were prepared as described in Materials and Methods. Transcripts from these templates are shown by filled arrows indicating the direction of transcription. bp, Base pairs; b, bases. (B) Nucleotide sequences of the uxuAB, lac, pBR-P4, gal, ompC and pstS promoter regions. Transcription initiation sites are marked by stars. Basic promoter elements (-10 and -35 signals) are underlined by black bars. The binding sites for CRP, OmpR, and PhoB are underlined by gray bars. The nucleotides homologous to the consensus CRP-binding site, OmpR-binding site, and PhoB-binding sites (pho box) are indicated by dashed overlines. The predicted lengths of the run-off transcripts are 45 and mutant RNA polymerases containing a-235 or a-256. Basal 50 nucleotides from the gal P1 and P2 promoters, respec- transcription from P2 in the absence of cAMP/CRP was tively; 241 nucleotides from the pBR-P4; 98 nucleotides from weaker with the mutant enzymes than that with the wild-type the ompC promoter; 41 nucleotides from the pstS promoter; enzymes. When the DNA fragment was preincubated with and 63 nucleotides from the lacUV5 promoter (see Fig. 1). cAMP/CRP, transcription from P1 was markedly stimulated In Vitro Transcription. In vitro transcription from the gal not only for the wild-type but also the mutant enzymes. and pBR-P4 promoters was carried out according to Kajitani Although the basal transcription from P2 was weaker for the and Ishihama (27) with some modifications (9). Transcription mutant enzymes, the level of induced P1 transcription was from the ompC promoter was carried out essentially as almost the same as that by the wild-type enzymes. Inhibition described by Aiba and Mizuno (28), while transcription from ofinitiation at P2 is due to interference with RNA polymerase the pstS promoter was carried out as described by Makino et binding by cAMP/CRP, which attaches to the -35 region of al. (22). RNA products were purified by ethanol precipitation the P2 promoter. and analyzed by electrophoresis in polyacrylamide gels con- Fig. 2B shows transcription from the pBR-P4 promoter. taining 8 M urea. This promoter resembles gal P1 with respect to the location ofthe CRP site relative to the transcription start site (see Fig. 1). Again, transcription from pBR-P4 was stimulated by RESULTS cAMP/CRP with all the enzymes used, although
Load more

Recommended publications
  • Evolutionary and Functional Aspects of Two-Component Signalling Systems
    IMPERIAL COLLEGE LONDON Evolutionary and Functional Aspects of Two-Component Signalling Systems by Xia Sheng in the Theoretical System Biology Group Division of Molecular Biosciences February 2013 IMPERIAL COLLEGE LONDON Abstract Theoretical System Biology Group Division of Molecular Biosciences by Xia Sheng Two-component systems (TCSs) are critical for bacteria to interact with their extra- cellular environment. They define a type of signalling system that is composed of a transmembrane histidine kinase (HK) and a cytoplasmic response regulator (RR). In this thesis we have studied the evolutionary and functional aspects of these important signalling systems. By studying the distribution of the TCS orthologues of E.coli across more than 900 bacterial organisms, we have found that a pair of TCS proteins does not always coexist in one organism. The genomic localisation map of TCSs reveals a possi- ble translocation mechanism for TCS evolution. The alignments of HKs and RRs have shown that HKs are genetically more divergent, probably due to their signal recognizing role. An analysis of the steady states of TCS dynamics has shown that the outputs of the TCSs are bistable if they have positive auto-regulation feedback loops in their tran- scriptional regulation. Our analysis has also shown that the phosphorylation process of a TCS is always monostable and the factors that affect steady states have been studied. For both orthodox and non-orthodox TCSs, autophosphorylation rates of the HKs are the most important factor to affect the steady states of the TCSs' outputs. To study the phosphorelay mechanism of the non-orthodox TCS ArcB/A, we constructed plasmids carrying different copy numbers of ArcB mutants with different phosphorylation sites ablated.
    [Show full text]
  • A Nanobody Targeting the Translocated Intimin Receptor Inhibits the Attachment of Enterohemorrhagic E
    RESEARCH ARTICLE A nanobody targeting the translocated intimin receptor inhibits the attachment of enterohemorrhagic E. coli to human colonic mucosa 1,2 3,4 1 2 David Ruano-GallegoID , Daniel A. YaraID , Lorenza Di IanniID , Gad Frankel , 3,4 1 Stephanie SchuÈ llerID , Luis AÂ ngel FernaÂndezID * a1111111111 1 Department of Microbial Biotechnology, Centro Nacional de BiotecnologõÂa, Consejo Superior de Investigaciones CientõÂficas (CSIC), Campus UAM-Cantoblanco, Madrid, Spain, 2 MRC Centre for Molecular a1111111111 Bacteriology and Infection, Life Sciences Department, Imperial College London, London, United Kingdom, a1111111111 3 Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, United Kingdom, a1111111111 4 Quadram Institute Bioscience, Norwich Research Park, Norwich, United Kingdom a1111111111 * [email protected] Abstract OPEN ACCESS Citation: Ruano-Gallego D, Yara DA, Di Ianni L, Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic Frankel G, SchuÈller S, FernaÂndez LAÂ (2019) A colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently avail- nanobody targeting the translocated intimin able to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by receptor inhibits the attachment of injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In enterohemorrhagic E. coli to human colonic mucosa. PLoS Pathog 15(8): e1008031. https:// this project, we identified a camelid single domain antibody (nanobody), named TD4, that doi.org/10.1371/journal.ppat.1008031 recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin Editor: Brian K. Coombes, McMaster University, with high affinity and stability.
    [Show full text]
  • Recombinant Protein Expression in Escherichia Coli François Baneyx
    411 Recombinant protein expression in Escherichia coli François Baneyx Escherichia coli is one of the most widely used hosts for the co-overexpression of additional gene products is desired, production of heterologous proteins and its genetics are far ColE1 derivatives are usually combined with compatible better characterized than those of any other microorganism. plasmids bearing a p15A replicon and maintained at Recent progress in the fundamental understanding of about 10–12 copies per cell. Under laboratory conditions, transcription, translation, and protein folding in E. coli, together such multicopy plasmids are randomly distributed dur- with serendipitous discoveries and the availability of improved ing cell division and, in the absence of selective genetic tools are making this bacterium more valuable than pressure, are lost at low frequency (10–5–10–6 per gener- ever for the expression of complex eukaryotic proteins. ation) primarily as a result of multimerization [2•]. Nevertheless, plasmid loss can increase tremendously in Addresses the case of very high copy number plasmids, when plas- Department of Chemical Engineering, University of Washington, mid-borne genes are toxic to the host or otherwise Box 351750, Seattle, WA 98195, USA; significantly reduce its growth rate, or when cells are cul- e-mail: [email protected] tivated at high density or in continuous processes. Current Opinion in Biotechnology 1999, 10:411–421 The simplest way to address this problem is to take 0958-1669/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. advantage of plasmid-encoded antibiotic-resistance mark- ers and supplement the growth medium with antibiotics Abbreviations to kill plasmid-free cells.
    [Show full text]
  • Engineering Escherichia Coli to Sense Acidic Amino Acids by Introduction of a Chimeric Two-Component System
    Korean J. Chem. Eng., 32(10), 2073-2077 (2015) pISSN: 0256-1115 DOI: 10.1007/s11814-015-0024-z eISSN: 1975-7220 INVITED REVIEW PAPER INVITED REVIEW PAPER Engineering Escherichia coli to sense acidic amino acids by introduction of a chimeric two-component system Sambandam Ravikumar*,‡, Irisappan Ganesh**,‡, Murali Kannan Maruthamuthu**, and Soon Ho Hong**,† *Department of Pharmaceutical Science and Technology, Catholic University of Daegu, 330, Geumrak 1-ri, Hayang-eup, Gyeongsan, Gyeongbuk 712-702, Korea **Department of Chemical Engineering, University of Ulsan, 93, Daehak-ro, Nam-gu, Ulsan 680-749, Korea (Recevied 28 September 2014 • accepted 27 January 2015) Abstract−In an attempt to create an acidic amino acid-sensing Escherichia coli, a chimeric sensor kinase (SK)-based biosensor was constructed using Pseudomonas putida AauS. AauS is a sensor kinase that ultimately controls expression of the aau gene through its cognate response regulator AauR, and is found only in P. puti d a KT2440. The AauZ chi- mera SK was constructed by integration of the sensing domain of AauS with the catalytic domain of EnvZ to control the expression of the ompC gene in response to acidic amino acids. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as the concentration of acidic amino acids in- creased. These data suggest that AauS-based recombinant E. coli can be used as a bacterial biosensor of acidic amino acids. By employing the chimeric SK strategy, various bacteria biosensors for use in the development of biochemical- producing recombinant microorganisms can be constructed. Keywords: Acidic Amino Acids, Chimera Two-component System, Green Fluorescent Protein, Escherichia coli INTRODUCTION the transcription of related genes [6].
    [Show full text]
  • Complete Article
    JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 619-622 Vol. 174, No. 2 0021-9193/92/020619-04$02.00/0 Copyright X 1992, American Society for Microbiology NOTES Bacteriophage T7 RNA Polymerase Travels Far Ahead of Ribosomes In Vivo ISABELLE IOST, JEAN GUILLEREZ, AND MARC DREYFUS* Laboratoire de Genetique Moleculaire (Centre National de la Recherche Scientifique D1302), Ecole Normale Superieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France Received 2 July 1991/Accepted 24 October 1991 We show that in Escherichia coli at 32°C, the T7 RNA polymerase travels over the lacZ gene about eightfold faster than ribosomes travel over the corresponding mRNA. We discuss how the T7 phage might exploit this high rate in its growth optimization strategy and how it obviates the possible drawbacks of uncoupling transcription from translation. In Escherichia coli, transcription occurs simultaneously sequences and on the other by a terminator for T7 RNA with translation, and both processes are often tightly cou- polymerase (5). Because it is present as a single copy, the pled. Thus, the average rate of transcription matches that of lacZ gene can be transcribed constitutively from the very translation, even though it can theoretically be higher (3, active T7 promoter without affecting cell growth. For the lla). Moreover, in the absence of translation, transcription purpose of this study, we replaced the gene JO leader usually terminates prematurely (1). Based on in vitro stud- sequence by a synthetic DNA fragment encompassing the ies, it is believed that this coupling reflects the existence in lac operator (Fig. 1). Thereby, not only the transcription of most genes of specific pause sites where RNA polymerase the T7 gene I but also that of the target lacZ gene can be waits for the first translating ribosome (12).
    [Show full text]
  • The Irga Homologue Adhesin Iha Is an Escherichia Coli Virulence Factor in Murine Urinary Tract Infection
    Washington University School of Medicine Digital Commons@Becker Open Access Publications 2005 The rI gA homologue adhesin Iha is an Escherichia coli virulence factor in murine urinary tract infection James R. Johnson University of Minnesota - Twin Cities Srdjan Jelacic Children's Hospital and Regional Medical Center, Seattle Laura M. Schoening Children's Hospital and Regional Medical Center, Seattle Connie Clabots University of Minnesota - Twin Cities Nurmohammad Shaikh Washington University School of Medicine in St. Louis See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Johnson, James R.; Jelacic, Srdjan; Schoening, Laura M.; Clabots, Connie; Shaikh, Nurmohammad; Mobley, Harry L.T.; and Tarr, Phillip I., ,"The rI gA homologue adhesin Iha is an Escherichia coli virulence factor in murine urinary tract infection." Infection and Immunology.73,2. 965-971. (2005). https://digitalcommons.wustl.edu/open_access_pubs/2157 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Authors James R. Johnson, Srdjan Jelacic, Laura M. Schoening, Connie Clabots, Nurmohammad Shaikh, Harry L.T. Mobley, and Phillip I. Tarr This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/open_access_pubs/2157 The IrgA Homologue Adhesin Iha Is an Escherichia coli Virulence Factor in Murine Urinary Tract Infection James R. Johnson, Srdjan Jelacic, Laura M. Schoening, Connie Clabots, Nurmohammad Shaikh, Harry L. T. Mobley and Phillip I.
    [Show full text]
  • Tuning Escherichia Coli for Membrane Protein Overexpression
    Tuning Escherichia coli for membrane protein overexpression Samuel Wagner*†‡, Mirjam M. Klepsch*, Susan Schlegel*, Ansgar Appel*, Roger Draheim*, Michael Tarry*, Martin Ho¨ gbom*, Klaas J. van Wijk§, Dirk J. Slotboom¶, Jan O. Perssonʈ, and Jan-Willem de Gier*†** *Center for Biomembrane Research, Department of Biochemistry and Biophysics, ʈDepartment of Mathematics and Statistics, and †Xbrane Bioscience AB, Arrhenius Laboratories, Stockholm University, SE-106 91 Stockholm, Sweden; §Department of Plant Biology, Cornell University, Ithaca, NY 14853; and ¶Department of Biochemistry, University of Groningen, Nyenborg 4, 9747 AG Groningen, The Netherlands Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved July 30, 2008 (received for review April 28, 2008) A simple generic method for optimizing membrane protein over- produce low amounts of T7Lys, whereas pLysE hosts produce expression in Escherichia coli is still lacking. We have studied the much more enzyme and, therefore, provide a more stringent physiological response of the widely used ‘‘Walker strains’’ control (6). C41(DE3) and C43(DE3), which are derived from BL21(DE3), to Recently, we studied the physiological response of E. coli membrane protein overexpression. For unknown reasons, overex- BL21(DE3)pLysS to membrane protein overexpression (7). Our pression of many membrane proteins in these strains is hardly aim was to identify potential bottlenecks that hamper membrane toxic, often resulting in high overexpression yields. By using a protein overexpression and to use this information to engineer combination of physiological, proteomic, and genetic techniques strains with improved overexpression characteristics. We found we have shown that mutations in the lacUV5 promoter governing that membrane protein overexpression resulted in accumulation expression of T7 RNA polymerase are key to the improved mem- of cytoplasmic aggregates containing the overexpressed protein brane protein overexpression characteristics of the Walker strains.
    [Show full text]
  • Tailoring the Evolution of BL21(DE3) Uncovers a Key Role for RNA Stability in Gene Expression Toxicity ✉ Sophia A
    ARTICLE https://doi.org/10.1038/s42003-021-02493-4 OPEN Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity ✉ Sophia A. H. Heyde1 & Morten H. H. Nørholm 1 Gene expression toxicity is an important biological phenomenon and a major bottleneck in biotechnology. Escherichia coli BL21(DE3) is the most popular choice for recombinant protein production, and various derivatives have been evolved or engineered to facilitate improved yield and tolerance to toxic genes. However, previous efforts to evolve BL21, such as the Walker strains C41 and C43, resulted only in decreased expression strength of the 1234567890():,; T7 system. This reveals little about the mechanisms at play and constitutes only marginal progress towards a generally higher producing cell factory. Here, we restrict the solution space for BL21(DE3) to evolve tolerance and isolate a mutant strain Evo21(DE3) with a truncation in the essential RNase E. This suggests that RNA stability plays a central role in gene expression toxicity. The evolved rne truncation is similar to a mutation previously engineered into the commercially available BL21Star(DE3), which challenges the existing assumption that this strain is unsuitable for expressing toxic proteins. We isolated another dominant mutation in a presumed substrate binding site of RNase E that improves protein production further when provided as an auxiliary plasmid. This makes it easy to improve other BL21 variants and points to RNases as prime targets for cell factory optimisation.
    [Show full text]
  • Highly Efficient Primed Spacer Acquisition from Targets Destroyed by the Escherichia Coli Type I-E CRISPR-Cas Interfering Complex
    Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex Ekaterina Semenovaa, Ekaterina Savitskayab,c, Olga Musharovab,d,e, Alexandra Strotskayaa,b,c,e, Daria Vorontsovaa,b,e, Kirill A. Datsenkoa,f, Maria D. Logachevag, and Konstantin Severinova,b,c,d,e,1 aWaksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854; bSkolkovo Institute of Science and Technology, Skolkovo 143025, Russia; cInstitute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia; dInstitute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia; ePeter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia; fDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907; and gM.V. Lomonosov Moscow State University, Moscow 119991, Russia Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved May 9, 2016 (received for review February 16, 2016) Prokaryotic clustered regularly interspaced short palindromic repeat complex alone (type II) or through recruitment of an additional (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acqui- endonuclease Cas3 (type I systems) (4). Cas1 and Cas2 are not sition of spacers—short fragments of foreign DNA. For the type I-E required for interference in vivo (2) and in vitro (15, 16). CRISPR-Cas system from Escherichia coli, efficient “primed” adapta- Point mutations in protospacer or associated PAM decrease the tion requires Cas effector proteins and a CRISPR RNA (crRNA) whose affinity of crRNA–effector complex binding to protospacer (17). spacer partially matches a segment (protospacer) in target DNA. Under pressure from CRISPR-Cas, mobile genetic elements ac- Primed adaptation leads to selective acquisition of additional spacers cumulate such mutations, which allows them to escape CRISPR from DNA molecules recognized by the effector–crRNA complex.
    [Show full text]
  • A Plasmid-Encoded Papb Paralogue Modulates Autoaggregation of Escherichia Coli Transconjugants
    A plasmid-encoded papB paralogue modulates autoaggregation of Escherichia coli transconjugants Ruben Monarrez Haverford College Iruka Okeke ( [email protected] ) Haverford College, University of Ibadan https://orcid.org/0000-0002-1694-7587 Research note Keywords: plasmid, PapB, PefB, mbrial regulation, autoaggregation Posted Date: December 7th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-36006/v3 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on December 14th, 2020. See the published version at https://doi.org/10.1186/s13104-020-05405-7. Page 1/17 Abstract Objective: Plasmids are key to antimicrobial resistance transmission among enteric bacteria. It is becoming increasingly clear that resistance genes alone do not account for the selective advantage of plasmids and bacterial strains that harbor them. Deletion of a 32 Kb tness-conferring region of pMB2, a conjugative resistance plasmid, produced a hyper-autoaggregation phenotype in laboratory Escherichia coli. This study sought to determine the genetic basis for hyper-autoaggregation conferred by the pMB2- derived mini-plasmid. Results: The 32 Kb fragment deleted from pMB2 included previously characterized nutrient acquisition genes as well as putative transposase and integrase genes, a 272 bp papB/ pefB-like gene, and several open-reading frames of unknown function. We cloned the papB/ pefB paralogue and found it sucient to temper the hyper-autoaggregation phenotype. Hyper-autoaggregation conferred by the mini-plasmid did not occur in a m-negative background. This study has identied and characterized a gene capable of down-regulating host adhesins and has shown that trans-acting papB/pefB paralogues can occur outside the context of an adhesin cluster.
    [Show full text]
  • RNA Remodeling by Bacterial Global Regulator Csra Promotes Rho-Dependent Transcription Termination
    Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination Nara Figueroa-Bossi,1 Annie Schwartz,2 Benoit Guillemardet,1 Francxois D’Heyge`re,2 Lionello Bossi,1,3 and Marc Boudvillain2,3 1CNRS UPR3404, Centre de Ge´ne´tique Mole´culaire, Gif-sur-Yvette 91198, France, affilie´ a` l’Universite´ Paris Sud XI, Orsay 91405, France; 2CNRS UPR4301, Centre de Biophysique Mole´culaire, Orle´ans 45071, France, affilie´ a` l’Universite´ d’Orle´ans, Orle´ans 45100, France RNA-binding protein CsrA is a key regulator of a variety of cellular processes in bacteria, including carbon and stationary phase metabolism, biofilm formation, quorum sensing, and virulence gene expression in pathogens. CsrA binds to bipartite sequence elements at or near the ribosome loading site in messenger RNA (mRNA), most often inhibiting translation initiation. Here we describe an alternative novel mechanism through which CsrA achieves negative regulation. We show that CsrA binding to the upstream portion of the 59 untranslated region of Escherichia coli pgaA mRNA—encoding a polysaccharide adhesin export protein—unfolds a secondary structure that sequesters an entry site for transcription termination factor Rho, resulting in the premature stop of transcription. These findings establish a new paradigm for bacterial gene regulation in which remodeling of the nascent transcript by a regulatory protein promotes Rho-dependent transcription attenuation. [Keywords: CsrA; Rho factor; transcription attenuation; bacterial gene regulation] Supplemental material is available for this article. Received February 17, 2014; revised version accepted April 18, 2014.
    [Show full text]
  • Part One Expression Systems
    11 Part One Expression Systems 13 1 Bacterial Systems James Samuelson 1.1 Introduction The study of membrane protein structure and function is limited by various chal- lenges. In native cells, membrane protein copy number is often very low, so the study of individual proteins is often not feasible. Alternatively, overexpression of these hydrophobic molecules in heterologous hosts is not a routine endeavor as it is for many water - soluble proteins. Most modern bacterial expression systems have been engineered for maximal output of recombinant protein. This character- istic is ideal for well - behaved soluble proteins, but less desirable when the target protein normally resides within a lipid environment. A compounding problem in the study of membrane proteins is that the isolated target protein may exhibit polydispersity, meaning that diverse oligomeric complexes can spontaneously accumulate. This latter concern may be infl uenced by the expression method, but primarily depends on the detergent/lipid and buffer used for solubilization. This chapter highlights preferred strategies for membrane protein expression in bacte- ria that will increase the likelihood of isolating adequate amounts of homogenous target protein. Many sections will also detail the features of expression strains that are relevant to the yield and quality of expressed protein. In this chapter, the term membrane protein will generally be used to represent α - helical membrane proteins that reside within a phospholipid bilayer environ- ment of either eukaryotic or prokaryotic cells. Such integral membrane proteins are the most diffi cult to manipulate since each contains hydrophobic transmem- brane ( TM ) regions as well as hydrophilic extramembrane regions or domains.
    [Show full text]