DOCSLIB.ORG

Cloning and Mutagenesis of the Rhizobium Meliloti Isocitrate Dehydrogenase Gene

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cloning and Mutagenesis of the Rhizobium Meliloti Isocitrate Dehydrogenase Gene JOURNAL OF BACTERIOLOGY, July 1992, p. 47904797 Vol. 174, No. 14 0021-9193/92/144790-08$02.00/0 Copyright X 1992, American Society for Microbiology Cloning and Mutagenesis of the Rhizobium meliloti Isocitrate Dehydrogenase Gene TIMOTHY R. McDERMOTT' AND MICHAEL L. KAHN1,2* Institute ofBiological Chemistry' and Department ofMicrobiology,2 Washington State University, Pullman, Washington 99164-6340 Received 18 February 1992/Accepted 21 April 1992 The gene encoding Rhizobium meliloti isocitrate dehydrogenase (ICD) was cloned by complementation of an Escherichia coli icd mutant with an R. meliloti genomic library constructed in pUC18. The complementing DNA was located on a 4.4-kb BamHI fragment. It encoded an ICD that had the same mobility as R. meliloti ICD in nondenaturing polyacrylamide gels. In Western immunoblot analysis, antibodies raised against this protein reacted with R. meliloti ICD but not with E. coli ICD. The complementing DNA fragment was mutated with transposon TnS and then exchanged for the wild-type allele by recombination by a novel method that employed the Bacillus subtilis levansucrase gene. No ICD activity was found in the two R. meliloti icd::Tn5 mutants isolated, and the mutants were also found to be glutamate auxotrophs. The mutants formed nodules, but they were completely ineffective. Faster-growing pseudorevertants were isolated from cultures of both R. melloti icd::Tn5 mutants. In addition to lacking all ICD activity, the pseudorevertants also lacked citrate synthase activity. Nodule formation by these mutants was severely affected, and inoculated plants had only callus structures or small spherical structures. In the symbiosis between legumes and rhizobia, the host tarate. Although no data were reported, an R meliloti plant provides the bacteria with reduced carbon as an energy ax-ketoglutarate dehydrogenase mutant was said to be Fix- source. The bacteria use this energy to reduce atmospheric (10). nitrogen to ammonia, which they release to the plant. The We are interested in how the bacteroid TCA cycle is qualitative nature of this energy source has been the focus of regulated and have selected ICD for our initial studies much research. Sucrose is the major photosynthate trans- because it is a regulated enzyme at a branch point in the TCA ported from the shoot to the nodule (41), but biochemical cycle (15), because it is regulated by aerobiosis in other evidence suggests that neither sucrose nor hexoses obtained gram-negative bacteria (23, 24), and because it is a relatively from sucrose degradation are important sources of energy simple enzyme with a single type of subunit that, in other for the bacteroids (reviewed in references 7 and 34). In bacteria, is encoded by a single gene (2). In this study, we support of this conclusion, mutants of various species of report the isolation of the gene that encodes ICD in R rhizobia with defects in sugar metabolism have been found meliloti and the symbiotic properties of mutants with defects to be effective in symbiosis (7, 34). By contrast, dicarboxylic in this gene. acids appear to be important carbon sources in the estab- lishment of an effective symbiosis. Succinate and malate are found at high concentrations in nodules (14, 45, 55), are MATERIALS AND METHODS actively transported across the peribacteroid membrane (20, Bacterial strains and plasmids. The strains of R. meliloti 56), are taken up by bacteroids (11, 20, 42, 50), and are and Escherichia coli, phage, and plasmids used in this study quickly oxidized to CO2 after uptake (47). Dicarboxylic acid are shown in Table 1. R meliloti was grown on yeast transport (dct) mutants of Rhizobium meliloti (5) and R. extract-mannitol medium (YMB [53]) or on minimal manni- leguminosarum biovars viciae (3, 12) and trifolii (44) are all tol medium (53) supplemented with arabinose (5 g/liter), Fix-. glutamate (1.1 g/liter), and filter-sterilized antibiotics as Succinate and malate are intermediates in the tricarboxy- indicated. Strains of E. coli were grown on either LB or M9 lic acid (TCA) cycle, and thus the TCA cycle is implicated as mineral salts (49) medium. M9 medium contained glucose as a major catabolic sequence. Enzymes of the TCA cycle, the carbon source and ammonium chloride as the primary such as citrate synthase (CS) (31), isocitrate dehydrogenase nitrogen source plus histidine (15 ,ug/ml), tryptophan (40 (ICD) (22, 28, 31), fumarase (28), malate dehydrogenase (22, p,g/ml), and thiamine (2 ,ug/ml). Filter-sterilized antibiotics 28), and a-ketoglutarate dehydrogenase (48), have been and glutamate were added as needed. For long-term storage, found in bacteroids of various species of Rhizobium and strains ofR. meliloti and E. coli were grown to mid-log phase Bradyrhizobium. Radioactive metabolite conversion studies in appropriate selective medium, diluted with an equal by Stovall and Cole (54) implied a fully functional TCA volume of sterile 50% glycerol, and stored at -70°C. cycle, but evidence for the decarboxylating leg of the TCA Cloning of R. meliloti icd and isolation of mutants. Con- cycle was not obtained until Salminen and Streeter (47) struction of the R meliloti gene bank and subsequent DNA reported that significant amounts of label from [2,3-14C]suc- manipulations followed the protocols of Sambrook et al. cinate accumulated in glutamate, indicating that in soybean (49). Aliquots of chromosomal DNA were partially digested bacteroids the TCA cycle is complete, at least to oa-ketoglu- with restriction enzyme Sau3A, and the pooled digests were fractionated by sucrose gradient centrifugation. The 4- to 8-kb fragments were then ligated to pUC18 (60) that had * Corresponding author. been digested with BamHI and calf intestinal alkaline phos- 4790 VOL. 174, 1992 R MELILOTI ISOCITRATE DEHYDROGENASE GENE 4791 TABLE 1. Strains of E. coli and R melioti, phage, and plasmids used Strain, phage, Relevant genotype or characteristics Relevant phenotype" Reference or source or plasmid RI meliloti 104A14 Wild type 53 A39S 104A14::TnSicd ICD- Glut- Kanr This study B49S 104A14::TnSicd ICD- Glut- Kanr This study A39L 104A14::TnSicd gItA (?) ICD- Glut- Kanr CS- This study B49L 104A14::TnSicdgkA (?) ICD- Glut- Kan' CS This study E. coli S17-1 Pro- Mob' 52 DL39 ilvE12 tyrB507 aspC13 Sup' 4 DEK2038 F- A- his-4 thi-1 rspL31 lacBKI rfiB recA ICD- Glut- Peter Thorsness aceKi icd-11 RR1 supE44 hsdS20 ara-14 proA2 lacYI galK2 High-efficiency transformation 49 rpsL20 xyl-5 mtl-l Phage X467 TnS Kanr 8 Plasmids pUC18 Penr 60 pTK509 pBR322 with E. coli icd Penr Peter Thorsness pTM1 pUC18 with R meliloti icd Pen' This study pRK311 tetA Tetr 9 pUM24 nptI sacB sacR Kanr Suc, 43 pMK413 tetA sacB sacR Tetr Suc, This study pTM2 pMK413 with R. meliloti icd Tetr Suc, This study pTM3 pRK311 with R melioti icd Tetr This study pTM2::TnSA39 icd::TnS mutant of pTM2 Suc5 Tetr Kanr This study pTM2::Tn5B49 icd::TnS mutant of pTM2 Suc5 Tetr Kanr This study I Glut, glutamate; Pro, proline; Suc, sucrose. phatase (Boehringer). Transformation ofE. coli RR1 yielded mM DL-isocitrate, 0.4 mM NADP+, 3.3 mM MgCl2, 10 mM approxmately 10,000 independent transformants that con- potassium phosphate buffer (pH 7.4), and enzyme extract in tained inserts. Plasmid DNA was purified from these colo- a final reaction volume of 1.0 ml. nies and used as the gene bank in the initial selection. For (ii) CS. CS was assayed by measuring the formation of transposon TnS mutagenesis, we used X467::TnS as outlined coenzyme A-sulfhydryl by the increase in A412 as described by de Bruijn and Lupski (8). by Sere (51). Assay mixes contained 200 mM Tris (pH 8.1), A mutated allele is often recombined into R meliloti by first using a broad-host-range plasmid to bring the allele into the strain and then using a second plasmid of the same incompatibility group to destabilize the first plasmid (46). By EOR continuing to select for the mutated allele, the desired p_iEcoRI recombinant can be recovered, since it will stably maintain / ~~~~onV ~% Hindlll the selected marker. Although the technique works in R sac meliloti 104A14, plasmid incompatibility is not very strong, Ps and this procedure can be tedious, especially if the mutant grows more slowly than the wild type (26). An alternative method was developed in which a vector that allows direct selection is used to obtain cells that have lost the plasmid carrying the mutated allele. pMK413 (Fig. 1) contains the pMK413 SetA Bacillus subtilis levansucrase genes, which inhibit the LSmal growth of gram-negative bacteria when they are incubated in 23kb media that contain sucrose (17, 21). To construct pMK413, the 1.7-kb cos fragment of pRK311 (9) was replaced by the 3.8-kb BamHI fragment from pUM24 (43), which contains \_oriT/ the B. subtilis sacR and sacB genes, which encode levansu- crase, and the nptl gene, which confers kanamycin resis- ESol la a2 tance (KanT). The nptI gene was subsequently deleted by SaiHl using PstI. E. coli DEK2038(pMK413) and R meliloti PstI 104A14(pMK413) do not grow on media that contain tetra- Hindlil cycline (to select for plasmid retention) and 5% sucrose. FIG. 1. Restriction map of pMK413. The cos fragment was Enzyme assays. (i) ICD. ICD activity in cell extracts was deleted from pRK311 (9) and replaced with the sac cassette from measured spectrophotometrically. The rate of NADP+ re- pUM24 (49) as described in Materials and Methods. Arrow indicates duction was measured at 340 nm. Assay mixes contained 0.5 orientation of lacZ relative to the polylinker site. 4792 McDERMOTT AND KAHN J. BACTERIOL. 0.2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 0.1 mM containing 0.02 M Tris base and 0.1 M NaCl (pH 8.0), and acetyl-S-CoA, 0.5 mM potassium oxaloacetate, and enzyme stored at -20°C.
Load more

Recommended publications
  • Part Ii - Molecular Biology
    PART II - MOLECULAR BIOLOGY Module VII Nature of Genetic Materials Modern concept of gene (Cistron, muton, recon, viral genes). Brief account of the following-- Split genes (introns and exons), Junk genes, Pseudogenes, Overlapping genes, Transposons The term gene was introduced by Johanssen in 1909. Prior to him Mendel had used the word factor for a specific, distinct, particulate unit of inheritance that takes part in expression of a trait. Johanssen has defined gene as an elementary unit of inheritance which can be assigned to a particular trait. Morgan’s work suggested gene to be the shortest segment of chromosome which can be separated through crossing over, can undergo mutation and influence expression of one or more traits. Presently, a gene is defined as a unit of inheritance composed of a segment of DNA or chromosome situated at a specific locus (gene locus) which carries coded information associated with a specific function and can undergo crossing over as well as mutation. A gene is: A unit of genetic material which is able to replicate, It is a unit of recombination, i.e., capable of undergoing crossing over, A unit of genetic material which can undergo mutation, A unit of heredity connected with somatic structure or function that leads to a phenotypic expression. Modern concept of gene (Cistron, muton, recon, viral genes). After the discovery of DNA, the gene has been defined as cistron, recon and muton. The classical gene is the smallest unit that could undergo a mutational change. A gene further divided into smaller units of function, mutation and recombination.
    [Show full text]
  • 5 Complementation Analysis: How Many Genes Are Involved?
    Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic) 5 Complementation Analysis: How Many Genes are Involved? Complementation analysis is used to determine whether two independent mutations arealterations in the same gene; that is, they are alleles, orare alterations in different genes. In essence, a complementation analysis is a functional test used to define a gene. If a researcher has isolated anumber of mutants with a similar phenotype, the next question asked is: ‘How many genes have I identified?’. If there are 10 mutant strains, are they each in different genes, does each mutant carry a different mutation (allele) in the same gene, or something in between such as two genes one with six alleles and the otherwith four alleles? Complementation analysis will help answer this question. Seymour Benzer’s study of the rZZ locus of phage T4 is a most elegant example of the power of complementation analysis (Benzer, 1955). Benzer had several hundred mutations that gave the same phenotype, large plaques on one host strain of E. coli and no plaques on another host strain, and mapped to the same region of the T4 chromosome. Using the host strain in which rll mutants formed no plaques, he found that when host cells were coinfected with different mutant pairs some pairs produced a normal phage burst while others did not. Those that produced a normal burst he concluded were in different functional genetic units that he called cistrons, a term that is synonymous with gene.
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [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]
  • Polymorphisms of the 5' Leader Cistron of the Human Beta2- Adrenergic Receptor Regulate Receptor Expression
    Polymorphisms of the 5' leader cistron of the human beta2- adrenergic receptor regulate receptor expression. D W McGraw, … , L A Kramer, S B Liggett J Clin Invest. 1998;102(11):1927-1932. https://doi.org/10.1172/JCI4862. Research Article Cellular expression of the beta2-adrenergic receptor (beta2AR) is controlled in part by a 19-amino acid peptide that regulates mRNA translation. This peptide is encoded by a short open reading frame, termed the 5' leader cistron (5'LC), which is 102 bp upstream of the beta2AR coding block. In 176 normal subjects we found a single nucleotide polymorphism resulting in either Arg (previously denoted wild-type) or Cys at position 19 of this peptide. Allele frequencies were 0.37 for Arg and 0.63 for Cys. To determine if these variants altered beta2AR expression, COS-7 cells were transfected with polymorphic constructs consisting of 1,989 bp encompassing the 5'LC and the beta2AR coding block exactly as found in the human gene. beta2AR density, as determined by [125I]CYP radioligand binding, was 72% higher in cells transfected with the 5'LC-Cys19 construct as compared with those transfected with the 5'LC-Arg19 construct and 110% higher when a cotransfection technique with a luciferase construct was used to control for transfection efficiency. Levels of the two mRNA transcripts were not different, confirming in vitro studies that the upstream peptide regulates receptor expression at the translational level. In human airway smooth muscle cells that natively express beta2AR, receptor expression was approximately twofold higher in those bearing the Cys versus the Arg polymorphism, confirming the phenotype in a relevant cell type.
    [Show full text]
  • STRUCTURE of the Trpc CISTRON SPECIFYING INDOLEGLYCEROL PHOSPHATE SYNTHETASE, and ITS LOCALIZATION in the TRYPTOPHAN OPERON of ESCHERICHIA COLP
    STRUCTURE OF THE trpC CISTRON SPECIFYING INDOLEGLYCEROL PHOSPHATE SYNTHETASE, AND ITS LOCALIZATION IN THE TRYPTOPHAN OPERON OF ESCHERICHIA COLP OLIVER H. SMITH Department of Biology, Marquette University, Milwaukee, Wisconsin 53233 Received March 22, 1967 HE definition of an operon (JACOBand MONOD1961) implies that it consti- Ttutes a unit of both gene structure and gene expression. In Escherichia coli the synthesis of the enzymes specific to the tryptophan pathway has been shown to be coordinate (ITOand CRAWFORD1965) and subject to polarized regulation ( SOMERVILLEand YANOFSKY1965; YANOFSKYand ITO1966). These observations have confirmed that the earlier reported closely linked sequence of trp genes (YANOFSKYand LENNOX1959) constitute an operon. Since this latter report the complete sequence of enzymatic reactions in tryptophan synthesis by E. coli, under the control of five structural genes (A-E),has been elucidated (e.g. see YANOFSKYand ITO1966). YANOFSKYet al. (1 964) have reported that the E. coli trpA and B genes which determine the a and j3 subunits of tryptophan synthetase are adjacent. One of the purposes of this stud:y was to determine whether the trpC gene and the other genes of the tryptophan toperon were directly adjacent to one another. The enzyme indoleglycerol phosphate (InGP) synthetase is the gene product of the trpC locus (SMITHand YANOFSKY1960; SMITH1965). The single poly- peptide chain of which InGP synthetase consists has recently been purified to homogeneity in the ultracentrifuge ( CREIGHTONand YANOFSKY1966; SMITH, unpublished) and shown to carry out the following consecutive reactions in tryptophan biosynthesis: (1) Phosphoribosyl anthranilic acid (PRA) -1- (0-Carboxylphenylamino) -1-deoxyribu- lose 5-phosphate (CDRP) (2) CDRP- InGP This report offers evidence that the ability to catalyze each of the above reactions is altered by mutations affecting different parts of the InGP synthetase poly- peptide chain.
    [Show full text]
  • Seymour Benzer and T4 Rii Running the Map Into the Ground
    GENERAL ARTICLE Seymour Benzer and T4 rII Running the Map into the Ground R Jayaraman One of the goals of genetics is to understand genes in as much detail as possible. For instance, with respect to a given gene, one would like to know its chromosomal location, its physical and genetic size, its neighbours, the number of mutations/ alleles defining the gene, the order of mutations, the genetic/ physical distance between them, etc. Thus, rather than focus- R Jayaraman is an ing on the whole genome, one focuses on the finer details of a Emeritus Professor of given genetic segment. The exercise of probing such details is Molecular Biology at the School of Biological called ‘fine structure genetic analysis’. There are several Sciences, Madurai pioneers who have contributed enormously to this area in Kamaraj University, many bacterial and phage systems. Two stalwarts, Seymour Madurai, where he taught BenzerandCharles Yanofsky,standpreeminentamong them. and carried out research in the area of microbial In the following pages I present briefly Benzer’s outstanding genetics for three decades. workonthefinestructureofthe rII region of bacteriopage T4. These path-breaking studies contributed significantly to our understanding of the structure, organization and function of genes. Seymour Benzer (1921–2007) is hailed as a scientist who, “more than any other single individual, enabled geneticists adapt to the molecular age” [1]. After graduating from Brooklyn College with a degree in physics, he went to Purdue University, Lafayette, Indiana, to continue his education in physics. His work at Purdue on germanium semiconductors was a forerunner to the invention of the transistor by Brattain, Bardeen and Shockley, a work which fetched them the Nobel Prize.
    [Show full text]
  • Downloaded October, 2015) 83
    bioRxiv preprint doi: https://doi.org/10.1101/522607; this version posted January 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Induction of an alternative 5’ leader enhances translation of Inpp5e and resistance to oncolytic virus infection Huy-Dung Hoang1,Δ, Tyson E. Graber1,2,Δ, Jian-Jun Jia1, Nasana Vaidya1, Victoria Gilchrist1, Wencheng Li3, Christos G. Gkogkas4, Maritza Jaramillo5, Seyed Mehdi Jafarnejad6, and Tommy Alain1* 1 Children's Hospital of Eastern Ontario Research Institute, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada 2 Department of Biochemistry and Goodman Cancer Center, McGill University, Montreal, Quebec, Canada 3 Department of Biochemistry and Molecular Biology, Rutgers New Jersey Medical School, Newark, NJ, USA 4 Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK. 5 INRS Institut Armand-Frappier Research Centre, Laval, Quebec, Canada 6 Centre for Cancer Research and Cell Biology, The Queen’s University of Belfast, Belfast, UK *correspondence: Tommy Alain Children’s Hospital of Eastern Ontario Research Institute 401 Smyth Road Ottawa, Ontario, Canada Δ These authors contributed equally to this work. Short Title: Oncolytic viruses trigger an innate RNA translation response ABSTRACT variant that lacks repressive upstream open reading frames (uORFs) within its 5’ leader and is consequently Residual cell-intrinsic innate immunity in cancer cells efficiently translated. Furthermore, we show that INPP5E hampers infection with oncolytic viruses. mRNA contributes to antiviral immunity by altering virus translation is an important component of innate attachment. These findings uncover a role for immunity, yet the targeted cellular mRNAs remain ill- translational control through alternative 5’ leader defined.
    [Show full text]
  • Degradation of Mrna Mk2 and Enhanced Transcription Termination Before the Galk Gene
    Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene Xun Wang, Sang Chun Ji1, Heung Jin Jeon, Yonho Lee, and Heon M. Lim2 Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea Edited by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved May 11, 2015 (received for review December 29, 2014) The Escherichia coli gal operon has the structure Pgal-galE-galT- that there is an independent molecular mechanism other than galK-galM. During early log growth, a gradient in gene expression, transcription termination that operates to regulate galK expression named type 2 polarity, is established, as follows: galE > galT > galK > (1) and that the regulation of the amount of mK2 determines the galM. However, during late-log growth, type 1 polarity is established polarity type. in which galK is greater than galT,asfollows:galE > galK > galT > Small RNAs (sRNAs) in bacteria bind to their target mRNA galM. We found that type 2 polarity occurs as a result of the down- molecules and regulate their expression. Often, the binding of an regulation of galK, which is caused by two different molecular mech- sRNA to a target mRNA is mediated by Hfq, a homo-hexameric, anisms: Spot 42-mediated degradation of the galK-specific mRNA, bacterial Sm-like protein (3). In the case of an sRNA whose mK2, and Spot 42-mediated Rho-dependent transcription termination binding site on the target mRNA overlaps with the Shine- at the end of galT.
    [Show full text]
  • Identification and Characterization of RP1 Tra1 Cistrons Involved in Pilus Function and Plasmid Mobilization
    JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 448-456 Vol. 175, No. 2 0021-9193/93/020448-09$02.00/0 Copyright © 1993, American Society for Microbiology Identification and Characterization of RP1 Tral Cistrons Involved in Pilus Function and Plasmid Mobilization S. T. FONGt AND VILMA A. STANISICH* Department ofMicrobiology, La Trobe University, Bundoora 3083, Australia Received 21 January 1992/Accepted 6 November 1992 Transfer-defective mutants of the Tral region of RP1 were isolated. Complementation studies involving stable heterozygotes combined with the mapping of TnS insertion mutations revealed two pilus cistrons, pi4 and pilB, at positions 46.9 to 48.2 kb and 46.0 to 46.4 kb, respectively. All pilB mutants were Dps- (i.e., resistant to donor-specific phages PR4 and PRR1), whereas piL4 mutants were Dps- (promoter-proximal mutations), Dps+'- (sensitive only to PR4 [more centrally located mutations]), or Dps' (sensitive to both phages [promoter-distal mutations]). The correlation between the site mutated and the Dps phenotype, together with the finding that certain Dps+ piL4 mutants continued to mobilize nonconjugative plasmids, suggested that piL4 is bifunctional, contributing both to pilus function (at the promoter-proximal end) and to RP1 mobilization. It was also shown that the 43.5- to 49.5-kb region that includes pilA and pilB encodes all of the Tral pilus functions required for propagation of donor-specific phages and hence, probably, for pili that are active in conjugation. Finally, three cistrons that specifically affect RP1 mobilization were identified. Two of these, mobA and mobB, occur immediately anticlockwise to oriT and probably correspond to the traJ and traI genes characterized by other workers.
    [Show full text]
  • Working with Molecular Genetics Chapter 1. Fundamental Properties of Genes CHAPTER 1 FUNDAMENTAL PROPERTIES of GENES Species
    Working with Molecular Genetics Chapter 1. Fundamental Properties of Genes CHAPTER 1 FUNDAMENTAL PROPERTIES OF GENES Species share many traits in common from generation to generation. The bluebird nestlings in the box in my yard will look much like their parents when they are full-grown. The tomato plants that we set out will produce fruits that look, and hopefully taste, like those of their parents. Observable features of organisms, like color, size, and shape, comprise their phenotype. Adult male bluebirds share the phenotype of blue wings and a red breast. A phenotype can be determined by inherited factors, by the environment, and often by both. For example, you are similar to your parents in many aspects of your appearance, your intelligence, and your susceptibility to some diseases, but you are not identical to them in all aspects of these traits. These three traits are clearly the product of both inherited and environmental factors. Considering appearance, I have crooked lower teeth and thinning gray hair, just like my father, but unlike me, neither of my parents has a scar on their knee from a childhood cut. The hair phenotype is inherited, whereas scars are from environmental influences. Quantitative studies show that intellectual capacity is about equally influenced by genetic and environmental factors. Susceptibility to diabetes is partially inherited, but a viral infection may trigger the autoimmune response at its core. The genetic determinants of the inherited component of a phenotype are called genes. The set of genes that make up an organism is its genotype. In practice, we will consider only a small subset of the genes in an organism, which comprise a partial genotype.
    [Show full text]
  • Noncoding RNA Control of the Making and Breaking of Sugars
    Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Noncoding RNA control of the making and breaking of sugars Boris Görke1 and Jörg Vogel2,3 1Department of General Microbiology, Georg August University Göttingen, D-37077 Göttingen, Germany; 2RNA Biology Group, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany Noncoding RNA regulators have been implicated in al- drate transporters or of intracellular glycolytic metabo- most all imaginable cellular processes. Here we review lites. These cues modulate the synthesis of intracellular how regulatory small RNAs such as Spot42, SgrS, GlmY, signaling molecules such as cAMP, or the activity of and GlmZ and a cis-encoded ribozyme in glmS mRNA protein kinases, which in turn control the activity of control sugar metabolism. Besides discussing the physi- global regulatory proteins; e.g., Crp (cAMP receptor pro- ological implications, we show how the study of these tein) in enteric bacteria or cAMP-controlled protein ki- molecules contributed to our understanding of the nase A in eukaryotic cells (Deutscher 2008; Gancedo mechanisms and of general principles of RNA-based 2008; Görke and Stülke 2008). regulation. These include the post-transcriptional re- Once inside the cell, carbohydrates enter a central pression or activation of gene expression within poly- metabolic pathway i.e., glycolysis, the pentose phos- cistronic mRNAs; novel ribonucleoprotein complexes phate pathway, or the Krebs (TCA) cycle. Flux through composed of small RNA, Hfq, and/or RNase E; and the these pathways must be tightly coordinated with all hierarchical action of regulatory RNAs. other processes in the cell to avoid deleterious accumu- lation or depletion of metabolites.
    [Show full text]