DOCSLIB.ORG

Structure-Function Studies of the Co2 Uptake Complex In

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structure-Function Studies of the Co2 Uptake Complex In STRUCTURE-FUNCTION STUDIES OF THE CO2 UPTAKE COMPLEX IN CYANOBACTERIA By JULIANA ARTIER Bachelor of Science in Biological Science Universidade Federal de São Carlos São Carlos, SP, Brazil 2007 Master of Science in Genetics and Evolution Universidade Federal de São Carlos São Carlos, SP, Brazil 2010 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY July, 2018 STRUCTURE-FUNCTION STUDIES OF THE CO2 UPTAKE COMPLEX IN CYANOBACTERIA Dissertation Approved: Robert L. Burnap Dissertation Adviser Rolf A. Prade Marianna A. Patrauchan Wouter D. Hoff William J. Henley ii ACKNOWLEDGEMENTS I want to thank my advisor and mentor Robert Burnap for all the support, in the past, present, and future. I also greatly appreciate the time and efforts my committee members kindly devoted towards my academic development. I am grateful to Dean G. Price for detailed help with the Ci affinity assays and collaboration in this study. Thanks to Anthony D. Kappell and Steven Holland for contribution in the molecular design of the CCM knockout constructs. Also, I am extremely thankful to all involved in this project: Steven Holland for assistance constructing the CCM knockout mutants and with PAM analysis, Minquan Zhang for collaboration on protein expression experiments, Neil T. Miller for help with Western blots, and Prachi Zawar for collaboration in constructing the Arthrospira heterologous expression systems. Also, I appreciate the kind gifts of vectors: the integration vectors provided by H. Wang and Wim Vermaas; MBP containing vectors from Junpeng Deng. I want to thank the assistance of Janet Rogers and Steven Hartson for DNA sequencing and mass spectrometry analyses within the DNA/Protein Resource Facility at Oklahoma State University, supported by the NSF MRI and EPSCoR programs (award #0722494). This dissertation was only possible due to financial support by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences, grant no. DE- iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. FG02-08ER15968. During all the years of my PhD studies, my family and friends were essential, although not directly involved in the project, but in my daily life, sharing the good and the bad. I am extremely grateful to all the emotional support and loving moments. At the Burnap lab I had the pleasure to share a great, professional and fun environment by the side of Han Bao, Steven Holland, Preston Dilbeck, Aparna Nagarajan, Neil Miller and Anton Avramov, making the daily work so much more enjoyable, and I am so grateful. Also, I am thankful to the friends I made along the way, Abhaya Ranganathan, Amanda Behar, Ruth Weidman, Rachana Rathod, Sharmily Khanam, Prachi Zawar, Shaikh Saad. I am grateful for the family environment and amazing moments shared with the Brazilian community in Stillwater, including Hevila Brognaro, Mariane Zubieta, Zeyna Abramson, with a special thanks to Beatriz Solera and family for opening their hearts and home. I am also grateful to Bruno Sauce, for pushing me towards better worlds. Special thanks to my loving and kind husband Raid Alzaher, who I met at OSU, for all the support, patience and for never letting me give up. I am grateful to my family, my brother Alexandre, aunts, uncles, grandparents, for the emotional support and understanding. I dedicate this dissertation to my parents, Marli and Irineu, who always stand by my side, even when an ocean away. iv Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: JULIANA ARTIER Date of Degree: JULY, 2018 Title of Study: STRUCTURE-FUNCTION STUDIES OF TH CO2 UPTAKE COMPLEX IN CYANOBACTERIA Major Field: MICROBIOLOGY & MOLECULAR BIOLOGY Abstract: Increase of CO2 atmospheric level has led to an intense search for solutions to mitigate the problem. A natural pathway exists in photosynthesis where CO2 is fixed into biomass. Cyanobacteria, which constitute a large phylum of natural oxygenic photosynthetic bacteria, have a huge potential for bioengineering. This includes candidates for use in diverse CO2 capture and storage projects and the potential to redirect its energy for the production of valuable compounds. Our aim in this project is to understand the structure-function of Cup (CO2 uptake) proteins in the NDH-13,4 complex, part of the cyanobacterial CO2 concentrating mechanism (CCM). Cyanobacteria have five CCM systems responsible for increasing inorganic carbon (Ci) concentration inside of the cell, an effort to raise CO2 level close to Rubisco, the main enzyme responsible for carbon fixation. Among them, two CO2 uptake systems are specialized NDH-1 complexes, NDH- 13 (NdhF3/NdhD3/CupA/CupS) and NDH-14 (NdhF4/NdhD4/CupB), which have very little known about their mechanism. CO2 is usually regulated in living organisms by - carbonic anhydrases (CAs), enzymes that catalyzes the interconversion of CO2 and HCO3 . The hypothesis is that Cup, in the NDH-13,4 complex, is involved in this (non)reversible reaction, possibly coupled with release of a proton across the membrane. We constructed a double knockout mutant, where no NDH-13,4 is produced by the bacteria, and introduced the whole cupA operon under Rubisco promoter control in Synechocystis 6803. Physiological analysis of Ci depleted mutants with chlorophyll fluorescence traces and O2 evolution dependent on Ci, show a high Ci requirement feature on the double knockout mutants. However, the strain expressing constitutively NDH-13 lost its high CO2-requiring phenotype, displaying restored cell CO2 uptake. In parallel, we also analyzed Synechococcus 7942 mutants with no functional CO2 uptake systems, later complemented with only chpX (cupB). These systems were used in studies directed to analyze the effect of point mutations replacing amino acids (His/Cys) of CupA/CupB proteins to evaluate their potential role on CO2 uptake in cyanobacteria. v TABLE OF CONTENTS Chapter Page 1 CHAPTER I ........................................................................................................... 1 INTRODUCTION AND LITERATURE REVIEW .................................................... 1 1.1 INTRODUCTION ................................................................................................. 1 1.2 CYANOBACTERIA, PHOTOSYNTHESIS AND CO2 ADAPTATION............................... 5 1.3 PHOTOSYNTHESIS AND THE PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN ...... 7 1.4 CARBON FIXATION IN CYANOBACTERIA ........................................................... 15 1.5 CO2 CONCENTRATING MECHANISMS IN CYANOBACTERIA .................................. 16 1.5.1 Carboxysome ............................................................................................. 19 1.5.2 Bicarbonate transporters: BCT1, BicA and SbtA ........................................ 21 1.6 NDH-1 COMPLEX AND CUP PROTEINS .............................................................. 23 1.7 CARBONIC ANHYDRASES, STRUCTURE-FUNCTION OVERVIEW AND IMPORTANCE IN CYANOBACTERIA ........................................................................................................ 29 1.8 QUESTIONS AND GOALS ................................................................................... 33 2 CHAPTER II ....................................................................................................... 35 EXPERIMENTAL PROCEDURES ........................................................................... 35 2.1 MATERIALS AND METHODS ............................................................................. 35 2.2 CULTURE AND GROWTH CONDITIONS ............................................................... 35 2.3 STRAINS AND MOLECULAR CONSTRUCTS .......................................................... 37 2.4 CELL SAMPLE PREPARATION ............................................................................ 40 2.5 CI AFFINITY AND OXYGEN EVOLUTION ASSAYS. ................................................ 41 2.6 CHL AND NADPH FLUORESCENCE AND P700 SPECTROSCOPY .......................... 42 2.6.1 Chl and NADPH fluorescence .................................................................... 42 vi 2.6.2 P700 spectroscopy ..................................................................................... 43 2.7 SDS-PAGE AND IMMUNOBLOT ANALYSIS ....................................................... 44 3 CHAPTER III ...................................................................................................... 47 HETEROLOGOUS EXPRESSION OF CUPA ......................................................... 47 3.1 INTRODUCTION ............................................................................................... 47 3.2 MATERIAL AND METHODS .............................................................................. 48 3.2.1 Molecular Construction.............................................................................. 48 3.2.2 Expression of CupA protein in E. coli ......................................................... 49 3.2.3 Protein purification, ................................................................................... 50 3.2.4 Antibody production ................................................................................... 52 3.2.5 CD spectroscopy ........................................................................................ 52 3.2.6 Zn binding evaluation and CA activity .......................................................
Load more

Recommended publications
  • Comparative Chloroplast Genomes of Four Lycoris Species (Amaryllidaceae) Provides New Insight Into Interspecific Relationship and Phylogeny
    biology Article Comparative Chloroplast Genomes of Four Lycoris Species (Amaryllidaceae) Provides New Insight into Interspecific Relationship and Phylogeny Fengjiao Zhang 1,2, Ning Wang 1,2, Guanghao Cheng 1,2, Xiaochun Shu 1,2, Tao Wang 1,2 , Weibing Zhuang 1,2, Ruisen Lu 1,2,* and Zhong Wang 1,2,* 1 Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun Yat-Sen), Nanjing 210014, China; [email protected] (F.Z.); [email protected] (N.W.); [email protected] (G.C.); [email protected] (X.S.); [email protected] (T.W.); [email protected] (W.Z.) 2 The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun Yat-Sen), Nanjing 210014, China * Correspondence: [email protected] (R.L.); [email protected] (Z.W.) Simple Summary: The genus Lycoris (Amaryllidaceae) comprises about 20 species with high orna- mental and medicinal value. However, germplasm identification is still difficult due to frequent interspecific hybridization and intraspecific morphological variation within this genus. Plastid genome sequencing has been proven to be a useful tool to identify closely related species and is widely used in the field of plant evolution and phylogeny. In the present study, we provided four Citation: Zhang, F.; Wang, N.; chloroplast genomes of Lycoris and retrieved seven published species in the genus for comparative Cheng, G.; Shu, X.; Wang, T.; Zhuang, genomics and phylogenetic analyses. All these chloroplast genomes possess the typical quadripartite W.; Lu, R.; Wang, Z.
    [Show full text]
  • Complete Chloroplast Genomes Shed Light on Phylogenetic
    www.nature.com/scientificreports OPEN Complete chloroplast genomes shed light on phylogenetic relationships, divergence time, and biogeography of Allioideae (Amaryllidaceae) Ju Namgung1,4, Hoang Dang Khoa Do1,2,4, Changkyun Kim1, Hyeok Jae Choi3 & Joo‑Hwan Kim1* Allioideae includes economically important bulb crops such as garlic, onion, leeks, and some ornamental plants in Amaryllidaceae. Here, we reported the complete chloroplast genome (cpDNA) sequences of 17 species of Allioideae, fve of Amaryllidoideae, and one of Agapanthoideae. These cpDNA sequences represent 80 protein‑coding, 30 tRNA, and four rRNA genes, and range from 151,808 to 159,998 bp in length. Loss and pseudogenization of multiple genes (i.e., rps2, infA, and rpl22) appear to have occurred multiple times during the evolution of Alloideae. Additionally, eight mutation hotspots, including rps15-ycf1, rps16-trnQ-UUG, petG-trnW-CCA , psbA upstream, rpl32- trnL-UAG , ycf1, rpl22, matK, and ndhF, were identifed in the studied Allium species. Additionally, we present the frst phylogenomic analysis among the four tribes of Allioideae based on 74 cpDNA coding regions of 21 species of Allioideae, fve species of Amaryllidoideae, one species of Agapanthoideae, and fve species representing selected members of Asparagales. Our molecular phylogenomic results strongly support the monophyly of Allioideae, which is sister to Amaryllioideae. Within Allioideae, Tulbaghieae was sister to Gilliesieae‑Leucocoryneae whereas Allieae was sister to the clade of Tulbaghieae‑ Gilliesieae‑Leucocoryneae. Molecular dating analyses revealed the crown age of Allioideae in the Eocene (40.1 mya) followed by diferentiation of Allieae in the early Miocene (21.3 mya). The split of Gilliesieae from Leucocoryneae was estimated at 16.5 mya.
    [Show full text]
  • Ndhf Sequence Evolution and the Major Clades in the Sunflower Family KI-JOONG KIM* and ROBERT K
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10379-10383, October 1995 Evolution ndhF sequence evolution and the major clades in the sunflower family KI-JOONG KIM* AND ROBERT K. JANSENt Department of Botany, University of Texas, Austin, TX 78713-7640 Communicated by Peter H. Raven, Missouri Botanical Garden, St. Louis, MO, June 21, 1995 ABSTRACT An extensive sequence comparison of the either too short or too conserved to provide adequate numbers chloroplast ndhF gene from all major clades of the largest of characters in recently evolved families. A number of alter- flowering plant family (Asteraceae) shows that this gene native genes have been suggested as potential candidates for provides -3 times more phylogenetic information than rbcL. phylogenetic comparisons at lower taxonomic levels (9). The This is because it is substantially longer and evolves twice as phylogenetic utility of one of these, matK, has been recently fast. The 5' region (1380 bp) ofndhF is very different from the demonstrated (10). Comparison of sequences of two chloro- 3' region (855 bp) and is similar to rbcL in both the rate and plast genomes (rice and tobacco), however, revealed only two the pattern of sequence change. The 3' region is more A+T- genes, rpoCl and ndhF, that are considerably longer and evolve rich, has higher levels of nonsynonymous base substitution, faster than rbcL (9, 11). We selected ndhF because it is longer and shows greater transversion bias at all codon positions. and evolves slightly faster than rpoCl (11), because rpoCl has These differences probably reflect different functional con- an intron that may require additional effort in DNA amplifi- straints on the 5' and 3' regions of nduhF.
    [Show full text]
  • Sequencing of Cdna
    ORIGINAL RESEARCH ARTICLE published: 07 March 2011 doi: 10.3389/fmicb.2011.00041 Transcription profiling of the model cyanobacterium Synechococcus sp. strain PCC 7002 by Next-Gen (SOLiD™) sequencing of cDNA Marcus Ludwig and Donald A. Bryant* Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA Edited by: The genome of the unicellular, euryhaline cyanobacterium Synechococcus sp. PCC 7002 encodes Thomas E. Hanson, University of about 3200 proteins. Transcripts were detected for nearly all annotated open reading frames Delaware, USA by a global transcriptomic analysis by Next-Generation (SOLiD™) sequencing of cDNA. In the Reviewed by: Martin Hagemann, University Rostock, cDNA samples sequenced, ∼90% of the mapped sequences were derived from the 16S and Germany 23S ribosomal RNAs and ∼10% of the sequences were derived from mRNAs. In cells grown Jack Meeks, University of California, photoautotrophically under standard conditions [38°C, 1% (v/v) CO2 in air, 250 μmol photons USA m−2 s−1], the highest transcript levels (up to 2% of the total mRNA for the most abundantly *Correspondence: transcribed genes; e.g., cpcAB, psbA, psaA) were generally derived from genes encoding Donald A. Bryant, Department of Biochemistry and Molecular Biology, structural components of the photosynthetic apparatus. High-light exposure for 1 h caused The Pennsylvania State University, changes in transcript levels for genes encoding proteins of the photosynthetic apparatus, Type-1 S-235 Frear Building, University Park, NADH dehydrogenase complex and ATP synthase, whereas dark incubation for 1 h resulted PA 16802, USA. in a global decrease in transcript levels for photosynthesis-related genes and an increase in e-mail: [email protected] transcript levels for genes involved in carbohydrate degradation.
    [Show full text]
  • 82167915.Pdf
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1837 (2014) 954–963 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbabio Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines☆ Lici A. Schurig-Briccio a, Takahiro Yano b,HarveyRubinb,RobertB.Gennisa,⁎ a Department of Biochemistry, University of Illinois, 600 S. Mathews Street, Urbana, IL 61801, USA b Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA article info abstract Article history: Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant Received 21 March 2014 bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic Received in revised form 27 March 2014 targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine Accepted 28 March 2014 dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects Available online 5 April 2014 of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic fl Keywords: membrane proteins that catalyze the transfer of electrons from NADH via avin adenine dinucleotide (FAD) to + Bioenergetics/electron transfer complex the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD ) Enzyme inhibitor redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in Respiratory chain mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S.
    [Show full text]
  • Molecular and Functional Analysis of Nicotinate Catabolism in Eubacterium Barkeri
    Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri Ashraf Alhapel, Daniel J. Darley, Nadine Wagener, Elke Eckel, Nora Elsner, and Antonio J. Pierik* Laboratorium fu¨r Mikrobielle Biochemie, Fachbereich Biologie, Philipps Universita¨t, D-35032 Marburg, Germany Edited by Perry A. Frey, University of Wisconsin, Madison, WI, and approved June 29, 2006 (received for review March 1, 2006) The anaerobic soil bacterium Eubacterium barkeri catabolizes nic- complex (see Fig. 1). Based on the identified intermediates, otinate to pyruvate and propionate via a unique fermentation. A several anticipated enzymes were purified and characterized: full molecular characterization of nicotinate fermentation in this nicotinate dehydrogenase (12), 6-hydroxynicotinate reductase organism was accomplished by the following results: (i) A 23.2-kb (7), 2-methyleneglutarate mutase, and 3-methylitaconate DNA segment with a gene cluster encoding all nine enzymes was isomerase (13, 14). These findings outlined the nicotinate fer- cloned and sequenced, (ii) two chiral intermediates were discov- mentation pathway and placed the identified intermediates in an ered, and (iii) three enzymes were found, completing the hitherto enzymatic framework. unknown part of the pathway. Nicotinate dehydrogenase, a (non- The nicotinate dehydrogenase contains [2Fe-2S] clusters (15), selenocysteine) selenium-containing four-subunit enzyme, is en- FAD and molybdopterin cytosine dinucleotide (16), and has an coded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by unusual subunit composition [50, 37, 33, and 23 kDa (17)]. It has the ndhL͞ndhM genes. In contrast to all enzymes of the xanthine labile (nonselenocysteine) selenium (18) also identified in pu- dehydrogenase family, the latter two encode a two-subunit mo- rine dehydrogenase from Clostridium purinolyticum and xanthine lybdopterin protein.
    [Show full text]
  • Physiological and Proteomic Responses of Diploid and Tetraploid Black Locust (Robinia Pseudoacacia L.) Subjected to Salt Stress
    Int. J. Mol. Sci. 2013, 14, 20299-20325; doi:10.3390/ijms141020299 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article Physiological and Proteomic Responses of Diploid and Tetraploid Black Locust (Robinia pseudoacacia L.) Subjected to Salt Stress Zhiming Wang 1,†, Mingyue Wang 1,†, Likun Liu2 and Fanjuan Meng 1,* 1 College of Life Science, Northeast Forestry University, Harbin 150040, China; E-Mails: [email protected] (Z.W.); [email protected] (M.W.) 2 Department of Medical Biotechnology, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon-do 200-701, Korea; E-Mail: [email protected] † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +86-451-8219-2170; Fax: +86-451-8643-3905. Received: 17 June 2013; in revised form: 31 August 2013 / Accepted: 9 September 2013 / Published: 14 October 2013 Abstract: Tetraploid black locust (Robinia pseudoacacia L.) is adaptable to salt stress. Here, we compared morphological, physiological, ultrastructural, and proteomic traits of leaves in tetraploid black locust and its diploid relatives under salt stress. The results showed that diploid (2×) plants suffered from greater negative effects than those of tetraploid (4×) plants. After salt treatment, plant growth was inhibited, photosynthesis was reduced, reactive oxygen species, malondialdehyde content, and relative electrolyte leakage increased, and defense-related enzyme activities decreased in 2× compared to those in 4×. In addition, salt stress resulted in distorted chloroplasts, swollen thylakoid membranes, accumulation of plastoglobules, and increased starch grains in 2× compared to those in 4×.
    [Show full text]
  • Interplay Between Non-Coding RNA Transcription, Stringent/Relaxed Phenotype and Antibiotic Production in Streptomyces Ambofaciens
    antibiotics Article Interplay between Non-Coding RNA Transcription, Stringent/Relaxed Phenotype and Antibiotic Production in Streptomyces ambofaciens Eva Pinatel 1,†, Matteo Calcagnile 2,† , Adelfia Talà 2, Fabrizio Damiano 2 , Luisa Siculella 2 , Clelia Peano 3,4, Giuseppe Egidio De Benedetto 5 , Antonio Pennetta 5 , Gianluca De Bellis 1 and Pietro Alifano 2,* 1 Institute of Biomedical Technologies, National Research Council, 20090 Segrate, Italy; [email protected] (E.P.); [email protected] (G.D.B.) 2 Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Monteroni, 73100 Lecce, Italy; [email protected] (M.C.); adelfi[email protected] (A.T.); [email protected] (F.D.); [email protected] (L.S.) 3 Institute for Genetic and Biomedical Research, Operative Unit (UoS) of Milan, National Research Council, 20089 Rozzano, Italy; [email protected] 4 Human Technopole, 20157 Milan, Italy; [email protected] 5 Department of Cultural Heritage, University of Salento, Via Monteroni, 73100 Lecce, Italy; [email protected] (G.E.D.B.); [email protected] (A.P.) * Correspondence: [email protected] † These authors contributed equally to this work. Citation: Pinatel, E.; Calcagnile, M.; Abstract: While in recent years the key role of non-coding RNAs (ncRNAs) in the regulation of gene Talà, A.; Damiano, F.; Siculella, L.; expression has become increasingly evident, their interaction with the global regulatory circuits is Peano, C.; De Benedetto, G.E.; still obscure. Here we analyzed the structure and organization of the transcriptome of Streptomyces Pennetta, A.; De Bellis, G.; Alifano, P.
    [Show full text]
  • (Yvmb) Regulates Expression of Genes Involved in Pulcherriminic Acid
    Randazzo et al. BMC Microbiology (2016) 16:190 DOI 10.1186/s12866-016-0807-3 RESEARCH ARTICLE Open Access The MarR-like protein PchR (YvmB) regulates expression of genes involved in pulcherriminic acid biosynthesis and in the initiation of sporulation in Bacillus subtilis Paola Randazzo, Anne Aubert-Frambourg, Alain Guillot and Sandrine Auger* Abstract Background: Cyclodipeptides and their derivatives constitute a large class of peptide natural products with noteworthy biological activities. In some yeasts and bacterial species, pulcherriminic acid derived from cyclo-L- leucyl-L-leucyl is excreted and chelates free ferric ions to form the pulcherrimin. In Bacillus subtilis, the enzymes YvmC and CypX are known to be involved in pulcherriminic acid biosynthesis. However, the mechanisms controlling the transcription of the yvmC-cypX operon are still unknown. Results: In this work, we demonstrated that the B. subtilis YvmB MarR-like regulator is the major transcription factor controlling yvmC-cypX expression. A comprehensive quantitative proteomic analysis revealed a wide and prominent effect of yvmB deletion on proteins involved in cellular processes depending on iron availability. In addition, expression of yvmB depends on iron availability. Further analysis with real-time in vivo transcriptional profiling allowed us to define the YvmB regulon. We identified yvmBA, yvmC-cypX and yvnB for negative regulation and yisI for positive regulation. In combination with genetic approaches, gel mobility shift assays indicated that a 14-bp palindromic motif constitutes the YvmB binding site. It was unexpected that YvmB controls expression of yisI, whose encoding protein plays a negative role in the regulation of the sporulation initiation pathway.
    [Show full text]
  • Supplemental Table S1: Comparison of the Deleted Genes in the Genome-Reduced Strains
    Supplemental Table S1: Comparison of the deleted genes in the genome-reduced strains Legend 1 Locus tag according to the reference genome sequence of B. subtilis 168 (NC_000964) Genes highlighted in blue have been deleted from the respective strains Genes highlighted in green have been inserted into the indicated strain, they are present in all following strains Regions highlighted in red could not be deleted as a unit Regions highlighted in orange were not deleted in the genome-reduced strains since their deletion resulted in severe growth defects Gene BSU_number 1 Function ∆6 IIG-Bs27-47-24 PG10 PS38 dnaA BSU00010 replication initiation protein dnaN BSU00020 DNA polymerase III (beta subunit), beta clamp yaaA BSU00030 unknown recF BSU00040 repair, recombination remB BSU00050 involved in the activation of biofilm matrix biosynthetic operons gyrB BSU00060 DNA-Gyrase (subunit B) gyrA BSU00070 DNA-Gyrase (subunit A) rrnO-16S- trnO-Ala- trnO-Ile- rrnO-23S- rrnO-5S yaaC BSU00080 unknown guaB BSU00090 IMP dehydrogenase dacA BSU00100 penicillin-binding protein 5*, D-alanyl-D-alanine carboxypeptidase pdxS BSU00110 pyridoxal-5'-phosphate synthase (synthase domain) pdxT BSU00120 pyridoxal-5'-phosphate synthase (glutaminase domain) serS BSU00130 seryl-tRNA-synthetase trnSL-Ser1 dck BSU00140 deoxyadenosin/deoxycytidine kinase dgk BSU00150 deoxyguanosine kinase yaaH BSU00160 general stress protein, survival of ethanol stress, SafA-dependent spore coat yaaI BSU00170 general stress protein, similar to isochorismatase yaaJ BSU00180 tRNA specific adenosine
    [Show full text]
  • The Complete Genome Sequence of the Gram-Positive Bacterium Bacillus Subtilis
    articles The complete genome sequence of the Gram-positive bacterium Bacillus subtilis F. Kunst1, N. Ogasawara2, I. Moszer3, A. M. Albertini4, G. Alloni4, V. Azevedo5, M. G. Bertero3,4, P. Bessie` res5, A. Bolotin5, S. Borchert6, R. Borriss7, L. Boursier3, A. Brans8, M. Braun9, S. C. Brignell10,S.Bron11, S. Brouillet3,12, C. V. Bruschi13, B. Caldwell14, V. Capuano5, N. M. Carter10, S.-K. Choi15, J.-J. Codani16, I. F. Connerton17, N. J. Cummings17, R. A. Daniel18, F. Denizot19, K. M. Devine20,A.Du¨sterho¨ ft9, S. D. Ehrlich5, P.T. Emmerson21, K. D. Entian6, J. Errington18, C. Fabret19, E. Ferrari14, D. Foulger18, C. Fritz9, M. Fujita22, Y.Fujita23,S.Fuma24, A. Galizzi4, N. Galleron5, S.-Y.Ghim15, P.Glaser3, A. Goffeau25, E. J. Golightly26, G. Grandi27, G. Guiseppi19,B.J.Guy10, K. Haga28, J. Haiech19, C. R. Harwood10,A.He´naut29, H. Hilbert9, S. Holsappel11, S. Hosono30, M.-F. Hullo3, M. Itaya31, L. Jones32, B. Joris8, D. Karamata33, Y.Kasahara2, M. Klaerr-Blanchard3, C. Klein6, Y.Kobayashi30, P.Koetter6, G. Koningstein34, S. Krogh20, M. Kumano24, K. Kurita24, A. Lapidus5, S. Lardinois8, J. Lauber9, V. Lazarevic33, S.-M. Lee35, A. Levine36, H. Liu28, S. Masuda30, C. Maue¨ l33,C.Me´digue3,12, N. Medina36, R. P. Mellado37, M. Mizuno30, D. Moestl9, S. Nakai2, M. Noback11, D. Noone20, M. O’Reilly20, K. Ogawa24, A. Ogiwara38, B. Oudega34, S.-H. Park15, V. Parro37,T.M.Pohl39, D. Portetelle40, S. Porwollik7, A. M. Prescott18, E. Presecan3, P. Pujic5, B. Purnelle25, G. Rapoport1, M. Rey26, S. Reynolds33, M. Rieger41, C. Rivolta33, E. Rocha3,12,B.Roche36, M.
    [Show full text]
  • (10) Patent No.: US 6593114 B1
    USOO6593114B1 (12) United States Patent (10) Patent No.: US 6,593,114 B1 Kunsch et al. (45) Date of Patent: Jul. 15, 2003 (54) STAPHYLOCOCCUS AUREUS OTHER PUBLICATIONS POLYNUCLEOTIDES AND SEQUENCES Lewin, in Genes IV, Oxford University Press, p. 816, 1990.* (75) Inventors: Charles A. Kunsch, Norcross, GA Sharrocks, in “PCR Technology Current Innovations”, Grif (US); Gil H. Choi, Rockville, MD fin et al eds. CRC Press Inc, pp. 5-11, 1994.* (US); Steven Barash, Rockville, MD American Type Culture (ATCC), Catalogue of Bacteria & (US); Patrick J. Dillon, Carlsbad, CA Bacteriophages 17th Edition, pp. 202-204, 1989.* (US); Michael R. Fannon, Silver Walkenhorst et al, Microbiol Res, 150:347–361, 1995.* Spring, MD (US); Craig A. Rosen, Dorrell et al., Photochem. Photobiol, 58:831-835, 1993.* Laytonsville, MD (US) Sambrook et al Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor pp. 17.1-17.44, 1989.* (73) Assignee: Human Genome Sciences, Inc., Kennell et al, Proc. Nucl. Acid Res. Mol. Biol. 11:259-301, Rockville, MD (US) 1971.* (*) Notice: Subject to any disclaimer, the term of this Herzog et al., DNA and Cell Biology 12(6): 465-471, 1993.* ject y Jazin et al, Regulatory Peptides 47:247–258, 1993.* past Sh adjusted under 35 Rudinger et al., in “Peptide Hormones”, ed. Parsons J.A. a -- (b) by 0 days. University Park Press, pp. 1–6, 1976.* Burgess et al. et al., The Journal of Cell Biology (21) Appl. No.: 08/956,171 111:2129-2138, 1990.* (22) Filed: Oct. 20, 1997 Lazar et al., Molecular and Cellular Biology 8(3):1247–1252, 1988.* Related U.S.
    [Show full text]