DOCSLIB.ORG

Structure and Mechanism of a Group-I Cobalt Energy Coupling Factor Transporter

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structure and Mechanism of a Group-I Cobalt Energy Coupling Factor Transporter Cell Research (2017) 27:675-687. © 2017 IBCB, SIBS, CAS All rights reserved 1001-0602/17 $ 32.00 ORIGINAL ARTICLE www.nature.com/cr Structure and mechanism of a group-I cobalt energy coupling factor transporter Zhihao Bao1, 2, 3, *, Xiaofeng Qi1, 3, *, Sen Hong1, 3, Ke Xu1, 3, Fangyuan He1, Minhua Zhang1, Jiugeng Chen1, Daiyin Chao1, Wei Zhao4, Dianfan Li4, Jiawei Wang5, Peng Zhang1 1National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; 2Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; 3University of Chinese Academy of Sciences, Beijing 100049, China; 4State Key Laboratory of Molecular Biology, National Center for Protein Sciences, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chi- nese Academy of Sciences, Shanghai 200031, China; 5State Key Laboratory of Membrane Biology, School of Life Sciences, Tsing- hua University, Beijing 100084, China Energy-coupling factor (ECF) transporters are a large family of ATP-binding cassette transporters recently iden- tified in microorganisms. Responsible for micronutrient uptake from the environment, ECF transporters are mod- ular transporters composed of a membrane substrate-binding component EcfS and an ECF module consisting of an integral membrane scaffold component EcfT and two cytoplasmic ATP binding/hydrolysis components EcfA/A’. ECF transporters are classified into groups I and II. Currently, the molecular understanding of group-I ECF transport- ers is very limited, partly due to a lack of transporter complex structural information. Here, we present structures and structure-based analyses of the group-I cobalt ECF transporter CbiMNQO, whose constituting subunits CbiM/ CbiN, CbiQ, and CbiO correspond to the EcfS, EcfT, and EcfA components of group-II ECF transporters, respec- tively. Through reconstitution of different CbiMNQO subunits and determination of related ATPase and transporter activities, the substrate-binding subunit CbiM was found to stimulate CbiQO’s basal ATPase activity. The structure of CbiMQO complex was determined in its inward-open conformation and that of CbiO in β, γ-methyleneadenosine 5′-triphosphate-bound closed conformation. Structure-based analyses revealed interactions between different compo- nents, substrate-gating function of the L1 loop of CbiM, and conformational changes of CbiO induced by ATP bind- ing and product release within the CbiMNQO transporter complex. These findings enabled us to propose a working model of the CbiMNQO transporter, in which the transport process requires the rotation or toppling of both CbiQ and CbiM, and CbiN might function in coupling conformational changes between CbiQ and CbiM. Keywords: ECF transporter; cobalt transporter; ATPase activity; ABC transporter; transport mechanism Cell Research (2017) 27:675-687. doi:10.1038/cr.2017.38; published online 21 March 2017 Introduction protein complexes that transport a variety of substrates across cell membranes. On the basis of the transport di- Powered by ATP hydrolysis, ATP-binding cassette rection, ABC transporters are categorized into importers (ABC) transporters comprise a large superfamily of and exporters, both of which are composed of two trans- membrane domains, and two nucleotide-binding domains (NBDs). Importers, usually found in prokaryotes, contain *These two authors contributed equally to this work. a periplasmic solute-binding protein (SBP) for substrate Correspondence: Peng Zhanga, Jiawei Wangb capture. Through extensive studies, a two state rock-and- aTel: +86 21 54924219 switch model has been suggested for ABC transporters E-mail: [email protected] b [1-8]. E-mail: [email protected] Received 23 December 2016; revised 8 February 2017; accepted 15 Febru- In recent years, a new family of ABC transporters, ary 2017; published online 21 March 2017 known as energy-coupling factor (ECF) transporters, Structure of a cobalt ECF transporter 676 have been identified in bacteria [9-12]. ECF transport- transporters are the widely distributed CbiMNQO and ers mediate micronutrient uptake and are composed of NikMNQO that uptake Co2+ and/or Ni2+, which are co- four components, two cytoplasmic ATPase components factors of a number of essential metabolic enzymes in EcfA and EcfA’, and two transmembrane components bacteria [28-30]. CbiMNQO/NikMNQO are composed namely EcfT for energy coupling and EcfS for substrate of M/N, Q, and O subunits, corresponding to the EcfS, recognition. The EcfT-EcfA-EcfA’ components com- EcfT, and EcfA components of group-II ECF transport- prise the ECF module (Figure 1A). To date two groups ers, respectively (Figure 1A). The structure of NikM2 in of ECF transporters have been identified, with group-I complex with Ni2+ has been reported [31], and compared ECF transporters being an EcfS protein associated with to group-II EcfS proteins, NikM2 contains an additional a dedicated ECF module and the encoding genes located N-terminal transmembrane helix and an extended loop within one operon. Group-II ECF transporters include essential for substrate coordination. This study suggests several EcfS proteins; their coding genes are distributed that group-I ECF transporters possess different sub- throughout the genome and they can bind to a common strate-binding mechanisms from that of group-II ECF ECF module [12]. In some group-I ECF transporters, the transporters. In addition, although the M subunit alone EcfS component is comprised of two proteins, M and N, can bind substrate, the transporter activity of CbiMN- which fuse together into one polypeptide MN in some QO/NikMNQO depends on the N subunit [32, 33]. The species [9]. The most striking divergence that sets ECF molecular understanding of group-I ECF transporters transporters apart from canonical ABC importers lies remains elusive, in part due to the lack of structural in- in the SBPs, such that ECF transporters utilize integral formation for the complete transporter complex. Here, membrane EcfS proteins for substrate binding, whereas structural and biochemical analyses of the CbiMNQO canonical ABC transporters utilize periplasmic SBPs to transporter complex are presented. Our results support capture substrates [13-16]. a rotation/toppling model and shed new lights on the Extensive studies have been carried out to dissect the mechanism of group-I ECF transporters. mechanism of ECF transporters, especially that of group- II transporters. The substrate-specific binding proteins Results of group-II ECF transporters, such as RibU, ThiT, BioY, and FolT, have very low sequence similarity, but they Characterization of CbiMNQO transporter adopt similar six-helical-bundle 3D structures [17-20]. The CbiMNQO transporter was characterized by ex- The high binding affinities of these EcfS proteins for pression of the transporter complex and combinations of their substrates [10, 21] are attributed to buried sub- different subunits in E. coli. Transporter activity was as- strate-binding pockets within the middle of transmem- sayed using inductively coupled plasma mass spectrom- brane helices near the periplasmic side. Residues consti- etry (ICP-MS) (Figure 1B). The results showed that the tuting the substrate-binding pockets dictate the substrate complete CbiMNQO transporter possessed high cobalt specificity, and are highly conserved within a specific uptake activity, whereas a complex composed of CbiM EcfS across different species [22]. This forms a basis for and CbiN subunits exhibited low but significant activi- predicting the substrates for ECF transporters from dif- ty. Removal of CbiN demolished cobalt uptake activity, ferent species. consistent with a previous report [32]. CbiMNQO trans- The assembly of the EcfS component with the Ec- porter exhibited very low nickel uptake activity, about fAA’T module was not known until the inward-open 8% of that of cobalt, indicating that CbiMNQO is highly structures of ECF transporters were captured [23-25]. specific for cobalt. These structures reveal that EcfA/A’ components have By adding a 6× His tag to the CbiQ subunit, the trans- two NBDs that form a dimer required for ATP hydrolysis, porter complex was purified using affinity chromatog- and EcfT component acts like a scaffold that connects raphy. CbiM, CbiQ, and CbiO subunits were present in EcfS component with EcfA/A’ components, whereas substantial amounts (1/1/2 molar ratio), but amounts of EcfS components within the structures, without excep- the CbiN subunit were reduced after each purification tion, adopt conformations parallel to membrane planes. step (Figure 1C; Supplementary information, Figure S1). These studies provided a snapshot toward elucidating These results suggest that CbiM, CbiQ, and CbiO form scenarios of transport processes, and laid the foundation a stable subcomplex (CbiMQO) and that the interaction for a rotation/toppling model of group-II ECF transport- of CbiN with the subcomplex is weak and dynamic. The ers [22, 26, 27]. formation of a stable CbiMQO subcomplex was verified Compared to group-II ECF transporters, limited when the three subunits were coexpressed and purified in progress has been made in the understanding of group-I the absence of CbiN (Figure 1C). Interestingly, the pres- ECF transporters [9, 11]. The best known group-I ECF ence of CbiN appears
Load more

Recommended publications
  • Examining the Mechanism of a Heavy- Metal ABC Exporter Dennis Hicks University of San Francisco, [email protected]
    The University of San Francisco USF Scholarship: a digital repository @ Gleeson Library | Geschke Center Master's Theses Theses, Dissertations, Capstones and Projects Spring 3-31-2019 NaAtm1: Examining the mechanism of a Heavy- metal ABC Exporter Dennis Hicks University of San Francisco, [email protected] Follow this and additional works at: https://repository.usfca.edu/thes Part of the Biochemistry Commons Recommended Citation Hicks, Dennis, "NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter" (2019). Master's Theses. 1204. https://repository.usfca.edu/thes/1204 This Thesis is brought to you for free and open access by the Theses, Dissertations, Capstones and Projects at USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. It has been accepted for inclusion in Master's Theses by an authorized administrator of USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. For more information, please contact [email protected]. NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter A thesis presented to the faculty of the Department of Chemistry at the University of San Francisco in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Written by Dennis Hicks Bachelor of Science in Biochemistry San Francisco State University August 2019 NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter Thesis written by Dennis Hicks This thesis is written under the guidance of the Faculty Advisory Committee, and approved by all its members, has been accepted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at the University of San Francisco Thesis Committee Janet G.
    [Show full text]
  • Reca and DNA Recombination: a Review of Molecular Mechanisms
    Biochemical Society Transactions (2019) 47 1511–1531 https://doi.org/10.1042/BST20190558 Review Article RecA and DNA recombination: a review of molecular mechanisms Downloaded from https://portlandpress.com/biochemsoctrans/article-pdf/47/5/1511/859295/bst-2019-0558.pdf by Tata Institute of Fundamental Research user on 20 January 2020 Elsa del Val1,2, William Nasser1, Hafid Abaibou2 and Sylvie Reverchon1 1Microbiologie, Adaptation et Pathogénie, Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, 11 Avenue Jean Capelle, F-69621 Villeurbanne, France; 2Molecular Innovation Unit, Centre Christophe Mérieux, BioMérieux, 5 rue des Berges, 38024 Grenoble Cedex 01, France Correspondence: Sylvie Reverchon ([email protected]) or Hafid Abaibou ([email protected]) Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combin- ation of DNA synthesis, ligation and resolution.
    [Show full text]
  • The Universal Stress Proteins of Bacteria
    The Universal Stress Proteins of Bacteria Dominic Bradley Department of Life Sciences A thesis submitted for the degree of Doctor of Philosophy and the Diploma of Imperial College London The Universal Stress Proteins of Bacteria Declaration of Originality I, Dominic Bradley, declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institute of tertiary education. Information derived from the published and unpublished work of others has been acknowledged in the text and a list of references is given in the bibliography. 1 The Universal Stress Proteins of Bacteria Abstract Universal stress proteins (USPs) are a widespread and abundant protein family often linked to survival during stress. However, their exact biochemical and cellular roles are incompletely understood. Mycobacterium tuberculosis (Mtb) has 10 USPs, of which Rv1636 appears to be unique in its domain structure and being the only USP conserved in M. leprae. Over-expression of Rv1636 in M. smegmatis indicated that this protein does not share the growth arrest phenotype of another Mtb USP, Rv2623, suggesting distinct roles for the Mtb USPs. Purified Rv1636 was shown to have novel nucleotide binding capabilities when subjected to UV crosslinking. A range of site-directed mutants of Rv1636 were produced, including mutations within a predicted nucleotide binding motif, with the aim of identifying and characterising key residues within the Rv1636 protein. Further putative biochemical activities, including nucleotide triphosphatase, nucyleotidylyation and auto-phosphorylation were also investigated in vitro; however Rv1636 could not be shown to definitively possess these activities, raising the possibility that addition factors may be present in vivo.
    [Show full text]
  • Mismatch Repair Inhibits Homeologous Recombination Via Coordinated Directional Unwinding of Trapped DNA Structures
    Molecular Cell Article Mismatch Repair Inhibits Homeologous Recombination via Coordinated Directional Unwinding of Trapped DNA Structures Khek-Chian Tham,1 Nicolaas Hermans,1 Herrie H.K. Winterwerp,3 Michael M. Cox,4 Claire Wyman,1,2 Roland Kanaar,1,2 and Joyce H.G. Lebbink1,2,* 1Department of Genetics, Cancer Genomics Netherlands 2Department of Radiation Oncology Erasmus Medical Center, Rotterdam 3000 CA, The Netherlands 3Division of Biochemistry and Center for Biomedical Genetics, Netherlands Cancer Institute, Amsterdam 1066 CX, The Netherlands 4Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.07.008 SUMMARY transduction (Zahrt and Maloy, 1997), transformation (Majewski et al., 2000), and conjugational E. coli-E. coli crosses that involve Homeologous recombination between divergent the creation of mismatches (Feinstein and Low, 1986). Likewise, DNA sequences is inhibited by DNA mismatch repair. in eukaryotes, individual MMR proteins have roles of varying In Escherichia coli, MutS and MutL respond to DNA magnitude in the prevention of homeologous recombination mismatches within recombination intermediates (Selva et al., 1995; Datta et al., 1996; Nicholson et al., 2000; de and prevent strand exchange via an unknown mech- Wind et al., 1995). Inhibition of homeologous reactions is thought anism. Here, using purified proteins and DNA sub- to protect the genome from recombination events between divergent repeats (de Wind et al., 1999). This is important in hu- strates, we find that in addition to mismatches within mans, in which the amount of repetitive DNA is as high as 50%. the heteroduplex region, secondary structures within An intriguing question is how MMR and recombination path- the displaced single-stranded DNA formed during ways are integrated at the molecular level.
    [Show full text]
  • Recbcd-Dependent Joint Molecule Formation Promoted by The
    Proc. Nail. Acad. Sci. USA Vol. 88, pp. 3367-3371, April 1991 Biochemistry RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins (single-stranded DNA-binding protein/genetic recombination/homologous pairing) LINDA J. ROMAN, DAN A. DIXON, AND STEPHEN C. KOWALCZYKOWSKI* Department of Molecular Biology, Northwestern University Medical School, Chicago, IL 60611 Communicated by Bruce M. Alberts, December 18, 1990 (receivedfor review September 22, 1990) ABSTRACT We describe the formation of homologously We previously described a reaction in which heteroduplex paired joint molecules in an in vitro reaction that is dependent DNA formation between ssDNA and homologous linear on the concerted actions ofpurified RecA and RecBCD proteins dsDNA required both the dsDNA-unwinding activity of and is stimulated by single-stranded DNA-binding protein RecBCD and the ssDNA-renaturation activity of RecA (15). (SSB). RecBCD enzyme initiates the process by unwinding the In this paper, we expand upon the previous in vitro studies to linear double-stranded DNA to produce single-stranded DNA, include DNA substrates that are more representative of the which is trapped by SSB and RecA. RecA uses this single- substrates encountered in vivo (i.e., homologous linear ds- stranded DNA to catalyze the invasion of a supercoiled double- DNA and supercoiled dsDNA) and that require the joint stranded DNA molecule, forming a homologously paired joint molecule formation activity of RecA protein. RecA alone is molecule. At low RecBCD enzyme concentrations, the rate- unable to catalyze pairing between two dsDNA molecules limiting step is the unwinding of duplex DNA by RecBCD, unless one of them contains ssDNA in a region of homology whereas at higher RecBCD concentrations, the rate-limiting (16).
    [Show full text]
  • High Fidelity of Reca-Catalyzed Recombination: a Watchdog of Genetic Diversity
    High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity Dror Sagi, Tsvi Tlusty and Joel Stavans Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot 76100, Israel ABSTRACT delicately balanced, to give the highest chance to the functional maintenance of coded proteins. Furthermore, the Homologous recombination plays a key role in extent of recombination between related organisms, such as generating genetic diversity, while maintaining Escherichia coli and Salmonella, controls their genetic isola- protein functionality. The mechanisms by which tion and speciation (3). Therefore, inhibition of chromosomal RecA enables a single-stranded segment of DNA gene transfer isolates related species. to recognize a homologous tract within a whole In prokaryotes, recombination is carried out by RecA (4), a genome are poorly understood. The scale by which protein that also plays a fundamental role during the repair homology recognition takes place is of a few tens of and bypass of DNA lesions, enabling the resumption of base pairs, after which the quest for homology is DNA replication at stalled replication forks. According to over. To study the mechanism of homology recogni- the prevailing view, the RecA-catalyzed recombination tion, RecA-promoted homologous recombination process proceeds along the following steps. First, a nucleo- protein complex is formed by the polymerization of RecA between short DNA oligomers with different degrees along a single-stranded DNA (ssDNA) substrate. ssDNA, of heterology was studied in vitro, using fluores- which is accepted to be a prerequisite, is produced by the cence resonant energy transfer. RecA can detect recombination-specific helicases RecBCD and RecG at single mismatches at the initial stages of recombi- double strand breaks (5), or as a byproduct of DNA repair nation, and the efficiency of recombination is processes at stalled replication forks (6).
    [Show full text]
  • The Specificity of the Secondary DNA Binding Site of Reca Protein
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10673-10678, October 1996 Biochemistry The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange (genetic recombination/homologous pairing/DNA-protein interactions) ALEXANDER V. MAZIN* AND STEPHEN C. KOWALCZYKOWSKIt Division of Biological Sciences, Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665 Communicated by Frank Stahl, University of Oregon, Eugene, OR, July 1, 1996 (received for review April 17, 1996) ABSTRACT The RecA protein-single-stranded DNA They suggested that the observed binding specificity is deter- (ssDNA) filament can bind a second DNA molecule. Binding mined by non-Watson-Crick bonds that are formed between of ssDNA to this secondary site shows specificity, in that ssDNA in the primary site and ssDNA bound to the secondary polypyrimidinic DNA binds to the RecA protein-ssDNA fila- site of the nucleoprotein filament, so-called "self-recognition," ment with higher affinity than polypurinic sequences. The as a part of a homology search process. affinity of ssDNA, which is identical in sequence to that bound Despite rather extensive characterization, the precise nature in the primary site, is not always greater than that of and function of the secondary DNA binding site remains nonhomologous DNA. Moreover, this specificity of DNA bind- unknown. Consequently, we sought to determine its role in ing does not depend on the sequence of the DNA bound to the DNA strand exchange and the basis for the apparently novel RecA protein primary site. We conclude that the specificity sequence-specificity attributed to this site.
    [Show full text]
  • Mismatch Repair Proteins Muts and Mutl Inhibit Reca-Catalyzed Strand
    Proc. Natd. Acad. Sci. USA Vol. 91, pp. 3238-3241, April 1994 Biochemistry Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs LEROY WORTH, JR.*, SUSANNA CLARK*, MIROSLAV RADMANt, AND PAUL MODRICH*I *Department of Biochemistry, Duke University Medical Center, Durham, NC 27710; and tLaboratoire de Mutagenese, Institut Jacques Monod, 2 Place Jussieu, Tour 43, 75251 Paris-C6dex 05, France Contributed by Paul Modrich, December 29, 1993 ABSTRACT Bacterial mutS and mutL mutatons confer are 20%o divergent at the sequence level (6), and also controls large increases in recombination between sequences that are the frequency of chromosomal rearrangements occurring in divergent by several percent at the nucleotide level, an effect E. coli as a consequence ofrecombination between rhs repeat attributed to a role for products of these genes In control of elements, which are about 1% divergent at the sequence level recombinatlon fidelity. Since MutS and MutL are proteins (8). Recombination between homeologous sequences scored involved in the eriest steps of mismatch repair, Inclug in either of these systems is increased 1-2 orders of magni- mismatch recognition by MutS, we have tested the psibility tude in mutL or mutS mutants. that they may affect strand exchange in response to occurrence In regular genetic crosses between isogenic genomes, of mspairs within the recombation heteroduplex. We show where the only sources of mismatches are the genetic mark- that MutS aboies RecA-catalyzed strand transfer between fd ers used to score recombinants, one can observe so-called and M13 bacteriophage DNAs, which vary by 3% at the marker effects, which depend on the activity of mismatch- nucleoide level, but Is without effect on M13-M13 or fd-fd repair systems, but appear only when the markers are close exchange.
    [Show full text]
  • Mismatch Repair This Is Backup to Replicative Proofreading By
    Mismatch repair This is backup to replicative proofreading by correcting mismatched nucleotides remaining in DNA after replication. Mismatches often involve the normal four bases in DNA. For example, a T may be mis-paired with a G. Because both T and G are normal components of DNA, mismatch repair systems need some way to determine whether the T or the G is the correct base at a given site. The repair system makes this distinction by identifying the template strand, which contains the original nucleotide sequence, and the newly synthesized strand, which contains the misincorporated base (the error). In bacteria, this distinction can be made based on the pattern of methylation in newly replicated DNA. In E. coli, the A in GATC sequences is methylated subsequent to its synthesis. Thus, a time interval occurs during which the template strand is methylated, and the newly synthesized strand is unmethylated. The mismatch repair system uses this difference in methylation state to excise the mismatched nucleotide in the nascent strand and replace it with the correct nucleotide by using the methylated parental strand of DNA as template. • In E. coli, mismatch repair requires the products of four genes, mutH, mutL, mutS, and mutU (uvrD). • The MutS protein recognizes mismatches and binds to them to initiate the repair process. • MutH and MutL proteins then join the complex. • MutH contains a GATC-specific endonuclease activity that cleaves the unmethylated strand at hemimethylated (that is, half methylated) GATC sites either 5’ or 3’ to the mismatch. The incision sites may be 1000 nucleotide pairs or more from the mismatch.
    [Show full text]
  • Dissecting the Reca-(In)Dependent Response to Mitomycin C in Mycobacterium Tuberculosis Using Transcriptional Profiling and Proteomics Analyses
    cells Article Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses Anna Brzostek 1,*,† , Przemysław Płoci ´nski 1,2,† , Alina Minias 1 , Aneta Ciszewska 1, Filip G ˛asior 1 , Jakub Pawełczyk 1 , Bozena˙ Dziadek 3, Marcin Słomka 4 and Jarosław Dziadek 1,* 1 Institute of Medical Biology of the Polish Academy of Sciences, Lodowa 106, 93-232 Łód´z,Poland; [email protected] (P.P.); [email protected] (A.M.); [email protected] (A.C.); fi[email protected] (F.G.); [email protected] (J.P.) 2 Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Łód´z,Banacha 12/16, 90-237 Łód´z,Poland 3 Department of Molecular Microbiology, Faculty of Biology and Environmental Protection, University of Łód´z, Banacha 12/16, 90-237 Łód´z,Poland; [email protected] 4 Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łód´z,Pomorska 139, 90-235 Łód´z,Poland; [email protected] * Correspondence: [email protected] (A.B.); [email protected] (J.D.) † These authors equally contributed to this work. Abstract: Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, Citation: Brzostek, A.; Płoci´nski,P.; such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of Minias, A.; Ciszewska, A.; G ˛asior, F.; infection while residing inside host macrophages.
    [Show full text]
  • Role of Reca in the Protection of DNA Damage by UV-A in Escherichia Coli
    Journal of Experimental Microbiology and Immunology Vol. 12:39-44 Copyright © April 2008, M&I UBC Role of RecA in the Protection of DNA Damage by UV-A in Escherichia coli. Amanda Chau, Karen Giang, Marcus Leung and Natalie Tam Department of Microbiology and Immunology, UBC Solar Water Disinfection (SODIS) is a simple, yet effective method for water decontamination by utilizing heat and UV-A from sunlight to kill microbes. However, the mechanism for UV-A mediated cell death is unknown. UV-A has been shown to cause DNA damage by the induction of reactive oxygen species (ROS). This damage might cause cell death in SODIS. Therefore, we investigated whether limiting the repair of DNA damaged by UV-A can increase bacterial susceptibility to UV-A. RecA is a DNA repair regulator protein that is activated upon stresses such as heat and reactive oxygen. In this study, we used Escherichia coli strains with and without RecA to determine survival frequencies after UV-A irradiation. recA+ cells with functional RecA showed a higher survival frequency compared to RecA- mutants after 120 minutes of UV-A exposure, while a smaller difference was observed when cells were cultured without light. These results suggest that the absence of RecA is correlated with an increased vulnerability of cells under UV-A exposure. This difference in sensitivity is most likely due to the inability of mutant bacteria to repair DNA damages. Further studies are required to determine not only the effects of RecA on repairing DNA damage, but also other bactericidal mechanisms exerted by UV-A because the presence of RecA was insufficient to fully protect the treated cells.
    [Show full text]
  • Quantification of Reca Gene Expression As an Indicator of Repair Potential in Marine Bacterioplankton Communities of Antarctica
    AQUATIC MICROBIAL ECOLOGY Vol. 24: 51–59, 2001 Published March 28 Aquat Microb Ecol Quantification of recA gene expression as an indicator of repair potential in marine bacterioplankton communities of Antarctica Melissa G. Booth1,*, LeAnna Hutchinson2, Margaret Brumsted2, Peter Aas2, Richard B. Coffin3, Roswell C. Downer Jr4, Cheryl A. Kelley5, M. Maille Lyons2, J. Dean Pakulski2, Sonya L. Holder Sandvik6, Wade H. Jeffrey2, Robert V. Miller1,** 1Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078, USA 2Center for Environmental Diagnosis and Bioremediation, University of West Florida, Pensacola, Florida 32514, USA 3Naval Research Laboratory, Washington, DC, Washington 20375, USA 4Department of Oceanography, Texas A&M University, College Station, Texas 77840, USA 5Department of Geological Sciences, University of Missouri, Columbia, Missouri 65211, USA 6National Research Council, c/o U.S. EPA, Gulf Breeze, Florida 32561, USA ABSTRACT: Marine bacteria in surface waters must cope daily with the damaging effects of expo- sure to solar radiation (containing both UV-A and UV-B wavelengths), which produces lesions in their DNA. As the stratospheric ozone layer is depleted, these coping mechanisms are likely to play an even more important role in the viability of marine bacterial communities. The recA gene is ubiq- uitous among eubacteria and is highly conserved both in nucleotide and amino acid sequence. Besides its role in generalized recombination, the gene’s translational product, RecA, is the regulator of ‘dark repair’ activity (DNA-repair mechanisms that do not require visible light as a cofactor). We have taken advantage of this function and used recA gene expression as a barometer of the DNA- damage repair capacity of bacterial assemblages in the Southern Ocean.
    [Show full text]