title: Construction and characterization of a Borrelia burgdorferi strain with conditional expression of the essential telomere resolvase, ResT.
abstract: Borrelia species are unique in the bacterial world in possessing segmented genomes which sometimes contain over 20 genetic elements. Most elements are linear and contain covalently closed hairpin ends requiring a specialized process, telomere resolution, for their generation. Hairpin telomere resolution is mediated by the telomere resolvase, ResT. Although the process has been studied extensively in vitro, the essential nature of the resT gene has precluded biological studies to further probe the role of ResT. In this work, we have generated a B. burgdorferi strain that carries an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible resT gene controlled by a tightly regulated promoter. ResT is expressed in this strain at ~14,000 monomers per cell, similar to the ~15,000 monomers observed for the parental strain. We demonstrate ResT depletion with a half-life of 16 h upon IPTG washout. ResT depletion resulted in arrested growth 48 h after washout. Interestingly, not all spirochetes died after ResT washout, and at least 15% remained quiescent and could be resuscitated even at 2 weeks postwashout. Significant levels of DNA synthesis were not observed upon growth arrest, suggesting that ResT might interact directly or indirectly with factors controlling the initiation or elongation of DNA synthesis. Analysis of the linear plasmids lp17 and lp28-2 showed that the linear forms of these plasmids began to disappear and be replaced by higher-molecular-weight forms by 24 h post-IPTG washout. Treatment of DNA from the ResT-depleted strain with ResT in vitro revealed the presence of replicated telomeres expected in replication intermediates.
authors.affiliation: Department of Biochemistry & Molecular Biology, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Canada.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Bandy
authors.affiliation: Department of Biochemistry & Molecular Biology, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Canada Department of Microbiology, Immunology & Infectious Diseases, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Canada.
authors.forename: Aydan
authors.initials: A
authors.lastname: Salman-Dilgimen
authors.affiliation: Department of Biochemistry & Molecular Biology, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Canada Department of Microbiology, Immunology & Infectious Diseases, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Canada chaconas@ucalgary.ca.
authors.forename: George
authors.initials: G
authors.lastname: Chaconas
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D025065:Borrelia burgdorferi / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D009154:Mutation
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004706:Endodeoxyribonucleases; C446515:ResT protein, Borrelia
references.citation: Annu Rev Genet. 2012;46:121-43
references.pmid: 22934648
references.citation: J Clin Invest. 2004 Apr;113(8):1093-101
references.pmid: 15085185
references.citation: J Bacteriol. 1991 Nov;173(22):7233-9
references.pmid: 1938918
references.citation: J Biol Chem. 2009 Aug 28;284(35):23293-301
references.pmid: 19561077
references.citation: Annu Rev Genet. 2012;46:515-36
references.pmid: 22974303
references.citation: J Bacteriol. 2013 May;195(10):2220-31
references.pmid: 23475971
references.citation: J Biol Chem. 2009 Mar 13;284(11):7264-72
references.pmid: 19122193
references.citation: Lancet. 2012 Feb 4;379(9814):461-73
references.pmid: 21903253
references.citation: Yale J Biol Med. 1984 Jul-Aug;57(4):521-5
references.pmid: 6393604
references.citation: Mol Microbiol. 2005 Jul;57(1):132-42
references.pmid: 15948955
references.citation: J Biol Chem. 2010 Dec 24;285(52):41010-8
references.pmid: 20952394
references.citation: Science. 1982 Jun 18;216(4552):1317-9
references.pmid: 7043737
references.citation: Mol Microbiol. 2009 Mar;71(5):1117-30
references.pmid: 19170885
references.citation: EMBO J. 2001 Jun 15;20(12):3229-37
references.pmid: 11406599
references.citation: J Bacteriol. 2004 Jun;186(11):3561-9
references.pmid: 15150244
references.citation: J Bacteriol. 2012 Jul;194(13):3336-42
references.pmid: 22544267
references.citation: Nature. 1997 Dec 11;390(6660):580-6
references.pmid: 9403685
references.citation: Appl Environ Microbiol. 2009 Oct;75(20):6553-8
references.pmid: 19700541
references.citation: J Biol Chem. 2004 Dec 17;279(51):53699-706
references.pmid: 15471873
references.citation: Appl Environ Microbiol. 2007 Mar;73(5):1501-13
references.pmid: 17220265
references.citation: Mol Microbiol. 2003 May;48(4):901-11
references.pmid: 12753185
references.citation: Mol Microbiol. 2007 Feb;63(4):1259-73
references.pmid: 17257307
references.citation: Genome Res. 2000 Oct;10(10):1594-604
references.pmid: 11042157
references.citation: Mol Microbiol. 2000 Feb;35(3):490-516
references.pmid: 10672174
references.citation: Nat Rev Microbiol. 2012 Jan 09;10(2):87-99
references.pmid: 22230951
references.citation: J Bacteriol. 2000 May;182(10):2909-18
references.pmid: 10781562
references.citation: Antimicrob Agents Chemother. 2009 Oct;53(10):4441-9
references.pmid: 19596868
references.citation: PLoS Pathog. 2009 Dec;5(12):e1000680
references.pmid: 19997508
references.citation: Mol Cell. 2002 Jan;9(1):195-201
references.pmid: 11804598
references.citation: Mol Cell. 2005 Mar 18;17(6):783-91
references.pmid: 15780935
references.citation: Annu Rev Microbiol. 2010;64:185-202
references.pmid: 20536352
references.citation: Science. 1987 Jul 24;237(4813):409-11
references.pmid: 3603026
references.citation: Mol Microbiol. 1999 Apr;32(2):437-45
references.pmid: 10231498
references.citation: Mol Microbiol. 2006 Nov;62(3):895-905
references.pmid: 16999829
references.citation: J Biol Chem. 2005 Jul 22;280(29):26788-95
references.pmid: 15917226
references.citation: FEMS Microbiol Lett. 2003 Apr 11;221(1):1-6
references.pmid: 12694903
references.citation: J Bacteriol. 2010 Dec;192(23):6105-15
references.pmid: 20870761
references.citation: Mol Microbiol. 2007 Nov;66(4):975-90
references.pmid: 17919281
references.citation: J Microbiol Methods. 2011 Aug;86(2):243-7
references.pmid: 21605603
references.citation: PLoS Biol. 2006 Jun;4(6):e185
references.pmid: 16719561
references.citation: Mol Microbiol. 2005 Nov;58(3):625-35
references.pmid: 16238614
references.citation: J Bacteriol. 2006 Nov;188(21):7378-86
references.pmid: 16936037
references.citation: Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13768-73
references.pmid: 15365172
references.citation: Mol Microbiol. 2007 May;64(3):580-90
references.pmid: 17462009
references.citation: J Bacteriol. 2000 May;182(9):2445-52
references.pmid: 10762244