title: Development of a single-plasmid-based regulatable gene expression system for Borrelia burgdorferi.
abstract: We developed a single-plasmid-based regulatable protein expression system for Borrelia burgdorferi. Expression of a target gene is driven by P(ost), a hybrid B. burgdorferi ospA-tetO promoter, from a recombinant B. burgdorferi plasmid constitutively expressing TetR. The system was tested using the green fluorescent protein (GFP) as a reporter. Under noninducing conditions, recombinant B. burgdorferi cells were nonfluorescent, no GFP protein was detected, and residual, small amounts of transcript were detectable only by reverse transcription-PCR but not by Northern blot hybridization. Upon induction with anhydrotetracycline, increasing levels of GFP transcript, protein, and fluorescence were observed. This tight and titratable promoter system will be invaluable for the study of essential borrelial proteins. Since target protein, operator, and repressor are carried by a single plasmid, the system's application is independent of a particular strain background.
authors.affiliation: Department of Microbiology, University of Kansas Medical Center, Mail Stop 3019, 3025 Wahl Hall West, 3901 Rainbow Boulevard, Kansas City, KS 66160-7420,USA.
authors.forename: Christine R
authors.initials: CR
authors.lastname: Whetstine
authors.forename: Joyce G
authors.initials: JG
authors.lastname: Slusser
authors.forename: Wolfram R
authors.initials: WR
authors.lastname: Zückert
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D025065:Borrelia burgdorferi / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013753:Tetracycline Resistance / Q000235:genetics; D013754:Tetracyclines / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C083139:Tet O resistance protein, Bacteria; D013754:Tetracyclines; D049452:Green Fluorescent Proteins; C016229:4-epianhydrotetracycline
references.citation: Infect Immun. 1993 Sep;61(9):3590-6
references.pmid: 8359881
references.citation: J Bacteriol. 2004 Jun;186(11):3561-9
references.pmid: 15150244
references.citation: J Bacteriol. 1994 Oct;176(19):6045-9
references.pmid: 7928965
references.citation: Infect Immun. 1986 May;52(2):549-54
references.pmid: 3516878
references.citation: Yale J Biol Med. 1984 Jul-Aug;57(4):521-5
references.pmid: 6393604
references.citation: Nat Rev Microbiol. 2005 Feb;3(2):129-43
references.pmid: 15685224
references.citation: Infect Immun. 2004 Mar;72(3):1463-9
references.pmid: 14977951
references.citation: Nature. 1997 Dec 11;390(6660):580-6
references.pmid: 9403685
references.citation: Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):425-30
references.pmid: 16407165
references.citation: Infect Immun. 1999 Jul;67(7):3181-7
references.pmid: 10377088
references.citation: Appl Environ Microbiol. 2007 Mar;73(5):1501-13
references.pmid: 17220265
references.citation: Mol Microbiol. 2007 Feb;63(4):1259-73
references.pmid: 17257307
references.citation: J Bacteriol. 1998 Sep;180(18):4850-5
references.pmid: 9733687
references.citation: Mol Microbiol. 2006 Mar;59(5):1473-84
references.pmid: 16468989
references.citation: Infect Immun. 1993 May;61(5):2192-5
references.pmid: 8478109
references.citation: Mol Microbiol. 2000 Feb;35(3):490-516
references.pmid: 10672174
references.citation: Gene. 1994 Dec 30;151(1-2):131-5
references.pmid: 7828861
references.citation: Infect Immun. 1999 Jul;67(7):3257-66
references.pmid: 10377099
references.citation: Gene. 2000 Sep 19;255(2):297-305
references.pmid: 11024290
references.citation: Science. 2007 Jan 26;315(5811):509-13
references.pmid: 17255510
references.citation: Curr Protoc Microbiol. 2007 Feb;Chapter 12:Unit 12C.1
references.pmid: 18770608
references.citation: J Bacteriol. 2004 May;186(9):2745-56
references.pmid: 15090516
references.citation: Gene. 1989 Apr 15;77(1):51-9
references.pmid: 2744487
references.citation: Mol Microbiol. 2001 Feb;39(3):714-21
references.pmid: 11169111
references.citation: Nucleic Acids Res. 1993 Sep 11;21(18):4411-2
references.pmid: 8415012
references.citation: Mol Microbiol. 1999 Jul;33(1):41-51
references.pmid: 10411722
references.citation: Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10268-73
references.pmid: 8816789
references.citation: Microbiology (Reading). 2003 Jul;149(Pt 7):1819-1828
references.pmid: 12855733
references.citation: Mol Microbiol. 2007 Nov;66(4):975-90
references.pmid: 17919281
references.citation: Infect Immun. 1995 Nov;63(11):4535-9
references.pmid: 7591099
references.citation: Nucleic Acids Res. 2004 Feb 04;32(2):842-7
references.pmid: 14764926
references.citation: Cell Microbiol. 2007 Sep;9(9):2230-41
references.pmid: 17490405
references.citation: J Bacteriol. 2000 May;182(9):2445-52
references.pmid: 10762244
references.citation: Science. 1982 Jun 18;216(4552):1317-9
references.pmid: 7043737
references.citation: Nat Struct Biol. 2000 Mar;7(3):215-9
references.pmid: 10700280
references.citation: Gene. 1999 Jan 21;226(2):243-51
references.pmid: 9931496
references.citation: Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
references.pmid: 9092630