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There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel(r)) and Escherichia coli aspartate-semialdehyde dehydrogenase (asd(Ec)), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn7-derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ, gfp, and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Deltabla-Tel(r)-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Tel(r)-based transposon vector (mini-Tn7-Tel(r)-betBA) and a transposase-containing helper plasmid (pTNS3-asd(Ec)) to complement the B. thailandensis DeltabetBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Tel(r)) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asd(Ec)) to construct the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing beta-galactosidase assays on the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) fusion strain. Finally, we engineered B. thailandensis DeltabetBA::FRT-gfp-Tel(r) and DeltabetBA::FRT-lux-Tel(r) fusion strains by utilizing fusion vectors pFRT1-gfp-Tel(r) and pFRT1-lux-Tel(r), respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.

Titel:	Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the Burkholderia thailandensis betBA operon.
Autor/in / Beteiligte Person:	Kang, Y ; Norris, MH ; Barrett, AR ; Wilcox, BA ; Hoang, TT
Link:	Zum Volltext
Zeitschrift:	Applied and environmental microbiology, Jg. 75 (2009-06-01), Heft 12, S. 4015
Veröffentlichung:	Washington, American Society for Microbiology., 2009
Medientyp:	academicJournal
ISSN:	1098-5336 (electronic)
DOI:	10.1128/AEM.02733-08
Schlagwort:	Artificial Gene Fusion Aspartate-Semialdehyde Dehydrogenase genetics Aspartate-Semialdehyde Dehydrogenase metabolism Choline metabolism DNA Transposable Elements DNA, Bacterial chemistry DNA, Bacterial genetics Escherichia coli Proteins genetics Escherichia coli Proteins metabolism Gene Deletion Gene Expression Profiling Gene Expression Regulation, Bacterial Genes, Reporter Genetic Complementation Test Molecular Sequence Data Osmotic Pressure Selection, Genetic Sequence Analysis, DNA Anti-Bacterial Agents pharmacology Bacterial Proteins genetics Burkholderia genetics Drug Resistance, Bacterial Molecular Biology methods Operon Tellurium pharmacology
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S. Language: English [Appl Environ Microbiol] 2009 Jun; Vol. 75 (12), pp. 4015-27. <i>Date of Electronic Publication: </i>2009 Apr 17. MeSH Terms: Drug Resistance, Bacterial* ; Operon* ; Anti-Bacterial Agents / pharmacology ; Bacterial Proteins / genetics ; Burkholderia / genetics ; Molecular Biology / methods ; Tellurium / *pharmacology ; Artificial Gene Fusion ; Aspartate-Semialdehyde Dehydrogenase / genetics ; Aspartate-Semialdehyde Dehydrogenase / metabolism ; Choline / metabolism ; DNA Transposable Elements ; DNA, Bacterial / chemistry ; DNA, Bacterial / genetics ; Escherichia coli Proteins / genetics ; Escherichia coli Proteins / metabolism ; Gene Deletion ; Gene Expression Profiling ; Gene Expression Regulation, Bacterial ; Genes, Reporter ; Genetic Complementation Test ; Molecular Sequence Data ; Osmotic Pressure ; Selection, Genetic ; Sequence Analysis, DNA References: Gene. 2002 May 15;290(1-2):153-61. (PMID: 12062810) ; Antimicrob Agents Chemother. 1999 Sep;43(9):2332. (PMID: 10577350) ; J Bacteriol. 1990 Nov;172(11):6557-67. (PMID: 2172216) ; Microbiology (Reading). 2008 Jun;154(Pt 6):1584-1598. (PMID: 18524913) ; Gene. 2009 Feb 1;430(1-2):123-31. (PMID: 19010402) ; Biotechniques. 2005 Feb;38(2):225-9. (PMID: 15727129) ; Trends Microbiol. 1999 Mar;7(3):111-5. (PMID: 10203839) ; Nat Methods. 2005 Jun;2(6):443-8. (PMID: 15908923) ; Protein Expr Purif. 2007 Oct;55(2):325-33. (PMID: 17716915) ; Mol Microbiol. 2006 Dec;62(6):1643-54. (PMID: 17116241) ; Antimicrob Agents Chemother. 2004 Apr;48(4):1128-35. (PMID: 15047512) ; Mol Plant Microbe Interact. 2003 Aug;16(8):709-19. (PMID: 12906115) ; Antimicrob Agents Chemother. 1999 Mar;43(3):465-70. (PMID: 10049252) ; Appl Environ Microbiol. 2008 Jul;74(14):4498-508. (PMID: 18502918) ; Plasmid. 2000 Jan;43(1):12-23. (PMID: 10610816) ; J Bacteriol. 1988 Nov;170(11):5208-15. (PMID: 3141381) ; J Bacteriol. 1997 Apr;179(7):2116-25. (PMID: 9079894) ; Nat Protoc. 2006;1(1):162-9. (PMID: 17406228) ; Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14240-5. (PMID: 15377794) ; Environ Microbiol. 2004 Jul;6(7):726-32. (PMID: 15186351) ; Mol Microbiol. 2005 Jan;55(2):368-80. (PMID: 15659157) ; Microbiol Immunol. 1992;36(12):1251-75. (PMID: 1283774) ; Appl Environ Microbiol. 1998 Oct;64(10):4040-6. (PMID: 9758838) ; Protein Expr Purif. 2006 Oct;49(2):219-27. (PMID: 16737826) ; Nat Rev Microbiol. 2005 Feb;3(2):144-56. (PMID: 15643431) ; Appl Environ Microbiol. 2008 Feb;74(4):1064-75. (PMID: 18156318) ; Appl Environ Microbiol. 2008 Dec;74(24):7529-35. (PMID: 18952878) ; Microbiology (Reading). 1995 Apr;141 ( Pt 4):1045-50. (PMID: 7773382) ; Gene. 1991 Dec 20;109(1):167-8. (PMID: 1661697) ; Gene. 1998 May 28;212(1):77-86. (PMID: 9661666) ; Plasmid. 2007 May;57(3):286-95. (PMID: 17207855) ; Biotechnol Bioeng. 2006 May 5;94(1):151-8. (PMID: 16496398) ; Emerg Infect Dis. 2008 Nov;14(11):1689-92. (PMID: 18976550) ; J Bacteriol. 2005 Nov;187(21):7351-61. (PMID: 16237018) ; Int J Syst Bacteriol. 1998 Jan;48 Pt 1:317-20. (PMID: 9542103) ; Plasmid. 2008 Mar;59(2):111-8. (PMID: 18221997) Grant Information: P20 RR018727 United States RR NCRR NIH HHS; R21 AI074608 United States AI NIAID NIH HHS; R21-AI074608 United States AI NIAID NIH HHS; P20RR018727 United States RR NCRR NIH HHS Molecular Sequence: GENBANK EU626135; EU626136; EU626138; EU626139; EU626140; FJ455408; FJ455409; FJ455410; FJ455411; FJ797680 Substance Nomenclature: 0 (Anti-Bacterial Agents) ; 0 (Bacterial Proteins) ; 0 (DNA Transposable Elements) ; 0 (DNA, Bacterial) ; 0 (Escherichia coli Proteins) ; EC 1.2.1.11 (Aspartate-Semialdehyde Dehydrogenase) ; IVA6SGP6QM (tellurous acid) ; N91BDP6H0X (Choline) ; NQA0O090ZJ (Tellurium) Entry Date(s): Date Created: 20090421 Date Completed: 20090625 Latest Revision: 20211020 Update Code: 20240513 PubMed Central ID: PMC2698364

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Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the Burkholderia thailandensis betBA operon.