title: Hygromycin B and apramycin antibiotic resistance cassettes for use in Campylobacter jejuni.
abstract: Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Andrew
authors.initials: A
authors.lastname: Cameron
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Erin C
authors.initials: EC
authors.lastname: Gaynor
mesh_terms: D001192:Arylsulfatases / Q000235:genetics; D016123:Campylobacter jejuni / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D004352:Drug Resistance, Microbial / Q000187:drug effects* / Q000235:genetics*; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D006921:Hygromycin B / Q000494:pharmacology*; D016254:Mutagenesis, Insertional / Q000187:drug effects / Q000235:genetics*; D009328:Nebramycin / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D009328:Nebramycin; C011666:apramycin; D006921:Hygromycin B; D001192:Arylsulfatases
references.citation: Appl Environ Microbiol. 1999 Mar;65(3):1036-44
references.pmid: 10049860
references.citation: J Bacteriol. 1996 Jun;178(11):3335-8
references.pmid: 8655516
references.citation: Nature. 1991 Jun 6;351(6326):456-60
references.pmid: 1904554
references.citation: PLoS Biol. 2009 Aug;7(8):e1000171
references.pmid: 19688030
references.citation: Antimicrob Agents Chemother. 1990 Oct;34(10):1915-20
references.pmid: 1963287
references.citation: Appl Environ Microbiol. 2005 Jul;71(7):4004-13
references.pmid: 16000815
references.citation: Mol Microbiol. 2001 Apr;40(1):214-24
references.pmid: 11298288
references.citation: Cell. 2000 Dec 22;103(7):1143-54
references.pmid: 11163189
references.citation: J Infect Dis. 1985 Sep;152(3):592-6
references.pmid: 4031557
references.citation: J Bacteriol. 1995 Jul;177(14):4121-30
references.pmid: 7608087
references.citation: J Bacteriol. 1988 Apr;170(4):1704-8
references.pmid: 2832375
references.citation: J Bacteriol. 2004 Jan;186(2):503-17
references.pmid: 14702320
references.citation: J Bacteriol. 1987 Nov;169(11):5320-3
references.pmid: 2822671
references.citation: J Gen Microbiol. 1985 Jun;131(6):1289-98
references.pmid: 2995542
references.citation: J Bacteriol. 2010 Apr;192(8):2182-92
references.pmid: 20139192
references.citation: Curr Protoc Microbiol. 2008 Aug;Chapter 8:Unit 8A.2.1-8A.2.17
references.pmid: 18729059
references.citation: Gene. 1983 Nov;25(2-3):179-88
references.pmid: 6319235
references.citation: J Biol Chem. 2012 Jan 2;287(1):299-310
references.pmid: 22069311
references.citation: J Bacteriol. 1993 Sep;175(18):5899-906
references.pmid: 8376337
references.citation: J Bacteriol. 1996 Nov;178(21):6357-60
references.pmid: 8892841
references.citation: Infect Immun. 2000 Sep;68(9):5450-3
references.pmid: 10948182
references.citation: J Bacteriol. 2001 Apr;183(7):2384-8
references.pmid: 11244083
references.citation: J Antimicrob Chemother. 1978 Jul;4(4):309-13
references.pmid: 690031
references.citation: Gene. 1990 Sep 28;94(1):23-8
references.pmid: 2227449
references.citation: J Bacteriol. 2012 Nov;194(22):6116-30
references.pmid: 22961853
references.citation: Nat Rev Microbiol. 2007 Sep;5(9):665-79
references.pmid: 17703225
references.citation: Mol Microbiol. 2003 Oct;50(2):687-702
references.pmid: 14617189
references.citation: BMC Microbiol. 2011 Jul 25;11:166
references.pmid: 21787430
references.citation: Gene. 1993 Aug 16;130(1):127-30
references.pmid: 8344519
references.citation: Infect Immun. 2009 Nov;77(11):4912-24
references.pmid: 19703978
references.citation: Biochemistry. 2005 Dec 13;44(49):16275-83
references.pmid: 16331988