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Applied and Environmental Microbiology, July 2007, p. 4619-4630, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.02407-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of Experimental Genetic Tools for Campylobacter fetus{triangledown} ,{dagger}

Sabine Kienesberger,1 Gregor Gorkiewicz,1,2 Martina M. Joainig,1 Sylvia R. Scheicher,1 Eva Leitner,1 and Ellen L. Zechner1*

Institut für Molekulare Biowissenschaften, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria,1 Institut für Pathologie, Medizinische Universität Graz, Auenbruggerplatz 25, A-8036 Graz, Austria2

Received 13 October 2006/ Accepted 13 May 2007

Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.

* Corresponding author. Mailing address: Karl-Franzens-Universität, Institut für Molekulare Biowissenschaften, Universitätsplatz 2, A-8010 Graz, Austria. Phone: 43 316 380 5624. Fax: 43 316 380 9898. E-mail: ellen.zechner{at}uni-graz.at

{triangledown} Published ahead of print on 18 May 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, July 2007, p. 4619-4630, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.02407-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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