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Applied and Environmental Microbiology, October 2004, p. 5853-5858, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.5853-5858.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Plasmid pOR1 from Ornithobacterium rhinotracheale and Construction of a Shuttle Plasmid

Ruud Jansen, Niwat Chansiripornchai, Wim Gaastra, and Jos P. M. van Putten*

Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands

Received 27 February 2004/ Accepted 2 June 2004

The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.

* Corresponding author. Mailing address: Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands. Phone: 31 30 253 4344. Fax: 31 30 254 0784. E-mail: j.vanputten{at}vet.uu.nl.

Applied and Environmental Microbiology, October 2004, p. 5853-5858, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.5853-5858.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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