Identification and Mutagenesis by Allelic Exchange of choE, Encoding a Cholesterol Oxidase from the Intracellular Pathogen Rhodococcus equi

doi:10.1128/JB.183.16.4796-4805.2001

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Journal of Bacteriology, August 2001, p. 4796-4805, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4796-4805.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification and Mutagenesis by Allelic Exchange of choE, Encoding a Cholesterol Oxidase from the Intracellular Pathogen Rhodococcus equi

Jesús Navas,¹ Bruno González-Zorn,² Néstor Ladrón,¹ Patricia Garrido,² and José A. Vázquez-Boland²^,^*

Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, 39011 Santander,¹ and Grupo de Patogénesis Molecular Bacteriana, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid,² Spain

Received 6 February 2001/Accepted 26 May 2001

The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidatevirulence factors of this pathogenic actinomycete is a secretedcholesterol oxidase, a putative membrane-damaging toxin. We identifiedand characterized the gene encoding this enzyme, the choE monocistron.Its protein product, ChoE, is homologous to other secreted cholesteroloxidases identified in Brevibacterium sterolicum and Streptomycesspp. ChoE also exhibits significant similarities to putative cholesteroloxidases encoded by Mycobacterium tuberculosis and Mycobacteriumleprae. Genetic tools for use with R. equi are poorly developed.Here we describe the first targeted mutagenesis system availablefor this bacterium. It is based on a suicide plasmid, a selectablemarker (the aacC4 apramycin resistance gene from Salmonella),and homologous recombination. The choE allele was disrupted byinsertion of the aacC4 gene, cloned in pUC19 and introduced byelectroporation in R. equi. choE recombinants were isolated atfrequencies between 10² and 10³. Twelve percent of the recombinants were double-crossover choEmutants. The choE mutation was associated with loss of cooperative(CAMP-like) hemolysis with sphingomyelinase-producing bacteria(Listeria ivanovii). Functional complementation was achieved byexpression of choE from pVK173-T, a pAL5000 derivative conferringhygromycin resistance. Our data demonstrate that ChoE is an importantcytolytic factor for R. equi. The highly efficient targeted mutagenesisprocedure that we used to generate choE isogenic mutants willbe a valuable tool for the molecular analysis of R. equivirulence.

^* Corresponding author. Mailing address: Grupo de Patogénesis Molecular Bacteriana, Departamento de Patología Animal I, Facultadde Veterinaria, Universidad Complutense, 28040 Madrid, Spain.Phone: 34-91-394.37.04. Fax: 34-91-394.39.08. E-mail: vazquez{at}eucmax.sim.ucm.es.

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