Site-specific integration of StreptomycesΦC31 integrase-based vectors in the chromosome of Rhodococcus equi

Authors


  • Editor: Wolfgang Schumann

Correspondence: Mary K. Hondalus, Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA. Tel.: +1 706 542 5778; fax: +1 706 542 5771; e-mail: hondalus@uga.edu

Abstract

StreptomycesΦC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the ΦC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.

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