Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

Laboratory of Recombinant Technology, Bio-Manguinhos, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil 1 Biotechnology Centre, Federal University of Pelotas, Pelotas, Brazil 2 Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil 3 School of Biological Sciences, University of Surrey, Guildford, Surrey, UK 4 Author for correspondence: Douglas McIntosh. Tel: +55 21 2598 4284. Fax: +55 21 2260 4727. e-mail: mcintosh{at}bio.fiocruz.br Mycobacterium vaccae represents an alternative mycobacterial cloning host that has been largely overlooked to date. The main reason for this may be the reported non-transformability of this species, specifically the so-called Stanford strain (NCTC 11659), with expression vectors that use kanamycin resistance as a selection method. However, this strain can be transformed using hygromycin resistance as an alternative selectable phenotype. The present study has shown that in contrast to previous reports, M. vaccae (ATCC 15483) is capable of being transformed with a range of vectors encoding kanamycin resistance as the selectable marker. Thereafter, the expression of the lacZ reporter gene in M. vaccae , Mycobacterium bovis BCG and Mycobacterium smegmatis mc 2 155 was evaluated using a range of characterized mycobacterial promoter sequences ( hsp60 , hsp70 , P AN, 18kDa and 16S rRNA ) cloned in the same promoter probe vector. In general, the promoters showed similar levels of activity in the three species, demonstrating that existing expression systems can readily be employed with M. vaccae (ATCC 15483). This was further confirmed by the observation that M. vaccae was capable of stable, in vitro expression of recombinant S1 subunit of pertussis toxin at levels equivalent to those obtained with BCG and M. smegmatis . Analysis of structural and functional stability of a range of vectors demonstrated that the incidence of instability noted for M. vaccae was lower than that recorded for M. smegmatis . Taken together, the results indicate that M. vaccae is an additional cloning host which may prove useful for specific aspects of mycobacterial biology and provide increased flexibility to the field of recombinant protein technology for mycobacteria. Keywords: mycobacteria, shuttle vectors, expression, stability