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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-17915

Keller, P M. Generation of unmarked mycobacterium bovis BCG recA mutants. 2008, University of Zurich, Faculty of Medicine.




Mycobacterium bovis BCG is used as a live vaccine against tuberculosis. RecA-dependent homologous recombination is a major driving force of bacterial evolution. Therefore a RecA phenotype is recommended for all live vaccines. RecA- life vaccines demonstrate increased genetic stability and are favourable because of a reduced risk of reversion to virulence. Characterization of marked recA mutants provided proof of principle that persistence in the host, which is a major criterion of vaccine efficiency, is not affected by the deletion of recA. However, due to the presence of antibiotic resistance markers such a recombinant strain may not be used in human vaccination trials. The aim of this study was to generate unmarked recA deletion mutants in phylogenetically different Mycobacterium bovis BCG substrains.


Six BCG vaccine substrains were selected based on the following criteria: The substrains had to feature a complete historical documentation with date of lyophilization for the primary seed lot, protective efficiency in case control studies, an official WHO, FDA or government issued permission for human use, and a molecular description (regions of difference, and single nucleotide polymorphisms in comparison to M. bovis AF2122/97). Identity of the substrains was confirmed by molecular techniques. Mixed cultures with more than one genotype were excluded. Unmarked ,inactivation of recA was achieved with a suicide (i.e. non-replicative) knock-out plasmid containing a partially deleted recA allele flanked by a hygromycin and a kanamycin selectable marker and a levansucrase (sacB)counterselectable marker. SacB induces sucrose sensitivity in mycobacteria and allows the isolation of allelic exchange mutants in a two-step selective process. M. tuberculosis H37Rv recA was cloned into the suicide vector together with approximately 1.2 kbs of flanking genomic DNA. The genomic sequences of the cloned region are identical in M. bovis AF2122/97 and M. tuberculosis H37Rv. An in-frame stop codon (following amino acid residue 169) was introduced in the cloned recA instead of an internal 1.2-kb PstI fragment. BCG substrains were grown in Middlebrook media; electrocompetent cells were produced and transformed with the suicide knock-out plasmid.
Following selection of single-crossover transformants at the recA locus on hygromycin B containing media, deletion
mutants resulting from intramolecular recombination were isolated after a counterselective step on 10% sucrose
containing media. The respective genotypes were determined by PCR and Southern blot analyses.


Unmarked recA deletion mutants were obtained in BCG substrains Frappier, Pasteur, and Denmark. In BCG Tice
and BCG Phipps, single-crossover mutants for the recA locus could be isolated. After counterselection, only
reversion to wild-type allele or persistence of the single-crossover genotype was noted. In BCG Russia only
illegitimate recombinant mutants were obtained. Illegitimate integration excludes generation of recA deletion mutants as an intramolecular recombination does not occur. Sequencing of the recA locus revealed a mechanistic explanation for the inability to obtain homologous recombination in BCG Russia. This substrain has an insertional single ,nucleotide polymorphism (SNP) in recA causing translational frame shift and truncation of RecA. Thus, tuberculosis vaccine strain BCG Russia is a natural recA mutant.


The presented strategy allows generation of unmarked allelic exchange mutants for the recA locus in M. bovis BCG
and delivered new recA- vaccine candidate strains that base on three well-documented BCG vaccine substrains. The mutants are free of genomic selectable and ounterselectable markers. The constructed suicide vector backbone for the generation of unmarked allelic exchange mutants may be used for other allelic exchange experiments in mycobacteria. BCG Russia recA SNP provides an explanation for the observed high degree of in vitro evolutional stasis.


The deletion of recA in M. bovis BCG leads to a stable genome in this live vaccine reducing the risk of further
attenuation or reversion of virulence in the process of vaccine production and in vaccinees. The availability of recA mutants in M. bovis BCG substrains facilitates the construction of heterologous antigen expression vectors for the intracellular compartment with stable expression of recombinant antigens profiting from BCG’s long generation time and persistence in the host.


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Item Type:Dissertation
Referees:Böttger E C
Communities & Collections:04 Faculty of Medicine > Institute of Medical Microbiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Deposited On:24 Mar 2009 09:36
Last Modified:05 Apr 2016 13:11
Number of Pages:91
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=005581294

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