A new site-specific integration system for mycobacteria
Introduction
Mycobacterium tuberculosis is an important pathogen that infects close to 2 billion people worldwide (WHO). Numerous genetic tools have recently become available to study M. tuberculosis and other mycobacteria. In particular, protein expression of plasmid-encoded genes can now be easily performed. However, stable transformation with episomal plasmids requires continuous antibiotic selection. Such selection is not practical when organisms are used for infection and, therefore, bacteria may lose plasmids as infection progresses.
Two strategies have been used to produce stable expression. One is the use of homologous recombination to create stable double-crossover strains. This has proven difficult in slow-growing mycobacterium such as M. tuberculosis and M. bovis BCG, where problems have been encountered due to low levels of homologous recombination, high levels of non-homologous recombination, and high levels of variation in recombination frequencies at different loci.1
An alternative method to produce chromosomal recombinants is to use site-specific recombination. This approach has three advantages. First, genes reproducibly integrate at a single site (or, at least, a limited number of sites). Any effects of the integration sites (their influence on gene expression and bacterial biology) will be equally applicable to any insertion. Second, genes are integrated in single copy, thus mitigating artifacts that can occur with multicopy plasmids. Finally, integrants are generally much more stable than episomes.
Site-specific recombination has been used extensively in mycobacteria using the integrase produced by phage L5.2 This protein catalyzes the insertion of DNA into a tRNA locus and produces recombinants at high frequency. The L5 integrase has become one of the central tools in mycobacterial molecular biology. However, the integration site used by this phage has one principal drawback. This locus has proven unfavorable for transcription of integrated genes and thus, in our hands, few genes are expressed efficiently from their native promoters.
Recent studies in bacterial and mammalian systems have demonstrated the usefulness of a new group of large serine recombinases for site-specific recombination. Characterization of the integrase gene of φC31, a bacteriophage that infects Streptomyces coelicolor, has demonstrated that this enzyme catalyzes recombination between the phage attachment site (attP) and the attachment site on the bacterial chromosome (attB) independent of host factors or other phage-encoded products.3 Similar to the reaction catalyzed by the well-studied λ integrase, φC31 integrase catalyzes recombination between non-identical attP and attB sites, creating the hybrid sites attR and attL (Fig. 1). In vitro studies with φC31 integrase have shown that this protein will not independently catalyze the reverse excision reaction, which would recombine attR and attL to recreate attP and attB.4 This specificity indicates that integration products created by φC31 integrase are stably maintained when no additional excision factors are present.
Despite the directionality of this integration reaction, the recognition sites characteristic of the φC31 integrase contain unusually small core regions. The minimal recognition sites, which are merely 39 and 34 bp for attP and attB, respectively,5 share a 5′-TTG-3′ sequence at the core region in S. coelicolor and only 5′-TT-3′ in other Streptomyces species.6, 7 This small core region allows variation in the recognition site specificity of the φC31 integrase. Studies using S. coelicolor strains in which the standard attB site is not present have found that φC31 integrase can use alternative pseudo-attB sites to facilitate recombination.7 In addition, recent studies have demonstrated that φC31 integrase can catalyze recombination between the standard attB site and pseudo-attP sites in mammalian cells, demonstrating the diversity of organisms in which the φC31 integrase can function effectively.8
The above studies indicate that the φC31 integrase may be useful in the development of new integration vectors for use in mycobacteria. Both Streptomyces and Mycobacterium are in the same Actinomycete family and both are characterized by GC-rich genomes. This characteristic, as well as the demonstrated diversity of integration sites that have been observed with the φC31 integration system make this system attractive for the development of new mycobacterial integrating vectors. We show here that the φC31 integrase/attP system facilitates integration into both rapidly and slowly growing mycobacteria. This will most likely be useful in future genetic studies with mycobacteria.
Section snippets
Bacterial strains, growth conditions and plasmids
Mycobacterium smegmatis mc2-155, M. bovis BCG Pasteur, and M. tuberculosis H37Rv were maintained by standard methods.9 When necessary apramycin was added to medium (30 μg/ml). The plasmids pIJ8600 and pIJ865510 were kindly provided by Daniel Kearns. pMV306 was obtained from Adrie Steyn. pMV306 is a derivative of pMV3612 in which the expression cassette was replaced by a multiple cloning site. pPE207 was obtained from Julian Davies.11 Plasmids were introduced into M. smegmatis, M. bovis BCG, and
Plasmid integration in mycobacteria
To test whether φC31 integrase could facilitate integration into a mycobacterial genome, we used the pIJ8600 plasmid. This plasmid contains the φC31 attP, the φC31 integrase, an Escherichia coli origin of replication, and an apramycin resistance marker that allows selection in both E. coli and mycobacteria.10 M. smegmatis, M. bovis BCG, and M. tuberculosis were each electroporated with pIJ8600 and selected for resistance to apramycin.
The efficiency of M. bovis BCG transformation with pIJ8600
Discussion
In this study we used the φC31 integrase and attP to facilitate site-specific integration into M. smegmatis, M. bovis BCG, and M. tuberculosis, demonstrating that this system functions efficiently in mycobacteria. The attB sites that were found in M. smegmatis and M. bovis BCG show that the φC31 recombinase system tolerates significant sequence variation at the integration sites as expected, but they also share some conserved regions. Specifically, each site has a GGnG or GnGG motif six or
Acknowledgments
We thank Daniel Kerns for providing the φC31 integrase system on the vectors pIJ8600 and pIJ8655. This work was supported in part by NIH Grants AI48704 and AI51929.
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Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress
2018, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Initially, complementation was attempted using the native promoter; however, the production of ERG was restored only in the ΔegtA mutant (ΔegtA+ in Table 1). This difficulty has been observed in previous studies, demonstrating that complementation of operonic genes by fusing the operon's promoter to the gene is usually problematic unless it is the first gene of the operon [34]. Nevertheless, when genes were expressed from the hsp60 promoter, ERG production was restored in all mutant except the ΔegtB complemented strain (Table 1).
The Secret Lives of Mycobacteriophages
2012, Advances in Virus ResearchCitation Excerpt :Vectors derived from plasmid pSAM2 integrate site specifically into at attB overlapping tRNApro gene Msmeg_6204 and the tRNApro gene located between Rv3684 and Rv3685c in M. tuberculosis H37Rv (Martin et al., 1991; Seoane et al., 1997). Phage phiC31-derived vectors (using a serine-integrase) integrate into an attB site located within the putative glutamyl-tRNA(Gln) amidotransferase gene Msmeg_3400 and presumably inactivate it; there are three potential attB sites in M. tuberculosis (Murry et al., 2005). L5 integration-proficient plasmids have also been manipulated such as to carry an additional attB site that will accept secondary integration events (Saviola and Bishai, 2004), and the Ms6 system has been manipulated so as to use alternative tRNAala genes as integration sites (Vultos et al., 2006).