Elsevier

Tuberculosis

Volume 87, Issue 6, November 2007, Pages 481-488
Tuberculosis

Creation and characterisation of a high-copy-number version of the pAL5000 mycobacterial replicon

https://doi.org/10.1016/j.tube.2007.08.003Get rights and content

Abstract

The majority of mycobacterial plasmid vectors are derived from the pAL5000 replicon and maintained at approximately five copies per cell. We have devised a method that directly selects for high-copy-number plasmids. This involves enriching for high copy number plasmids by repeatedly isolating and retransforming plasmids from a mutant library. Using this method we have selected a copy-up version of the pAL5000 replicon. In Mycobacterium smegmatis the copy-number was shown to have increased 7-fold to between 32 and 64 copies/cell, and the plasmid remained relatively stable after 100 generations in the absence of antibiotic selection. The plasmid also has a high-copy-number phenotype in M. bovis BCG and can be used to increase expression of cloned genes, as we have demonstrated with the green fluorescent protein. The mutation was found to be the deletion of an alanine residue in the C-terminal end of the RepA replication protein. We argue that the mutation exerts its effect through altered RNA folding, thereby affecting the translationally coupled RepA-RepB expression.

Introduction

The global TB epidemic has fuelled world-wide efforts in mycobacterial research aimed at better understanding pathogenesis, identification of potential drug targets and the development of improved vaccines. Live attenuated strains of Mycobacterium bovis BCG, used globally as childhood vaccines against TB and leprosy, are being modified to promote improved protection.1 Such approaches include the construction of recombinant BCG strains expressing immunodominant TB antigens. In addition, BCG is a very attractive vaccine vehicle for the delivery of antigens for other diseases as well.

The best-characterised mycobacterial replicon for the expression of recombinant proteins is that of pAL5000.2 The majority of mycobacterial plasmid vectors utilise the pAL5000 replication machinery; its advantage lies in its ability to replicate within both the fast growing non-pathogenic model organism Mycobacterium smegmatis as well as the slow growing pathogenic mycobacteria.3 The sequence of pAL5000 consists of five large open reading frames (ORF).4, 5 Three of these, repA, repB and rap (originally ORFs 1, 2 and 5) appear to be involved in replication. The genes repA and repB overlap by one base pair and are expressed from a common transcript.6 A potential SD sequence for repB constitutes part of the C-terminus of repA. Both RepA and RepB, acting in trans, are necessary for replication, and the region upstream of repA carries the origin of replication and influences compatibility.6, 7 Within RepB there is a helix-turn-helix motif, typical of DNA-binding regulatory proteins4 and it has been shown that RepB binds specifically to two upstream regions with different affinities designated the H- and L-sites (for high and low affinity). The H-site is upstream of the repA/B transcription start site and overlaps the promoter, suggesting that RepB acts as an autoregulator of its own expression. The importance of the H-site has been demonstrated by functional assays involving an extensive series of mutant H-sites.8 Basu et al.9 have demonstrated that RepB binding to the ori is stimulated in the presence of RepA and host factors, but that the stimulatory effect of RepA required the coupled expression of the two genes in their native overlapping arrangement. Later it was shown that coupling affects the final structure adopted by RepB and it was suggested that ribosomes that engage via re-initiation at translation coupling sites are better able to promote protein folding10 due to the activity of ribosome-tethered chaperones.11

Rap is orientated in the opposite direction to repA and repB and encodes a 115aa protein with no convincing similarity to any reported gene. Stolt and Stoker7 have observed that M. smegmatis transformants carrying the minimal replicon (ori, repA and repB) form colonies more slowly than those that carry rap in addition. It was later shown that rap encodes an auxiliary factor, which improves replication ability but which is either not essential or can be substituted for by a host factor. The transcription start point of rap was found to lie within the H-site implying that the promoters overlap.12

The natural copy-number of pAL5000 is estimated at 3–5 copies per cell.13 A high-copy-number mycobacterial plasmid would be a very useful tool for a number of reasons, including ease of isolation and for increased expression of cloned genes, particulary in vaccine design. To date, attempts to convert pAL5000 to a high-copy-number phenotype by specific modification of targets in the replicon, either by expressing different levels of RepA and RepB or by mutation of the H-site, have been unsuccessful.8

We have devised a novel method to select high-copy-number plasmids from a library of random mutants that relies on direct selection for copy-number increases. The method involves repeatedly isolating plasmid from the pooled library and re-transforming the mix, thus enriching for the high-copy-number mutants until eventually they take over the library and can be isolated. We have applied this method to the mycobacterial plasmid pAL5000. Here we describe the construction, isolation and characterisation of a high-copy-number mutant pAL5000 replicon.

Section snippets

Strains, growth conditions and transformation

E. coli strains JM109,14 LKIII,15M. smegmatis mc215516 and Mycobacterium bovis BCG (Pasteur) were used. E. coli cells were grown in LB broth or on agar plates17 with the appropriate antibiotic (100 μg/ml). M. smegmatis and M. bovis BCG cells were grown in Middelbrooks 7H9/ADC broth with shaking or on Middelbrooks 7H11/ADC agar plates18 with antibiotics as appropriate (100 μg/ml). Electrotransformation of M. bovis BCG, M. smegmatis and E. coli was as previously described.19

Reference plasmids

Plasmid pADM4 carries an

Construction and isolation of a high-copy-number plasmid

Our strategy for selecting a high copy number mutant plasmid was based on the principal that, although the initial mutagenesis step will result in a very low ratio of high-copy-number plasmid carrying clones, after repeated plasmid isolation and retransformation of bacterial cells, the ratio of high-copy-number to low-copy-number plasmid carrying clones will be increased. This constitutes an enrichment process and, if repeated often enough, will result in high-copy-number plasmids dominating

Discussion

The method that we have used to create a high-copy-number plasmid is based on selection of a mutant by repeated rounds of transformation and plasmid isolation, which enriches for plasmids of the desired phenotype at each step. This has advantages over the usual method of selecting for increased expression of a plasmid-borne gene. The selection is not limited by the nature of the plasmid-borne genes and very large numbers of mutants can be screened. In addition, there is also a selection for

Acknowledgements

We thank Valerie Mizrahi for supplying us with the plasmid pADM4 and Ian Wiid for considerable technical advice.

Funding: This work was supported by the Glaxo-Smithkline Action TB initiative.

Competing interest: None declared.

Ethical approval: Not required.

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