Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality.

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells-a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality.

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis.

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis.