DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS) approach we have analyzed the polymorphism of a T7 RNAP-generated mRNA pool of the mboIIM2 gene. We find that the enzyme displays a relatively high level of template-dependent transcriptional infidelity. The nucleotide misincorporations and multiple insertions in A/T-rich tracts of homopolymers in mRNA (0.20 and 0.089%, respectively) cause epigenetic effects with significant impact on gene expression that is disproportionally high to their frequency of appearance. The sequence-dependent rescue of single and even double InDel frameshifting mutants and wild-type phenotype recovery is observed as a result. As a consequence, a heterogeneous pool of functional and non-functional proteins of almost the same molecular mass is produced where the proteins are indistinguishable from each other upon ordinary analysis. We suggest that transcriptional infidelity as a general feature of the most effective RNAPs may serve to repair and/or modify a protein function, thus increasing the repertoire of phenotypic variants, which in turn has a high evolutionary potential.

Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity. The production of R-M system components upon entering a new host cell must be finely tuned to confer protective methylation before the REase acts, to avoid host genome damage. Some type II R-M systems rely on a third component, the controller (C) protein, which is a transcription factor that regulates the production of REase and/or MTase. Previous studies have suggested C protein effects on the dynamics of expression of an R-M system during its establishment in a new host cell. Here, we directly examine these effects. By fluorescently labelling REase and MTase, we demonstrate that lack of a C protein reduces the delay of REase production, to the point of being simultaneous with, or even preceding, production of the MTase. Single molecule tracking suggests that a REase and a MTase employ different strategies for their target search within host cells, with the MTase spending much more time diffusing in proximity to the nucleoid than does the REase. This difference may partially ameliorate the toxic effects of premature REase expression.

The Virus-X—Viral Metagenomics for Innovation Value—project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.

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BackgroundThe viral or host systems for a gene expression assume repeatability of the process and high quality of the protein product. Since level and fidelity of transcription primarily determines the overall efficiency, all factors contributing to their decrease should be identified and optimized. Among many observed processes, non-programmed insertion/deletion (indel) of nucleotide during transcription (slippage) occurring at homopolymeric A/T sequences within a gene can considerably impact its expression. To date, no comparative study of the most utilized Escherichia coli and T7 bacteriophage RNA polymerases (RNAP) propensity for this type of erroneous mRNA synthesis has been reported. To address this issue we evaluated the influence of shift-prone A/T sequences by assessing indel-dependent phenotypic changes. RNAP-specific expression profile was examined using two of the most potent promoters, ParaBAD of E. coli and φ10 of phage T7.ResultsHere we report on the first systematic study on requirements for efficient transcriptional slippage by T7 phage and cellular RNAPs considering three parameters: homopolymer length, template type, and frameshift directionality preferences. Using a series of out-of-frame gfp reporter genes fused to a variety of A/T homopolymeric sequences we show that T7 RNAP has an exceptional potential for generating frameshifts and is capable of slipping on as few as three adenine or four thymidine residues in a row, in a flanking sequence-dependent manner. In contrast, bacterial RNAP exhibits a relatively low ability to baypass indel mutations and requires a run of at least 7 tymidine and even more adenine residues. This difference comes from involvement of various intrinsic proofreading properties. Our studies demonstrate distinct preference towards a specific homopolymer in slippage induction. Whereas insertion slippage performed by T7 RNAP (but not deletion) occurs tendentiously on poly(A) rather than on poly(T) runs, strong bias towards poly(T) for the host RNAP is observed.ConclusionsIntrinsic RNAP slippage properties involve trade-offs between accuracy, speed and processivity of transcription. Viral T7 RNAP manifests far greater inclinations to the transcriptional slippage than E. coli RNAP. This possibly plays an important role in driving bacteriophage adaptation and therefore could be considered as beneficial. However, from biotechnological and experimental viewpoint, this might create some problems, and strongly argues for employing bacterial expression systems, stocked with proofreading mechanisms.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1034-4) contains supplementary material, which is available to authorized users.