The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.
Summary Bacteriocins are a distinct family of antimicrobial proteins postulated to porate bacterial membranes. However, direct experimental evidence of pore formation by these proteins is lacking. Here we report a multi-mode poration mechanism induced by four-helix bacteriocins, epidermicin NI01 and aureocin A53. Using a combination of crystallography, spectroscopy, bioassays, and nanoscale imaging, we established that individual two-helix segments of epidermicin retain antibacterial activity but each of these segments adopts a particular poration mode. In the intact protein these segments act synergistically to balance out antibacterial and hemolytic activities. The study sets a precedent of multi-mode membrane disruption advancing the current understanding of structure-activity relationships in pore-forming proteins.
BackgroundThe secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity.ResultsHere we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres.ConclusionsThe methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1047-z) contains supplementary material, which is available to authorized users.
We investigated the relevance of gene expression heterogeneity to virulence properties of a major fungal pathogen, Candida glabrata. The organism's key virulence-associated factors include glycosylphosphatidylinositol-anchored adhesins, encoded subtelomerically by the EPA gene family. Individual-cell analyses of expression revealed very striking heterogeneity for Epa1, an adhesin that mediates ϳ95% of adherence to epithelial cells in vitro. The heterogeneity in Epa1 was markedly greater than that known for other yeast genes. Sorted cells expressing high or low levels of Epa1 exhibited high and low adherence to epithelial cells, indicating a link between gene expression noise and potential virulence. The phenotypes of sorted subpopulations reverted to mixed phenotypes within a few generations. Variation in single-cell Epa1 protein and mRNA levels was correlated, consistent with transcriptional regulation of heterogeneity. Sir-dependent transcriptional silencing was the primary mechanism driving heterogeneous Epa1 expression in C. glabrata BG2, but not in CBS138 (ATCC 2001). Inefficient silencing in the latter strain was not due to a difference in EPA1 sequence or (sub)telomere length and was overcome by ectopic SIR3 expression. Moreover, differences between strains in the silencing dependence of EPA1 expression were evident across a range of clinical isolates, with heterogeneity being the greatest in strains where EPA1 was subject to silencing. The study shows how heterogeneity can impact the virulence-related properties of C. glabrata cell populations, with potential implications for microbial pathogenesis more broadly.
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