微生物学

The fission yeast Schizosaccharomyces pombe is an excellent genetically tractable model organism used in the study of conserved eukaryotic cellular biology. One genetic tool in the assessment of gene function is the in vivo overexpression of proteins. Existing overexpression tools have limitations of induction kinetics, dynamic range, and/or system-wide changes due to the induction conditions or inducer. Here, I describe the methodology for the use of a plasmid-based long non-coding RNA (lncRNA)-regulated overexpression system that is induced by the addition of thiamine. This system, termed the pTIN-system (thiamine inducible), utilizes the fast repression kinetics of the thiamine-regulated nmt1⁺ promoter integrated with the lncRNA regulated tgp1⁺ promoter. The advantages of the pTIN-system are rapid induction kinetics of gene expression, broad dynamic range, and tunable expression.

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. Here, we describe an optimized conjugation protocol enabling the transfer of autonomously replicating plasmids from an E. coli donor strain into Roseburia inulinivorans and Eubacterium rectale. The modular nature of the plasmids and their ability to be maintained in the recipient bacterium by autonomous replication makes them ideal for investigating heterologous gene expression, and as a platform for other genetic tools including antisense RNA silencing or mobile group II interon gene disruption strategies.

Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is further used to confirm the absence of the plasmid in rhamnose grown bacteria.