Combining Transposon Mutagenesis and Reporter Genes to Identify Novel Regulators of the Topa Promoter in Streptomyces

Abstract

BackgroundIdentifying the regulatory factors that control transcriptional activity is a major challenge of gene expression studies. Here, we describe the application of a novel approach for in vivo identification of regulatory proteins that may directly or indirectly control the transcription of a promoter of interest. ResultsA method based on the combination of Tn5 minitransposon-driven random mutagenesis and lux reporter genes was applied for the first time for the Streptomyces genus. As a proof of concept, we studied the topA supercoiling-sensitive promoter, whose activity is dependent on unknown regulatory factors. We found that the sco4804 gene product positively influences topA transcription in S. coelicolor, demonstrating SCO4804 as a novel player in the control of chromosome topology in these bacteria. ConclusionsOur approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.

Screening transcriptional connections in Staphylococcus aureus using high-throughput transduction of bioluminescent reporter plasmids

“…mutants of regulatory genes) can be labour intensive and therefore prevents high-throughput screens. To overcome these challenges, various studies have combined reporter plasmids with the use of transposon mutagenesis [20][21][22]. Specifically, mutant generation is frequently performed in the parental strain harbouring the reporter plasmid, presenting technical challenges during library preparation, and necessitating sequencing of strains identified in the screen.…”