Combining transposon mutagenesis and reporter genes to identify novel regulators of the topA promoter in Streptomyces

Abstract

Background Identifying 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 in Streptomyces. Results A 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. Conclusions Our approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.

Discovery and Biosynthesis of the Cytotoxic Polyene Terpenomycin in Human Pathogenic Nocardia

“…As we were unable to find any previous studies that had utilized transposon mutagenesis in the genus Nocardia, we looked to systems that had been used in the closely related and better studied Streptomyces. Previous work showed that a codon-optimized hyperactive variant of the transposase Tn 5 could be used to successfully mutagenize Streptomyces species. − Due to our previous work adapting Streptomyces genetic elements to function in Nocardia, , Tn5’s preference for high-GC regions of DNA, and similarity in codon usage and GC content between Streptomyces and Nocardia, we hypothesized that this Streptomyces codon-optimized Tn 5 may also function in the Nocardia. Utilizing the vector pHL734, we performed multiple rounds of transformation of N.…”

Development of a native-locus dual reporter system for the efficient screening of the hyper-production of natural products in Streptomyces

“…However, the biggest challenge of the mutagenesis strategy is the high throughput but efficient screening of strains for hyper-production of the desired natural products. To overcome this obstacle, many reporter systems have been developed in Streptomyces , such as chromogenic strategies with enzymes IdgS and XylE to produce the blue and yellow pigments ( Li et al, 2015 ; Li et al, 2018 ), respectively, and antibiotic resistance to kanamycin ( Li et al, 2018 ; Gongerowska-Jac et al, 2021 ). However, false positives frequently arise in single-reporter systems, because different disturbances might result in the same reporter phenotypes.…”

“…Thus, dual reporter systems have been preferentially used to more efficiently obtain the desired mutants ( Xiang et al, 2009 ). The reporter systems are usually designed linked to the target promoters for screening of improved transcriptional strength, but mostly on the replicative plasmids or integrated in the genome distant from the native promoters of the biosynthetic gene cluster ( Gongerowska-Jac et al, 2021 ). Probably due to the influence of genomic locations or the incompleteness of transcriptional elements on the cloned promoter ( Bilyk et al, 2017 ), the strength of the reporter system does not always correlate well with the productivity of natural products.…”

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 [20–22]. 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.…”