Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions.

Yuriy Rebets,Andriy Luzhetskyy,Liliya Horbal,Jörn Kalinowski,Marco Oldiges,Maksym Myronovskyi,Joachim Koepff,Christian Rückert,Elísabet Eik Guðmundsdóttir,Wolfgang Wiechert,Anastassios Economou,Arne Bleidt,Tobias Busche,Beata Wawiernia,Bohdan Bilyk,Jozef Anné,Yousra Ahmed,Konstantinos C Tsolis,Ólafur H Friðjónsson ,Mohamed Belal Hamed

doi:10.3389/fmicb.2018.03033

Abstract

Alternative sigma factors control numerous aspects of bacterial life, including adaptation to physiological stresses, morphological development, persistence states and virulence. This is especially true for the physiologically complex actinobacteria. Here we report the development of a robust gene deletions system for Streptomyces lividans TK24 based on a BAC library combined with the λ-Red recombination technique. The developed system was validated by systematically deleting the most highly expressed genes encoding alternative sigma factors and several other regulatory genes within the chromosome of S. lividans TK24. To demonstrate the possibility of large scale genomic manipulations, the major part of the undecylprodigiosin gene cluster was deleted as well. The resulting mutant strains were characterized in terms of morphology, growth parameters, secondary metabolites production and response to thiol-oxidation and cell-wall stresses. Deletion of SLIV_12645 gene encoding S. coelicolor SigR1 ortholog has the most prominent phenotypic effect, resulted in overproduction of actinorhodin and coelichelin P1 and increased sensitivity to diamide. The secreted proteome analysis of SLIV_12645 mutant revealed SigR1 influence on trafficking of proteins involved in cell wall biogenesis and refactoring. The reported here gene deletion system will further facilitate work on S. lividans strain improvement as a host for either secondary metabolites or protein production and will contribute to basic research in streptomycetes physiology, morphological development, secondary metabolism. On the other hand, the systematic deletion of sigma factors encoding genes demonstrates the complexity and conservation of regulatory processes conducted by sigma factors in streptomycetes.

Highlights

Actinobacteria are large group of soil-dwelling bacteria occupying diverse ecological niches, ranging from different types of soils to the specialized glands of insects (Scott et al, 2008; Kaltenpoth et al, 2012)
We identified 19 homologues of the characterized S. coelicolor M145 anti-sigma factor genes being present in S. lividans TK24 and located in the corresponding operons as in S. coelicolor M145
The affected proteins fall in Streptomyces coelicolor and S. lividans are two closely related species that are used for decades as models for basic genetic, physiological, biochemical and morphological research of actinobacteria

Summary

Introduction

Actinobacteria are large group of soil-dwelling bacteria occupying diverse ecological niches, ranging from different types of soils to the specialized glands of insects (Scott et al, 2008; Kaltenpoth et al, 2012) Such habitat diversity entails that these bacteria are facing an enormous diversity of conditions that are not always ideal for growth. These conditions often change rapidly, imposing new challenges for bacterial survival This led to the development in Actinobacteria of a highly branched regulatory network sensing environmental changes and responding by re-tuning metabolic pathways, modulating growth rates and activating morphological processes (Bruijn, 2016). These regulatory networks mainly act by mediating transcription of specific sets of genes. It is not surprising that actinobacterial genomes are enriched in genes coding for alternative RNA polymerase sigma factors and diverse families of transcriptional regulators (Bentley et al, 2002; Ruckert et al, 2015)

Methods

Results

Conclusion