Multi-Omics and Targeted Approaches to Determine the Role of Cellular Proteases in Streptomyces Protein Secretion.

Tobias Busche,Jozef Anné,Wolfgang Wiechert,Arne Bleidt,Mohamed B Hamed,Joachim Koepff,Yuriy Rebets,Mariia Lopatniuk,Christian Rückert,Spyridoula Karamanou,Andriy Luzhetskyy,Marco Oldiges,Konstantinos C Tsolis,Ahmed Yousra,Jörn Kalinowski,Anastassios Economou

doi:10.3389/fmicb.2018.01174

Abstract

Gram-positive Streptomyces bacteria are profuse secretors of polypeptides using complex, yet unknown mechanisms. Many of their secretory proteins are proteases that play important roles in the acquisition of amino acids from the environment. Other proteases regulate cellular proteostasis. To begin dissecting the possible role of proteases in Streptomyces secretion, we applied a multi-omics approach. We probed the role of the 190 proteases of Streptomyces lividans strain TK24 in protein secretion in defined media at different stages of growth. Transcriptomics analysis revealed transcripts for 93% of these proteases and identified that 41 of them showed high abundance. Proteomics analysis identified 57 membrane-embedded or secreted proteases with variations in their abundance. We focused on 17 of these proteases and putative inhibitors and generated strains deleted of their genes. These were characterized in terms of their fitness, transcriptome and secretome changes. In addition, we performed a targeted analysis in deletion strains that also carried a secretion competent mRFP. One strain, carrying a deletion of the gene for the regulatory protease FtsH, showed significant global changes in overall transcription and enhanced secretome and secreted mRFP levels. These data provide a first multi-omics effort to characterize the complex regulatory mechanisms of protein secretion in Streptomyces lividans and lay the foundations for future rational manipulation of this process.

Highlights

Microorganisms of the family Streptomycetaceae are filamentously growing key players in soil habitats all around the planet, where they vitally contribute as decomposers to recycle organic material (Hopwood, 2007; Barka et al, 2016; Ranjani et al, 2016)
To determine whether proteases play a role in the secretory processes of TK24, we first classified potential proteases based on several parameters, as follows: (I) the presence of a Secreted protein (Sec) or Tat secretion signal using multiple bioinformatics tools (Tsolis et al, 2018), (II) whether the protein in question belongs to the set of core genes of S. lividans TK24, based on the comparison to the core genome of 13 Streptomyces species, and (III) the maximal transcript level, determined by RNAseq
TK24 was grown under two media regimes and cells were harvested at three different growth phases

Summary

Introduction

Microorganisms of the family Streptomycetaceae are filamentously growing key players in soil habitats all around the planet, where they vitally contribute as decomposers to recycle organic material (Hopwood, 2007; Barka et al, 2016; Ranjani et al, 2016). Streptomyces lividans, a well-investigated member of the Streptomyces family, already innately displays a rather low extracellular protease activity in comparison to other related species (Butler et al, 1993; Gilbert et al, 1995; Liu et al, 2013), while at the same time maintaining a highly active secretion machinery (Anné et al, 2017) and being manipulated genetically This strain has become an important model organism for heterologous protein production (Gilbert et al, 1995; Anné et al, 2012; Chater, 2016)

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Results

Conclusion