Die dritte Säule bakterieller Signaltransduktion: Regulatorische Vielfalt und Evolution von Extracytoplasmic function (ECF) Sigmafaktoren
Zusammenfassung der Projektergebnisse
Extracytoplasmic function σ factors (ECFs) are the third most abundant fundamental principle of bacterial signal transduction that enables microbes to respond to environmental changes. Comparative genomics analyses, performed by our group, demonstrated a surprisingly wide distribution and diversity of these regulatory devices. Our data indicated the existence of numerous novel mechanisms for controlling σ factor activity that have not been experimentally studied. The goal of this project was therefore to (i) expand and refine the ECF classification to develop a comprehensive and systematic understanding of ECF-dependent regulation and signal transduction, and (ii) apply the predictive power of the classification for unravelling the regulatory and signaling mechanisms of novel, conserved and phylogenetically widespread ECF groups that had been identified by comparative genomics, but for which no experimental evidence was available at the time. On the comparative genomics end of the project, we expanded our ECF classification efforts to deal with the ‘explosion’ in the bacterial genome sequence space that has taken place in recent years. While analyzing large numbers of genome sequences poses a technical challenge, it also holds the promise of uncovering hidden information, e.g. identifying and classifying novel ECF groups that are only found in ‘rare’, underrepresented bacterial phyla. Our recent and partially still ongoing classification projects therefore specifically focused on this hidden minority: of the approx. 130.000 bacterial genome sequences currently in public databases, 121.000 are derived from only four bacterial phyla, while the remaining 9.000 genomes are distributed over 128 (the underrepresented) phyla. Comprehensive studies of 150 genomes from only one of those underrepresented phylum, the Planctomycetes, has already increased the total number of conserved ECF groups by 25% (from 94 groups to 124) and hence significantly expanded the known ECF diversity, including altogether new mechanisms of regulating ECF activity that have never been found before. A large comprehensive study to cover the remaining underrepresented phyla is currently under way. The value of the ECF classification lies in its predictive power. It enables researchers interested in a particular ECF to postulate (i) its mechanism of signal transduction, (ii) the target promoter and hence regulon, and (iii) the physiological role, based on sequence conservation and the associated knowledge of the group-specific promoter motifs and genomic context conservation. This greatly simplifies and facilitates the experimental studies by allowing to design a very directed strategy for analyzing new ECFs. We applied this knowledge for two novel and so far uncharacterized ECF groups, ECF41 and ECF42 that, in fact, represent the largest and most widespread ECF groups known to date. Both groups are particularly ubiquitous in Actinobacteria. Hence, we studied representatives of them in a model organism for these high G+C Gram-positive bacteria, Streptomyces venezuelae. While typical ECFs are regulated by cognate anti-σ factors, both groups lack such proteins. Instead, we could demonstrate that the activity of both ECF41 and ECF42 σ factors is controlled by group-specific and conserved Cterminally fused regulatory extensions. Using a combination of statistical covariance analyses and experimental validation, we could identify amino acids crucially important for regulating ECF function. Moreover, we could predict and experimentally verify the group-specific target promoters and hence identify the regulons, by combining genome-wide motif scans with global expression profiling studies. Studies on the regulatory mechanism and physiological role of these ECFs can now be performed. Such comprehensive and mechanistic understanding of (the diversity of) ECF-dependent gene regulation, as gained from our studies, is a prerequisite for subsequently applying these σ factors, e.g. as building blocks in Synthetic Biology projects: ECFs are ideally suited for genetic engineering purposes, since they are modular, inherently orthogonal, universal, and scalable. Therefore, they provide a perfect tool kit, e.g. as switches for designing and implementing synthetic ECF-based genetic circuits and expression programs.
Projektbezogene Publikationen (Auswahl)
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(2012). Identification of proteins likely to be involved in morphogenesis, cell division, and signal transduction in Planctomycetes by comparative genomics. J Bacteriol 194: 6419-30
Jogler C, Waldmann J, Huang X, Jogler M, Glöckner FO, Mascher T, Kolter R
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(2009). The third pillar of bacterial signal transduction: classification of the extracytoplasmic function (ECF) sigma factor protein family. Mol Microbiol 74: 55781
Staroń A, Sofia HJ, Dietrich S, Ulrich LE, Liesegang H, Mascher T
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(2010). Extracytoplasmic function σ factors come of age – the extracytoplasmic function σ factors provide bacteria a third mechanism for responding to extracellular stimuli. Microbe 5: 164-170
Staroń A, Mascher T
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(2010). General stress response in α-proteobacteria: PhyR and beyond. Mol Microbiol 78: 271-7
Staroń A, Mascher T
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(2012). Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a fused regulatory domain. MicrobiologyOpen 1: 194-213
Wecke T, Halang P, Staroń A, Dufour YS, Donohue TJ, Mascher T
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(2013). Signaling diversity and evolution of extracytoplasmic function (ECF) σ factors. Curr Opin Microbiol 16:14855
Mascher T
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(2015). Environmental sensing in Actinobacteria: a comprehensive survey on the signaling capacity of this phylum. J Bacteriol 197: 2517-35
Huang X, Pinto D, Fritz G, Mascher T
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(2016). The ECF classification: a phylogenetic reflection of the regulatory diversity in the extracytoplasmic function σ factor protein family. In “Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria" (2 Volume Set, editor De Bruijn FJ), John Wiley & Sons
Pinto D, Mascher T
