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Characterization of ECF42, a novel group of extracytoplasmic function (ECF) σ factors with C‑terminal regulatory extensions
Characterization of ECF42, a novel group of extracytoplasmic function (ECF) σ factors with C‑terminal regulatory extensions
Extracytoplasmic function (ECF) σ factors represent the third most abundant fundamental principle of bacterial signal transduction, outnumbered only by one- and two-component systems. A recent census of ECF σ factors revealed a large number of novel groups whose functions and regulatory mechanisms have not yet been elucidated. Here we report the characterization of members of the novel group ECF42. ECF42 σ factors are a highly abundant and widely distributed ECF group that is present in 11 phyla, but is predominantly found in Actinobacteria. Analysis of the genomic context conservation did not identify a putative anti-σ factor in the same operon of ECF σ factor as classical ones. Instead, ECF42 genes are co-transcribed with genes encoding a conserved so-called ‘DGPF protein’, which is not involved in the regulation of ECF42 σ factor activity. We have experimentally verified the target promoter of these ECF σ factors ("TGTCGA" in the -35 region and "CGA/TC" in the -10 region), which was found upstream of the ECF42-encoding operons in Streptomyces venezuelae, suggesting that ECF42 σ factors are positively auto-regulated. ECF42 triple deletion mutant of S. venezuelae (Δsven_0747 Δsven_4377 Δsven_7131) was generated and submitted for Phenotype Microarrays to identify ECF42-related phenotypes. RNA sequencing (RNA-seq) was performed to define the regulons of the three ECF42 proteins in S. venezuelae, which identified mostly genes encode DGPF proteins with unknown function. In contrast to typical ECF σ factors, ECF42 σ factors are characterized by a long C-terminal extension containing a tetratricopeptide repeat (TPR) domain, which is postulated to mediate protein-protein interactions. An interaction between the C-terminal extension and the N-terminal σ domains was hypothesized and supported by experimental evidence from a mutational analysis. The putative interaction, mediated by the co-variable residues, plays a positive regulatory role on the activity of the ECF42 σ factor. Truncations of the C-terminal extension of ECF42 abolished σ factor activity completely, suggesting that it is necessary for σ factor activity. In conclusion, this work provides the first insights into the function and mechanism behind ECF42 σ factors’ activation.
Not available
Liu, Qiang
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Liu, Qiang (2018): Characterization of ECF42, a novel group of extracytoplasmic function (ECF) σ factors with C‑terminal regulatory extensions. Dissertation, LMU München: Faculty of Biology
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Abstract

Extracytoplasmic function (ECF) σ factors represent the third most abundant fundamental principle of bacterial signal transduction, outnumbered only by one- and two-component systems. A recent census of ECF σ factors revealed a large number of novel groups whose functions and regulatory mechanisms have not yet been elucidated. Here we report the characterization of members of the novel group ECF42. ECF42 σ factors are a highly abundant and widely distributed ECF group that is present in 11 phyla, but is predominantly found in Actinobacteria. Analysis of the genomic context conservation did not identify a putative anti-σ factor in the same operon of ECF σ factor as classical ones. Instead, ECF42 genes are co-transcribed with genes encoding a conserved so-called ‘DGPF protein’, which is not involved in the regulation of ECF42 σ factor activity. We have experimentally verified the target promoter of these ECF σ factors ("TGTCGA" in the -35 region and "CGA/TC" in the -10 region), which was found upstream of the ECF42-encoding operons in Streptomyces venezuelae, suggesting that ECF42 σ factors are positively auto-regulated. ECF42 triple deletion mutant of S. venezuelae (Δsven_0747 Δsven_4377 Δsven_7131) was generated and submitted for Phenotype Microarrays to identify ECF42-related phenotypes. RNA sequencing (RNA-seq) was performed to define the regulons of the three ECF42 proteins in S. venezuelae, which identified mostly genes encode DGPF proteins with unknown function. In contrast to typical ECF σ factors, ECF42 σ factors are characterized by a long C-terminal extension containing a tetratricopeptide repeat (TPR) domain, which is postulated to mediate protein-protein interactions. An interaction between the C-terminal extension and the N-terminal σ domains was hypothesized and supported by experimental evidence from a mutational analysis. The putative interaction, mediated by the co-variable residues, plays a positive regulatory role on the activity of the ECF42 σ factor. Truncations of the C-terminal extension of ECF42 abolished σ factor activity completely, suggesting that it is necessary for σ factor activity. In conclusion, this work provides the first insights into the function and mechanism behind ECF42 σ factors’ activation.