AUTHOR=Amin Rafat , Franz-Wachtel Mirita , Tiffert Yvonne , Heberer Martin , Meky Mohamed , Ahmed Yousra , Matthews Arne , Krysenko Sergii , Jakobi Marco , Hinder Markus , Moore Jane , Okoniewski Nicole , Maček Boris , Wohlleben Wolfgang , Bera Agnieszka 

TITLE=Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=3

YEAR=2016

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2016.00038

DOI=10.3389/fmolb.2016.00038

ISSN=2296-889X

ABSTRACT=<p>Soil-dwelling <italic>Streptomyces</italic> bacteria such as <italic>S</italic>.coelicolor have to constantly adapt to the nitrogen (<italic>N</italic>) availability in their habitat. Thus, strict transcriptional and post-translational control of the <italic>N</italic>-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the <italic>N</italic>-assimilation in <italic>S. coelicolor</italic> and other members of the <italic>Actinomycetales</italic>. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in <italic>S. coelicolor</italic> M145. The expression of <italic>glnR</italic> and GlnR-target genes was revisited under four different <italic>N</italic>-defined conditions and a complex <italic>N</italic>-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing <italic>N-concentrations</italic>, the <italic>glnR</italic> expression itself was independent of the <italic>N</italic>-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex <italic>N</italic>-rich medium GlnR was phosphorylated on six serine/threonine residues and acetylated on one lysine residue. Under defined <italic>N</italic>-excess conditions only two phosphorylated residues were detected whereas under defined <italic>N</italic>-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with <italic>N</italic>-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the <italic>N</italic>-concentration in the defined media and on only one lysine residue in the complex <italic>N</italic>-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex. This study clearly demonstrated that GlnR DNA-binding affinity is modulated by post-translational modifications in response to changing <italic>N</italic>-conditions in order to elicit a proper transcriptional response to the latter.</p>