Abstract
Soil-dwelling Streptomyces bacteria such as S.coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. 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 S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-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 N-rich medium GlnR was phosphorylated on six serine/threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-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 N-conditions in order to elicit a proper transcriptional response to the latter.
Highlights
Streptomycetes, like other microorganisms, need to accurately modulate their regulatory network according to their developmental stage while simultaneously responding to environmental changes, such as the continuous variation in nutrient availability, including nitrogen (N), in the soil habitat
Our results showed that expression of all tested genes glnA, glnII, amtB, and nirB was strongly induced in condition of low nitrate concentration in S. coelicolor M145 (Figure 1B)
Transcriptional analysis revealed that the glnA, glnII, amtB, and nirB were not expressed in S. coelicolor M145 grown in S-medium (Figure 1C)
Summary
Streptomycetes, like other microorganisms, need to accurately modulate their regulatory network according to their developmental stage while simultaneously responding to environmental changes, such as the continuous variation in nutrient availability, including nitrogen (N), in the soil habitat. Transcriptional regulation of the N-assimilation in streptomycetes is accomplished by GlnR (global nitrogen response regulator; Tiffert et al, 2008; Pullan et al, 2011). The GlnR regulator in S. coelicolor controls at least 10 genes which are directly involved in N-metabolism and seven additional genes encoding proteins of unknown function (Reuther and Wohlleben, 2007; Tiffert et al, 2008; Wang and Zhao, 2009; Amin et al, 2012). Regulation of the N-metabolism in S. coelicolor is very complex and depending on the conditions, involves additional control by other regulators such as: GlnRII (Fink et al, 2002; Reuther and Wohlleben, 2007), PhoP (Rodríguez-García et al, 2009; Martín et al, 2011; Sola-Landa et al, 2013), Crp (Gao et al, 2012), and AfsQ1 (Wang R. et al, 2013)
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