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

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.

Keywords: GlnR; Streptomyces coelicolor; acetylation; nitrogen assimilation; phosphorylation; post-translational modifications; regulation.

Figure 1

Transcriptional analysis of the selected GlnR target genes under variable nitrogen and carbon conditions. (A) RT-PCR of glnA, glnII, amtB, nirB, and glnR in S. coelicolor M145, and glnR mutant cultivated in defined Evans medium with low (5 mM) or high (100 mM) concentrations of ammonium chloride as sole nitrogen source. (B) RT-PCR of glnA, glnII, amtB, nirB, and glnR in S. coelicolor M145 and glnR mutant cultivated in defined Evans medium with low (5 mM) or high (100 mM) sodium nitrate as sole nitrogen source. (C) RT-PCR of glnA, glnII, amtB, nirB, and glnR in S. coelicolor M145 and glnR mutant cultivated under complex nitrogen rich conditions (S-medium). Total RNA was isolated from mycelium harvested after 24 h of cultivation.

Figure 2

Detection of the native GlnR protein in the cell lysate from S. coelicolor M145 cultivated under different defined nitrogen conditions. Western blot analysis of GlnR probed with anti-GlnR antibodies generated in rabbit (1:5000) and the secondary goat anti-rabbit antibodies conjugated with HRP (1:3000). Lane 1: cell lysate from glnR mutant grown in S-medium (200 μg of total protein)—negative control, Lane 2–6: S. coelicolor M145 cell lysates (200 μg of total protein, each). Cell lysates generated from cells grown in complex S-medium (Lane 2), in defined Evans medium with 5 mM NH₄Cl (Lane 3), 100 mM NH₄Cl (Lane 4), 5 mM NaNO₃ (Lane 5), and 100 mM NaNO₃ (Lane 6). Lane 7: cell lysate from the Strep-GlnR overexpression strain grown in S-medium—positive control. Lane 8: PageRuler Prestained Protein Ladder (Fermentas). Location of the GlnR with the estimated molecular masses of ~35 and 38 kDa.

Figure 3

EMSAs demonstrating different binding behavior of Strep-GlnR depending on differential modification patterns. Lane (−), control, promoter regions of the known GlnR target genes (Cy5—labeled probe without Strep-GlnR protein). Lane (+), Cy5—labeled probe with 4 μg of the Strep-GlnR_N+ from complex, rich condition (phosphorylated on the six Ser/Thr residues and acetylated on the one Lys residue), Strep-GlnR_N−from nitrate limited conditions (acetylated on four Lys residues), and Strep-GlnR_N+ from the nitrate excess conditions (acetylated on four Lys residues and additionally phosphorylated on two Ser/Thr residues). The differential modification pattern of Strep-GlnR was confirmed by LC-MS/MS prior EMSAs. EMSAs were performed in the presence of the 300 × excess of the unlabeled, unspecific salmon sperm DNA.

Figure 4

EMSAs demonstrating different binding behavior of the acetylated Strep-GlnR and in vitro deacetylated Strep-GlnR. Lane (−), control, promoter regions of the known GlnR target genes (Cy5—labeled probe without Strep-GlnR protein). Lane (+), Cy5—labeled probe with 4 μg of the acetylated Strep-GlnR (Ac+) or deacetylated Strep-GlnR (Ac−). The loss of the acetylation on Strep-GlnR was confirmed by Western blot analysis prior EMSAs. EMSAs were performed in the presence of the 300 × excess of the unlabeled, unspecific salmon sperm DNA.

Figure 5

Schematic model of the regulation of the GlnR-target genes under N-rich and N-limited conditions by the differentially modified GlnR regulator.