Mechanism Of Nitrogen Regulator GlnR-mediate Protein Lysine Acetylation In Saccharopolyspora Erythraea

Lysine acylation is a dynamic, reversible, and conserved post-translational modification ?PTM?. Lysine acylation and its enzymes have been shown to influence several fundamental cellular pathways including cell apoptosis, cellular differentiation and metabolism. Up to now, mechanism between acylation and intracellular metabolism reported in actinomycetes, the major source of antibiotic, was limited. In the last decade, studies of lysine acylation in actinomycetes only focused on a few species of streptomyces such as Streptomyces coelicolor. The mechanism of acylation in Saccharopolyspora erythraea who is close relative of Streptomyces coelicolor is poorly understood. Acyl-CoA is the donor of acylation as well as the precursor of secondary metabolism, further understanding of the acylation regulatory mechanism in these species has a number of potential applications because of the diversity of their natural products. This thesis mainly concentrated on the regulatory mechanism between lysine acetylation and metabolism in high G+C-content Gram-positive bacteria Saccharopolyspora erythraea. The main research includes three parts:?1? Identification of reversible lysine acetylation ?RLA? system and its transcriptional regulatory mechanism. According to the bioinformatics analysis, we identified Gcn5-like protein acetyltransferase AcuA ?SACE₅148? revealed a unique feature that acetylated multiple lysine sites, and sirtuin-type NAD+-dependent deacetylase SrtN ?SACE₃798? in Saccharopolyspora erythraea. Further studies showed that nitrogen regulator GlnR controlled the RLA system composed of AcuA and SrtN by directly activating the expression of acuA and srtN, which combined RLA with nitrogen metabolism.?2? GlnR regulated Ac-CoA synthetases at the transcriptional and acetylation levels. Acyl-CoA synthetases are the key enzymes involved in erythromycin synthesis and fatty acid metabolism. There are three AMP-forming Ac-CoA synthetases with highly sequence identity ?> 98%? encoded by acsAl ?SACE₀337?, acsA2 ?SACE₂375? and acsA3 ?SACE₄729? in Saccharopolyspora erythraea, among which AcsAl may play a major role in acetate or other fatty acid assimilation. The acetylation levels and activities of three Acs enzymes were all tightly regulated by RLA system. Acs could also occur AcP-dependent acetylation, while the key site of catalyzation was only acetylated by AcuA according to the MS results.The nitrogen regulator GlnR regulated AcsAl, AcsA2 and AcsA3 at both transcriptional and post-translational levels in response to nitrogen changes. A glnR-deleted mutant ??glnR? caused a growth defect in acetate, which resulted from the imbalance on Acs acetylation state. These results enriched the field of intracellular acetylation regulatory networks, and revealed the mechanism of GlnR regulated RLA as well as acetate metabolism.?3? Nitrogen regulator GlnR regulated glutamine synthetase ?GS? at the transcriptional and acetylation levels. We further identified new RLA substrates that central enzyme of nitrogen metabolism-GSs ?GlnAl and GlnA4? of Saccharopolyspora erythraea. The two GSs were both under control of RLA catalyzed by AcuA and SrtN. AcuA acetylated K179 and K357 of GlnAl ?GSI-??, and K319 of GlnA4 ?GSII?. The AcuA-dependent lysine acetylation exerted different effects on the two GSs:Acetylation inactivated GSII enzyme ?GlnA4? while had no effect on GSI-? ?GlnAl? activity. However, the acetylated GlnAl gained the chaperone activity interacting with GlnR regulator, which enhanced GlnR-DNA binding ability. Thus, the RLA had influence on the nitrogen utilization, while GlnR controlled the catalytic and regulatory activities ?intracellular acetylation levels? of GS at the transcriptional and post-translational levels. This mechanism indicated an autofeedback loop that regulated nitrogen metabolism in response to environmental changes. The GlnR-mediated signal transduction of protein acetylation provides a possible regulatory mechanism of feedback loop ?GlnR-AcuA-GS? at multiple levels controlling nitrogen metabolism.