title: GlnR negatively regulates the transcription of the alanine dehydrogenase encoding gene ald in Amycolatopsis mediterranei U32 under nitrogen limited conditions via specific binding to its major transcription initiation site.
abstract: Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT) and alanine dehydrogenase (AlaDH) in Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs) of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites (a1, b1 sites in region I and a2 site in region II) were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts.
authors.affiliation: Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; Department of Microbiology, School of Life Science, Fudan University, Shanghai, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Microbiology, School of Life Science, Fudan University, Shanghai, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Li
authors.affiliation: Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Na
authors.initials: N
authors.lastname: Duan
authors.affiliation: Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Microbiology, School of Life Science, Fudan University, Shanghai, China.
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Ding
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Yao
authors.affiliation: Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.affiliation: Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; Department of Microbiology, School of Life Science, Fudan University, Shanghai, China; Department of Microbiology and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhao
authors.affiliation: Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D050788:Alanine Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D009584:Nitrogen / Q000172:deficiency / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D050788:Alanine Dehydrogenase; D009584:Nitrogen