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.

GlnR Dominates Rifamycin Biosynthesis by Activating the rif Cluster Genes Transcription Both Directly and Indirectly in Amycolatopsis mediterranei

“…In actinomycetes, GlnR was typically viewed as a global nitrogen metabolism regulator before, governing many nitrogen metabolism related processes (Fink et al, 2002;Tiffert et al, 2008;Wang and Zhao, 2009;Wang et al, 2012Wang et al, , 2013Wang et al, , 2014. Recently, several studies have reported that the GlnR homologs in Streptomyces regulate the biosynthesis of some antibiotics, e.g., actinorhodin, avermectin and validamycin (Qu et al, 2015;He et al, 2016).…”

“…In actinomycetes, GlnR is an OmpR-type transcriptional regulator, and those early studies show that most GlnR-regulated genes (e.g., amtB, nirB, nasA, ureA, glnA, and ald) are involved in the assimilation and utilization of a variety of nitrogen sources, such as ammonium, nitrate and urea (Fink et al, 2002;Tiffert et al, 2008;Wang and Zhao, 2009;Wang et al, 2012Wang et al, , 2013Wang et al, , 2014. Accordingly, the GlnR in actinomycetes is mainly considered as a global nitrogen metabolism regulator.…”

GlnR-Mediated Regulation of ectABCD Transcription Expands the Role of the GlnR Regulon to Osmotic Stress Management

“…Production of His-tagged Amycolatopsis mediterranei GlnR (AME_GlnR) was the same as described before (31,33). For purification of Streptomyces avermitilis GlnR (SAV_GlnR), primers PglnRsav-fw and PglnRsav-rv were used to amplify the structure gene of S. avermitilis glnR, which was further cloned into the NdeI and EcoRI sites of pET28a, yielding the expression plasmid pET-glnRSav.…”

“…The ectoine and hydroxyectoine biosynthetic genes of ect-ABCD are present in most actinomycetes (25). As GlnR-mediated regulation of nitrogen metabolism is also very conserved in actinomycetes (29,31,33,35,36), it is thus hypothesized that GlnR regulates the expression of ect genes in actinomycetes that contain both GlnR and ect genes.…”

“…Therefore, it is possible that the regulation of the biosynthesis of ectoine and hydroxyectoine is closely connected to the nitrogen metabolism. In S. coelicolor and many other actinomycetes, nitrogen metabolism is governed by the OmpR family regulator GlnR, which is a global regulator and regulates most nitrogen metabolism-associated genes (29)(30)(31)(32)(33)(34)(35)(36). Therefore, it is easily speculated that GlnR may regulate the biosynthesis of ectoine and hydroxyectoine in S. coelicolor.…”