Both organic and inorganic forms of nitrogen sources are essential for bacterial growth and important components of cellular biomolecules, such as nucleotides, amino acids, and amino sugars (Merrick and Edwards, 1995). Actinomycetes are gram-positive bacteria with a high GC content (Berdy, 2005) that are able to produce, usually late in growth, a great variety of different bio-active molecules such as anti-bacterial, anti-fungi, herbicide, insecticide, immunosuppressants, immunomodulators, and anti-cancer drugs etc. (Berdy, 2005). The biosynthesis of these molecules is regulated by nitrogen, carbon, and phosphate sources availability (Hodgson, 2000; Reuther and Wohlleben, 2007). Among Actinomycetes, Streptomyces are major active metabolite producers, able to synthetize a great variety of bioactive metabolites of industrial interest with potential medical and agricultural applications (Ossai et al., 2022). Streptomyces are soil-dwelling bacteria with a unique and complicated life cycle, including vegetative mycelium formation, aerial mycelium differentiation, and sporulation (Chater, 2001). This complex morphological differentiation process, that is usually accompanied by the synthesis of bio-active specialized metabolites, involves temporally and spatially regulated regulatory networks (Hopwood et al., 1995). In soil habitats, Streptomyces compete with other bacteria for nutrients and especially for nitrogen sources (Reuther and Wohlleben, 2007), that are used to synthesize almost all important biological molecules (Arcondeguy et al., 2001) and also play a crucial role in the regulation specialized metabolism (Reuther and Wohlleben, 2007). Actinomycetes have thus developed complex transcriptional, translational and post-translationnal regulatory systems to regulate the expression of genes and activity of proteins involved in nitrogen metabolism in response to changes in environmental nitrogen levels (Leigh and Dodsworth, 2007). Diverse nitrogen resources, either mineral (nitrate, ammonium, etc.) or organic (urea, glutamine, etc.), are present in the environment. When the bacteria is facing nitrogen organic source shortage in its environment, it can assimilate inorganic nitrogen resources into organic ones (Sun et al., 2017). The two amino acids, glutamate and glutamine, that are the main intracellular nitrogen donors, can be synthesized from various mineral nitrogen sources and especially ammonium (Reitzer and Schneider, 2001; Sun et al., 2017). The nitrogen of glutamate is used for the biosynthesis of numerous amino acids (Fisher, 1989) whereas glutamine is used for the biosynthesis of both purines and pyrimidines, as well as other nitrogenous metabolites (Fisher, 1992). Depending on nitrogen availability, there are two key pathways of ammonium assimilation: the glutamate dehydrogenase (GDH) and the glutamine synthetase/glutamate synthase (GS/GOGAT) pathways (Fisher, 1989; Fisher, 1992) (Figure 1). In Streptomyces coelicolor, when the nitrogen supply is high, glutamine is synthesized from ammonium and 2-oxoglutarate by NADPH-dependent GDH whereas under low nitrogen concentrations, the GS of the GS/GOGAT system catalyzes the formation of glutamine from ammonium and glutamate, consuming ATP whereas GOGAT catalyzes the formation of two glutamate molecules from glutamine and 2-oxoglutarate (Magasanik, 1982; Tiffert et al., 2008). Most studies on microbial nitrogen metabolism and its regulatory network come from enterobacter (Magasanik, 1982; Merrick and Edwards, 1995; Oelze and Klein, 1996; Dubbs and Tabita, 2004; Zhang et al., 2014). In gram-negative Escherichia coli, ammonium assimilation is regulated by the two-component system NtrB/NtrC (Weiss et al., 2002) that requires σ⁵⁴ to activate the transcription of target genes (Zimmer et al., 2000; Reitzer and Schneider, 2001). When bacteria are starved of nitrogen sources, the kinase NtrB phosphorylates NtrC that stimulates the transcriptional expression of downstream genes involved into nitrogen metabolism (Weiss et al., 2002). In the gram-positive bacteria, Bacillus subtilis, with a low GC content, the intracellular nitrogen availability is mainly regulated by two regulators of the MerR family, TnrA and GlnR (GlnR^bsu) (Schreier et al., 1989; Wray et al., 1996). In excess of nitrogen, GlnR^bsu represses the transcription of genes of nitrogen metabolism (Tiffert et al., 2008) whereas in condition of nitrogen limitation, TnrA activates the expression of the nrgAB, nasB, gabP, and ureA genes and represses that of glnRA (Wray et al., 1996). GlnR senses nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS). FBI-GS, as a GlnR chaperone, binds the GlnR C-terminal domain within its active-site cavity, promoting the formation of a DNA-binding-competent GlnR dimer and leading to GlnR DNA-binding activation (Travis et al., 2022).

In gram-positive Actinomycetes, with high GC content, the regulation mechanism of nitrogen metabolism evolved completely differently from that of E. coli and B. subtilis (Burkovski, 2003). The nitrogen metabolism of most Actinomycetes (such as S. coelicolor, Amycolatopsis mediterranei) is mediated by GlnR (Amon et al., 2009), a regulator of the OmpR family that is an orphan response regulator, significantly different from GlnR^bsu of B. subtilis (Hutchings et al., 2004). In contrast, a small number of Actinomycetes (such as Mycobacterium smegmatis, Streptomyces avermitilis) bear two regulatory proteins, GlnR and AmtR, involved in the regulation of nitrogen metabolism whereas only AmtR regulates nitrogen metabolism in Corynebacterium (Burkovski, 2003; Beckers et al., 2005). In 1988, the glnA gene encoding glutamine synthase GSI was located in S. coelicolor, and GSI enzyme activity was determined (Wray and Fisher, 1988). In the 1990s, the research mainly focused on GlnR and GSI/GSII. In 1990, a second gene, glnII, encoding glutamine synthase GSII was identified in Frankia and Streptomyces viridochromogenes (Behrmann et al., 1990; Rochefort and Benson, 1990). In 1991, the isolation of six glutamine-deficient mutants of S. coelicolor led to the identification of the glnR encoding gene whose transcription start sites were determined (Wray et al., 1991). In 1993, the positive role that GlnR plays in the regulation of glnA expression was demonstrated (Wray and Fisher, 1993) as well as the post-translational modification of GSI (Fisher, 1992). With the completion of the genome sequence of S. coelicolor in 2002 and that of other Streptomyces subsequently, the conserved GlnR binding sites were identified (Tiffert et al., 2008) and used to identify GlnR target genes. Moreover, cross-regulation between GlnR and other transcription regulators was demonstrated (Rodriguez-Garcia et al., 2007; Yang et al., 2009; Wang R. et al., 2013). Since 2000, the functional identification of GlnR was achieved in other Actinomycetes (such as A. mediterranei U32, M. smegmatis, Saccharopolyspora erythraea, Streptomyces variabilis). In 2011, Tiffert et al. performed a proteomic analysis of the glnR deletion mutant of S. coelicolor and found that GlnR affects the expression of numerous genes belonging to different metabolic pathways (Tiffert et al., 2011).

This review focuses on the functional studies of GlnR mainly in S. coelicolor but also in other antibiotic producing Streptomyces species as well as in other Actinomycetes. This review reports what is known on the role played by GlnR in the regulation of nitrogen as well as of carbon and phosphate metabolisms and on the cross-regulation between GlnR and other regulators. The structure of the protein GlnR and of that of its target sites is also commented. This review thus provides a systematic understanding of the critical role played by the global regulator GlnR in the regulation of both primary and specialized metabolism in Actinomycetes.

Tiffert et al. performed a comparative proteome analysis of the wild type strain of S. coelicolor M145 and a glnR-deficient mutant and discovered that GlnR regulates the expression of numerous proteins (Tiffert et al., 2011) involved (1) amino acid metabolism, GlnR inhibits the biosynthesis and degradation of certain types of amino acids (methionine, tryptophan, serine), (2) carbon metabolism, GlnR enhances the synthesis of acetyl-CoA and inhibits the pentose phosphate pathway, (3) stress response, GlnR promotes the transcription of numerous stress-response genes (Tiffert et al., 2011), and (4) antibiotic biosynthesis. Whether GlnR directly or indirectly affects these processes requires further experimental demonstration, but it is clear that GlnR plays a pleiotropic role in the regulation of S. coelicolor M145 metabolism.

The essential nutrients, such as carbon, nitrogen, and phosphorus, usually limit the growth of soil-dwelling bacteria. The deficiency of a particular nutrient will trigger a series of interrelated reactions governed by specific regulators to maintain metabolic balance (Martin et al., 2011).

In Actinomycetes, GlnR belongs to the OmpR family of proteins (Amon et al., 2010). OmpR-like proteins are response regulators of the bacterial two-component system. They are primarily transcriptional activators with typical DNA domain at the C-terminal and a signal-receiving domain at the N-terminal end of the protein (MartinezHackert and Stock, 1997). The comparison of GlnR amino acid sequences of 10 Actinomycetes, with the exception of that of Corynebacterium glutamicum, revealed that the GlnR proteins share 62 to 82% similarity. The DNA binding domain (α2-loop-α3) was relatively conserved and the α3 sequence, was identical (Tiffert et al., 2008). OmpR-like proteins typically contain a conserved Asp residue as their specific phosphorylation site (Delgado et al., 1993; Lee et al., 2002) whereas two residues, serine/threonine and tyrosine, are involved in phosphate transfer (Hengge, 2001). The OmpR crystal structure of E. coli shows that the α2-loop-α3 of the DNA binding domain forms the helix-turn-helix (HTH), where the α3 helix recognizes the specific DNA sequence through direct interaction with the major groove of DNA (Gross et al., 1989). The secondary structure of OmpR of E. coli showed that the conserved site of phosphorylation is an Asp residue (D-55) (Delgado et al., 1993), the serine/threonine residue is a Thr residue (T-83) and a tyrosine residue is present as Y-102 (Birck et al., 1999).

In S. coelicolor, GlnR is encoded by the glnR gene, which has three promoters: P1, P2, and P3. P2 has an identified −10 region, but P1 has no clearly identified −10 region (Wray and Fisher, 1993). The P2 promoter of glnR is preferentially recognized by a σ³¹-containing RNA polymerase during the stationary growth phase (Wray and Fisher, 1993). In bacteria, it is common that genes have several promoters and different σ factors of RNA polymerase selectively recognize these promoters, to adjust the transcription pattern to environmental changes (Storz and Toledano, 1994). GlnR is considered to be a typical orphan response regulator since glnR has no co-localized gene encoding a putative sensory component (Fink et al., 2002). GlnR of S. coelicolor contains a conserved Asp residue (D-50) that could constitute an alternative potential phosphorylation site. In GlnR, the other two conserved sites are replaced by a Tyr residue corresponding to the OmpR T-83 and a Val residue (V-95) corresponding to the OmpR Y-102 (Stock et al., 1989). Recently, Lin et al. characterized the structure of the GlnR-dependent transcription activation complex (GlnR-TAC). They reported the cryo-EM structure of GlnR-TAC and a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) (Shi et al., 2023). The GlnR-TAC includes RNA polymerase, GlnR, and a promoter containing four conserved GlnR binding sites. These structures elucidate that four GlnR_DBDs bind around the promoter DNA in a head-to-tail manner, and four N-terminal receiver domain of GlnRs (GlnR-RECs) are formed in a tetramer state. The tetramerization of GlnR-RECs coordinately bridge domains of RNAP core enzyme with GlnR_DBDs, promoting stabilization of GlnR-TACs (Shi et al., 2023).

A crystal structural analysis of the N-terminal region of GlnR of A. mediterranei U32 was performed by Wang et al. and this study revealed that the previously referred to as “phosphorylation pocket” was not conserved but replaced by an Arg-52 present in the β3-α3 loop (Lin et al., 2014). The potential phosphorylation residue Asp-50 is not phosphorylated but is involved in the homodimerization of GlnR. Additionally, Asp-50 is involved in charge interactions between the receiver domain and the highly conserved residues Arg-52 and Thr-9. These interactions are likely to play a crucial role to maintain the correct conformation for homodimerization (Lin et al., 2014) that affects GlnR DNA-binding capacity (Wang and Zhao, 2009). The receiver domain of the N-terminal end of GlnR forms a homodimer through the α4-β5-α5 dimer interface, while the aspartate residue at the N-terminal does not need to be phosphorylated (West and Stock, 2001). However, this site is necessary to maintain the stability of homodimerization (Lin et al., 2014).

In M. smegmatis, the aspartate residue D-48 is essential for GlnR activity under nitrogen limitation. When D-48 is changed to alanine, its phenotype is similar to that of a glnR mutant and showed blocked growth (Jenkins et al., 2012).

GlnRII, another regulator of nitrogen metabolism, is also an OmpR family protein whose C-terminal DNA domain is highly similar to that of GlnR (Fink et al., 2002). The α3-DNA recognition helix of GlnRII is almost identical to that of GlnR except for two amino acids Alanine (Ala-163 in GlnRII and Arg-195 in GlnR) and Arginine (Arg-167 in GlnRII and Ala-199 in GlnR) that may be related to the DNA binding specificity (Fink et al., 2002). This may explain why GlnR and GlnRII bind some common target genes. Interestingly, CheY, another regulator of the OmpR family, has α4 and β5 helixes (Fink et al., 2002) that are involved in the contact with the kinase CheA (Welch et al., 1998) whereas the N-terminal part of GlnRII does not contain such helixes (Fink et al., 2002). Moreover, GlnRII does not have the three conserved phosphorylation sites of OmpR family proteins, and it is not co-localized with a kinase (Fink et al., 2002). Therefore, the phosphorylation mode of GlnRII may differ from that of OmpR family proteins. As regulators of nitrogen metabolism, GlnR and GlnRII may receive different signals and have different target genes due to their structural differences. Since GlnR cannot be phosphorylated in vitro and its not co-localized with an histidine kinase, the post-translational modification of GlnR has been challenging to study. Amin et al. found that the transcriptional expression level of GlnR in S. coelicolor did not change significantly with nitrogen availability (Amin et al., 2016) whereas the transcriptional level of downstream target genes regulated by GlnR changes significantly according nitrogen source nature and availability. This indicated that GlnR binding activity might be modulated by post-translational modification. Indeed, it was demonstrated that GlnR is mainly phosphorylated on serine/threonine and acetylated on lysine (Amin et al., 2016). When nitrogen sources are abundant, GlnR is primarily modified by phosphorylation and cannot interact with the promoter region of its target genes whereas under nitrogen limitation, the acetylation of GlnR takes place but does not alter its DNA-binding capacity (Amin et al., 2016).

GlnR of S. coelicolor can bind to the glnA promoter regions of different Actinomycetes (Tiffert et al., 2008). Sequence comparisons of glnA promoter regions from various Actinomycetes including S. coelicolor, S. avermitilis, Bifidobacterium longum, Frankia sp. EAN1, M. bovis, M. tuberculosis, C. glutamicum, Propionibacterium acnes, Rhodococcus sp. RHA1, Nocardia farcinica, Nocardioides sp. JS614 showed that all Actinomycetes except C. glutamicum, whose nitrogen metabolism is regulated by AmtR, contain conserved GlnR binding sites (Tiffert et al., 2008). In S. coelicolor, the promoter sequences of the 13 GlnR target genes were compared, and the conserved GlnR binding sequence was determined to be gTnAc-n₆-GaAAc-n₆-GtnAC-n₆-GAAAc-n₆ (a-b-a-b), with each GlnR binding box having both a-site and b-site (Tiffert et al., 2008). Additionally, DNase I footprinting analysis accurately verifies the binding sequences on glnA and gdhA promoter regions. As predicted, two binding boxes were needed for GlnR binding in the glnA promoter region, while only one was necessary for GlnR binding in the gdhA promoter region (Tiffert et al., 2008). The GlnR binding box is frequently found on the coding strand of the target gene (Wang et al., 2012). However, there are exceptions. For example, SCO0888’s GlnR binding box is on the non-coding strand (Tiffert et al., 2008). Generally, when two GlnR binding boxes are present, they are continuous on the DNA sequence (Shi et al., 2023) but Wang et al. found that the two binding boxes of GlnR in the nasA promoter region were separated by 1 bp (Wang and Zhao, 2009). There are also three GlnR binding boxes on the promoter of the amtB gene, which is responsible for ammonium transport (Wang et al., 2012). A PhoP protected sequence also exists between the first and the second GlnR binding box (Wang et al., 2012). Wang et al. confirmed that the three binding boxes are necessary for amtB regulation by GlnR and revealed the molecular mechanism by which GlnR and PhoP cross-regulate amtB expression (Wang et al., 2012). Subsequently, Sola-landa et al. comprehensively analyzed the promoter regions of 14 GlnR target genes in S. coelicolor. They confirmed these targets by footprinting analysis, and proposed a novel 22-nt long consensus for GlnR DNA binding motifs (Sola-Landa et al., 2013). In this 22-nt long consensus, the second 11-nt long repeat is more highly conserved than the first one (Sola-Landa et al., 2013). In GlnR of S. venezuelae, a 16-bp GlnR box was proposed, which contained 5-bp a-site, 6-bp spacer and 5-bp highly conserved b-site (5 + 6 + 5-bp): GTnAC-n₆-GTnAC (Pullan et al., 2011) but this consensus is fairly similar to the GlnR/22 nt long consensus. In addition, GlnR conserved binding sites were also identified in M. smegmatis (t/gTAAC-n₆-Gc/aAAC) (Amon et al., 2008) and S. erythraea (t/gna/cAC-n₄cn-GnAAc) (Yao et al., 2014) (Table 2). In A. mediterranei U32, the nasACKBDEF operon involved in nitrate assimilation (Shao et al., 2011), is directly regulated by GlnR (Wang Y. et al., 2013). Four GlnR binding sites were predicted by bioinformatics analysis in the promoter region of this operon (Wang Y. et al., 2013) but the studies revealed that only three sites (a1-b1-b2 sites) were necessary for GlnR binding: while a2-site was not required. EMSA (Electrophoretic Mobility Shift Assay) results demonstrated that GlnR yielded two binding bands with the nas promoter region forming two complexes (complexes I and II), where the complex with a lower mobility rate was designated complex II (Wang Y. et al., 2013). In this binding site pattern, a1-b1 sites were “5 + 6 + 5-bp,” just enough for the GlnR homodimer to combine and generate complex I. The generation of complex II required the b2-site. A 16-bp spacer between the b1-site and b2-site may cause a bend between the b1-site and b2-site upon GlnR binding as observed in other prokaryotic systems (PerezMartin and deLorenzo, 1997). Wang et al. determined the GlnR conserved DNA binding motif by changing the nucleotides of GlnR binding site on the glnA promoter in U32 one by one. This study revealed that the adenine at the fourth position was highly conserved and irreplaceable and proposed a 5-nt GlnR box as the fundamental motif for GlnR binding (Wang et al., 2015). Altogether these studies demonstrated that the GlnR binding mode is more adaptable than anticipated.

Under the stimulation of an unknown nitrogen signal(s), unknown sensory kinase(s) may transmit the signal(s) to GlnR, which then interacts with the promoter region of its target genes to regulate nitrogen metabolism. In addition, nitrogen metabolism is also cross-regulated by other regulators (Figure 3) such as PhoP, the regulatory protein of phosphate metabolism that can also control the expression of glnR and of its target genes belonging to nitrogen metabolism; MtrA and AfsQ1, which regulate antibiotic production but can also negatively regulate nitrogen metabolism, while AfsR, NdgR and ScbR positively regulate glnR expression. Genes and proteins in interaction with GlnR are summarized in Table 3. There are still many things to be revealed concerning GlnR regulatory role. Continuous research efforts should lead to the identification of the kinase(s) involved in the phosphorylation of GlnR as well as of the enzymes involved in its post-translational modification. The implementation of systems biology approaches including proteomics, transcriptomics, metabolomics, and bioinformatics will surely lead to the characterization of new direct and indirect GlnR target genes and novel cross-regulatory networks with other regulators.

BG: Investigation, Validation, Writing – original draft, Writing – review & editing. GL: Investigation, Validation, Writing – review & editing. DG: Funding acquisition, Validation, Writing – review & editing. JW: Conceptualization, Funding acquisition, Validation, Writing – review & editing.