Nitrogen Degradation Pathways in Actinomycetes: Key Components of Primary Metabolism Ensuring Survival in the Environment

Abstract

Nitrogen is an essential element required for bacterial homeostasis. It serves as a building block for the biosynthesis of macromolecules and provides precursors for secondary metabolites. Actinomycetes have developed the ability to use various nitrogen sources to ensure their survival in ecological niches with fluctuating nutrient availability. A complex nitrogen metabolism of Actinobacteria allows the utilization of various compounds as N sources, including ammonium, nitrate, urea, amino acids, amino sugars, and amines. One such adaptation is the ability to acquire nitrogen from alternative amine sources like monoamines or polyamines putrescine, cadaverine, spermidine, and spermine, ensuring both nutrient availability (C and N sources) and resistance against high polyamine concentrations. Actinobacterial nitrogen degradation, including the catabolism of amines, is not only important under low nitrogen availability, but also required to survive under high concentrations of these compounds. The purpose of this review is to summarize the knowledge on nitrogen degradation and, more specifically, catabolism of amines in Actinobacterial survival and its role in nitrogen metabolism. Applying critical analysis of the recent available literature and sequencing data, this work aims to explore strategies of pathogenic and non-pathogenic Actinobacteria to survive in the presence of different nitrogen sources, and their impact on primary and secondary metabolism. The knowledge about nitrogen degradation pathways in Actinobacteria including mono- and polyamine catabolism collected in the scope of this review paper is brought in connection with possibilities to combat pathogens by using their capability to metabolize polyamines as an antibiotic drug target. This might offer new directions for target-based drug design to combat Actinobacterial infections.

1. Introduction

1.1. Nitrogen Metabolism in Prokaryotes

In addition to carbon, oxygen, and hydrogen, the main component of all living cells is nitrogen (N). It makes up about 14% of the cell’s dry weight [1]. It is an essential component for bacterial metabolism needed for the synthesis of purines and pyrimidines that are the basic building blocks of DNA and RNA, of amino acids that are required for protein synthesis, and of amino sugars that are components of cell walls. The basic building blocks often serve as precursor molecules for the formation of secondary metabolites [2,3,4], which may contain atoms of nitrogen. For example, Streptomyces produced molecules of the antibiotic undecylprodigiosin (Red) contain three nitrogen atoms, while the calcium-dependent antibiotic contains 14 N atoms [5,6]. The availability of nitrogen plays a crucial role in primary metabolism and in secondary metabolite formation [7].

While soil bacteria can utilize both animal and plant remains, most enterobacteria depend on metabolites of animal metabolism. These include organic substances such as amino acids, creatinine, urea, or simple inorganic substances such as ammonium or nitrate [8]. The preferred nitrogen source for optimal growth in most bacteria is ammonium, which is absorbed via the central assimilation pathway [3]. For the uptake and utilization of nitrogen, bacteria have developed specific transporters along with mechanisms of assimilation and regulation. This allows them to respond quickly to changes in the environment and rapidly optimize their metabolism [9].

Due to their importance for antibiotics production and development of new medicines against human pathogens, bacteria from the phylum Actinobacteria are of special research interest. Actinomycetes can obtain the nitrogen they need for cellular metabolism from various sources to supply precursors in their complex primary and secondary metabolism. This allows them to adapt to habitats with fluctuating nitrogen conditions. Nitrogen metabolism in Actinobacteria, its regulation, and its importance for the central and secondary metabolism was covered in numerous reviews [10,11,12,13,14,15,16,17,18,19,20,21]. Metabolism of amines in Actinomyces has received a lot of attention, recently having been highlighted in several reviews [7,20,21,22]. However, a comprehensive review of the importance of nitrogen degradation pathways including catabolism in Actinomyces for pathogenicity and drug discovery efforts remained out of scope for these studies. This review provides the reader with a comprehensive overview of recent advancements in investigation of nitrogen degradation pathways in connection to possibilities to improve development of new relevant pharmaceuticals.

1.2. Nitrogen Uptake and Nitrogen Assimilation in Actinobacteria

Actinomycetes can utilize a wide range of substances as nitrogen sources. These include inorganic compounds such as ammonium, nitrate, and nitrite, as well as organic compounds, such as amino acids (e.g., histidine and arginine) and amino sugars [23,24]. Nitrogen uptake in Actinobacteria has been extensively investigated in the model bacterium Streptomyces coelicolor. S. coelicolor lives in the soil under variable N and C conditions and therefore possesses the ability to metabolize a variety of different C and N sources, including amino sugars, amino acids, amines (mono- and polyamines), peptides, urea, NO₃⁻, and NH₄. Ammonium is the preferred nitrogen source and leads to higher bacterial growth rates overall than other nitrogen sources. This is because ammonium can be directly absorbed into the cellular cycle [25,26].

All other inorganic compounds coming into the cell must first be reduced to ammonium at the expense of energy. Complex organic substances must be broken down by intracellular and/or extracellular enzymes. Various amino acids can be made available by deamination, which produces free ammonium, or transamination, i.e., the transfer of the amino group to, for example, 2-oxoglutarate [27]. Depending on the available nutrient, bacteria can specifically synthesize or activate the necessary proteins for transport and degradation of the substances [25]. Glutamine synthetases (GSs) play a key role in cellular nitrogen metabolism. Due to their high substrate specificity, these enzymes can assimilate ammonium at concentrations below 0.1 mM [23].

Under low ammonium concentration, glutamine is formed from ammonium and glutamate by glutamine synthetase (GS) using energy of adenosine triphosphate (ATP) (Figure 1A). Glutamate synthase (GOGAT) then catalyzes the NADPH-dependent formation of glutamate from glutamine and 2-oxoglutarate (Figure 1B). The GS/GOGAT pathway is ubiquitous in bacteria and is the only pathway for ammonium utilization in many organisms (Figure 1C). Glutamine serves as both an amino acid and a nitrogen donor in the synthesis of approximately 25% of all nitrogen-containing cellular components [3]. Some bacteria have an alternative route for ammonium assimilation: the direct formation of glutamate through reductive amination of 2-oxoglutarate by glutamate dehydrogenase (GDH), which is important during glucose-limited growth (Figure 1D). However, due to its low substrate specificity, this enzyme only works effectively under high ammonium concentrations [25].

2. Molecular Mechanisms of Assimilation of Different Nitrogen Sources in Actinobacteria

2.1. Nitrogen Assimilation in Actinomycetes: Ammonium Catabolism as Central Catabolic Route

Nitrogen assimilation was extensively studied in the model Gram-negative Enterobacterium Escherichia coli, in which catabolic pathways and their regulation were elucidated first [28]. The regulation of N assimilation in Actinobacteria differs from that in Enterobacteriaceae, but also has numerous similarities, particularly in the key enzymes. It can occur at the transcriptional and posttranslational levels. Proteins such as GlnR, GlnRII, AmtR, NnaR, and Crp play an important role in transcriptional regulation. Posttranslational regulation occurs through the signaling protein PII and by modulating the activity of important enzymes (GDH, GS) through adenylation/uridylation. GSI and GOGAT in Actinobacteria fulfill the same function in ammonium assimilation as GS and GOGAT in E. coli. In their overall reaction, which is well integrated into the TCA cycle, ammonium and α-ketoglutarate are converted to glutamate with the consumption of ATP and NADPH (Figure 2) [28,29,30,31].

GSI is regulated by GlnE via adenylation and deadenylation [32,33,34,35]. Interestingly, there is evidence of possible transcriptional regulation by GlnE, which would make GlnE a so-called “moonlighting” protein [36]. Another form of glutamine synthetase, GSII (encoded by glnII), was first discovered in Rhizobium [37]. GSII is a heat-labile octamer and shows sequence similarity to a eukaryotic GS. According to recent studies, GSII was introduced into the photosynthetic eukaryote Chloroplastida by horizontal gene transfer from gamma–proteobacteria at an early stage of plant evolution [38]. GSII has been found in many Actinobacteria [39,40,41], but it is absent in Amycolatopsis, Mycobacterium, and Corynebacterium [16]. These organisms do not have alternative GS enzymes but may contain GS-like enzymes that do not exhibit GS activity despite structural similarity [20]. The difference of GSII from GSI is the lack of posttranslational regulation by adenylation [42]. Instead, the GSII protein can degrade rapidly after N shock, suggesting control by proteolysis. In contrast to the dodecamer structure of GlnA (GSI), GlnII (GSII) is a homodecamer and possesses high sequence similarity to eukaryotic GSII enzymes [42]. In S. coelicolor, while GlnA (GSI) is responsible for primary metabolism, the GlnII (GSII) enzyme may be predominantly involved in secondary metabolism [16,42].

In addition to glutamine synthetases, bacteria possess GS-like proteins. GlnA2, GlnA3, and GlnA4 have been found in some Actinomycetes, e.g., in S. coelicolor [43] and in M. tuberculosis [44]. However, these proteins lack the amino acid residues conserved in GS, which form an adenylation motif. Therefore, a deficiency in the posttranslational regulation of GlnA2, GlnA3, and GlnA4 by adenylation was suspected. The transcription of glnA2, glnA3, and glnA4 is generally not regulated by GlnR or GlnRII in S. coelicolor, as is the case with glnA and glnII [45,46]. These proteins also lack the amino acid residues involved in the catalytic reaction of GS. Accordingly, GlnA2, GlnA3, and GlnA4 did not exhibit GS activity [47]. The function of the GS-like protein AtdA1 in Acinetobacter sp. strain YAA lies in the ATP-dependent reaction of glutamate and aniline to ɤ-glutamylanilide, which serves for aniline degradation [48].

On the other hand, transcription of glnA2 is upregulated in the glnR mutant of Mycobacterium smegmatis [49,50]. Not all bacteria function in parallel with GSI. For example, in another Gram-positive bacterium Bacillus subtilis, only the GS pathway is used [51]. In contrast, in Streptomyces hygroscopicus 155, another enzyme, alanine dehydrogenase (ADH), is used instead of GDH for ammonium assimilation [52]. ADH catalyzes the amination of pyruvate, consuming NAD+ and leading to alanine formation. Because ADH has a high Km value, it is particularly beneficial to bacteria at high N concentrations, such as 20–100 mM ammonium [52]. ADH activity has also been detected in Streptomyces clavuligerus [2,53] as well as in another soil bacterium Rhizobium leguminosarum [54]. In some Streptomyces, the activity of both enzymes, ADH and GDH, has been demonstrated [55].

In prokaryotes, ammonium assimilation is usually catalyzed by an anabolic, NADPH-dependent glutamate dehydrogenase (GDH) enzyme [56], which catalyzes the conversion of ammonium and α-ketoglutarate to glutamate (reductive amination) as well as the reverse reaction under high concentration of ammonium. This anabolic GDH enzyme was extensively investigated in the model organism E. coli. In contrast to E. coli, which possesses only the NADPH-dependent GDH [57], some Actinomycetes contain two GDHs (anabolic and catabolic). The anabolic GDH is a homohexamer with approximately 50 kDa subunits. The catabolic GDH2 was discovered later than the anabolic GDH. A characteristic feature of GDH2 is its large subunit of 180 kDa [58]. The GDH2 enzyme was described in Streptomyces as highly significant for glutamate catabolism, growth regulation, and most notably, the onset of antibiotic production [59]. Glutamate cleavage (oxidative deamination) during ammonium dissimilation is carried out by the catabolic, NADH-dependent glutamate dehydrogenase. The specificity of the enzymes toward their coenzymes NADP+ or NAD+ has been linked to conserved amino acid residues: an acidic amino acid at position 7 (P7) acts as an indicator of NADP+ specificity [60]. Anabolic glutamate dehydrogenase (GDH) activity is widely distributed among various prokaryotic organisms (both bacteria and archaea). Examples of NAD+-dependent, catabolic GDHs have been found in Actinobacteria, Staphylococcus aureus, and Bacillus subtilis [61] (Figure 3).

2.2. Nitrogen Assimilation in Actinobacteria: Catabolism of Poor Nitrogen Sources

The assimilation of poor nitrogen sources requires the use of specialized enzymes involved in sophisticated systems for nitrate assimilation and respiration, for the assimilation of urea, amino acids, and other nitrogen-containing substances (Figure 3,

Table 1. List of key enzymes in the central nitrogen metabolism of the model Actinobacterium S. coelicolor as well as pathogen Actinobacterium M. tuberculosis.

Annotated Function	Homologue in S. coelicolor	Homologue in M. tuberculosis
glnA4 gamma–glutamylethanolamide synthetase	SCO1613	Rv2860c
2,4-Diaminobutyric acid Acetyltransferase	SCO1864	Rv3225c
glnA (GSI) glutamine synthetase I	SCO2198	Rv2220
glnII (GSII) glutamine synthetase II	SCO2210	-
glnRII Response Regulator	SCO2213	Rv2884
glnE adenylyltransferase	SCO2234	Rv2221c
glnA2 gamma–glutamylpolyamine synthetase	SCO2241	Rv2222c
glnR Response Regulator	SCO4159	Rv0818
gdhA Glutamate dehydrogenase	SCO4683	Rv3726
ureA, B, C α-, β-, γ-subunits of urease	SCO5525-6	Rv1848-50
amtB ammonium transporter	SCO5583	Rv2920c
gluB glutamate transporter	SCO5776	Rv2919c
gltB glutamate synthase	SCO1977	Rv3859c
nirB2 nitrate reductase	SCO2486	-
nirB nitrate reductase	SCO2487	Rv0252
nirD nitrate reductase	SCO2488	Rv0253
nnaR Transcriptional regulator of nitrate/nitrite assimilation	SCO2958	Rv0260c
narK Nitrate Nitrite transporter	SCO2959	Rv2329c
narGHIJ respiratory nitrate reductase	SCO6532-5	Rv1161-4
glnA3 gamma–glutamylpolyamine synthetase	SCO6962	Rv1878
narB nitrate reductase	SCO7374	-
nasA nitrate reductase	SCO2473	-
narK2 Nitrate Nitrite transporter	SCO0213	Rv1737c

) [24,62].

Nitrate can be either assimilated (extracted nitrogen is incorporated into cellular components in anabolic reactions) or dissimilated (in so-called nitrate respiration, nitrate is used as an electron acceptor instead of oxygen). Some Actinobacteria can operate both nitrate reduction pathways, e.g., Actinomyces, Streptomyces, and Arthrobacter [26]. Nitrate uptake and utilization require special enzymes. First, nitrate molecules are transported into the cell by nitrite/nitrate transporters (NarK). There are two types of NarK transporters, NarK1 and NarK2. These are encountered as transport proteins in dissimilatory and assimilatory processes. The first is a nitrate/proton symporter, the second a nitrate/nitrite antiporter. Interestingly, many proteobacteria, which are dominant prokaryotes in soil, use a mixed transporter in which NarK1 and NarK2 are fused together [63,64].

After transporting into the cell, nitrate is reduced to nitrite. This is performed by a nitrate reductase. There are two classes of prokaryotic nitrate reductases: the Nar and Nas/Nap clades [65]. Cytosolic Nas reductase is involved in the assimilation of nitrate. In contrast, membrane-bound Nar and Nas reductases are involved in dissimilatory processes. The reducing equivalents for Nas can be derived from NAD(P)H, ferredoxin, or flavodoxin. Actinobacteria, including Streptomyces spp., primarily use the Nar (respiratory nitrate reductase) clade for nitrate reduction, which is important for maintaining membrane potential and viability under the oxygen-limiting conditions common in soil environments [66].

Transcription is induced by ammonium deficiency and nitrate presence (Stewart, 1994). Nas has the least conserved amino acid sequence among nitrate reductases, which is why it is considered a rapidly evolving protein [65]. Interestingly, the NarB belonging to the class of Nas reductases has also been found in Actinomycetes. For example, NasA and NarB are present in some Streptomyces [67,68,69].

Nas, a related protein, is more flexible in its function. In most cases, it catalyzes the first step in the reduction of nitrate to ammonium. Furthermore, it can also play a role in denitrification reactions, the maintenance of redox balance, and nitrate sensing and uptake under severe N deficiency [70]. Nar is involved in the establishment of the proton gradient during nitrate respiration [71].

Nitrite reductases convert nitrite produced in the reaction catalyzed by nitrate reductases into ammonium (assimilatory pathway) or into NO (dissimilatory pathway). Four types of nitrite reductases have been identified: Cu-containing NirK, found exclusively in denitrifying bacteria [71,72] and three other reductases with different heme groups (cytochrome cd1, siroheme, or multiheme) [73,74]. In prokaryotes, NirS with the heme group cytochrome cd1 has been found only in denitrifying bacteria, while two nitrite reductases, NirB with siroheme and NrfA with multiheme, were studied in E. coli [74,75]. In Streptomyces, the primary identified nitrite reductase is a siroheme-dependent enzyme NirBD (nitrite reductase, large and small subunits) [76] (Figure 3).

In S. coelicolor, NnaR together with the global regulator GlnR, regulates the gene expression of narK, nasA, and nirB [77,78,79]. The high sequence homology of NnaR in different Actinobacteria (particularly in the genera Streptomyces and Mycobacterium) and the genetic location of nnaR, which was always found near narK, nasA, and nirB, suggest that NnaR plays a role in nitrate assimilation in all Actinobacteria (Table 1) [79].

In Actinobacteria, the genes for the ammonium transporter (encoded by amtB), the signaling protein PII (unlike in E. coli, PII is encoded by glnK instead of glnB), and the uridylyltransferase (encoded by glnD) are combined in an amtB operon [45,74,80]. While the operon is inactive in E. coli under N excess [81], weakened expression of the amtB operon has been reported in Streptomyces under these conditions (Table 1) [77].

In Streptomyces and Mycobacteria, GlnK is adenylated by GlnD under N deficiency and deadenylated again under N excess [35,82,83]. In E. coli, GlnK is uridinylated and thus transmits the cellular N concentration to the ATase that regulates GSI activity [4,33,84,85,86,87,88]. In Actinobacteria, GlnK is modified by the glutamine synthetase adenylyltransferase GlnE (ATase equivalent in Actinobacteria) [33,89]. However, signaling from GlnK to GlnE is absent in Actinobacteria [35], and the significance of GlnK adenylation is currently unknown. In addition, GlnK is Streptomyces by cleavage of the first three amino acids at the N terminus after ammonium shock [82] (Table 1).

The functional versatility of GlnK in Actinomycetes is also evident in N metabolism systems such as the AmtR regulon of Corynebacterium [80] or the TnrA regulon of Bacillus [90]. The AmtR repressor in Corynebacteria is not regulated by small effector molecules, as is common in the TetR family. Instead, the repressor dissociates from the DNA after forming a complex with GlnK adenylated at Tyr51 [34,80,91,92]. Interestingly, AmtB plays a role in this process [91]. TnrA, the transcriptional regulator of N metabolism in Gram-positive bacteria from the genus Bacillus, is bound to a membrane-bound GlnK-AmtB complex in the absence of ATP [93] (Figure 3).

2.3. Assimilation of Amines in Actinomycetes

Amines are organic compounds that contain carbon–nitrogen bonds, whereas one or more hydrogen atoms in ammonia are replaced by alkyl or aryl groups. The functional group −NH2, present in primary amines, is called the amino group, which is present in biologically occurring amines, such as monoamines (e.g., ethanolamine) and polyamines (e.g., putrescine). Polyamines are cationic charged molecules with a hydrocarbon chain and multiple amino groups [94]. They fulfill a lot of physiological functions, e.g., cell growth, maturation and proliferation, cell signaling, gene expression, and others [95,96,97,98]. The monoamine ethanolamine is a short molecule which is both a primary amine and a primary alcohol and is a nitrogenous base in phospholipids and a building block of biomembranes (e.g., phosphoethanolamine) [99]. Excess of polyamines or ethanolamine is very toxic for bacterial cells and can lead to cell death [98]. Hence, polyamine and ethanolamine utilization represent bacterial survival strategies that allow survival of S. coelicolor in the soil environment. Cationic charged molecules like polyamines and ethanolamine, which are toxic in high concentrations due to interaction with negatively charged molecules like DNA or RNA, can be modified by glutamylation or acetylation by specialized enzymes. This leads to the generation of glutamylated/acetylated mono-/polyamines, which are neutrally charged molecules that can be further used in primary metabolism as sources of carbon and nitrogen [7].

In S. coelicolor putrescine, spermidine and diaminopropan biosynthesis have been described in the late-stationary phase in NMMP-medium, but cadaverine synthesis occurred only under iron limitation [100]. Proteins with high similarity to the polyamine binding lipoprotein (PotD) and/or putrescine-binding periplasmic protein (PotF), to the amino acids/polyamine permease (PuuP) and/or putrescine importer (PlaP) were found in many phyla including Actinobacteria. SCO5667 is a predicted homolog of the putrescine-binding periplasmic protein (PotF) from E. coli with the sequence identity/similarity of 29/47%. However, transcriptional analysis of expression patterns of these genes revealed only weak expression of sco5667 and sco5671 in presence of spermidine [20,47].

Polyamine assimilation has been extensively investigated in model system E. coli and Pseudomonas aeruginosa, and until recently remained largely unstudied in Actinobacteria The most likely steps of the polyamine utilization pathway in S. coelicolor were reported to be like that known in E. coli [101,102,103] and P. aeruginosa [104]. The initial step of the polyamine utilization is catalyzed by gamma–glutamylpolyamine synthetases GlnA2 and/or GlnA3 resulting in glutamylated polyamines [47,105]. These glutamylated products can likely be further reduced by the predicted gamma–glutamylpolyamine oxidoreductase (SCO5671). The reaction results in the production of the gamma–glutamyl–gamma–aminopentanal or gamma–glutamyl-gamma–aminobutyraldehyde. The SCO5671 enzyme is a close ortholog of the gamma–glutamylpolyamine oxidoreductases PuuB from E. coli and PauB1-B4 from P. aeruginosa. The gene sco5671 showed expression in presence of spermidine, but no expression in the presence of polyamines putrescine and cadaverine. In the next step of the utilization pathway, the predicted dehydrogenases (SCO5666 and SCO5657) might be involved. These proteins are predicted homologs of PuuC and PatD from E. coli, which are (gamma–glutamyl-) gamma–aminobutyraldehyde dehydrogenases. This step of the pathway may result in the production of the gamma–glutamyl–aminovalerate or gamma–glutamyl–GABA. The next step of polyamine utilization might require a predicted hydrolase (SCO6961) and result in the production of aminovalerate or GABA. Enhanced expression of sco5666, sco5657 and sco6961 in presence of polyamines was observed [20,47]. These results suggest the role of SCO5666, SCO5657 and SCO6961 in polyamine utilization. Afterwards, SCO5676, which is a predicted homolog of the GABA aminotransferase GabT from E. coli, may be involved and catalyze the production of glutarate semialdehyde or succinate semialdehyde [20]. It has been shown that the expression of the sco5676 gene is strong in presence of arginine [106]. Arginine is a precursor of putrescine in S. coelicolor M145. This finding as well as the observation of enhanced expression of sco5676 in the presence of polyamines suggest the possible role of SCO5676 in polyamine utilization. SCO5679, which is a predicted homolog of the succinic semialdehyde dehydrogenase GabD from E. coli, may be involved in the last step of the polyamine utilization pathway. The polyamine utilization pathway ends with the succinate or glutarate that feeds the tricarboxylic acid (TCA) cycle (Figure 2 and Figure 4) [20].

A predicted amidotransferase (SCO5655) was identified, which is a homolog of the putrescine amidotransferase (PatA) from E. coli. In transcriptional analysis, the expression of sco5655, sco6960, and sco6961 was enhanced in presence of polyamines. Moreover, sco5655 was reported to be induced by a diamide and not by arginine [106]. No orthologs of SCO6961 and SCO6960 were found in E. coli or P. aeruginosa. These findings suggest the possibility of an alternative polyamine utilization pathway in S. coelicolor (Figure 4,

Table 2. List of predicted proteins involved in polyamine and ethanolamine utilization in E. coli, P. aeruginosa, S. coelicolor and M. tuberculosis (modified after [7,107,108,109]).

Annotated Function	Homologue in E. coli	Homologue in P. aeruginosa	Homologue in S. coelicolor	Homologue in M. tuberculosis
Polyamine ABC transporter ATP-binding protein PotA-like	PotA (b1126)/YdcT (b1441)	PAO603/PAO326	SCO3453	Rv2397c
Polyamine ABC transporter ATP-binding protein PotC-like	PotC (b1124)/YdcV (b1443)	PAO324/PotC (PA3609)	SCO3454	Rv1237
Polyamine ABC transporter protein	PotB (b1125)/YdcU (b1442)	PotB (PA0205)/PA3252	SCO3455	Rv2040c
Polyamine ABC transporter protein—substrate binding protein	YnjB (b1754)	PA0203	SCO3456	Rv3869
Amino acid/polyamine permease	PuuP (b1296)/PlaP (b2014)	PA5510	SCO5057	Rv3253c
Lysine/ornithine decarboxylase-like enzyme	-	-	SCO5651	Rv1205
Pyruvate-polyamine aminotransferase	PatA (b3073)	SpuC (PA0299)	SCO5655	Rv3329
Lrp/AsnC family transcriptional regulator	-	-	SCO5656	Rv2779c
γ-aminobutyraldehyde or γ-glutamyl-γ-amino-butyraldehyde dehydrogenase	PatD (b1444)/PuuC (b1300)	BetB (PA5373)/PAO219	SCO5657	Rv0458
Polyamine-binding lipoprotein	PotF (b0854)	SpuD (PA0300)	SCO5658	Rv3484
γ-aminobutyraldehyde dehydrogenase or 4-guanidino-butyraldehyde dehydrogenase	PatD (b1444) PuuC (b1300)	PauC/KauB (PA5312)	SCO5666	Rv2858c
Polyamine ABC transporter substrate-binding protein	PotF (b0854)	SpuE (PA0301)	SCO5667	-
Polyamine ABC transporter substrate-binding protein	PotG (b0855)	SpuF (PA0302)	SCO5668	Rv2397c
Polyamine ABC-transporter integral membrane protein	PotH (b0856)	SpuG (PA0303)	SCO5669	Rv2399c
Polyamine ABC-transporter integral membrane protein	PotI (b0857)	SpuH (PA0304)	SCO5670	Rv2398c
γ-glutamyl-polyamine oxidoreductase	PuuB (b1301)	PauB3 (PA2776)	SCO5671	Rv3742c
γ-aminobutyrate aminotransferase gabT-like or puuE-like	GabT (b2662)/PuuE (b1302)	GabT (PA266)	SCO5676	Rv2589
Succinate-semialdehyde dehydrogenase gabD-like	GabD (b2661)	GabD (PA0265)	SCO5679	Rv0223c
Amino acids/polyamine permease	PuuP (b1296)	PA5510/PAO322	SCO5977	Rv2320c
Hydrolase	-	-	SCO6960	Rv0193c
Amidohydrolase	-	-	SCO6961	Rv1879
γ-glutamyl-polyamine synthetase	PuuA (b1297)	PauA7 (PA5508)/SpuI (PA0296)	SCO6962	Rv1878
γ-glutamyl ethanolamine synthetase/ethanolamine γ-glutamylase	-	-	SCO1613	Rv2860c
γ-glutamyl ethanolamine dehydrogenase/iron-dependent dehydrogenase	-	-	SCO1611	Rv1941
γ-glutamyl aldehyde dehydrogenase	-	-	SCO1612	Rv2858c
γ-glutamyl glycine amidohydrolase /formylglutamate amidohydrolase	-	-	SCO1615	Rv2859c

) [47].

The metabolism of monoamine ethanolamine has been investigated in such Actinobacteria as Streptomyces and Mycobacterium (Table 2) [7]. In S. coelicolor, predicted ethanolamine permeases (SCO6014 and SCO5977) possess likely other functions, because the expression of the genes sco6014 and sco5977 was not induced by ethanolamine [107]. Likely, an ethanolamine permease encoding gene was not required and lost in S. coelicolor during the evolution process and enough ethanolamine may enter the cells very probably through diffusion. It is also possible that S. coelicolor uses ethanolamine utilization pathway to control intracellular ethanolamine level during decomposition of membranes and to recycle the N source.

In order to utilize ethanolamine as an N and C sources and balance the intracellular ethanolamine pool, the bacteria had to develop metabolic pathways for ethanolamine utilization. The canonical ethanolamine utilization pathway was studied in S. typhimurium and E. coli. However, alternative ethanolamine utilization pathways have been reported in M. tuberculosis and C. salexigens. These pathways do not require a metabolosome and do not result in the production of toxic intermediates, such as acetaldehyde [7].

It was shown in a recent study that diverse organisms ranging from the Actinobacteria to the Proteobacteria possess the capability for ethanolamine metabolism, which does not require eut genes [108,109]. The most likely steps of the ethanolamine utilization pathway in S. coelicolor may be similar to the pathway described in studies of Brian et al. and Gerlt et al. as well as in detail in C. salexigens by Gerlt et al. [108,109]. After the glutamylation of ethanolamine by GlnA4 (SCO1613), a predicted gamma–glutamylethanolamine dehydrogenase (SCO1611) may be required, producing gamma–glutamylacetaldehyde. In the next step of the pathway, a predicted gamma–glutamylaldehyde dehydrogenase (SCO1612) may be involved, producing gamma–glutamylglycine. The last step of the pathway may require a predicted gamma–glutamylglycine amidohydrolase (SCO1615). The pathways may end in the production of glycine and glutamate (Table 2). Further studies of SCO1611, SCO1612, and SCO1615 are required to determine their functionality in the gamma–glutamylation pathway of ethanolamine in S. coelicolor [20,107].

3. Nitrogen Assimilation in Actinobacteria: Transcriptional Regulation

3.1. Regulation of Central Pathways

Nitrogen assimilation was very well studied in Enterobacteriaceae, in which it is regulated by the Ntr system. However, most Actinobacteria possess the global transcriptional regulator GlnR for the same purpose [77,110]. Homodimerization of this regulator is necessary for DNA binding and thus for regulation. In the Ntr system, phosphorylation of the conserved Asp residue by NtrB ensures NtrC dimerization. Since a GlnR with a conserved Asp residue in the N terminal receiver domain was found in Actinobacteria, but no kinase phosphorylating this Asp residue, GlnR was long considered an “orphan” regulator. Then, it was discovered that the conserved Asp residue is not phosphorylated and that the activating GlnR dimerization in Amycolatopsis mediterranei is caused by the ionic interaction of unphosphorylated Asp50 with the conserved Arg52 and Thr9 [111]. Due to the lack of phosphorylation of Asp, GlnR is considered an atypical OmpR-like regulator. The receiver domain of one GlnR molecule forms an α4-β5-α5 surface, through which interaction with the receiver domain of the second GlnR molecule takes place [111]. More than 15 genes are under the control of GlnR, most of which regulate nitrogen metabolism [77]. GlnR exerts its activating effect on gene transcription under nitrogen deficiency by binding to the promoter regions of the regulated genes, thereby altering transcription [78]. Thus, the amtB operon in S. coelicolor is controlled by GlnR activation [45], with a correlation between transcription and the amount of nitrogen found [34,45]. This again differs from the regulation in E. coli, where glnD expression has been described as constitutive [81]. In addition to GlnR, Actinomycetes also contain GlnRII, which is less common in bacteria and has not been studied as extensively as GlnR. Like GlnR, GlnRII plays a role a regulatory role in the genes of N metabolism. However, so far, GlnRII has only been shown to affect the genes glnA, amtB, and glnII [45].

Another transcriptional regulator of N metabolism that is widespread among Actinomycetes is AmtR. In Corynebacteria, AmtR, a regulator of the TetR family that acts as a repressor of gene expression [80], replaces the GlnR regulator. In N excess, AmtR is bound to DNA, thus preventing the expression of key genes involved in N metabolism, such as glnA, gltB, and the amtB operon [34,80,92]. In N deficiency, an adenylated GlnK gene product binds to AmtR and leads to the dissociation of the repressor from the DNA, allowing the released genes to be transcribed [92]. It was recently discovered that some members of the Corynebacterineae and Streptomyces, which possess a GlnR regulator, also possess an AmtR protein that shows low homology to the AmtR in Corynebacteria [110]. Even though TetR regulators possess a highly conserved DNA-binding domain, AmtR regulators exhibit individual recognition features in the amino acid sequence [112]. Subsequent investigation of these AmtR regulators revealed that GlnR plays the main role in the regulation of nitrogen assimilation, while AmtR regulates only a subset of genes and its regulation is GlnR-dependent [50]. This AmtR regulon includes genes for amidase, urea carboxylase, and amino acid permease [50].

3.2. Regulation of Catabolism of Amines in Actinomycetes

In S. coelicolor, the global regulator of the nitrogen metabolism GlnR can undergo posttranslational modifications by phosphorylation and/or acetylation that affect its binding affinity to DNA leading to interaction with different target promoter sequences and change in transcription levels of target genes as response to changing N conditions [113]. It was shown that the phosphorylation of Ser/Thr occurs under N-excess conditions. In agreement with these results, lack of phosphorylation on the Asp50 and lack of any Ser/Thr phosphorylation in GlnR isolated from S. coelicolor grown under N-limiting conditions was also demonstrated [111]. Phosphorylation and acetylation were shown to influence the DNA-binding affinity of GlnR [113]. GlnR regulates the glutamine synthetase encoding genes glnA and glnII at transcriptional level in dependence of N conditions. However, no binding GlnR in promoter regions of glnA2, glnA3, and glnA4 were reported [45,46]. However, the acetylated version of GlnR binds better to the glnA2 promoter region [114]. Interestingly, studies in the actynomycete Saccharopolyspora erythraea revealed that GlnR activates the expression of glnA3 (SACE_3095) [115].

In the genome of S. coelicolor, the sco5656 (epuRII) gene is localized close to the genes that encode predicted enzymes of polyamine utilization pathway [114] and is annotated as putative regulator [43]. The expression of the epuRII gene in presence of putrescine, cadaverine, and spermidine was enhanced [47]. EMSA analysis of regulatory targets of EpuRII revealed hints towards a complex regulation of several polyamine associated genes. These include glnA3, as well as sco5676 encoding a putative homolog of the 4-amino-butyrate aminotransferase GabT of E. coli K12 and sco5977, encoding a putative polyamine antiporter. The tests with EpuRII resulted in eight positive hits for EpuRII-interacting promoter sequences. The following genes seem to be regulated by EpuRII: glnA3, sco5676, coding for a putative homologue of the 4-amino-butyrate aminotransferase GabT, sco5977 encoding a putative polyamine antiporter, and sco6960 with unknown function (

Table 3. List of regulatory proteins directly or indirectly involved in nitrogen primary and secondary metabolism regulation in Stretpomyces.

Regulator Name	Function	Reference
GlnR	Central regulator of nitrogen metabolism regulating glnA, glnII, gdhA, nirB, ureA, and amtB-glnK-glnD	[78,116,117,118]
GlnRII	A GlnR homologue that recognizes glnA, amtB, and glnII	[45]
Crp	Regulates the interplay of primary and secondary metabolism, activating glnA, glnII, and amtB-glnK-glnD	[119]
ArgR	Controls the expression of glnR in response to nutrient stress stimuli	[7,105]
PhoP	Represses the amtB-glnK-glnD operon and glnA, glnII, and glnR under conditions of phosphate limitation	[7,105]
AfsR	Controls expression of glnR in response to unknown nutrient stress stimulus	[7,105]
AmtR	Regulates key genes involved in N metabolism, such as glnA, gltB, and amtB	[50]
AfsQ1	Required for regulation of carbon, nitrogen, and phosphate metabolism in the presence of glutamate	[7,105]
EpuRI	Regulates genes for ethanolamine utilization glnA4, sco1612, sco1611, and sco1610	[107]
EpuRII	Regulates genes for polyamine utilization glnA3, sco5676, sco5977, and sco6960	[114]

) [114].

There are two known regulatory mechanisms of ethanolamine utilization genes: the EutR system and the EutVW system. Regulation of ethanolamine utilization genes was well studied in S. typhimurium, E. coli and E. faecalis. In S. coelicolor, the sco1614 (epuRI) gene was annotated as putative regulator in the genome of S. coelicolor that is localized close to the gene glnA4 (sco1613) [43]. EMSA analysis revealed glnA4 gene as a potential target of EpuRI. Other genes located downstream of glnA4, namely sco1612, sco1611, and sco1610, encode predicted enzymes of the ethanolamine utilization pathway and are organized in one putative operon together with glnA4. Thus, these genes may be transcribed together with glnA4. The role of EpuRI as a negative transcriptional regulator of the ethanolamine utilization associated genes was shown in a transcriptional analysis [107]. Interestingly, in preliminary EMSA analysis also interactions of EpuRI with promoter sequences of sco5652 and with the adjacent operon including sco5654 were observed. The sco5652 gene encodes a protein of unknown function, sco5654 encodes a putative ABC transporter. EpuRI demonstrated interactions with the promoter region of sco5657 (encoding a putative aldehyde dehydrogenase), sco1616 (a putative regulator), and the promoter sequence of epuRI itself (Table 3) [107,114].

4. Summary and Discussion

In their natural habitat, Actinobacteria must cope with very changeable nutrient conditions. Nitrogen is available in low concentrations most of the time. Depending on conditions at a given point of time, the nutrient availability varies and may range from low to high concentrations. Nitrogen metabolism is essential for bacterial survival under nutrient limitation conditions in competitive ecological niches [116,117,118,120,121]. Bacteria have developed complex metabolic networks and regulatory machinery to control intracellular pools of nitrogen. Nitrogen metabolism, comprising transport, biosynthesis, utilization, and regulation, is a central part of bacterial primary metabolism, ensuring the supply of building blocks for biomolecules and biomass generation.

Because of complex developmental cycle and metabolism, Actinobacteria including Streptomyces spp. and Mycobacterium spp. can adapt to fluctuating nitrogen levels by using their complex nitrogen catabolism. Utilization of a variety of nitrogen sources allows these organisms to survive under nutrient stress in specific ecological niches, e.g., in soil or intracellularly. Research on nitrogen degradation in Streptomyces and Mycobacterium has been connected to investigations designed to better understand physiology and metabolic changes during adaptation for survival [11,13,19,20]. Recently, enzymatic and regulatory machinery involved in these processes was studied from perspective of drug development to target parts of nitrogen metabolism, e.g., metabolism of polyamines [22,122,123]. This review focuses on the link between nitrogen degradation in Actinobacteria and studies on identification and validation of drug targets in the enzymatic machinery that allow survival of pathogenic Actinobacteria during the infection process.

5. Conclusions and Future Perspectives

The catabolism of nitrogen-containing compounds, like ammonium, nitrate, amino acids, amino sugars, urea, and amines. has been investigated in Actinobacteria due to its connection to secondary metabolism, specifically in regard to precursor supply for antibiotics production. However, nitrogen catabolism also ensures bacterial survival and pathogenicity. Studies in pathogenic bacterial species including Salmonella typhimurium, Brucella abortus, Mycobacterium tuberculosis, Chlamydia pneumoniae, Legionella pneumophila, Listeria monocytogenes, and some others prove the crucial role of nitrogen and, more specifically, polyamines for their proliferation [124,125].

The interconnection between the biosynthesis, uptake, and assimilation of amines remains crucial in finding new therapeutic drug targets [22,123]. Most human pathogens rely on both polyamine biosynthesis and on polyamine detoxification to proliferate and maintain infection; targeting the metabolism of amines can extend the options for combating bacterial infections [125,126]. There is an urgent need to find new anti-bacterial drugs with novel modes of action that would be efficient on bacterial infections, especially in the light of the emergence of resistances. Investigation of the pathways required for bacterial growth, survival, and pathogenicity in nitrogen metabolism can provide new drug target candidates for the development of more effective agents. Proposal of drug candidates to target nitrogen degradation pathways, especially polyamine utilization, has gained attention during the last decade.