Nitrogen is an essential element required for bacterial growth. It serves as a building block for the biosynthesis of macromolecules and provides precursors for secondary metabolites. Bacteria have developed the ability to use various nitrogen sources and possess two enzyme systems for nitrogen assimilation involving glutamine synthetase/glutamate synthase and glutamate dehydrogenase. Microorganisms living in habitats with changeable availability of nutrients have developed strategies to survive under nitrogen limitation. One adaptation is the ability to acquire nitrogen from alternative sources including the polyamines putrescine, cadaverine, spermidine and spermine, as well as the monoamine ethanolamine. Bacterial polyamine and monoamine metabolism is not only important under low nitrogen availability, but it is also required to survive under high concentrations of these compounds. Such conditions can occur in diverse habitats such as soil, plant tissues and human cells. Strategies of pathogenic and non-pathogenic bacteria to survive in the presence of poly- and monoamines offer the possibility to combat pathogens by using their capability to metabolize polyamines as an antibiotic drug target. This work aims to summarize the knowledge on poly- and monoamine metabolism in bacteria and its role in nitrogen metabolism.

The natural soil environment of Streptomyces is characterized by variations in the availability of nitrogen, carbon, phosphate and sulfur, leading to complex primary and secondary metabolisms. Their remarkable ability to adapt to fluctuating nutrient conditions is possible through the utilization of a large amount of substrates by diverse intracellular and extracellular enzymes. Thus, Streptomyces fulfill an important ecological role in soil environments, metabolizing the remains of other organisms. In order to survive under changing conditions in their natural habitats, they have the possibility to fall back on specialized enzymes to utilize diverse nutrients and supply compounds from primary metabolism as precursors for secondary metabolite production. We aimed to summarize the knowledge on the C-, N-, P- and S-metabolisms in the genus Streptomyces as a source of building blocks for the production of antibiotics and other relevant compounds.

Soil-dwelling Streptomyces bacteria such as S. coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex N-rich medium GlnR was phosphorylated on six serine/ threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex. This study clearly demonstrated that GlnR DNA-binding affinity is modulated by post-translational modifications in response to changing N-conditions in order to elicit a proper transcriptional response to the latter.

Until now, knowledge of the utilization of ethanolamine in Streptomyces was limited. Our work represents the first attempt to reveal a novel ethanolamine utilization pathway in the actinobacterial model organism S. coelicolor through the characterization of the key enzyme gamma-glutamylethanolamide synthetase GlnA4, which is absolutely required for growth in the presence of ethanolamine. The novel ethanolamine utilization pathway is dissimilar to the currently known ethanolamine utilization pathway, which occurs in metabolome. The novel ethanolamine utilization pathway does not result in the production of toxic by-products (such as acetaldehyde); thus, it is not encapsulated. We believe that this contribution is a milestone in understanding the ecology of Streptomyces and the utilization of alternative nitrogen sources. Our report provides new insight into bacterial primary metabolism, which remains complex and partially unexplored.

Actinobacteria from the genus Streptomyces feature complex primary and secondary metabolism, developmental cycle, and ability to produce a variety of natural products. These soil bacteria are major producers of antibiotics and other bioactive compounds and have been extensively investigated due to the medical and industrial relevance of Streptomyces-derived secondary metabolites. However, the genetic toolbox for Streptomyces engineering as well as yield optimization strategies for the production of relevant metabolites are limited. On the one hand, the genetic potential of these organisms has not been fully utilized due to many “silent” or poorly expressed biosynthetic gene clusters, whose activation depends on environmental stimuli and nutrient availability. On the other hand, these GC-rich Gram-positive bacteria are difficult to manipulate, and traditional genetic manipulation strategies are time-consuming and have low efficiency. Recent studies of Streptomyces metabolism and genomes provided new insights into possibilities to overcome these challenges. In this review, advances and approaches for Streptomyces manipulations and secondary metabolite production optimization are discussed. Special focus is given to understanding the interplay between primary and secondary metabolism in Streptomyces and the supply of nitrogen-containing compounds into secondary metabolism. Existing strategies to manipulate cellular metabolism in Streptomyces are reviewed.

S. coelicolor M145 was shown to be able to grow in the presence of high concentrations of polyamines, such as putrescine, cadaverine, spermidine or spermine, as a sole nitrogen source. However, hardly anything is known about polyamine utilization and its regulation in streptomycetes. In this study we demonstrated that only one of the three proteins annotated as glutamine synthetase-like protein, GlnA3 (SCO6962), was involved in the catabolism of polyamines. Transcriptional analysis revealed that the expression of glnA3 was strongly induced by exogenous polyamines and repressed in the presence of ammonium. The ∆glnA3 mutant was shown to be unable to grow on defined Evans agar supplemented with putrescine, cadaverine, spermidine and spermine as sole nitrogen source. HPLC analysis demonstrated that the ∆glnA3 mutant accumulated polyamines intracellularly, but was unable to degrade them. In a rich complex medium supplemented with a mixture of the four different polyamines, the ∆glnA3 mutant grew poorly showing abnormal mycelium morphology and decreased life span in comparison to the parental strain. These observations indicated that the accumulation of polyamines was toxic for the cell. An in silico analysis of the GlnA3 protein model suggested that it might act as a gamma-glutamylpolyamine synthetase catalyzing the first step of polyamine degradation. GlnA3-catalyzed glutamylation of putrescine was confirmed in an enzymatic in vitro assay and the GlnA3 reaction product, gamma-glutamylputrescine, was detected by HPLC/ESI-MS. In this work, the first step of polyamine utilization in S. coelicolor has been elucidated and the putative polyamine utilization pathway has been deduced based on the sequence similarity and transcriptional analysis of homologous genes expressed in the presence of polyamines.