Initial Metabolic Step of a Novel Ethanolamine Utilization Pathway and Its Regulation in Streptomyces coelicolor M145

Abstract

Streptomyces coelicolor is a Gram-positive soil bacterium with a high metabolic and adaptive potential that is able to utilize a variety of nitrogen sources. However, little is known about the utilization of the alternative nitrogen source ethanolamine. Our study revealed that S. coelicolor can utilize ethanolamine as a sole nitrogen or carbon (N/C) source, although it grows poorly on this nitrogen source due to the absence of a specific ethanolamine permease. Heterologous expression of a putative ethanolamine permease (SPRI_5940) from Streptomycespristinaespiralis positively influenced the biomass accumulation of the overexpression strain grown in defined medium with ethanolamine. In this study, we demonstrated that a glutamine synthetase-like protein, GlnA4 (SCO1613), is involved in the initial metabolic step of a novel ethanolamine utilization pathway in S. coelicolor M145. GlnA4 acts as a gamma-glutamylethanolamide synthetase. Transcriptional analysis revealed that expression of glnA4 was induced by ethanolamine and repressed in the presence of ammonium. Regulation of glnA4 is governed by the transcriptional repressor EpuRI (SCO1614). The ΔglnA4 mutant strain was unable to grow on defined liquid Evans medium supplemented with ethanolamine. High-performance liquid chromatography (HPLC) analysis demonstrated that strain ΔglnA4 is unable to utilize ethanolamine. GlnA4-catalyzed glutamylation of ethanolamine was confirmed in an enzymatic in vitro assay, and the GlnA4 reaction product, gamma-glutamylethanolamide, was detected by HPLC/electrospray ionization-mass spectrometry (HPLC/ESI-MS). In this work, the first step of ethanolamine utilization in S. coelicolor M145 was elucidated, and a putative ethanolamine utilization pathway was deduced based on the sequence similarity and genomic localization of homologous genes. IMPORTANCE 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.

Poly- and Monoamine Metabolism in <b><i>Streptomyces coelicolor</i></b>: The New Role of Glutamine Synthetase-Like Enzymes in the Survival under Environmental Stress

“…and Clostridium sp. [Tsoy et al, 2009;Garsin, 2010], and recently in S. coelicolor [Krysenko et al, 2019].…”

“…Since GS-like proteins are not able to synthesize glutamine from ammonium and glutamate, it was hypothesized that they may not accept ammonium as substrate, but other nitrogen containing compounds, which are typically present in soil. A phenotypic analysis of glnA2, glnA3 and glnA4 knockout mutants in the presence of different sole nitrogen sources finally revealed that GS-like enzymes can accept poly-and monoamines as substrates [Krysenko et al, 2017;2019].…”

Antibiotic Production and Antibiotic Resistance: The Two Sides of AbrB1/B2, a Two-Component System of Streptomyces coelicolor

“…The operon SCO1611-13 was up-regulated in the mutant strain at 36 h. Although the entire operon is related with ethanolamine metabolism, only glnA4 ( SCO1613 ) has been studied more in depth. GlnA4 is a γ-glutamyl-ethanolamine synthetase, an enzyme that allows S. coelicolor to use ethanolamine as an alternative nitrogen/carbon source ( Rexer et al, 2006 ; Krysenko et al, 2019 ).…”

Novel Glutamate–Putrescine Ligase Activity in Haloferax mediterranei: A New Function for glnA-2 Gene

“…Other examples of GS-like proteins have been described in Pseudomonas sp. KIE171, in which the IpuC protein annotated as GS-like, is involved in the degradation of isopropylamine [57]; in E. coli K-12, the puuA gene was initially annotated as GS, subsequently demonstrating that this gene encodes a glutamate-putrescine ligase [54]; and in S. coelicolor M145, the GlnA3 protein is involved in the degradation of polyamines encoding a γ-glutamylpolyamine synthetase [22], and the GlnA4 protein in the degradation of ethanolamine [23].…”

“…In S. coelicolor M145, it has been demonstrated that the GlnA3 protein (SCO6962), first annotated as GS, is involved in the catabolism of polyamines, and is expressed under ammonium limitation conditions and with low glucose concentration [22]. Another GS-like protein from S. coelicolor M145, GlnA4 (SCO1613), is involved in the metabolism of a new ethanolamine pathway, in which the GlnA4 protein acts as a γ-glutamyl-ethanolamide synthetase [23].…”