Gamma-Glutamylpolyamine Synthetase GlnA3 Is Involved in the First Step of Polyamine Degradation Pathway in Streptomyces coelicolor M145

Abstract

Streptomyces 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 ΔglnA3 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 ΔglnA3 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 ΔglnA3 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.

Keywords: GS-like enzyme; GlnA3; Streptomyces coelicolor; gamma-glutamylpolyamine synthetase; nitrogen assimilation; polyamine utilization.

FIGURE 1

Combined model of polyamine utilization pathways within prokaryotes based on published studies (Yao et al., 2011; Schneider and Reitzer, 2012; Foster et al., 2013; Campilongo et al., 2014; Kusano and Suzuki, 2015). GGP, gamma-glutamylation pathway; AMTP, aminotransferase pathway; DOP, direct oxidation pathway; SPDP, spermine/spermidine dehydrogenase pathway; ACP, acetylation pathway. Dashed arrows represent not entirely investigated metabolic pathways. In gray, deduced steps of the polyamine degradation pathway in S. coelicolor.

FIGURE 2

Phenotypic analysis of parental strain S. coelicolor M145 and mutants: ΔglnA, ΔglnII and ΔglnAΔglnII (1, 2, 3 – independent clones) on defined Evans medium supplemented with 50 mM ammonium chloride (A) or with 50 mM ammonium chloride and 50 mM L-glutamine (B). Deletion of both genes, glnA and glnII, for GSs, resulted in a glutamine auxotrophic phenotype indicating that GlnA2, GlnA3, and GlnA4 were incapable to substitute GSI and GSII metabolic function in the cell.

FIGURE 3

Physiological role of the glnA3 gene product in S. coelicolor M145 cells grown in the presence of polyamines. (A) Phenotypic comparison of parental strain S. coelicolor M145 and ΔglnA2, ΔglnA3, and ΔglnA4 mutants grown on defined Evans medium supplemented with Put – putrescine dihydrochloride (200 mM), Cad – cadaverine dihydrochloride (50 mM), Sm – spermine tetrahydrochloride (25 mM), Sd – spermidine trihydrochloride (25 mM) as well as on Glu – monosodium glutamate (50 mM), Gln – glutamine (50 mM), NH₄Cl – ammonium chloride (50 mM) and NaNO₃ – sodium nitrate (50 mM) as sole nitrogen source. Each panel represents observations on a single agar plate, except the phenotypic analysis of the glnA3 mutant and parental strain in the presence of glutamate has been documented on two separate agar plates as indicated by the dotted line. Deletion of glnA3 resulted in the no-growth phenotype in the presence of high polyamine concentrations.

FIGURE 4

Transcriptional analysis of glnA3 in the presence of polyamines and ammonium as a sole nitrogen sources. Reverse transcriptase/PCR of glnA3 and hrdB (control) from S. coelicolor M145 cultivated in defined Evans medium with low (5 mM) or high (50 mM) concentrations of ammonium chloride or polyamines (Put – putrescine; Cad – cadaverine and Spd – spermidine, 25 mM of each) and glucose as a sole carbon source high Glc+ (25 g/l) or low Glc– (2.5 g/l). Total RNA was isolated from mycelium harvested after 24 h of cultivation in the defined Evans medium.

FIGURE 5

Transcriptional analyses of putative gene homologs encoding predicted proteins involved in polyamine degradation in S. coelicolor. Reverse transcriptase/PCR of genes and hrdB (control) from S. coelicolor M145 cultivated in defined Evans medium with polyamines (Put – putrescine; Cad – cadaverine or Spd – spermidine, 25 mM of each) and glucose as a sole carbon source Glc (25 g/l). Total RNA was isolated from mycelium harvested after 24 h of cultivation in the defined Evans medium.

FIGURE 6

Effect of glnA3 deletion on intracellular polyamine concentration. The intracellular polyamine level of parental strain S. coelicolor M145 and ΔglnA3 mutant was monitored after 24 and 96 h of cultivation in defined Evans medium supplemented with polyamines (Put – putrescine; Cad – cadaverine or Sd – spermidine, 25 mM of each). The mean value of three biological replicates was calculated in μmol per 1 g of wet cells. Error bars indicate standard error of three biological replicates.

FIGURE 7

HPLC-based detection of polyamines in supernatant from the 96 h culture of parental strain M145 and ΔglnA3 mutant. Both strains were grown in defined Evans medium supplemented with either (A) putrescine (25 mM), (B) cadaverine (25 mM) or (C) spermidine (25 mM). Upper HPLC chromatogram: polyamine standard, middle HPLC chromatogram: detection of polyamines remained in the supernatant of the M145 culture, lower HPLC chromatogram: detection of polyamines remained in the supernatant of the ΔglnA3 mutant culture. Results indicate that only parental strain S. coelicolor M145 was able to completely utilize whole polyamines from the medium after 96 h of incubation.

FIGURE 8

Effect of the glnA3 deletion on a biomass accumulation in a rich complex medium supplemented with and without polyamines. Monitoring of changes in biomass accumulation of the parental strain S. coelicolor M145 and ΔglnA3 mutant in the YEME:TSB (1:1) medium supplemented with polyamines total concentration 100 mM (putrescine, cadaverine, spermidine, and spermine, 25 mM of each) after 72 and 168 h of incubation. Error bars indicate standard error of three biological replicates.

FIGURE 9

Effect of the glnA3 deletion on cell morphology and its viability in the presence of polyamines. Parental strain S. coelicolor M145 and ΔglnA3 mutant were cultivated in YEME:TSB (1:1) medium with or without polyamines total concentration 100 mM (putrescine, cadaverine, spermidine, and spermine, 25 mM of each). Phase contrast microscopic pictures of parental strain M145 and ΔglnA3 mutant mycelium stained with SYTO9/PI were taken after 72 and 168 h of growth (under 400× enlargement).

FIGURE 10

Comparison of the hyphae thickness of parental strain S. coelicolor M145 and ΔglnA3 mutant grown in the presence of polyamines. Parental strain S. coelicolor M145 and ΔglnA3 mutant were cultivated in YEME:TSB (1:1) medium with or without polyamines. The hyphae thickness was measured after 72 and 168 h of growth. Error bars indicate standard error of n = 100 biological replicates.

FIGURE 11

HPLC/ESI-MS analysis of the glutamylated product generated by GlnA3 in in vitro assay. Two samples were analyzed in the MS positive mode: reaction mixtures without addition of GlnA3 (A) and with addition of GlnA3 (B). Extracted ion chromatograms for the GlnA3 reaction product corresponding to gamma-glutamylputrescine with charge to mass ratio of 218 m/z was shown (B), and no product in the sample without addition of GlnA3 could be detected (A).