A Second Gamma-Glutamylpolyamine Synthetase, GlnA2, Is Involved in Polyamine Catabolism in Streptomyces coelicolor

Abstract

Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.

Keywords: GS-like enzymes; GlnA; GlnA2; GlnA3; Streptomyces coelicolor; nitrogen assimilation; polyamine catabolism.

Figure 1

Combined model of polyamine utilization pathways within prokaryotes and in S. coelicolor based on published studies [2,4,12,13,14,15,17]. GGP, gamma-glutamylation pathway; AMTP, aminotransferase pathway; DOP, direct oxidation pathway; SPDP, spermine/spermidine dehydrogenase pathway; ACP, acetylation pathway. Dashed arrows represent predicted metabolic pathways. Enzymes in red: confirmed gamma-glutamylpolyamine synthetases in S. coelicolor. Proteins in blue: confirmed role in polyamine utilization in S. coelicolor. Proteins in grey: predicted role in polyamine utilization in S. coelicolor.

Figure 2

Model structure of GlnA2 from Streptomyces coelicolor. The overall dodecamer structure is based on the GS1 from S. typhimurium (PDB: 1FPY) that was used as a template (A). The sub-units A and B are zoomed out and depicted in light red and are superposed with the 1FPY template (yellow) (B). The active site is shown with the identified key amino acid substitutions in GlnA2 (red) compared with GS1 from S. typhimurium (dark yellow) (C).

Figure 3

HPLC/ESI-MS analysis of the glutamylated reaction product generated by His-GlnA2. Two samples were analyzed in the MS positive mode (EIC—extracted ion chromatogram): reaction mixtures without addition of GlnA2 (below) and with addition of GlnA2 (above). Extracted ion chromatograms for the GlnA2 reaction product corresponding to gamma-glutamylputrescine with charge to mass ratio of 218 m/z is shown (above), and no product in the sample without GlnA2 could be detected (below). The chromatogram demonstrates detected peaks at the retention time 18 min and later (for full chromatogram see Figure S2).

Figure 4

Effect of different amines on the activity of GlnA2 and GlnA3. All substrates were at a concentration of 50 mM. The mean value of n = 6 biological replicates from different cultures with n = 3 technical replicates each with standard error is shown.

Figure 5

Transcription analysis of glnA2 in S. coelicolor M145 under different N-conditions. RT-PCR of glnA2 and hrdB in S. coelicolor M145 cultivated in minimal Evans medium with: (A) low 5 mM (Gln−) or high 50 mM (Gln+) glutamine concentration; (B) high 25 mM polyamine concentration (Put: putrescine, Cad: cadaverine, Sd: spermidine); (C) low 5 mM (NH₄⁻) or high 50 mM (NH₄⁺) ammonium/low 2.5 g/L (Glc−) or high 25 g/L (Glc+) glucose concentration. Total RNA was isolated from mycelium harvested after 0–48 h (A) or 24 h (B,C) of cultivation in the defined Evans medium.

Figure 6

Growth of S. coelicolor M145 as well as glnA2, glnA3, and glnA4 mutants on polyamine containing media. (A) Phenotypic analysis of strains on the rich LB-medium in the presence of 100 mM putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Sp) or polyamine mixture (25 mM each). (B) Phenotypic analysis of strains on defined Evans medium with glutamine (50 mM) (Gln), glutamate (50 mM) (Glu), ammonium (100 mM) (NH₄Cl), nitrate (100 mM), urea (50 mM). (C) Phenotypic analysis of strains on defined Evans medium with putrescine (100 mM), cadaverine (100 mM), spermidine (100 mM), and spermine (50 mM). Each panel represents observations on a single agar plate; observations on separate agar plates are indicated by the dashed line.

Figure 7

Intracellular polyamine concentration in S. coelicolor strains. The polyamine level of the ΔglnA2 mutant and parental strain S. coelicolor M145 was monitored in samples taken after 24, 48, 72, and 96 h of cultivation in defined Evans medium supplemented with polyamines (Put—putrescine; Cad—cadaverine, or Spd—spermidine, 25 mM of each) as a sole nitrogen source. The mean value of three biological replicates was calculated in μmol per 1 g of wet cells. Error bars indicate standard error of two biological replicates.

Figure 8

Band shift assays performed with GlnR and EpuRII. (A) 1 ng fluorescence-labeled glnA2 promoter region was incubated without (−) or with (+) 0.5–2 μg Strep-GlnR (increasing arrow). As a control 1000-fold amount of unmarked specific (K1) or non-specific DNA (K2) was added. (B) 1 ng fluorescence-labeled promoter regions of polyamine associated genes were incubated without (−) or with (+) 2 μg His-EpuRII. As a control 1000-fold amount of specific unlabeled DNA fragments (K) was added.