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TrpM, a Small Protein Modulating Tryptophan Biosynthesis and Morpho-Physiological Differentiation in Streptomyces coelicolor A3(2)

View Article: PubMed Central - PubMed

ABSTRACT

In the model actinomycete Streptomyces coelicolor A3(2), small open reading frames encoding proteins with unknown functions were identified in several amino acid biosynthetic gene operons, such as SCO2038 (trpX) in the tryptophan trpCXBA locus. In this study, the role of the corresponding protein in tryptophan biosynthesis was investigated by combining phenotypic and molecular analyses. The 2038KO mutant strain was characterized by delayed growth, smaller aerial hyphae and reduced production of spores and actinorhodin antibiotic, with respect to the WT strain. The capability of this mutant to grow on minimal medium was rescued by tryptophan and tryptophan precursor (serine and/or indole) supplementation on minimal medium and by gene complementation, revealing the essential role of this protein, here named TrpM, as modulator of tryptophan biosynthesis. His-tag pull-down and bacterial adenylate cyclase-based two hybrid assays revealed TrpM interaction with a putative leucyl-aminopeptidase (PepA), highly conserved component among various Streptomyces spp. In silico analyses showed that PepA is involved in the metabolism of serine, glycine and cysteine through a network including GlyA, CysK and CysM enzymes. Proteomic experiments suggested a TrpM-dependent regulation of metabolic pathways and cellular processes that includes enzymes such as GlyA, which is required for the biosynthesis of tryptophan precursors and key proteins participating in the morpho-physiological differentiation program. Altogether, these findings reveal that TrpM controls tryptophan biosynthesis at the level of direct precursor availability and, therefore, it is able to exert a crucial effect on the morpho-physiological differentiation program in S. coelicolor A3(2).

No MeSH data available.


Actinorhodin antibiotic production of S. coelicolor 2038KO.Production of actinorhodin in 2038KO and WT on MM, and in 2038KO on MM-Trp after 4 days of incubation. Histograms report values from three different cultivations. Vertical bars represent standard deviations.
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pone.0163422.g003: Actinorhodin antibiotic production of S. coelicolor 2038KO.Production of actinorhodin in 2038KO and WT on MM, and in 2038KO on MM-Trp after 4 days of incubation. Histograms report values from three different cultivations. Vertical bars represent standard deviations.

Mentions: In S. coelicolor A3(2), Trp is used as an amino acid building block for protein biosynthesis and as carbon and nitrogen sources [10]. Consequently, it positively influences morphological differentiation and production of antibiotics by promoting the accumulation of: i) pleiotropic gene products associated with life-cycle progression; and ii) specific enzymes involved in many anabolic and catabolic process [10]. In order to evaluate the effect of the SCO2038 deletion, production of the antibiotic actinorhodin (ACT) was quantitatively analyzed in the 2038KO strain using a spectrophotometric assay [16]. Results of this analysis revealed that the amount of ACT produced by the mutant strain was more than ten-fold lower than that produced by the WT (Fig 3). The addition of Trp to the 2038KO mutant cultures considerably increased ACT production (Fig 3), confirming the positive effect of Trp on ACT biosynthesis [10]. Furthermore, Scanning Electron Microscopy (SEM) analysis of strain 2038KO showed that morphological development was compromised. In particular, after 5 days of incubation on MM, the mutant strain showed small aerial hyphae, and immature spore chains with few and faint septal constrictions (Fig 4A). Trp supplementation modestly improved mycelium morphology and sporulation. This evidence was further corroborated by spore quantification (Fig 4B), which suggested the involvement of TrpM in the morphological differentiation of S. coelicolor A3(2).


TrpM, a Small Protein Modulating Tryptophan Biosynthesis and Morpho-Physiological Differentiation in Streptomyces coelicolor A3(2)
Actinorhodin antibiotic production of S. coelicolor 2038KO.Production of actinorhodin in 2038KO and WT on MM, and in 2038KO on MM-Trp after 4 days of incubation. Histograms report values from three different cultivations. Vertical bars represent standard deviations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036795&req=5

pone.0163422.g003: Actinorhodin antibiotic production of S. coelicolor 2038KO.Production of actinorhodin in 2038KO and WT on MM, and in 2038KO on MM-Trp after 4 days of incubation. Histograms report values from three different cultivations. Vertical bars represent standard deviations.
Mentions: In S. coelicolor A3(2), Trp is used as an amino acid building block for protein biosynthesis and as carbon and nitrogen sources [10]. Consequently, it positively influences morphological differentiation and production of antibiotics by promoting the accumulation of: i) pleiotropic gene products associated with life-cycle progression; and ii) specific enzymes involved in many anabolic and catabolic process [10]. In order to evaluate the effect of the SCO2038 deletion, production of the antibiotic actinorhodin (ACT) was quantitatively analyzed in the 2038KO strain using a spectrophotometric assay [16]. Results of this analysis revealed that the amount of ACT produced by the mutant strain was more than ten-fold lower than that produced by the WT (Fig 3). The addition of Trp to the 2038KO mutant cultures considerably increased ACT production (Fig 3), confirming the positive effect of Trp on ACT biosynthesis [10]. Furthermore, Scanning Electron Microscopy (SEM) analysis of strain 2038KO showed that morphological development was compromised. In particular, after 5 days of incubation on MM, the mutant strain showed small aerial hyphae, and immature spore chains with few and faint septal constrictions (Fig 4A). Trp supplementation modestly improved mycelium morphology and sporulation. This evidence was further corroborated by spore quantification (Fig 4B), which suggested the involvement of TrpM in the morphological differentiation of S. coelicolor A3(2).

View Article: PubMed Central - PubMed

ABSTRACT

In the model actinomycete Streptomyces coelicolor A3(2), small open reading frames encoding proteins with unknown functions were identified in several amino acid biosynthetic gene operons, such as SCO2038 (trpX) in the tryptophan trpCXBA locus. In this study, the role of the corresponding protein in tryptophan biosynthesis was investigated by combining phenotypic and molecular analyses. The 2038KO mutant strain was characterized by delayed growth, smaller aerial hyphae and reduced production of spores and actinorhodin antibiotic, with respect to the WT strain. The capability of this mutant to grow on minimal medium was rescued by tryptophan and tryptophan precursor (serine and/or indole) supplementation on minimal medium and by gene complementation, revealing the essential role of this protein, here named TrpM, as modulator of tryptophan biosynthesis. His-tag pull-down and bacterial adenylate cyclase-based two hybrid assays revealed TrpM interaction with a putative leucyl-aminopeptidase (PepA), highly conserved component among various Streptomyces spp. In silico analyses showed that PepA is involved in the metabolism of serine, glycine and cysteine through a network including GlyA, CysK and CysM enzymes. Proteomic experiments suggested a TrpM-dependent regulation of metabolic pathways and cellular processes that includes enzymes such as GlyA, which is required for the biosynthesis of tryptophan precursors and key proteins participating in the morpho-physiological differentiation program. Altogether, these findings reveal that TrpM controls tryptophan biosynthesis at the level of direct precursor availability and, therefore, it is able to exert a crucial effect on the morpho-physiological differentiation program in S. coelicolor A3(2).

No MeSH data available.