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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).

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Related in: MedlinePlus

Growth of S. coelicolor 2038KO mutant strain on solid MM.Growth of 2038KO (1) and WT (2) on solid MM after 24, 48 and 72 h (A) and solid MM-Trp (B). (C) Growth of WT (2) (1), 2038KO (2), pKC796Hyg (3), pKC796Hyg-sco2038 (4) on solid MM with the antibiotic hygromycin.
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pone.0163422.g001: Growth of S. coelicolor 2038KO mutant strain on solid MM.Growth of 2038KO (1) and WT (2) on solid MM after 24, 48 and 72 h (A) and solid MM-Trp (B). (C) Growth of WT (2) (1), 2038KO (2), pKC796Hyg (3), pKC796Hyg-sco2038 (4) on solid MM with the antibiotic hygromycin.

Mentions: The SCO2038 smORF located in trpCXBA locus putatively encodes an unknown protein highly conserved among streptomycetes, and which does not show similarity to any protein having a known function (see S1 File). In order to assess the involvement of the SCO2038 product in Trp biosynthesis, we generated a SCO2038 knockout mutant strain (hereafter called 2038KO) using the ReDirect PCR-targeting technology [14], where the coding region was replaced with an apramycin resistance (apr) cassette. Interestingly, we observed that the SCO2038 deletion caused a 2 day-delay in growth of the 2038KO strain on minimal medium (MM), when compared to the wild type (WT) strain (Fig 1A). The phenotype of strain 2038KO was abolished by supplementation of Trp to the MM (MM-Trp) (Fig 1B) and by complementation of the mutation performed by introducing the SCO2038 gene into 2038KO using the pKC796Hyg vector (pKC796Hyg-SCO2038) (Fig 1C). The resulting complemented strain rescued the ability to grow on MM (Fig 1C). Moreover, this last result suggested that the SCO2038 downstream genes trpBA present in trpCXBA locus can be expressed independently from SCO2038, as also confirmed by (q)RT-PCR (data not shown), thus excluding the possibility that the delayed growth was caused by a polar effect.


TrpM, a Small Protein Modulating Tryptophan Biosynthesis and Morpho-Physiological Differentiation in Streptomyces coelicolor A3(2)
Growth of S. coelicolor 2038KO mutant strain on solid MM.Growth of 2038KO (1) and WT (2) on solid MM after 24, 48 and 72 h (A) and solid MM-Trp (B). (C) Growth of WT (2) (1), 2038KO (2), pKC796Hyg (3), pKC796Hyg-sco2038 (4) on solid MM with the antibiotic hygromycin.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163422.g001: Growth of S. coelicolor 2038KO mutant strain on solid MM.Growth of 2038KO (1) and WT (2) on solid MM after 24, 48 and 72 h (A) and solid MM-Trp (B). (C) Growth of WT (2) (1), 2038KO (2), pKC796Hyg (3), pKC796Hyg-sco2038 (4) on solid MM with the antibiotic hygromycin.
Mentions: The SCO2038 smORF located in trpCXBA locus putatively encodes an unknown protein highly conserved among streptomycetes, and which does not show similarity to any protein having a known function (see S1 File). In order to assess the involvement of the SCO2038 product in Trp biosynthesis, we generated a SCO2038 knockout mutant strain (hereafter called 2038KO) using the ReDirect PCR-targeting technology [14], where the coding region was replaced with an apramycin resistance (apr) cassette. Interestingly, we observed that the SCO2038 deletion caused a 2 day-delay in growth of the 2038KO strain on minimal medium (MM), when compared to the wild type (WT) strain (Fig 1A). The phenotype of strain 2038KO was abolished by supplementation of Trp to the MM (MM-Trp) (Fig 1B) and by complementation of the mutation performed by introducing the SCO2038 gene into 2038KO using the pKC796Hyg vector (pKC796Hyg-SCO2038) (Fig 1C). The resulting complemented strain rescued the ability to grow on MM (Fig 1C). Moreover, this last result suggested that the SCO2038 downstream genes trpBA present in trpCXBA locus can be expressed independently from SCO2038, as also confirmed by (q)RT-PCR (data not shown), thus excluding the possibility that the delayed growth was caused by a polar effect.

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.


Related in: MedlinePlus