PapR6, a Putative Atypical Response Regulator, Functions as a Pathway-Specific Activator of Pristinamycin II Biosynthesis in Streptomyces pristinaespiralis

Dun, Junling; Zhao, Yawei; Zheng, Guosong; Zhu, Hong; Ruan, Lifang; Wang, Wenfang; Ge, Mei; Jiang, Weihong; Lü, Yinhua

doi:10.1128/jb.02312-14

Cited by 13 publications

(23 citation statements)

References 29 publications

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“…pristinaespiralis fermentation and analysis of pristinamycin production. S. pristinaespiralis fermentation and analysis of pristinamycin production were carried out according to the previously described methods with some modifications (5,8). Briefly, the S. pristinaespiralis strains were grown on RP agar medium at 30°C for 4 to 5 days.…”

Section: Methodsmentioning

confidence: 99%

“…RNA reverse transcription was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, USA). Quantitative real-time reverse transcription (RT)-PCR (qPCR) analysis was performed as described previously (8). The primer pairs used for qPCR are listed in Table S2 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

“…In the bioassay, Bacillus subtilis ATCC 6633 was used as the indicator strain. HPLC analysis of pristinamycin production was performed as described previously (8). The standard curves for pristinamycin quantitative analysis were made using purified PIa and PIIa (98%) obtained from Laiyi Co. Ltd. (Shanghai, China).…”

Section: Methodsmentioning

confidence: 99%

“…pristinaespiralis HCCB10218 (CGMCC 5486), which was isolated after physical and chemical treatments of ATCC 25486, was used as the original strain. S. pristinaespiralis strains were grown at 30°C in RP liquid medium (peptone, 5; yeast extract, 5; valine, 0.5; NaCl, 2; KH 2 PO 4 , 0.5; MgSO 4 ·7H 2 O, 1[in grams per liter]; pH 6.4) for genomic DNA isolation (8). RP agar medium was used for the preparation of spore suspensions and conjugal transfer between E. coli and S. pristinaespiralis (12).…”

Section: Methodsmentioning

confidence: 99%

“…PI biosynthesis is catalyzed by a nonribosomal peptide synthetase (NRPS) complex composed of SnaA, SnaC, and SnaDE, which are responsible for the successive condensation of two proteinogenic amino acids, L-threonine and L-proline, and five aproteinogenic amino acids, including 3-hydroxypicolinic acid, L-aminobutyric acid, 4-oxo-L-pipecolic acid, L-phenylglycine (L-Phg), and 4-N,N-dimethylaminoL-phenylalanine (DMAPA) or N-methyl-4-(methylamino)-understood about the regulation of PI biosynthesis in S. pristinaespiralis. To our knowledge, until now, only seven cluster-situated regulatory genes (from papR1 to papR6 and spbR) and a serine/ threonine protein kinase gene, spy1, were identified as being involved in the regulation of PI biosynthesis (2,5,(7)(8)(9). For the genus Streptomyces, manipulating the regulatory network that controls antibiotic biosynthesis has been proven to be an efficient method for improving the production levels of desired antibiotics (10).…”

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Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis

et al. 2015

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Pristinamycin I (PI), produced by Streptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two proteinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of streptogramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is understood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded by SSDG_03033) was identified as playing a positive role in PI biosynthesis. Its homologue, PaaR, from Corynebacterium glutamicum serves as a transcriptional repressor of the paa genes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator as PaaR. Deletion of paaR led to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identical to the function of its homologue from C. glutamicum, PaaR is also involved in the suppression of paa expression. Given that phenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway of L-phenylglycine (L-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription of paa genes in a ⌬paaR mutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolic flux toward L-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI production of a ⌬paaR mutant was restored by L-Phg supplementation as well as by deletion of the paaABCDE operon in the ⌬paaR mutant. Altogether, this study provides new insights into the regulation of PI biosynthesis by S. pristinaespiralis. IMPORTANCEA better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues for Streptomyces strain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription of paa genes involved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) by affecting the supply of one of seven amino acid precursors, L-phenylglycine, in Streptomyces pristinaespiralis. To our knowledge, this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis in Streptomyces strains. Considering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, it could be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist. P ristinamycin I (PI), produced by Streptomyces pristinaespiralis, is a branched cyclohexadepsipeptide antibiotic and belongs to the B group of streptogramins. PI is coproduced with pristinamycin II (PII), a polyunsaturated cyclopeptidic macrolactone antibiotic, which is a member of the A group of streptogramin antibiotics (1, 2). Two substances are produced in a 30:70 ratio and show a potent synergistic effect with an approximate...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis

et al. 2015

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Identification of two novel regulatory genes involved in pristinamycin biosynthesis and elucidation of the mechanism for AtrA-p-mediated regulation in Streptomyces pristinaespiralis

Wang

Tian

Lei

et al. 2015

Appl Microbiol Biotechnol

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In this study, using a transposon-based strategy, two novel regulatory genes were identified as being involved in the biosynthesis of both pristinamycin I (PI) and II (PII) in Streptomyces pristinaespiralis, including a TetR-family regulatory gene atrA-p (SSDG_00466) and an orphan histidine kinase gene SSDG_02492. The mechanism by which AtrA-p exerted a positive role in pristinamycin production was elucidated. We showed that deletion of atrA-p resulted in a delayed production of both PI and PII as well as reduced PII production. Transcriptional analysis integrated with electrophoretic mobility shift assays (EMSAs) demonstrated that AtrA-p played a positive role in pristinamycin production by directly activating the transcription of two cluster-situated regulatory genes, spbR and papR5, which encode a γ-butyrolactone receptor protein and a TetR-family repressor, respectively. The precise AtrA-p-binding sites upstream of these two targets were determined, which allowed the identification of a relatively conserved binding motif comprising two 5-nt inverted repeats separated by a variable 5-nt sequence (5'-GGAAT-n5-ATTCC-3') possibly required for the regulation of AtrA-like regulators in Streptomyces. Base substitutions of the AtrA-p-binding sites on the genome caused similar decreases in spbR and papR5 transcription as those observed in ∆atrA-p. Taken together, herein, a novel mechanism for AtrA-dependent regulation of antibiotic biosynthesis was revealed in S. pristinaespiralis, which is distinct from those of its homologs, AtrA-c from Streptomyces coelicolor, AtrA-g from Streptomyces griseus, and AtrA from Streptomyces roseosporus that perform their effects in antibiotic biosynthesis directly via pathway-specific activator genes or the biosynthetic structural genes.

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MilR2, a novel TetR family regulator involved in 5-oxomilbemycin A3/A4 biosynthesis in Streptomyces hygroscopicus

Wei

Lei

et al. 2018

Appl Microbiol Biotechnol

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Milbemycins produced by several Streptomyces species are a group of 16-membered macrolides with potent insecticidal and anthelminthic activity. Milbemycin A3/A4, the main components of the milbemycins biosynthetic pathway, and 5-oxomilbemycin A3/A4, the analogs of milbemycin A3/A4 without the reduction of the C-5 keto group, have been developed as acaricides, insecticides, and anthelmintics. However, so far, little is known about the regulation of milbemycins biosynthesis, which has greatly hampered the generation of high producing strains by metabolic engineering. Herein, a TetR family regulator MilR2 (encoded by sbi_00792) was identified being involved in activation of 5-oxomilbemycin A3/A4 biosynthesis in a high 5-oxomilbemycins-producing strain Streptomyces hygroscopicus SIPI-KF. The ΔmilR2 mutant with an in-frame deletion of the MilR2 DNA-binding domain resulted in significantly reduced 5-oxomilbemycin A3/A4 production (approximately 36.9 and 39.7%) at tested two time points, and accordingly introduction of an extra copy of milR2 into SIPI-KF led to enhanced production by 12.6 and 34.4%. We further showed that MilR2 could directly repress the transcription of the gene sbi_00791 encoding a putative hydrolase, which is located divergently from milR2. The precise MilR2-binding site consisting of a 7-nt perfect inverted repeat separated by 10-nt (5'-ACCAACCAGCTGGTAAGGGTTGGT-3') was defined. In situ mutagenesis of the MilR2-binding site resulted in 19.7 and 13.5% decreases in 5-oxomilbemycin A3/A4 production, which is much lower than the decreased rates of ΔmilR2. Collectively, the results demonstrated that MilR2 serves as an activator for 5-oxomilbemycin A3/A4 production and the function of MilR2 is only partially mediated through its repression on the transcription of sbi_00791.

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PapR6, a Putative Atypical Response Regulator, Functions as a Pathway-Specific Activator of Pristinamycin II Biosynthesis in Streptomyces pristinaespiralis

Cited by 13 publications

References 29 publications

Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis

Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis

Identification of two novel regulatory genes involved in pristinamycin biosynthesis and elucidation of the mechanism for AtrA-p-mediated regulation in Streptomyces pristinaespiralis

MilR2, a novel TetR family regulator involved in 5-oxomilbemycin A3/A4 biosynthesis in Streptomyces hygroscopicus

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