Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

Dongmei Xu,Zixin Deng,Lin Cheng,Xinhua Lu,Wenqing Chen,Guang Liu,Bin He

doi:10.1371/journal.pone.0076068

Dongmei Xu, Zixin Deng + Show 5 more

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https://doi.org/10.1371/journal.pone.0076068

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Journal: PLoS ONE	Publication Date: Oct 15, 2013
Citations: 11	License type: CC BY 4.0

Affiliation: Shanghai Jiao Tong University, Wuhan University

Abstract

BackgroundMuraymycin, a potent translocase I (MraY) inhibitor, is produced by Streptomyces sp. NRRL30471. The muraymycin gene cluster (mur) was recently cloned, and bioinformatic analysis of mur34 revealed its encoding product exhibits high homology to a large family of proteins, including KanI and RacI in individual biosynthetic pathway of kanamycin and ribostamycin. However, the precise role of these proteins remains unknown.Principal FindingsHere we report the identification of Mur34 as the novel negative regulator involved in muraymycin biosynthesis. Independent disruption of mur34 on chromosome and cosmid directly resulted in significant improvement of muraymycin production by at least 10 folds, thereof confirming the negative function of Mur34 during muraymycin biosynthesis and realizing the engineered production of muraymycin in heterologous host. Gene expression analysis indicated that the transcription level of the mur genes in mur34 mutant (DM-5) was dramatically enhanced by ca. 30 folds. Electrophoretic mobility shift assay (EMSA) showed that Mur34 specifically bound to the promoter region of mur33. Further experiments showed that a 28-bp region downstream of the transcription start point (TSP) was protected by His6Mur34, and the −10 region is essential for the activity of mur33 promoter.ConclusionsMur34 plays an unambiguously negative role in muraymycin biosynthesis via binding to the upstream of mur33. More importantly, Mur34 represents a novel family of regulators acting in negative manner to regulate the secondary metabolites biosynthesis in bacteria.

Highlights

Streptomycetes are usually soil-living organisms with complex life cycle that includes formation of aerial mycelia and spores
Mur34 plays an unambiguously negative role in muraymycin biosynthesis via binding to the upstream of mur33
BlastP analysis of Mur34 shows that it displays significant homology to LivI (74% identities), RacA (76% identities), and KanI (74% identities) which are correspondingly involved in the biosynthesis of lividomycin, ribostamycin and kanamycin (Fig. S1, A), while the precise function of the proteins remains unraveled

Summary

Introduction

Streptomycetes are usually soil-living organisms with complex life cycle that includes formation of aerial mycelia and spores. Members of this genus have relatively large genomes and the capability of producing tremendous number of secondary metabolites, many of which have been used as antibiotics, anti-tumor agents, and immunosuppressants [1]. Muraymycins, a group of structurally related nucleoside antibiotics, are powerful translocase I (MraYs) inhibitors. This family of antibiotics including well-characterized pacidamycin and caprazamycin was recently pursued for their unusual structures and outstanding bioactivity with clinic potential [2] (Fig. 1A).

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