Abstract
Because of the remarkable efficacy in treating mycobacterial infections, rifamycin and its derivatives are still first-line antimycobacterial drugs. It has been intensely studied to increase rifamycin yield from Amycolatopsis mediterranei, and nitrate is found to provide a stable and remarkable stimulating effect on the rifamycin production, a phenomenon known as “nitrate-stimulating effect (NSE)”. Although the NSE has been widely used for the industrial production of rifamycin, its detailed molecular mechanism remains ill-defined. And our previous study has established that the global nitrogen regulator GlnR may participate in the NSE, but the underlying mechanism is still enigmatic. Here, we demonstrate that GlnR directly controls rifamycin biosynthesis in A. mediterranei and thus plays an essential role in the NSE. Firstly, GlnR specifically binds to the upstream region of rifZ, which leads us to uncover that rifZ has its own promoter. As RifZ is a pathway-specific activator for the whole rif cluster, GlnR indirectly upregulates the whole rif cluster transcription by directly activating the rifZ expression. Secondly, GlnR specifically binds to the upstream region of rifK, which is also characterized to have its own promoter. It is well-known that RifK is a 3-amino-5-hydroxybenzoic acid (AHBA, the starter unit of rifamycin) synthase, thus GlnR can promote the supply of the rifamycin precursor by directly activating the rifK transcription. Notably, GlnR and RifZ independently activate the rifK transcription through binding to different sites in rifK promoter region, which suggests that the cells have a sophisticated regulatory mechanism to control the AHBA biosynthesis. Collectively, this study reveals that GlnR activates the rif cluster transcription in both direct (for rifZ and rifK) and indirect (for the whole rif cluster) manners, which well interprets the phenomenon that the NSE doesn’t occur in the glnR null mutant. Furthermore, this study deepens our understanding about the molecular mechanism of the NSE.
Highlights
Being a broad-spectrum antibiotic with unique property of inhibiting the prokaryotic RNA polymerase activity (Wehrli et al, 1968; Campbell et al, 2001), rifamycin and its derivatives are still broadly used as the first-line antimycobacterial agents (Rothstein, 2016)
GlnR was integrated with the vector to obtain glnR/803 (Lin et al, 2014), which was used to investigate the influence of the empty plasmid on both bacterial growth and rifamycin production
The glnR showed a little higher growth rate than the wild type U32 when grown in liquid Bennet medium without nitrate, it had a significantly lower yield of rifamycin than the yield of U32 (Supplementary Figure S2), which was similar with the above results under nitrate condition
Summary
Being a broad-spectrum antibiotic with unique property of inhibiting the prokaryotic RNA polymerase activity (Wehrli et al, 1968; Campbell et al, 2001), rifamycin and its derivatives (e.g., rifampicin, rifabutin, rifapentine, and rifaximin) are still broadly used as the first-line antimycobacterial agents (Rothstein, 2016). Multiple strategies have been tested, including both engineering the producer Amycolatopsis mediterranei (a rare actinomycete) (Peano et al, 2014; Kumari et al, 2016) and optimizing fermentation conditions (Jiao et al, 1979; Lee et al, 1983; Mejia et al, 1998). Among those tested conditions, supplementation of nitrate was found to be able to remarkably stimulate rifamycin production, which has become known as the “nitratestimulating effect (NSE)” (Jiao et al, 1979). Our studies have revealed that RifZ is a pathway-specific regulator of the rif cluster and directly activates the transcription of all rif cluster genes (Li et al, 2017), and RifQ could reduce the intracellular toxicity of rifamycin via directly regulating the expression of the rifamycin exporter-encoding gene rifP (Lei et al, 2018)
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