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Identification and functional analysis of a nitrate assimilation operon nasACKBDEF from Amycolatopsis mediterranei U32

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Abstract

Nitrate assimilation has been well studied for Gram-negative bacteria but not so much in the Gram-positive actinomycetes up to date. In a rifamycin SV-producing actinomycete, Amycolatopsis mediterranei strain U32, nitrate not only can be used as a sole nitrogen source but also remarkably stimulates the antibiotic production along with regulating the related metabolic enzymes. A gene cluster of nasACKBDEF was cloned from a U32 genomic library by in situ hybridization screening with a heterogeneous nasB probe and confirmed later by whole genome sequence, corresponding to the protein coding genes of AMED_1121 to AMED_1127. These genes were co-transcribed as an operon, concomitantly repressed by ammonium while activated with supplement of either nitrate or nitrite. Genetic and biochemical analyses identified the essential nitrate/nitrite assimilation functions of the encoded proteins, orderly, the assimilatory nitrate reductase catalytic subunit (NasA), nitrate reductase electron transfer subunit (NasC), nitrate/nitrite transporter (NasK), assimilatory nitrite reductase large subunit (NasB) and small subunit (NasD), bifunctional uroporphyrinogen-III synthase (NasE), and an unknown function protein (NasF). Comparing rifamycin SV production and the level of transcription of nasB and rifE from U32 and its individual nas mutants in Bennet medium with or without nitrate indicated that nitrate assimilation function encoded by the nas operon played an essential role in the “nitrate stimulated” rifamycin production but had no effect upon the transcription regulation of the primary and secondary metabolic genes related to rifamycin biosynthesis.

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Acknowledgments

We thank Ying Wang for supplying the strain and Yi Zhong for helpful sequence analysis. The work was financially supported by NSFC (Grant No. 30830002) and Natural Science Foundation of Shanghai, China (Grant No. 11ZR1442900).

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Author notes

  1. Jin Gao

    Present address: University of California at Berkeley, 150 Morgan Hall#3104, Berkeley, CA, 94720, USA

Authors and Affiliations

  1. Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, China

    Zhihui Shao, Jin Gao, Juishen Chiao & Guoping Zhao

  2. State Key Laboratory of Genetic Engineering/Department of Microbiology, School of Life Science, Fudan University, 220 Handan Road, 200433, Shanghai, China

    Xiaoming Ding & Guoping Zhao

  3. Department of Microbiology and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China

    Jin Wang & Guoping Zhao

  4. Shanghai-MOST Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center at Shanghai and National Engineering Center for BioChip at Shanghai, 201203, Shanghai, China

    Guoping Zhao

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  1. Zhihui Shao
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Correspondence to Guoping Zhao.

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Communicated by Erko Stackebrandt.

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203_2011_690_MOESM1_ESM.ppt

Fig. S1 Time-course change of cell growth and nitrite extrusion of NK32 (nasK −) in Bennet medium with 20 mM KNO3 or 5 mM KNO3. (PPT 61 kb)

203_2011_690_MOESM2_ESM.ppt

Fig. S2 Time-course change of cell growth and nitrite extrusion of NE32 (nasE −) and U32 in minimal medium with 70 mM proline or 2 mM NaNO2. (PPT 60 kb)

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Shao, Z., Gao, J., Ding, X. et al. Identification and functional analysis of a nitrate assimilation operon nasACKBDEF from Amycolatopsis mediterranei U32. Arch Microbiol 193, 463–477 (2011). https://doi.org/10.1007/s00203-011-0690-0

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  • DOI: https://doi.org/10.1007/s00203-011-0690-0

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