Abstract
Mureidomycins (MRDs), a group of unique uridyl-peptide antibiotics, exhibit antibacterial activity against the highly refractory pathogen Pseudomonas aeruginosa. Our previous study showed that the cryptic MRD biosynthetic gene cluster (BGC) mrd in Streptomyces roseosporus NRRL 15998 could not be activated by its endogenous regulator 02995 but activated by an exogenous activator SsaA from sansanmycin’s BGC ssa of Streptomyces sp. strain SS. Here we report the molecular mechanism for this inexplicable regulation. EMSAs and footprinting experiments revealed that SsaA could directly bind to a 14-nt palindrome sequence of 5′-CTGRCNNNNGTCAG-3′ within six promoter regions of mrd. Disruption of three representative target genes (SSGG-02981, SSGG-02987 and SSGG-02994) showed that the target genes directly controlled by SsaA were essential for MRD production. The regulatory function was further investigated by replacing six regions of SSGG-02995 with those of ssaA. Surprisingly, only the replacement of 343–450 nt fragment encoding the 115–150 amino acids (AA) of SsaA could activate MRD biosynthesis. Further bioinformatics analysis showed that the 115–150 AA situated between two conserved domains of SsaA. Our findings significantly demonstrate that constitutive expression of a homologous exogenous regulatory gene is an effective strategy to awaken cryptic biosynthetic pathways in Streptomyces.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Alford, M.A., Baghela, A., Yeung, A.T.Y., Pletzer, D., and Hancock, R.E. W. (2020). NtrBC regulates invasiveness and virulence of Pseudomonas aeruginosa during high-density Infection. Front Microbiol 11, 773.
Bailey, T.L., Johnson, J., Grant, C.E., and Noble, W.S. (2015). The MEME suite. Nucleic Acids Res 43, W39–W49.
Bhukya, H., Jana, A.K., Sengupta, N., and Anand, R. (2017). Structural and dynamics studies of the TetR family protein, CprB from Streptomyces coelicolor in complex with its biological operator sequence. J Struct Biol 198, 134–146.
Blin, K., Shaw, S., Steinke, K., Villebro, R., Ziemert, N., Lee, S.Y., Medema, M.H., and Weber, T. (2019). antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res 47, W81–W87.
Breidenstein, E.B.M., de la Fuente-Núñez, C., and Hancock, R.E.W. (2011). Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol 19, 419–426.
Bugg, T.D.H., Lloyd, A.J., and Roper, D.I. (2006). Phospho-MurNAc-pentapeptide translocase (MraY) as a target for antibacterial agents and antibacterial proteins. Infect Disord Drug Targets 6, 85–106.
Chatterjee, S., Nadkarni, S.R., Vijayakumar, E.K.S., Patel, M.V., Gangul, B.N., Fehlhaber, H.W., and Vertesy, L. (1994). Napsamycins, new Pseudomonas active antibiotics of the mureidomycin family from Streptomyces sp. HIL Y-82, 11372. J Antibiot 47, 595–598.
Chen, R.H., Buko, A.M., Whittern, D.N., and McAlpine, J.B. (1989). Pacidamycins, a novel series of antibiotics with anti-Pseudomonas aeruginosa activity. II. Isolation and structural elucidation. J Antibiot 42, 512–520.
Du, D., Wang, L., Tian, Y., Liu, H., Tan, H., and Niu, G. (2015). Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces. Sci Rep 5, 8740.
Guan, H., Li, Y., Zheng, J., Liu, N., Zhang, J., and Tan, H. (2019). Important role of a LAL regulator StaR in the staurosporine biosynthesis and high-production of Streptomyces fradiae CGMCC 4.576. Sci China Life Sci 62, 1638–1654.
Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater, K.F. (2003). PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 100, 1541–1546.
Hopwood, D.A., Kieser, T., Bibb, M., Buttner, M., and Chater, K. (2000). Practical Streptomyces Genetics. Norwich: John Innes Foundation.
Huang, X., Ma, T., Tian, J., Shen, L., Zuo, H., Hu, C., and Liao, G. (2017). wblA, a pleiotropic regulatory gene modulating morphogenesis and daptomycin production in Streptomyces roseosporus. J Appl Microbiol 123, 669–677.
Inukai, M., Isono, F., Takahashi, S., Enokita, R., Sakaida, Y., and Haneishi, T. (1989). Mureidomycins A-D, novel peptidylnucleoside antibiotics with spheroplast forming activity. I. Taxonomy, fermentation, isolation and physico-chemical properties. J Antibiot 42, 662–666.
Isono, F., and Inukai, M. (1991). Mureidomycin A, a new inhibitor of bacterial peptidoglycan synthesis. Antimicrob Agents Chemother 35, 234–236.
Isono, F., Inukai, M., Takahashi, S., Haneishi, T., Kinoshita, T., and Kuwano, H. (1989a). Mureidomycins A-D, novel peptidylnucleoside antibiotics with spheroplast forming activity. II. Structural elucidation. J Antibiot 42, 667–673.
Isono, F., Katayama, T., Inukai, M., and Haneishi, T. (1989b). Mureidomycins A-D, novel peptidylnucleoside antibiotics with spheroplast forming activity. III. Biological properties. J Antibiot 42, 674–679.
Jiang, L., Wang, L., Zhang, J., Liu, H., Hong, B., Tan, H., and Niu, G. (2015). Identification of novel mureidomycin analogues via rational activation of a cryptic gene cluster in Streptomyces roseosporus NRRL 15998. Sci Rep 5, 14111.
Jin, Y.H., Zhan, Q.Y., Peng, Z.Y., Ren, X.Q., Yin, X.T., Cai, L., Yuan, Y.F., Yue, J.R., Zhang, X.C., Yang, Q.W., et al. (2020). Chemoprophylaxis, diagnosis, treatments, and discharge management of COVID-19: An evidence-based clinical practice guideline (updated version). Mil Med Res 7, 41.
Katz, L., and Baltz, R.H. (2016). Natural product discovery: past, present, and future. J Ind Microbiol Biotechnol 43, 155–176.
Kaysser, L., Tang, X., Wemakor, E., Sedding, K., Hennig, S., Siebenberg, S., and Gust, B. (2011). Identification of a napsamycin biosynthesis gene cluster by genome mining. Chembiochem 12, 477–487.
Khan, S., Muhammad, S., Rauf, A., Khan, A., Rizwan, M., Patel, S., Khan, H., Mahasneh, A.M., and Mubarak, M.S. (2018). Comprehensive review on Ebola (EBOV) virus: Future prospects. Infect Disord Drug Targets 18, 96–104.
Kim, D.E., Chivian, D., and Baker, D. (2004). Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res 32, W526–W531.
Laureti, L., Song, L., Huang, S., Corre, C., Leblond, P., Challis, G.L., and Aigle, B. (2011). Identification of a bioactive 51-membered macrolide complex by activation of a silent polyketide synthase in Streptomyces ambofaciens. Proc Natl Acad Sci USA 108, 6258–6263.
Le, T.B.K., Schumacher, M.A., Lawson, D.M., Brennan, R.G., and Buttner, M.J. (2011). The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding. Nucleic Acids Res 39, 9433–9447.
Li, C., He, H., Wang, J., Liu, H., Wang, H., Zhu, Y., Wang, X., Zhang, Y., and Xiang, W. (2019a). Characterization of a LAL-type regulator NemR in nemadectin biosynthesis and its application for increasing nemadectin production in Streptomyces cyaneogriseus. Sci China Life Sci 62, 394–405.
Li, D., Zhang, J., Tian, Y., and Tan, H. (2019b). Enhancement of salinomycin production by ribosome engineering in Streptomyces albus. Sci China Life Sci 62, 276–279.
Li, Q., Wang, L., Xie, Y., Wang, S., Chen, R., and Hong, B. (2013). SsaA, a member of a novel class of transcriptional regulators, controls sansanmycin production in Streptomyces sp. strain SS through a feedback mechanism. J Bacteriol 195, 2232–2243.
Li, Y., Li, J., Tian, Z., Xu, Y., Zhang, J., Liu, W., and Tan, H. (2016). Coordinative modulation of chlorothricin biosynthesis by binding of the glycosylated intermediates and end product to a responsive regulator ChlF1. J Biol Chem 291, 5406–5417.
Liu, G., Chater, K.F., Chandra, G., Niu, G., and Tan, H. (2013). Molecular regulation of antibiotic biosynthesis in Streptomyces. Microbiol Mol Biol Rev 77, 112–143.
Liu, X., Zheng, G., Wang, G., Jiang, W., Li, L., and Lu, Y. (2019). Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor. Sci China Life Sci 62, 1492–1505.
Lu, F., Hou, Y., Zhang, H., Chu, Y., Xia, H., and Tian, Y. (2017). Regulatory genes and their roles for improvement of antibiotic biosynthesis in Streptomyces. J Biotech 7, 250.
Mashalidis, E.H., Kaeser, B., Terasawa, Y., Katsuyama, A., Kwon, D.Y., Lee, K., Hong, J., Ichikawa, S., and Lee, S.Y. (2019). Chemical logic of MraY inhibition by antibacterial nucleoside natural products. Nat Commun 10, 2917.
McLean, T.C., Wilkinson, B., Hutchings, M.I., and Devine, R. (2019). Dissolution of the disparate: co-ordinate regulation in antibiotic biosynthesis. Antibiotics 8, 83.
Nguyen, C.T., Dhakal, D., Pham, V.T.T., Nguyen, H.T., and Sohng, J.K. (2020). Recent advances in strategies for activation and discovery/characterization of cryptic biosynthetic gene clusters in Streptomyces. Microorganisms 8, 616.
Niu, G., Li, Z., Huang, P., and Tan, H. (2019). Engineering nucleoside antibiotics toward the development of novel antimicrobial agents. J Antibiot 72, 906–912.
Niu, G., and Tan, H. (2015). Nucleoside antibiotics: biosynthesis, regulation, and biotechnology. Trends Microbiol 23, 110–119.
Paget, M.S.B., Chamberlin, L., Atrih, A., Foster, S.J., and Buttner, M.J. (1999). Evidence that the extracytoplasmic function sigma factor ζE is required for normal cell wall structure in Streptomyces coelicolor A3 (2). J Bacteriol 181, 204–211.
Pan, Y., Liu, G., Yang, H., Tian, Y., and Tan, H. (2009). The pleiotropic regulator AdpA-L directly controls the pathway-specific activator of nikkomycin biosynthesis in Streptomyces ansochromogenes. Mol Microbiol 72, 710–723.
Ray, S., Maitra, A., Biswas, A., Panjikar, S., Mondal, J., and Anand, R. (2017). Functional insights into the mode of DNA and ligand binding of the TetR family regulator TylP from Streptomyces fradiae. J Biol Chem 292, 15301–15311.
Wang, W., Zhang, J., Liu, X., Li, D., Li, Y., Tian, Y., and Tan, H. (2018). Identification of a butenolide signaling system that regulates nikkomycin biosynthesis in Streptomyces. J Biol Chem 293, 20029–20040.
Xie, Y., Chen, R., Si, S., Sun, C., and Xu, H. (2007). A new nucleosidylpeptide antibiotic, sansanmycin. J Antibiot 60, 158–161.
Xu, J., Zhang, J., Zhuo, J., Li, Y., Tian, Y., and Tan, H. (2017). Activation and mechanism of a cryptic oviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes. J Biol Chem 292, 19708–19720.
Yin, H., Wang, W., Fan, K., and Li, Z. (2019). Regulatory perspective of antibiotic biosynthesis in Streptomyces. Sci China Life Sci 62, 698–700.
Zhang, W., Ostash, B., and Walsh, C.T. (2010). Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics. Proc Natl Acad Sci USA 107, 16828–16833.
Zhang, X., Hindra, X., and Elliot, M.A. (2019). Unlocking the trove of metabolic treasures: activating silent biosynthetic gene clusters in bacteria and fungi. Curr Opin Microbiol 51, 9–15.
Zhang, Y.Y., Zou, Z.Z., Niu, G.Q., and Tan, H.R. (2013). jadR* and jadR2 act synergistically to repress jadomycin biosynthesis. Sci China Life Sci 56, 584–590.
Zou, Z., Du, D., Zhang, Y., Zhang, J., Niu, G., and Tan, H. (2014). A γ-butyrolactone-sensing activator/repressor, JadR3, controls a regulatory mini-network for jadomycin biosynthesis. Mol Microbiol 94, 490–505.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2020YFA0907800 and 2018YFA0901900) and the National Natural Science Foundation of China (81773615, 31771378 and 31800029). We are very grateful to Drs. Wang Wenxi, Liu Xiang and Zheng Jiazhen (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for their technical advice in EMSA and DNase I footprinting experiments.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Compliance and ethics The author(s) declare that they have no conflict of interest.
Supplementary Materials
Rights and permissions
About this article
Cite this article
Liu, N., Guan, H., Niu, G. et al. Molecular mechanism of mureidomycin biosynthesis activated by introduction of an exogenous regulatory gene ssaA into Streptomyces roseosporus. Sci. China Life Sci. 64, 1949–1963 (2021). https://doi.org/10.1007/s11427-020-1892-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11427-020-1892-3