Abstract
sigE in Streptomyces coelicolor encodes an extracytoplasmic function (ECF) sigma factor, σ E, which is part of a signal transduction system that senses and responds to general cell wall stress in S. coelicolor. Expression of sigE is induced by a wide variety of agents that stress the cell wall under the control of two-component signal transduction system, CseB/CseC encoded in the same operon where sigE was identified from. Here we describe a method developing a bioassay system in S. coelicolor via a transcriptional fusion in which the promoter of sigE operon and a reporter gene (neo) conferring resistance to kanamycin were used. The effectiveness of the resulting bioassay system was determined by monitoring various agents that cause bacterial cell wall stress such as lysozyme or some antibiotics that target cell wall. In consequence, the result confirms that the bioassay system has a potential to be a simple but effective screening tool for identifying novel extracellular agents targeting bacterial cell wall.
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References
Lonetto MA, Gribskov M, Gross CA (1992) The σ70 family: sequence conservation and evolutionary relationships. J Bacteriol 174: 3843–38491
Paget MSB, Chamberlin L, Atrih A, Foster SJ, Buttner MJ (1999) Evidence that the extracytoplasmic function sigma factor, σE, is required for normal cell wall structure in Streptomyces coelicolor A3(2). J Bacteriol 181:204–211
Hong H-J, Paget MS, Buttner MJ (2002) A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics. Mol Microbiol 44:1199–1211
Doyle RJ (1989) How cell walls of Gram-positive bacteria interact with metal ions. In: Beveridge TJ, Doyle RJ (eds) Metal ions and bacteria. John Wiley & Sons, New York, pp 275–293
Paget MSB, Leibovitz E, Buttner MJ (1999) A putative two-component signal transduction system regulates σE, a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2). Mol Microbiol 33:97–107
Ward JM, Janssen GR, Kieser T, Bibb MJ, Buttner MJ, Bibb MJ (1986) Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen Genet 203:468–478
Pearson H (2002) ‘Superbug’ hurdles key drug barrier. Nature 418:469–470
Bibb MJ, Cohen SN (1982) Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans. Mol Gen Genet 187: 265–277
Chakraburtty R, Bibb MJ (1997) The ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2) plays a conditional role in antibiotic production and morphological differentiation. J Bacteriol 179:5854–5861
Janssen GR, Bibb MJ (1993) Derivatives of pUC18 that have BglII sites flanking a multiple cloning site and that retain ability to identify recombinant clones by visual screening of Escherichia coli colonies. Gene 124:133–134
Acknowledgments
This work was supported by funding from the Medical Research council, UK (G0700141) and the Royal Society, UK (516002.K5877/ROG). The author would like to thank Andy Hesketh (University of Cambridge, Systems Biology Centre) for comments on the manuscript.
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Hong, HJ. (2016). Construction of a Bioassay System to Identify Extracellular Agents Targeting Bacterial Cell Envelope. In: Hong, HJ. (eds) Bacterial Cell Wall Homeostasis. Methods in Molecular Biology, vol 1440. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3676-2_10
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DOI: https://doi.org/10.1007/978-1-4939-3676-2_10
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