Abstract
MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.
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Acknowledgments
This work was made possible in part by National Institutes of Health grants 1 R15 GM070562-01 and P20 RR016480, the latter of which is from the NM-INBRE program of the National Center for Research Resources, a contribution from Calton Research Associates in honor of George and Clytie Calton, and an Internal Research Grant from ENMU. The authors are grateful to Dr. Jeffrey K. Griffith (University of New Mexico) for helpful comments and to Dr. Tomofusa Tsuchiya (Laboratory of Molecular Microbiology, University of Okayama, Japan) for kindly providing E. coli KAM32 used in this study. Ammini Parvathi is grateful to the Director, NIO, Goa and the SIC, NIO (RC), Kochi for their kind help and support. The work at NIO was supported by grant SIP 1302. This is NIO contribution no. 4514.
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Communicated by Jorge Membrillo-Hernández.
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Kumar, S., Parvathi, A., Hernandez, R.L. et al. Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli . Arch Microbiol 191, 425–429 (2009). https://doi.org/10.1007/s00203-009-0468-9
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DOI: https://doi.org/10.1007/s00203-009-0468-9