Abstract
A soil metagenomic library was constructed and screened for clones that conferred fosfomycin resistance. A novel protein with 46 % identity to UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Desulfuromonas acetoxidans DSM 684 (GenBank accession number: ZP_01311756) was identified. Multiple sequence alignment revealed that the novel protein was a natural MurA, in which an aspartic acid instead of a cysteine was located in the active site. An Asp120Cys mutant of Escherichia coli was constructed from the subclone through site-specific mutagenesis, and minimum inhibitory concentration of fosfomycin for the resistant subclone and its mutant were determined. These results showed that fosfomycin resistance was a result of the aspartic acid in the active site. Analysis of all existing MurA sequences revealed that MurAs with an active site aspartic acid that can confer fosfomycin resistance occur in ~14 % of bacteria.
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References
Allen HK, Moe LA, Rodbumrer J, Gaarder A, Handelsman J (2009) Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil. ISME J 3(2):243–251
Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J (2010) Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol 8(4):251–259
Arca P, Hardisson C, Suarez JE (1990) Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria. Antimicrob Agents Chemother 34(5):844–848
Clinical and Laboratory Standards Institute (CLSI) (2010) Performance standards for antimicrobial susceptibility testing: twentieth informational supplement M100–S20. CLSI, Wayne
Daniel R (2005) The metagenomics of soil. Nat Rev Microbiol 3(6):470–478
De Smet KA, Kempsell KE, Gallagher A, Duncan K, Young DB (1999) Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis. Microbiology 145(Pt 11):3177–3184
Donato JJ, Moe LA, Converse BJ, Smart KD, Berklein FC, McManus PS, Handelsman J (2010) Metagenomic analysis of apple orchard soil reveals antibiotic resistance genes encoding predicted bifunctional proteins. Appl Environ Microbiol 76(13):4396–4401
Falagas ME, Kastoris AC, Karageorgopoulos DE, Rafailidis PI (2009) Fosfomycin for the treatment of infections caused by multidrug-resistant non-fermenting Gram-negative bacilli: a systematic review of microbiological, animal and clinical studies. Int J Antimicrob Agents 34(2):111–120
Garcia JA, Prieto J, Saenz MC, Sanchez JE (1977) Sensitivity of bacteroidaceae to fosfomycin. Chemotherapy 23(Suppl 1):45–50
Horii T, Kimura T, Sato K, Shibayama K, Ohta M (1999) Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli O26. Antimicrob Agents Chemother 43(4):789–793
Jiang S, Gilpin ME, Attia M, Ting YL, Berti PJ (2011) Lyme disease enolpyruvyl-UDP-GlcNAc synthase: fosfomycin-resistant MurA from Borrelia burgdorferi, a fosfomycin-sensitive mutant, and the catalytic role of the active site Asp. Biochemistry 50(12):2205–2212
Kahan FM, Kahan JS, Cassidy PJ, Kropp H (1974) The mechanism of action of fosfomycin (phosphonomycin). Ann N Y Acad Sci 235:364–386
Kim DH, Lees WJ, Kempsell KE, Lane WS, Duncan K, Walsh CT (1996) Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin. Biochemistry 35(15):4923–4928
Lang KS, Anderson JM, Schwarz S, Williamson L, Handelsman J, Singer RS (2010) Novel florfenicol and chloramphenicol resistance gene discovered in Alaskan soil by using functional metagenomics. Appl Environ Microbiol 76(15):5321–5326
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948
McCoy AJ, Sandlin RC, Maurelli AT (2003) In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance. J Bacteriol 185(4):1218–1228
O’Hara K (1993) Two different types of fosfomycin resistance in clinical isolates of Klebsiella pneumoniae. FEMS Microbiol Lett 114(1):9–16
Skarzynski T, Mistry A, Wonacott A, Hutchinson SE, Kelly VA, Duncan K (1996) Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Structure 4(12):1465–1474
Takahata S, Ida T, Hiraishi T, Sakakibara S, Maebashi K, Terada S, Muratani T, Matsumoto T, Nakahama C, Tomono K (2010) Molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli. Int J Antimicrob Agents 35(4):333–337
Torres-Cortes G, Millan V, Ramirez-Saad HC, Nisa-Martinez R, Toro N, Martinez-Abarca F (2011) Characterization of novel antibiotic resistance genes identified by functional metagenomics on soil samples. Environ Microbiol 13(4):1101–1114
Uchiyama T, Miyazaki K (2009) Functional metagenomics for enzyme discovery: challenges to efficient screening. Curr Opin Biotechnol 20(6):616–622
Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62(2):316–322
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This work was supported in part by the National Basic Research Program of China (973 Program Grants 2007CB513002 and 2009CB522605).
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Cheng, G., Hu, Y., Lu, N. et al. Identification of a novel fosfomycin-resistant UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from a soil metagenome. Biotechnol Lett 35, 273–278 (2013). https://doi.org/10.1007/s10529-012-1074-5
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DOI: https://doi.org/10.1007/s10529-012-1074-5