Product of fosC, a gene from Pseudomonas syringae, mediates fosfomycin resistance by using ATP as cosubstrate

Abstract

Pseudomonas syringae PB-5123, a producer of fosfomycin, is resistant to high concentrations of the antibiotic. Two possible mechanisms of resistance have been detected: (i) impermeability to exogenous fosfomycin, even in the presence of sugar phosphate uptake inducers, and (ii) antibiotic phosphorylation. The gene responsible for this last activity, fosC, encodes a ca. 19,000-Da protein and is immediately followed by a second open reading frame, which shows sequence similarities to glutathione S-transferases. FosC uses ATP as a cosubstrate in an inactivation reaction that can be reversed with alkaline phosphatase. Other nucleotide triphosphates cannot be substituted for ATP in this reaction. No relationship between fosC and the previously described genes of fosfomycin resistance was found.Fosfomycin [L-(cis)-1,2-epoxy propyl phosphonic acid] is a broad-spectrum antibiotic produced by some strains of Streptomyces (15) and by Pseudomonas syringae (25). It enters cells by active transport through the L-␣-glycerophosphate and the hexose-6-phosphate uptake systems and blocks peptidoglycan biosynthesis through inhibition of the formation of N-acetylmuramic acid (18).Chromosomally encoded fosfomycin-resistant (Fo r ) strains have an impairment in fosfomycin uptake (17) or a low-affinity pyruvyl transferase (31). Plasmid-borne Fo r in gram-negative bacteria is mediated by a 141-amino-acid polypeptide (FosA) which catalyzes the fusion of fosfomycin and glutathione (for a comprehensive review, see reference 26). Another plasmidencoded Fo r determinant (fosB) was found in Staphylococcus epidermidis (10). The proteins FosA and FosB exhibit 38.3% sequence identity (35).It has been suggested that the genes encoding antibiotic resistance isolated from nosocomial strains may have originated from antibiotic-producing organisms, because analogous enzymatic activities (2, 33) and protein sequence similarities (27) have been found in both producers and clinical isolates. On the other hand, antibiotic resistance and biosynthetic genes are usually clustered, and consequently, the molecular cloning of resistance genes may also facilitate a molecular characterization of the biosynthetic pathways. In this report we describe the characterization of a DNA fragment from P. syringae PB-5123; when this fragment is introduced into Escherichia coli it leads to Fo r . The Fo r determinant, fosC, encodes a 19-kDa polypeptide that probably inactivates fosfomycin through the introduction of a phosphate group in the molecule, with ATP as a donor. MATERIALS AND METHODSBacterial strains, plasmids, and phages. P. syringae PB-5123 was used throughout the study (25). E. coli HB101 (23) and E. coli JM83 (34) were used as recipients for recombinant plasmids. E. coli XL1-Blue (24) was transfected with M13mp18 or M13mp19 phage DNA (34) containing the DNA fragments to be sequenced. Minicell experiments were conducted with E. coli AR1062 (F Ϫ leu minA minB rpsL thi thr). E. coli ESS, a fosfomycin-hypersusceptible strain, was used as an indicator organism in ...

Fosfomycin

“…FosX is a chromosomal enzyme of Listeria monocytogenes that catalyzes the reaction of fosfomycin with water (120). FosC, found in Pseudomonas syringae, is an enzyme similar to glutathione S-transferase that catalyzes phosphorylation with ATP and inactivation of fosfomycin (121). Finally, kinases that cause fosfomycin degradation through phosphorylation (FomA and FomB, which are structurally and functionally related to FosC) have been identified in Streptomyces spp.…”

Role of the CpxAR Two-Component Signal Transduction System in Control of Fosfomycin Resistance and Carbon Substrate Uptake

“…Mechanisms for fosfomycin resistance are encoded on plasmids and the chromosome. Resistance to fosfomycin is conferred by the production of the plasmid-encoded enzymes FosA, FosB, FosC, and FosX, which leads to inactivation of fosfomycin (13)(14)(15)(16). Chromosomal mutations that lead to the expression of MurA variants that have impaired fosfomycin binding or result in overexpression of MurA also confer resistance to fosfomycin.…”

Fosfomycin and Its Application in the Treatment of Multidrug-Resistant Enterobacteriaceae Infections

“…It has been described in P. syringae, a species that naturally produces fosfomycin. 29 A similar mechanism has been described with fomA and fomB in Streptomyces wedmorensis, 108 and with rare strains of P. aeruginosa. 109 • FosX leads to a water adduct.…”

“…1). Initially released by a few bacterial species, such as Streptomyces spp [26][27][28] or Pseudomonas syringae, 29 it is now produced synthetically for pharmaceutical purposes. The disodium salt is administered parenterally and the trometamine salt orally.…”

“…Serum protein binding is estimated at below 3%, which allows a large tissue availability. 29,44 The half life in serum is long, principally because of the slow renal clearance of fosfomycin. The apparent volume of distribution is large, suggesting good diffusion into interstitial fluid and access to infected tissues.…”