Gene Cassettes Potentially Encoding Fosfomycin Resistance Determinants

Gene cassettes are small mobile genetic elements that are found in arrays of one or more cassettes within an integron (5, 9). In addition to a single open reading frame, or sometimes two, each cassette contains a unique recombination site that belongs to a family of sites known as 59-be (59-base elements). The genes found in cassettes recovered from class 1 integrons found in plasmids or chromosomally integrated mobile regions are extremely diverse but include mainly genes that confer resistance to antibiotics, including aminoglycosides, β-lactams, chloramphenicol, trimethoprim, streptothricin, rifampin, lincomycin, and erythromycin, or resistance to antiseptics and disinfectants (5, 7, 9). However, a number of cassettes that include open reading frames whose function is not known have also been recovered from the same source. Our recent searches using the predicted sequences of the proteins encoded by cassette-encoded open reading frames revealed two that are significantly related to fosfomycin resistance proteins.

An open reading frame designated ORF1, found in a cassette in a class 1 integron from Pseudomonas aeruginosa (GenBank accession no. AY294333) (11), encodes a protein that is 52.3% identical to FosA, which is the product of a fosfomycin resistance determinant in the composite transposon Tn2921 found in Serratia marcescens (M85195) (8) and 40.5% identical to the FosB fosfomycin resistance proteins encoded by plasmids from Staphylococcus epidermidis (X54227) (12) and Staphylococcus haemolyticus (X89875) (1). The ORF1 product also exhibits 54.5 and 38.6% identity to proteins encoded in the chromosomes of P. aeruginosa PAO1 (PA1129 in AE004543) and Bacillus subtilis (yndH in Z99113) that have recently been cloned and shown to confer resistance to fosfomycin (3, 10). It is also more than 50% identical to predicted products of uncharacterized open reading frames found in the genomes of Chromobacterium violaceum (fofB/CV0163 in AE016910) and Nostoc species (AP003588). An alignment of some of these proteins is shown in Fig. 1. The level of identity between these proteins and the conservation of a number of key amino acids (2) suggest that the cassette gene is likely to confer resistance to fosfomycin.

FIG. 1.

Alignment of putative cassette-encoded fosfomycin resistance proteins with proteins known to confer resistance to fosfomycin. Completely conserved residues are shown as black on white, and the residues important in binding the divalent cations required for inactivation of fosfomycin (2, 10) are indicated by asterisks on the top line. The proteins are from the following sources: FosA, original FosA from Tn2921 (GenBank accession no. M85195); Pae, P. aeruginosa PAO1 (PA1129 in AE004543); ORF1, cassette encoded (ORF1 in AY294333); FosB, original FosB from S. epidermidis (X54227); Bsu, B. subtilis (yndH in Z99113); Lmo, L. monocytogenes (lmo1702 in AL591981); Mlr, M. loti (mlr3345 in AP003001); Orfi, cassette encoded (orf“i” in AY029772).

A second cassette-encoded open reading frame, designated orf“i”, predicts a protein that is 37.9% identical to ORF1 (Fig. 1). This cassette lies within a class 1 integron from P. aeruginosa (cassette at positions 2193 to 2654 in GenBank accession no. AY029772) (6), and the same open reading frame is also found in an integron in Pseudomonas putida (AY065966). The orf“i” product is 34.6 and 39.1% identical to FosA and FosB, respectively. It is also colinear with and more than 50% identical to products of open reading frames found in the genomes of Mesorhizobium loti (mlr3345 in AP003001), Listeria monocytogenes (mlo1702 in AL591981), Listeria innocua (lin1810 in AL596170), Brucella suis (BRA0578 in AE014554), and Brucella melitensis (BMEII0689 in AE009704). The genes from M. loti and L. monocytogenes have recently been cloned and designated fosX because they confer significant levels of resistance to fosfomycin (4). Thus, it is likely that orf“i” also determines resistance to fosfomycin.

FosA, FosB, and FosX confer resistance to fosfomycin by adding glutathione, l-cysteine, or a hydroxyl group, respectively, to the oxirane ring of this antibiotic, rendering it inactive (see references 2, 3, 4, and 10 and references therein).