Florfenicol Resistance Gene floR Is Part of a Novel Transposon

Abstract

The florfenicol/chloramphenicol resistance gene floR was found to be part of the novel 4,284-bp transposon TnfloR from Escherichia coli. TnfloR consists of the gene floR, a putative regulatory gene, and the transposase gene tnpA. A circular form of TnfloR was detected and suggested the potential mobility of this transposon.

Florfenicol is a fluorinated structural analogue of thiamphenicol and chloramphenicol approved exclusively for veterinary use (23). So far, three florfenicol resistance genes, cfr, fexA, and floR, which also mediate resistance to chloramphenicol, have been described (23). While cfr (24) and fexA (16) have been found exclusively in staphylococci, floR has been reported to occur in various gram-negative bacteria, e.g., in several Salmonella enterica subsp. enterica serovars as part of the Salmonella genomic island 1 (SGI1) (1, 3-5, 8, 10, 12, 13, 20, 21), in the SXT element from Vibrio cholerae (15), in the chromosomal DNA of Escherichia coli (2, 11), and on conjugative and nonconjugative plasmids in E. coli (2, 7), Klebsiella pneumoniae (6), Photobacterium damselae subsp. piscicida (19), S. enterica serovar Typhimurium, and S. enterica serovar Newport (9). The different locations of the gene floR suggest the involvement of a yet unidentified transposable element. A recent study showed that the florfenicol/chloramphenicol resistance gene fexA is part of a nonconjugative transposon whose intermediate circular forms were identified by inverse PCR (18). A similar approach was used in this study to identify the transposable element carrying floR.

During recent years, the floR gene areas present on the chromosome or on different-sized plasmids in a number of unrelated Salmonella and E. coli isolates had been characterized by hybridization studies and/or sequence analysis (1-4, 7-12). From this collection, we selected six isolates which revealed striking differences in the floR flanking regions. These isolates included one E. coli isolate with two chromosomal floR gene copies (11), one E. coli isolate with floR on a nonconjugative plasmid (2), an E. coli transconjugant with floR on a conjugative plasmid (7), and transconjugants of two S. enterica serovar Typhimurium isolates and one S. enterica serovar Newport isolate in which floR is located on conjugative plasmids (9). SGI1-carrying Salmonella isolates were not included, since sequence analysis of the floR flanking regions within the SGI1-associated multiresistance gene cluster gave no hints toward a transposable element. Whole-cell DNA of these six isolates was prepared (11) and subjected to inverse PCR to detect circular intermediate forms which may occur after resolution of the transposon from the donor molecule and precede integration into the new recipient molecule. For this, the two primers floRcirc1 (5′-ATCATAGCGGGCGTCGTGTT-3′) and floRcirc2 (5′-GCCGTCATTCCTCACCTTCA-3′) (annealing temperature of 62°C) and Taq DNA polymerase (Promega, Charbonnieres, France) were used. Only one of the six strains, namely the E. coli BN10660-1 transconjugant (7), yielded a PCR product and thus was considered as a suitable candidate to detect the transposable element carrying floR. In a previous study, the sequence of a 6,522-bp EcoRI-BamHI fragment of this conjugative plasmid of E. coli BN10660—including the floR gene—had already been reported (7). Analysis of the sequences upstream and downstream of the floR gene revealed the presence of two open reading frames, ΔtnpA and tnpA (formerly referred to as orfA′ and orfA [7]), whose products show considerable homology to transposase proteins. The sequence downstream of tnpA, missing in the former study (7), was determined by sequencing the terminus of the cloned 7-kb EcoRI fragment of the conjugative plasmid of E. coli BN10660 using the standard M13 reverse primer (MWG Biotech, Ebersberg, Germany). Thus, another 513 bp was added, and database entry AF231986 was updated accordingly.

Sequence analysis of the complete 7,035-bp EcoRI-fragment revealed the presence of a transposon-like element of 4,284 bp. This novel element, designated TnfloR, comprised the resistance gene floR, which codes for a membrane-associated protein of 404 amino acids (aa) that exports florfenicol and chloramphenicol from the bacterial cell. Further components of TnfloR are an open reading frame coding for a putative LysR-like transcriptional regulator of 101 aa and the gene tnpA coding for a putative transposase of 497 aa (Fig. 1a, b). TnfloR starts with a 7-bp sequence (TATACGT) located 903 bp upstream of the start codon of the floR gene, whereas 228 bp downstream of tnpA, the same sequence is found again and constitutes most likely the right-end junction of the transposon. TnfloR does not exhibit inverted or direct repeats at its termini. Inverse PCR using primers complementary to sequences in the 5′ and the 3′ end of the floR gene yielded a fragment of 3,528 bp (Fig. 1c). Sequence analysis of this amplicon revealed the presence of the 3′ end of the floR gene, the putative transcriptional regulator gene, the complete tnpA gene, and its downstream region, the 7-bp sequence (TATACGT), the upstream region of floR, and the 5′ end of the floR gene. This observation confirmed that the transposon-like element identified by sequence analysis is in fact a functionally active transposon which produces circular intermediate forms and that the 7-bp sequence is part of TnfloR. In this regard, TnfloR closely resembles several site-specific integrating transposons, such as Tn554 (22), Tn5406 (14), and Tn558 (18), which are known to produce circular intermediate forms, do not exhibit inverted repeats at their termini, and have 6- to 7-bp sequences at their left- and right-hand junctions, one of which is part of the transposon while the other represents the target sequence. The TnfloR-associated 7-bp target sequence is located within the phosphoglucosamine mutase gene glmM and has also been detected flanking the dfrA20 trimethoprim resistance gene area in the recently described Pasteurella multocida plasmid pCCK154 (17).

FIG. 1.

(a) Genetic organization of the floR transposon on plasmid from E. coli strain BN10660-1. The position and orientation of the genes are indicated by arrows. The 7-bp nucleotide sequences at the transposon junctions are shown in boxes. The positions of primers floRcirc1 and floRcirc2 used for the detection of a circular TnfloR intermediate are indicated by arrows. A distance scale in bp is given above the map. (b) Schematic view of the 4,284-bp circular form of the floR transposon. The integration site of TnfloR is shown in a box. (c) PCR product of the circular form of TnfloR (3,528 bp) obtained by using primers floRcirc1 and floRcirc2. Lane 1, Smart Ladder (Eurogentec, Angers, France); lane 2, E. coli BN10660-1 transconjugant.

Homology searches revealed that the complete TnfloR is not present in any other sequence deposited in the databases. However, there are several sequences where part of tnpA and the adjacent integration site are present (for a recent review, see reference 23). This might suggest either that there are other transposons that carry the same transposase gene and integrate at the same target site or that a TnfloR element was subject to recombination or deletion events after its integration into a new target sequence resulting in such largely truncated forms. Although TnfloR is a nonconjugative transposon, its location on a large conjugative plasmid—as in the present case—facilitates the spread of this transposon across species and genus borders. The observation that the TnfloR-carrying plasmid from E. coli BN10660 also mediates resistance to sulfonamides and trimethoprim (7), both of which are used in veterinary medicine at much higher quantities than florfenicol, indicate the potential of coselection of the floR gene by the use of other antimicrobial agents.