Genetic Environment of 16S rRNA Methylase Gene rmtD

Abstract

The genetic environment of the 16S rRNA methylase gene rmtD was investigated. rmtD was flanked by a novel ISCR motif located downstream of class I integron In163 in the original Pseudomonas aeruginosa strain. rmtD found in Klebsiella pneumoniae appeared to have been mobilized from P. aeruginosa by an IS26-mediated event.

rmtD is one of the recently discovered 16S rRNA methylase genes that confer high-level aminoglycoside resistance (2). It was initially identified in Pseudomonas aeruginosa from Brazil (3). rmtD has since been found in Klebsiella pneumoniae and other species in the family Enterobacteriaceae as well (6, 14), raising questions as to how the gene was mobilized across the species. In the present study, we analyzed the nucleotide sequences surrounding rmtD in these strains in an attempt to answer this question.

P. aeruginosa PA0905 (3) and K. pneumoniae R2 (14), both known to carry rmtD, were used in the study. For P. aeruginosa PA0905, genomic DNA was digested with SpeI and ligated with pBC-SK(−) (Stratagene, La Jolla, CA). For K. pneumoniae R2, plasmid DNA was digested with EcoRI, PstI, or Sau3AI and ligated with the same cloning vector. Escherichia coli DH10B was then transformed with the ligated products by electroporation. Transformants that possessed recombinant plasmids encoding rmtD were selected on Luria-Bertani agar plates containing chloramphenicol (25 μg/ml) and amikacin (50 μg/ml). As a result, rmtD-containing sequences stretching a total of 17.9 kb and 9.1 kb were obtained from P. aeruginosa PA0905 and K. pneumoniae R2, respectively. Sequencing of the nucleotides was performed with an ABI 3100 instrument (Applied Biosystems, Foster City, CA).

A schematic presentation of the genetic environment of rmtD from the two strains is shown in Fig. 1. In P. aeruginosa PA0905, rmtD was located downstream of In163, a class I integron which has been reported in an amikacin-susceptible P. aeruginosa strain from Brazil (1). As in the original report, the integron contained three antimicrobial resistance gene cassettes, aacA4, bla_OXA-56, and aadA7, and was interrupted by tnpA, a putative transposase gene. The transposase shared 97% identity with that of ISPa21 reported in P. aeruginosa (10) and was accompanied by 13-bp perfect inverted repeats, IRL and IRR. The integrase gene intI1 of In163 was interrupted by orf102, which had no significant similarity with known sequences. However, orf102 was accompanied by a putative 59-base element and thus may constitute a gene cassette. Since the initial report of In163 did not contain the intI1 sequence, it is not clear whether the defective gene is a common feature of this particular integron. rmtD, a putative tRNA ribosyltransferase gene, and truncated groEL, which likely originally encoded a heat shock protein, were located downstream of the 3′ conserved segment of In163. A putative tRNA ribosyltransferase gene has been reported to be located upstream of rmtA, the other 16S rRNA methylase gene found in P. aeruginosa (13). Though rmtD and rmtA share only 40% identity at the amino acid level, this similarity in alignment suggests that they may be derived from closely related ancestral species. This structure was bounded by two copies of orf494 in the same direction. The deduced amino acid sequence of orf494 was 96% identical to that of the putative transposase gene constituting ISCR3 and found adjacent to the 16S rRNA methylase gene rmtB in Serratia marcescens and E. coli (5). ISCRs are a group of IS91-like elements that are implicated in the accumulation of various antimicrobial resistance genes (12; http://www.cardiff.ac.uk/medic/aboutus/departments/medicalmicrobiology/genetics/iscr/iscr_elements.html).

FIG. 1.

Schematic presentation of the genetic environment of the rmtD gene. (A) The 17.9-kb section surrounding rmtD in P. aeruginosa PA0905. (B) The 9.1-kb section in K. pneumoniae R2. attI, attachment site of integrase; 59-be, 59-base element of gene cassette; IRR, right inverted repeat of ISPa21 or IS26; IRL, left inverted repeat of ISPa21 or IS26; oriIS, replication origin of ISCR14; terIS, replication terminator of ISCR14; IR1b, 22-bp inverted repeat of Tn5564.

Orf494 possessed all the key amino acid motifs that are conserved among the transposases constituting ISCRs. In addition, both copies of orf494 accompanied the consensus sequence for the 3′ ends of ISCRs (oriIS), 5′-GCGTTTGAACTTCCTATACXX-3′, 224 bp downstream of their 3′ ends. Thus, orf494 and its flanking sequences likely represented a novel ISCR element, here designated ISCR14. In fact, groEL was truncated at the 5′ end by a short punctuated inverted repeat of 4 bp, which may represent the 5′ end of the ISCR (terIS). terIS for the other copy of orf494, which likely played a role in mobilizing In163 adjacent to rmtD, could not be identified within the sequence studied. sul1 was located downstream of the second copy of orf494, followed by part of Tn5664 that likely originated from Corynebacterium species and contained cmx, a chloramphenicol export protein gene (11). The strain in which In163 was originally described likely did not possess rmtD downstream of the integron, given its susceptibility to amikacin (1). Additional sequence information from this strain may further clarify the role of ISCR14 in forming the resistance gene cluster downstream of In163. In any case, the results suggest that ISCR3/14 elements played a vital role in mobilizing rmtB and rmtD to gram-negative pathogens from yet-unidentified sources.

In K. pneumoniae R2, the sequence adjacent to rmtD was identical with that observed in P. aeruginosa PA0905. However, both copies of orf494 were interrupted by the insertion sequence IS26. Interestingly, both copies of IS26 were inserted at the same sequence, 5′-CGATCACC-3′, within each copy of orf494. Although IS26 is not known to show marked target specificity (9), this particular sequence may have served as a recognition site for IS26. The 5′ end of the upstream copy of IS26 was flanked by a class I integron. The 3′ end of intI1 was truncated by the insertion of IS26. dfrA22, a trimethoprim resistance dihydrofolate reductase gene cassette, was located downstream of intI1. The 3′ end of the downstream copy of IS26 was flanked by truncated orf26, which encoded an unknown protein and was reported in a conjugative plasmid encoding the 16S rRNA methylase gene armA (7). IS26 is known to give rise to cointegrates in which the donor and target replicons are separated by two directly repeated IS copies (8, 9). It is unlikely that the two copies of IS26 formed a composite transposon with each other, given the lack of direct repeat sequences and continuity of sequences at the outer ends. It may be possible, however, that additional IS copies are present further downstream of the integron which, along with the two copies described here, played a role in mobilization of rmtD from P. aeruginosa to K. pneumoniae.

Data in the literature to date suggest that rmtD is likely more prevalent in P. aeruginosa than Enterobacteriaceae in Brazil (4, 6). However, incorporation of rmtD by IS26-mediated recombinational events may facilitate future spread of the gene within Enterobacteriaceae. This has potential serious implications for the use of aminoglycosides in common infections caused by these species.

Nucleotide sequence accession numbers.

The sequences reported in this study have been deposited with GenBank/EMBL/DDBJ under accession numbers DQ914960 and EU269034.