Characterization of tet(32) Genes from the Oral Metagenome

Tetracycline resistance in the oral cavity is primarily mediated through the acquisition of genes encoding ribosomal protection proteins (RPPs) (5, 10, 13), which are often associated with mobile elements (9).

The RPP gene tet(32) was initially reported in the Clostridium-related human colonic anaerobe K10 (4) but was subsequently reported to be a mosaic, tet(O/32/0) (12). Recently, the proposed nonmosaic sequence of tet(32) has been reported (6). In this study we characterize two variants of tet(32) and their immediate flanking regions.

The strains and plasmids used in this study are shown in Table 1. Streptococcus salivarius FStet12 was isolated from a Finnish volunteer as part of a European study investigating antibiotic resistance in oral bacteria (ARTRADI; http://www.microfun.u-psud.fr/microfun/ ) and is the same isolate investigated by Patterson et al. (6). A tet(32) gene was cloned into pCC1BAC (Epicentre, Madison, WI) from metagenomic DNA isolated from saliva from healthy English volunteers (Table 1).

The sequences of tet(32) and its flanking regions were obtained using single-specific-primer PCR and a primer-walking strategy (Table 2). Analysis of the predicted amino acid sequences revealed two variants of Tet(32). The first, carried by 41.2T.2 and FStet12, shares 98% amino acid identity with the second variant carried by both 41.1T and ES2-K21. Interestingly, 41.1T and 41.2T.2A were isolated from the same subject (3), indicating that variants of Tet(32) can coexist. Tet(32) shows 69% amino acid identity with its closest relatives, Tet(M) in Tn916 and Tet(O) from Streptococcus pneumoniae (Fig. 1).

• Open in new tab
• Download powerpoint

FIG. 1.

Phylogenetic relationship between tet(32) and the other tetracycline-resistant RPPs. The Bacillus subtilis Fus protein for translation elongation factor EF-G was used as the outgroup. The protein names and their origins and GenBank accession numbers are as follows: Streptococcus salivarius FStet12, Tet(32), DQ647324; Bacteroides sp. strain 139, Tet(36), AJ514254; Clostridium acetobutylicum, TetB (P), AAK78830; Enterococcus faecalis Tn916, Tet(M), U09422; Streptococcus pneumoniae, Tet(O), P72533; Bacteroides thetaiotaomicron, Tet(Q), X58717; Listeria monocytogenes BM4210 pIP811, Tet(S), Q48791; Streptococcus pyogenes A498, Tet(T), L42544; Butyrivibrio fibrisolvens, Tet(W), AJ222769; Streptomyces coelicolor A3, Tet, CAC14348; Streptomyces rimosus, OtrA, CAA37477; Bacillus subtilis, Fus, P80868.

Genomic DNA from the three isolates, together with the DNA of pPJW1, was used as template in a PCR using the degenerate RPP primers RPP-F and RPP-R (see Fig. 2 for the primer binding sites). Sequence analysis of the PCR products showed that only tet(32) was amplified, indicating the absence of other RPP genes.

• Open in new tab
• Download powerpoint

FIG. 2.

Organization of the regions flanking tet(32) in 41.1T, ES2-K21, FStet12, and 41.2T.2 and similar regions from TnB1230 and ATE-3. ORFs are represented by arrows, with those conserved between species represented in the same shading. The dotted line indicates a region not present in FStet12/41.2T.2.

The presence of both variants of tet(32) in metagenomic DNA from Finland, Norway, and Scotland was demonstrated (data not shown) by PCR with specific primers, Tet(32) 21-F and Tet(32) 1898-R (Table 2). In addition, the input DNA used to make the clone ES2-K21 plus strains 41.1T and 41.2T.2 was derived from saliva taken from volunteers from England, demonstrating that tet(32) is present in different geographical locations within Europe.

Analysis of the flanking regions revealed that those from 41.2T.2 and FStet12 are identical, suggesting that tet(32) may be contained on a region of mobile DNA. The flanking regions in 41.1T and ES2-K21 shared 99% nucleotide homology over the majority of sequence, diverging at 1,164 bp upstream of tet(32) (Fig. 2). The region immediately flanking tet(32) is conserved in all four strains, suggesting a possible core structure surrounding the gene; a similar observation was reported for tet(W) (2).

The region immediately upstream of tet(32) shares 95% nucleotide identity with the region upstream of tet(W) in TnB1230 and ATE-3 (AJ222769 and DQ519395, respectively). The novel open reading frame (ORF) orf23, identified upstream of tet(32), is truncated upstream of tet(W) due to base pair substitutions resulting in an early stop codon.

Orf173, identified upstream of Orf23 in 41.1T and ES2-K21, shows 50% amino acid identity to a phosphotransferase (YP_001512320) reported to encode aminonucleoside antibiotic resistance, although strains 41.1T and ES2-K21 are not resistant to puromycin (data not shown). orf173 was not present in 41.2T.2 or FStet12 as a result of a deletion (dotted line, Fig. 2); however, the 200-bp region upstream was homologous to the sequences upstream of orf173 in 41.1T and ES2-K21.

Upstream of orf173, the sequences of 41.1T and ES2-K21 diverge, but two putative ORFs are identified in each sequence. In ES2-K21 one shared 97% amino acid identity with MafF (CAJ43240) from TnB1230, while Orf153 was not homologous to anything in the database. The two ORFs in 41.1T, Orf46 and Orf103, share conserved gene order with ORFs within the conjugal transfer region of CTn5 from Clostridium difficile 630 (11) and show homology with a putative conjugative transposon membrane protein (CAJ68724) and a single-stranded DNA binding protein (CAJ68723), respectively.

A single ORF was identified downstream of tet(32) with a predicted TTG start codon and an appropriately spaced Shine-Dalgarno sequence. In ES2-K21 and 41.1T, Orf151 has 96% amino acid identity to a hypothetical DNA binding protein from Bacteroides capillosus (ZP_02036259) and 83% identity to a helix-turn-helix domain protein from the ATE-3 element of Arcanobacterium pyogenes (ABF72130) (checked box in Fig. 2), although Orf151 has 102 amino acids more than Orf49. However, in 41.2T.2 and FStet12, a −1 frameshift within this ORF, at base pair 246, results in a stop codon and is designated orf114 to reflect this change. Orf114 still contains this initial homologous region, but no significant homology was seen after the deletion.

Recently, a genome fragment of Clostridium scindens ATCC 35704 (NZ_ABFY02000002), recovered from a human fecal sample (14), was reported to contain a tet(M)-like gene. However, tet(32) from ES2-K21 is identical to this gene, and the surrounding region from mafF to orf151 inclusive shows 98% nucleotide homology to the same region in C. scindens.

All three isolates were mated with rifampin-resistant Enterococcus faecalis JH2-2 or Streptococcus pyogenes ATCC 12202 as described previously (8). An average of 3.3 × 10¹⁰ donor and 5.7 × 10¹⁰ recipient cells were screened per mating, but no transconjugants were isolated. Bacillus subtilis BS34A carrying Tn916 was mated with E. faecalis JH2-2 as a control, and transconjugants arose at a frequency of 1 × 10⁻⁹ per donor. Although transfer of tet(32) could not be demonstrated, the presence of identical genes and flanking regions in Streptococcus and Eubacterium species suggests that transfer may have occurred between them or via one or more intermediates. The homology of these flanking regions to other conjugative transposons suggests that the resistance gene may be located on a mobile element. More work is required to understand how this gene is disseminated in bacterial populations.

In summary, we have characterized two variants of tet(32) from the oral cavity. Although transfer could not be demonstrated, the DNA flanking tet(32) revealed similarities to proven and putative conjugative transposons, in particular ATE-3, TnB1230 and CTn5.

• Copyright © 2009 American Society for Microbiology