Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(x) gene encoding for the degradation of tetracycline

Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(x) gene encoding for the degradation of tetracycline S. Ghosh 1, M.J. Sadowsky 2,3, M.C. Roberts 4, J.A. Gralnick 3,5 and T.M. LaPara 1 1 Department of Civil Engineering, University of Minnesota, Minneapolis, MN, USA 2 Department of Soil, Water, and Climate, University of Minnesota, St Paul, MN, USA 3 BioTechnology Institute, University of Minnesota, St Paul, MN, USA 4 Department of Environmental & Occupational Health Sciences, School of Public Health & Community Medicine, University of Washington, Seattle, WA, USA 5 Department of Microbiology, University of Minnesota, Minneapolis, MN, USA Keywords antibiotic resistance, Sphingobacterium, tet(x), tetracycline. Correspondence Timothy M. LaPara, Department of Civil Engineering, University of Minnesota, Minneapolis, MN 55455-0116, USA. E-mail: lapar001@umn.edu 2008 1330: received 30 July 2008, revised 29 September 2008 and accepted 1 October 2008 doi:10.1111/j.1365-2672.2008.04101.x Abstract Aims: The tet(x) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet(x) encodes for a NADP-dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram-negative Sphingobacterium sp. strain PM2-P1-29 that harbours a tet(x) gene. Methods and Results: Sphingobacterium sp. PM2-P1-29 demonstrated the ability to transform tetracycline compared with killed controls. The presence of the tet(x) gene was verified by PCR and nucleotide sequence analysis. Additional nucleotide sequence analysis of regions flanking the tet(x) gene revealed a mobilizable transposon-like element (Tn6031) that shared organizational features and genes with the previously described Bacteroides conjugative transposon CTnDOT. A circular transposition intermediate of the tet(x) region, characteristic of mobilizable transposons, was detected. However, we could not demonstrate the conjugal transfer of the tet(x) gene using three different recipient strains and numerous experimental conditions. Conclusions: This study suggests that Sphingobacterium sp. PM2-P1-29 or a related bacterium may be an ancestral source of the tet(x) gene. Significance and Impact of the Study: This study demonstrates the importance of environmental bacteria and lateral gene transfer in the dissemination and proliferation of antibiotic resistance among bacteria. Introduction Tetracyclines have been used to treat infectious diseases in humans and animals and as animal growth promoters for the last 60 years. Tetracyclines target the ribosome and inhibit protein synthesis in a wide range of aerobic, anaerobic, Gram-positive and Gram-negative bacteria. The effectiveness of tetracycline antibiotics, however, has been reduced because of the proliferation of acquired tetracycline (tet) resistance genes (Chopra and Roberts 2001). Currently, there are 42 known, different genes that confer resistance to tetracyclines, with 26 encoding for efflux proteins and 11 genes encoding for ribosomal protection proteins (http://faculty.washington.edu/marilynr/). Three other genes confer resistance to tetracyclines by encoding for enzymes that chemically modify tetracycline. The tet(x) gene encodes for an NADP-dependent monooxygenase that catalyses the degradation of virtually all tetracycline antibiotics including the recently introduced tigecycline (Yang et al. 2004; Moore et al. 2005). The history of tet(x) is curious because it has been exclusively identified in anaerobic Bacteroides spp., where it 1336 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1336 1342

S. Ghosh et al. tet(x) in Sphingobacterium sp. has been linked to an rrna methylase gene [erm(f)] that confers resistance to macrolides, lincosamides and streptogramin B in a single genetic element (Whittle et al. 2002). Because the TetX protein requires oxygen to transform tetracycline, the tet(x) gene does not confer tetracycline resistance in the host Bacteroides spp. The erm(f) and tet(x) genes have a G+C content of 36% and 37%, respectively, which suggests that they did not originate in Bacteroides spp. One hypothesis is that both antibiotic resistance genes were transferred into Bacteroides spp. as a single genetic element where the erm(f) gene is functional but the tet(x) gene is not (Speer et al. 1991). In our recent study of antibiotic resistance in agricultural soils, we isolated a tetracycline resistant, aerobic, Gram-negative bacterium, Sphingobacterium sp. strain PM2-P1-29, that contained the tet(x) gene (Ghosh and LaPara 2007). In this study, we more fully characterize the tet(x) and flanking gene sequences and show that this isolate has a genetic organization and synteny around tet(x) that is in common with other mobile elements in Bacteroides (Shoemaker et al. 2001; Whittle et al. 2001). However, unlike the tet(x) found in Bacteroides spp. strain, the Sphingobacterium sp. tet(x) gene is not linked to erm(f). Materials and methods Genomic DNA extraction and purification Bacterial cells were grown overnight in Luria Bertani broth (BD Diagnostics; Sparks, MD, USA), concentrated by centrifugation (10 000 g; 10 min) and suspended in lysis buffer (5% sodium dodecyl sulfate, 120 mmol l 1 sodium phosphate, ph 8). Cells were then subjected to three freeze thaw cycles, followed by incubation at 70 C for 90 min. Genomic DNA was then purified using a Fast DNA Spin Kit (MP Biomedicals LLC, Irvine, CA, USA). For direct genome nucleotide sequence analysis, genomic DNA was further purified and concentrated using the Fast DNA Spin Kit a second time followed by additional purification using a GeneClean kit (MP Biomedicals LLC). Purified genomic DNA had an OD 260 280 between 1Æ7 and 1Æ9 and an OD 200 260 of about 1Æ1. Degradation of tetracycline by Sphingobacterium sp. PM2-P1-29 Sphingobacterium sp. PM2-P1-29 was inoculated into LB broth medium and incubated overnight to allow growth into the late exponential phase (OD 600 3Æ0). The cells were then centrifuged, washed with phosphate-buffered saline (PBS; 10 mmol l 1, ph 7) and re-suspended in PBS at 10 the initial cell concentration. Tetracycline (final concentration = 20 mg l )1 ) was then added to the resting cells and incubated in the dark at 30 C. Tetracycline concentrations were monitored periodically by collecting aliquots of these resting cell suspensions, removing the cells by centrifugation (5 min, 16 000 g) and filtration (pore size = 0Æ2 lm) and measuring the absorbance at 363 nm using a UV-visible spectrophotometer (Beckman DU-530, Fullerton, CA, USA). Tetracycline molecules exhibit absorbance maxima at the wavelengths of 260 nm and 360 nm. Absorbance readings were taken at 363 nm because the degradation of tetracycline by the TetX enzyme leads to the disappearance of the absorbance at this wavelength (Yang et al. 2004). Absorbance readings were compared with analogous resting cell suspensions that were not spiked with tetracycline to account for background absorbance. Control incubations were prepared by sterilizing analogous resting cell suspensions by autoclaving (121 C, 15 min, 15 psig). Minimum inhibitory concentrations The minimum inhibitory concentration (MIC) for tetracycline was determined according to Andrews (2001) using E. coli DH5a as a tetracycline-susceptible control strain. Sphingobacterium sp. PM2-P1-29 and E. coli DH5a were inoculated into tubes containing 5 ml of Isosensitest broth (Oxoid Inc., Lenexa, KS, USA), supplemented with 0, 1, 4, 8, 16, 32, 64, 128 or 256 mg l )1 of a tetracycline antibiotic and incubated overnight at 30 C. The MICs were identified as the lowest antibiotic concentration that completely inhibited bacterial growth. Conjugation experiments Conjugation experiments were performed using E. coli HB101, Enterococcus faecalis JH2-2 and Pseudomonas putida KT2440 as recipient strains as described in the previous studies (Goldberg and Ohman 1984; Luna et al. 2002; Soge et al. 2006). An incubation temperature of 30 C was used in all conjugation experiments and the incubation times varied between 1 and 2 days, after which the conjugation was disrupted and cells were plated on selective media to select for transconjugants. Experiments with E. coli HB101 or Ent. faecalis JH2-2 as recipients were performed at donor : recipient ratios of 1 : 1 and 1 : 4; transconjugants were selected on tetracycline (25 mg l )1 ) and rifampicin (25 mg l )1 ). This technique can detect gene transfer frequencies of 10 )10 per recipient (Luna et al. 2002). Triparental mating experiments with E. coli HB101 were performed on solid agar plates using helper plasmid PRK2013 and in flasks containing liquid cultures at a donor : recipient : helper ratio of 1 : 1 : 1. Filter mating experiments with E. coli HB101 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1336 1342 1337

tet(x) in Sphingobacterium sp. S. Ghosh et al. were performed using donor : recipient : helper ratios of 2 : 1 : 1, 1 : 2 : 2 and 1 : 1 : 1. Experiments with P. putida were performed only on filters using 2 : 5 : 1 proportion of donor : recipient : helper (Goldberg and Ohman 1984); transconjugants were selected on minimal medium containing tetracycline and benzoate. USA.) and then ligated using T4 DNA ligase (Promega) following manufacturer s instructions. PCR primers (invf: 5 -GCA CAA GAA GAA TCA ACT CA-3 and invr: 5 -TAT GCC GTT TTG CTG TAA TA-3 ) were then used to amplify the ends of the sequenced tet(x) gene and elongate outward from the gene (Fig. 1). PCR The nearly complete 16S rrna gene was amplified using primers 27F (5 -AGA GTT TGA TCC TGG CTC AG-3 ) and 1522R (5 -AAG GAG GTG ATC CAN CCR CA-3 ) as described previously (Ghosh and LaPara 2007). The tet(x) gene was amplified using new primers (tetx-f: 5 -ATG ACA ATG CGA ATA GAT ACA GAC A-3 ; tetx-r: 5 - CAA TTG CTG AAA CGT AAA GTC-3 ) that were designed using the primer3 software (Rozen and Skaletsky 2000). Other genes in the vicinity of tet(x) were amplified using previously designed primers targeting portions of the Bacteroides conjugative transposon (CTn- DOT) as described previously (Whittle et al. 2001) (3R: 5 -CAA CAA GTA CAT CTC CAC GTT AAA GG-3 ; 6: 5 -TGA ATA TGT TGG CAG ATT ACG GAA TGC GT-3 ; 6R: 5 -ACG CAT TCC GTA ATC TGC CAA CAT ATT CA-3 ; 7:5 -CAT AAC TAC GTC GTA CAA CAT CGT ATT GG-3 ) (Fig. 1). Inverse PCR Inverse PCR was performed to initially amplify genes flanking the tet(x) gene. For inverse PCR, genomic DNA from the Sphingobacterium sp. PM2-P1-29 was digested using restriction enzyme HindIII (Promega, Madison, WI, Nucleotide sequence analysis Following PCR, amplicons were purified using a Gene- Clean kit (MP Biomedicals LLC) and sequenced. Nucleotide sequence analysis was performed at the Biomedical Genomics Center (BMGC) at the University of Minnesota using an ABI 3100 Genetic Analyzer (Applied Biosystems). All nucleotide sequences were fully determined in both directions; the reported nucleotide sequences represent the consensus of bi-directional sequence information. Direct genome sequencing was used to sequence regions not targeted by PCR or inverse PCR. For direct genome sequencing of nucleotides upstream and downstream from the tet(x) gene, sequencing primers were designed between 20 and 30 bases in length with melting temperature of approximately 55 60 C and a GC-clamp on the 3 -end (i.e., two bases at the 3 - end that were either G or C) (Fig. 1). Data analysis Sequence analysis was performed using the dnaman (Lynnon Corp., Vandreuil-Dorion, QC, Canada) and dnastar (DNASTAR, Inc., Madison, WI, USA) software packages. All sequences were submitted to the GenBank database to find best nucleotide amino acid sequence (i) kb 2 4 6 8 10 12 Direct genome seq. 3R 6R 6 HindIII inverse PCR HindIII 7 invr invf Direct genome seq. (ii) intf orf331 orf3f prmf mobb moba orf197 adds tet(x) orf290 orf280 tpnf orf61 orf99 adds tet(x2) tet(x1) ermf Left end Right end Figure 1 Comparison of: (i) the tet(x) region from Sphingobacterium sp. PM2-P1-29 with the (ii) the erm(f) region from the Bacteroides conjugative transposon CTnDOT. 3R, 6R, 6 and 7 denote the location of primers designed by Whittle et al. (2001) used during PCR to amplify and sequence parts of the tet(x) region. Areas of direct genome sequencing have been denoted by arrows. invr and invf denote locations of primers used for inverse PCR. 1338 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1336 1342

S. Ghosh et al. tet(x) in Sphingobacterium sp. matches. Known sequences were obtained from the Genbank database. The nucleotide sequences elucidated during this study has been deposited in GenBank under accession numbers EU864422 (Tn6031) and EU918344 (16S rrna gene). Results Characterization of Sphingobacterium sp. PM2-P1-29 The nearly complete 16S rrna gene (1426 bp) from the Sphingobacterium sp. PM2-P1-29 was sequenced. It grouped together with other members of the Bacteroidetes family, which include the Bacteroides, Flavobacteria and Sphingobacteria, while the closest type strain match in Genbank was Sphingobacterium faecium DSM 11690T (98Æ5%). The G+C content of the Sphingobacterium sp. PM2-P1-29 was 35Æ7 ± 0Æ4%, determined using the thermal denaturation method (Gonzalez and Saiz-Jimenez 2002), which is similar to the range of G+C content of other members of the genus Sphingobacterium (37Æ3 44Æ5%; Yoo et al. 2007). Tolerance to tetracyclines and tetracycline degradation Sphingobacterium sp. PM2-P1-29 was capable of growth in the presence of moderate concentrations of oxytetracycline, tetracycline, chlortetracycline and doxycycline with MIC values of 256 mg l )1, 128 mg l )1, 64 mg l )1 and 32 mg l )1, respectively. Sphingobacterium sp. PM2-P1-29 was able to degrade tetracycline leading to a approximately 50% decrease in OD 363 of filtered culture fluid during a 27-h incubation with live cells (Fig. 2). In contrast, the degradation of OD 363 0 6 0 4 0 2 0.0 0 5 10 15 20 25 Time (h) Figure 2 Degradation of tetracycline (starting concentration approximately 20 mg l )1 ) by the Sphingobacterium sp. PM2-P1-29. OD 363 readings are a measure of tetracycline concentration. Closed circles = killed cells, Open circles = live cells. tetracycline was not observed in flasks containing heatkilled cells, as OD 363 readings of the supernatant were unchanged. There was also a characteristic change in the colour of the live culture fluid from light yellow to reddish during the incubation, which is consistent with the previous studies on tetracycline degradation (Yang et al. 2004). This was not observed in the flask with dead cells or culture flasks not receiving Sphingobacterium sp. PM2- P1-29. Nucleotide sequence of Sphingobacterium tet(x) gene and flanking regions The tet(x) gene in Sphingobacterium sp. PM2-P1-29 was 100% identical to the tet(x) gene (1167 bp) found on transposon Tn4351 from Bacteroides fragilis, which has previously been cloned into E. coli and found to confer resistance to tetracycline (Speer et al. 1991). In addition, the tet(x) in Sphingobacterium sp. PM2-P1-29 had 99Æ8% and 99Æ5% nucleotide and amino acid sequence identity, respectively, to the tet(x) gene found on the conjugative transposon CTnDOT in Bacteroides thetaiotaomicron (Whittle et al. 2001). A 5-kb fragment flanking the tet(x) gene was initially amplified by inverse PCR and sequenced (Fig. 1). On the basis of the similarity of this sequence information to the erm(f) region of the Bacteroides CTnDOT element (Whittle et al. 2001), we used PCR primers targeting the erm(f) region to amplify additional fragments flanking the analogous region in Sphingobacterium sp. PM2-P1-29. This generated an additional 6 kb of nucleotide sequence information near the tet(x) gene. Finally, direct genome sequencing was used to generate a contiguous 12Æ9 kb fragment surrounding the tet(x) gene. Genes in this sequenced region matched the majority of the genes found in the Bacteroides erm(f) region from the Bacteroides conjugative transposon CTnDOT (Fig. 1; Table 1). The most striking differences between the region in the Sphingobacterium sp. PM2-P1-29 and the erm(f) region from Bacteroides are the absence of tet(x1) (a nonfunctional gene with 66% amino acid sequence identity to the tet(x) gene) and the absence of erm(f). Instead, this region in Sphingobacterium sp. PM2-P1-29 contained two unknown open reading frames (orf290 and orf280) that had low sequence similarity with the tet(x1) and erm(f) genes in CTnDOT (<30% similarity of inferred amino acid sequences). In Sphingobacterium sp. PM2-P1-29, 27 bp repeats (5 -TTT TGT CGGGGT GGC AGG ATT CGA ACC-3 ) were found at the ends of the sequenced region, suggesting that it was part of a mobilizable transposon (Fig. 3). Direct genome sequencing beyond the left end of the direct repeat was not possible because of overlapping Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1336 1342 1339

tet(x) in Sphingobacterium sp. S. Ghosh et al. Gene in Sphingobacterium sp. PM2-P1-29 Length (bp) % Amino acid similarity to protein encoded by CTnDOT Putative function intf 1335 100 Integrase orf331 1002 Unknown orf3f 1050 82Æ3 Unknown prmf 825 82Æ8 Unknown mobb 363 100 Mobilization moba 540 58Æ1 Mobilization orf197 675 86Æ7 Unknown aads 717 77Æ4 Aminoglycoside 6-adenylyltransferase tet(x) 1167 99Æ4 Tetracycline resistance orf290 843 Streptothricin resistance orf280 873 Unknown tpnf 840 99Æ6 Transposase Table 1 Nucleotide and amino acid similarity between the genes present in Sphingobacterium sp. PM2-P1-29 and the Bacteroides conjugative transposon CTnDOT described by Whittle et al. (2001) (a) aads orf197 moba mobb orit tet (x) PrmF Sphingobacterium tet(x) region map orf3f 12 909 kb orf290 orf280 tpnf cr 27 bp att cf intf orf331 (b) Sphingobacterium sp. DNA 27 bp att Putative trna gene Integration (c) 27 bp att intf tpnf 27 bp att Sphingobacterium tet(x) region Putative trna gene Figure 3 (a) Map of the circular intermediate within the Sphingobacterium sp. PM2-P1-29 showing the arrangement of genes and the putative 27 bp attachment (att) site. cf and cr are primers used in PCR. (b) Map of the att site and the location of the putative trna gene. (c) Map of the direct repeats formed at the two ends by the att sites following insertion of the sequenced region within Sphingobacterium sp. PM2-P1-29. 1340 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1336 1342

S. Ghosh et al. tet(x) in Sphingobacterium sp. nucleotide signals. Similarly, overlapping sequences were obtained 69 nucleotides downstream of the 27 bp repeat at the right end of the region; these 69 nucleotides, along with the direct repeat region, putatively encoded for a proline trna. In support of our hypothesis that the sequenced region excises from the genome to form a circular mobilizable transposon, we used PCR to detect a circular element that included the region between the tpnf and intf genes as well as one of the 27 bp direct repeats (this result was confirmed by nucleotide sequence analysis) (Fig. 3). In comparison, the Bacteroides erm(f) region, which was incapable of excising out of the Bacteroides conjugative transposon CTnDOT element, had a complete 27 bp repeat at the right end, but only part of the 27 bp direct repeat at the left end (Whittle et al. 2001). This novel mobilizable transposon-like element has been designated Tn6031 by the transposable element registry (http://www.ucl.ac.uk/eastman/tn/). The similarities between the Bacteroides erm(f) region and Tn6031 within the Sphingobacterium sp. PM2-P1-29 did not extend beyond the direct repeat sequences. Sphingobacterium sp. PM2-P1-29 did not have a CTn- DOT CTnERL-like element, as PCR primers targeting parts of the CTnERL element and sequences within the direct repeats failed to amplify. Similarly, the tet(q) gene, an essential feature of CTnDOT CTnERL conjugative transposons (Whittle et al. 2001; Moon et al. 2005), was absent in Sphingobacterium sp. PM2-P1-29. Conjugation experiments We were unable to conjugally transfer the tet(x) gene from Sphingobacterium sp. PM2-P1-29 to E. coli HB101, Enterococcus faecalis JH2-2, or Pseudomonas putida KT2440 using a myriad of growth conditions, ratios of donor, recipient and helper strains. Curiously, tetracycline-resistant transconjugants carrying an unknown tet gene were isolated from matings using Ent. faecalis recipients at frequencies of 5Æ2 10 )9 per recipient. PCR analyses indicated that the transferred tet gene was not tet(x) or any of the tet genes previously identified in bacteria. Discussion Aerobic cultures of Sphingobacterium sp. PM2-P1-29 degraded tetracycline in the same manner as a cloned tet(x) gene in E. coli (Speer et al. 1991), strongly suggesting that the tet(x) gene was functional in this bacterium (note: it is possible, though unlikely, that another gene encoding for tetracycline degradation exists in this bacterium). To our knowledge, Sphingobacterium sp. PM2- P1-29 is the first wild type bacterium to be isolated with a functionally expressed tet(x) gene, as the tet(x) gene is not expressed in anaerobic Bacteroides spp. from which it was originally cloned. The chromosomal G+C% content of Sphingobacterium sp. PM2-P1-29 is similar to that of the tet(x) gene, suggesting that it is an ancestral source of this gene. This hypothesis is substantiated by the genetic organization and synteny of the genes upstream and downstream of the tet(x) gene in Sphingobacterium sp. PM2-P1-29. These genes had numerous similarities with the previously described strains of Bacteriodes spp. containing nonfunctional tet(x) genes. Furthermore, the tet(x) region in the Sphingobacterium sp. PM2-P1-29 contained similarities with mobilizable transposons, also called nonreplicative Bacteroides Units (NBUs), suggesting that gene transfer from Bacteroides to Sphingobacterium sp. likely occurred some time in the past. However, we could not demonstrate the conjugal transfer of tet(x) from Sphingobacterium sp. PM2-P1-29 to other bacteria using multiple phylogentically distinct recipients and numerous experimental conditions, which theoretically had the ability to detect transfer at frequencies of at least 10 )10 per recipient. This suggests that other gene functions necessary for transfer of tet(x) to recipient bacteria may be lacking in Sphingobacterium sp. strain PM2-P1-29. On the other hand, we were able to conjugally transfer an unknown tetracycline resistance determinant to Ent. faecalis; we are currently attempting to more fully characterize this gene. In summary, this study characterizes a functional tet(x) gene in Sphingobacterium sp. strain PM2-P1-29. It is tempting to speculate that soil bacteria may be the ancestral source of the 12Æ9 kb region that surrounds the tet(x) gene and with further study, a bacterium capable of transferring this 12Æ9 kb region may be identified. This research also demonstrates that environmental bacteria are a potential reservoir of genetic material including antibiotic resistance genes against current antibiotics. Acknowledgements This work was financially supported by the National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education, and Extension Service (Grant number 2003-35107-13830). References Andrews, J.M. (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48(Suppl. 1), 5 16. Chopra, I. and Roberts, M. (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65, 232 260. Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1336 1342 1341

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