ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1992, p. 580-588 0066-4804/92/030580-09$02.00/0 Copyright X 1992, American Society for Microbiology

Vol. 36, No. 3

Nucleotide Sequence and Phylogeny of the tet(L) Tetracycline Resistance Determinant Encoded by Plasmid pSTE1 from Staphylococcus hyicus STEFAN SCHWARZ,`* MARISA CARDOSO,' AND HENRIK C. WEGENER2 Institut fiir Bakteriologie und Immunologie der Justus-Liebig-Universitat Giessen, Frankfurter Strasse 107, D-6300 Giessen, Germany, 1 and National Veterinary Laboratory, 1503 Copenhagen V, Denmark2 Received 22 July 1991/Accepted 27 November 1991

The nucleotide sequence of the tetracycline resistance (tet) gene and its regulatory region, encoded by the plasmid pSTE1 from Staphylococcus hyicus, was determined. The tet gene was inducible by tetracycline and encoded a hydrophobic protein of 458 amino acids. Comparisons between the predicted amino acid sequences of the pSTEl-encoded Tet from S. hyicus and the previously sequenced Tet K variants from Staphylococcus aureus, Tet L variants from Bacillus cereus, BaciUls stearothermophilus, and Bacillus subtilis, Tet M variants from Streptococcus faecalis and Staphylococcus aureus as well as Tet 0 from Streptococcus mutans were performed. An alignment of Tet amino acid sequences revealed the presence of 30 conserved amino acids among these Tet variants. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated large evolutionary distances between the Tet M and Tet 0 variants on one hand and the Tet K and Tet L variants on the other hand. The pSTE1-encoded Tet proved to be closely related to the Tet L proteins originally found on small BaciUus plasmids. The observed extensive similarities in the nucleotide sequences of the tet genes and in the deduced Tet amino acid sequences allowed the assignment of the pSTEl-encoded Tet to the Tet proteins of class L.

Tetracycline resistance (Tcr) is the most common antibiotic resistance observed in nature (25). The determinants for Tcr which occurred in most gram-positive and gram-negative bacterial species proved to be heterologous (6, 8, 26). They could be differentiated on the basis of DNA-DNA hybridization and sequence analyses (19, 27). Up to now, 13 different classes of Tcr determinants have been identified and designated A through G as well as K through P (27). The Tcr determinants in staphylococci were mostly assigned to the classes K, L, and M. Tet M determinants are known to protect ribosomes from the inhibitory effects of tetracycline, whereas Tet K and Tet L determinants specify membraneassociated efflux systems. In Staphylococcus aureus, Tcr determinants of the classes K and M had been studied in detail. The tet(M) gene is encoded chromosomally and its gene product mediates resistance to tetracycline and to the semisynthetic minocycline (26, 33). In contrast, the tet(K) gene is plasmid borne (26) and mediates inducible resistance only to tetracycline but not to its semisynthetic derivatives (31). Plasmids encoding Tet K have been isolated from several staphylococcal species such as S. aureus (20, 29, 31, 34), Staphylococcus epidernidis (9, 14, 48, 53), Staphylococcus hyicus (39, 44), Staphylococcus intermedius (49), and Staphylococcus haemolyticus (49). Restriction endonuclease analyses as well as DNA-DNA hybridization studies demonstrated that these plasmids were closely related (14, 44). Similar to Tet K, Tet L mediates resistance only to tetracycline (26). The tet(L) genes were found to be commonly located on small Bacillus plasmids. Although several reports described the presence of tet(L) genes in staphylococci and streptococci (26, 27), no sequence data of staphylococcal or streptococcal tet(L) genes have been published so far. In the current study, we investigated the 11.5-kb plasmid *

Corresponding

pSTE1 from S. hyicus for its tet gene and the respective regulatory region. pSTEl, which exhibited resistance only to tetracycline, demonstrated no restriction map homology to the Tcr plasmids previously identified in S. hyicus or other staphylococcal species. It also failed to hybridize with a tet(K) gene probe. The predicted amino acid sequence of the Tet protein from pSTEl was used for comparisons with those of the Tet proteins from S. aureus (20, 31, 34), Streptococcus faecalis (5, 30), Streptococcus mutans (23), Bacillus cereus (36), Bacillus subtilis (40, 41), and Bacillus stearothennophilus (19). A phylogenetic tree based on the alignment of the Tet amino acid sequences was constructed to provide insight into the origin and the evolutionary relationships of Tcr determinants found in gram-positive bacteria. MATERIALS AND METHODS Bacterial strains, plasmids, and phages. S. hyicus HW17 carrying the plasmid pSTEl was isolated from the skin of a piglet suffering from exudative epidermitis. S. aureus RN 4220 (35) served as a recipient strain in protoplast transformation experiments. Plasmid pBluescript II SK+ (Stratagene, Heidelberg, Germany) served as a cloning vector. Escherichia coli TG1 [supE hsd A5 thi A(lac-proAB) F'(traD36proAB+ lacIq lacZ AM15)] (Stratagene) was used as a recipient for plasmid pBluescript II SK+ and its recombinant derivatives. Growth conditions were as previously described (46). The production of single-stranded DNA with the helper phage M13K07 (Stratagene) was performed according to the recommendations of Vieira and Messing (54). Reagents and enzymes. Restriction endonucleases were purchased from Boehringer (Mannheim, Germany) or New England BioLabs (Schwalbach, Germany), T4 ligase and alkaline phosphatase were from Stratagene, and T7 DNA polymerase was from Pharmacia (Freiburg, Germany).

author. 580

tet(L) IN S. HYICUS

VOL. 36, 1992

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FIG. 1. Linear restriction map of the Tcr ML' plasmid pSTE1 from S. hyicus (a) and sequencing strategy for determination of the tet(L) nucleotide sequence within a 1.6-kb region (b). The large arrow shows the extent and the direction of transcription of the tet(L) gene. The small arrows indicate the extents and the directions of the respective sequence analyses.

[a-35S]dATP was a product of Amersham Corp. (Braunschweig, Germany), and ampicillin, tetracycline, and erythromycin were from Sigma (Deisenhofen, Germany). Sequencing reagents were purchased from Roth (Karlsruhe, Germany) or GIBCO (Paisley, Scotland, United Kingdom). Susceptibility testing, protoplast transformation, and plasmid preparation. The original pSTEl-containing S. hyicus strain was tested for its antimicrobial agent susceptibility patterns by the agar diffusion method (2) with disks containing the following (in micrograms): amoxicillin, 20-clavulanic acid, 10; ampicillin, 25; cephalotin, 30; chloramphenicol, 30; clindamycin, 10; erythromycin, 10; gentamicin, 10; kanamycin, 30; minocycline, 30; penicillin G, 10; streptomycin, 10; sulfamethoxazole, 23.75-trimethoprim, 1.25; and tetracycline, 30 (Oxoid, Basingstoke, United Kingdom). The antibiograms were evaluated after incubation for 24 h at 37°C. The antimicrobial agent susceptibility patterns of the respective pSTE1-transformed S. aureus RN 4220 clones were determined in the same manner. pSTE1 was transformed into S. aureus RN 4220 protoplasts by using a previously described modification (44) of the method of Chang and Cohen (7). The transformed S. aureus RN 4220 protoplasts were selected on DM3 regeneration plates (7) supplemented with 30 ,ug of tetracycline per ml and 30 ,ug of erythromycin per ml. Clones which appeared on these selective media after 48 to 72 h were screened for the presence of the plasmid pSTE1. pSTE1 DNA was prepared by using a commercially available plasmid preparation system (Qiagen Midi-Prep system; Diagen, Dusseldorf, Germany) modified for staphylococci as previously described (46). DNA-DNA hybridization. Double digests of pSTE1-DNA achieved with those restriction endonucleases for which

cleavage sites were indicated in Fig. la were separated in agarose gels and transferred to nitrocellulose filters by the capillary blot procedure (50). The tet(K) gene probe consisted of the 2.35-kb HindIII fragment of the previously described pT 181 analog Tcr plasmid pST1 from S. hyicus (44). Radioactive labelling of the probe, hybridization at 65°C, washing of the filters at 65°C, and autoradiography were as previously described (50). Determination of MIC of tetracycline. The MIC of tetracycline for pSTE1-transformed S. aureus RN 4220 was determined as previously described (45). To see whether the pSTE1-encoded tet gene was inducible, pSTEl transformants were treated with 0.5 ,ug of tetracycline per ml prior to MIC determination. Cloning and nucleotide sequence determination. For cloning experiments, plasmid pSTE1 was subjected to complete digestion with HindIII, HpaII, Bcll, and ClaL. The enzyme HinPl was used for cloning of the CfoI fragments. Although CfoI and HinPl had identical recognition sites, HinPl produced CiaI-compatible ends, in contrast to CfoI. Cloning of BclI-, ClaI-, HindIII-, HinPl-, and HpaII-digested pSTE1 DNA into pBluescript II SK+ was performed as described by Sambrook et al. (42). Transformation of E. coli TG1 with recombinant pBluescript II SK+ was conducted by the method of Dagert and Ehrlich (10). Sequence analyses were performed on both strands by using single-stranded phage DNA and double-stranded plasmid DNA as templates. The dideoxy chain termination method of Sanger et al. (43) was applied by using T7 polymerase (51) and [a-35S]dATP. T3, T7, SK, KS, and reverse primer (Stratagene) served for initial sequence determinations. Another two 14-mer and two 15-mer oligonucleotide primers, synthesized with an

582

SCHWARZ ET AL.

Applied Biosystems 380B DNA Synthesizer, were used to complete the tet gene sequence. Computer analysis. Alignment of the Tet amino acid sequences, determination of the percentages of Tet identity, and construction of the phylogenetic tree were performed by using the alignment program of Hein (17). The strategy of this alignment program was to use first pairwise comparisons. For each pair of aligned sequences one ancestral sequence was reconstructed. Thus, the total number of pairwise comparisons in our study was 54 = (10 x 9)/2 (distance tree construction) + (10 - 1) (number of ancestral sequences reconstructed, including one arbitrary root) (17). The relative evolutionary distances between two sequences were calculated on the basis of amino acid substitutions. These were weighted according to the minimum mutation matrix of Dayhoff (11). The gap penalty used was gk= 8 + 3k, which means an insertion-deletion of length k was weighted 8 + 3k. The percentages of Tet identity, as well as the branch lengths, were calculated on the basis of this alignment. Hydrophobicity values were calculated according to the method of Kyte and Dolittle (21) by using a window of 15 amino aclids. Nucleotide sequence accession number. The pSTE1 tet sequence has been submitted to the EMBL data bank and was assigned accession number X60828.

ANTIMICROB. AGENTS CHEMOTHER. GTGTAGAAATACTGAA?

ATATGGTGG'fU-GTATCAABGrrlCMGTATT ( 3) _ (31 ATGAAGTGTAATGAATGTAACAGGGTTCMAkTTAAAAGAGGGAAGCGTATCATTM-CCCTATAAACATCGTCT M K C N E C N R V Q L K E A S V S L T L (2) (21 AAACCTAGTT

GCCCTCATTATA0ZE2TGTG SD2

AATACATCCTATTCACAATCGAATTACGACACAACCAAATTTTA

143 215

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GCAAATGATTTTAATMACCACCTGCGAGTACAAACTGGGTGAACACAGCCTTTATGTTMCCTTTTCCATT A

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RESULTS Antimicrobial agent resistance patterns, plasmid content, restriction endonuclease analysis, DNA-DNA hybridization, and induction studies. The original S. hyicus HW17 strain proved to be resistant to ampicillin, clindamycin, erythromycin, streptomycin, and tetracycline but not to minocycline. It carried three plasmids of approximately 2.4, 4.5, and 11.5 kb. In interspecies protoplast transformation experiments, the 11.5-kb plasmid could be transferred into S. aureus RN 4220 in which it mediated Tcr and resistance to macrolide-lincosamide antibiotics (MLr). This plasmid, designated pSTE1, was considered to carry the genetic information for Tcr and MLr. A preliminary restriction map of the Tcr MLr plasmid pSTEl from S. hyicus was constructed (Fig. la). It differed distinctly from the restriction maps of the previously analyzed staphylococcal Tcr plasmids. Sequences homologous to the tet(K) probe could not be detected in pSTE1 by Southern blot hybridization under stringent conditions (data not shown). The MIC of tetracycline for the pSTE1-transformed S. aureus RN4220 was 50 ,ug/ml. Pretreatment of the pSTEl transformants with subinhibitory concentrations of tetracycline resulted in a fivefold increase to approximately 250 ,ug/ml in the MIC of tetracycline. Thus, the pSTE1-encoded tet gene was considered to be inducible by tetracycline. Cloning and sequencing of the pSTEl-encoded tet gene. Only the E. coli TG1 clones which harbored the 8,000-bp HindIl insert of pSTE1 exhibited Tcr. This observation led to the suggestion that the respective BclI, ClaI, HinPl (CfoI), and HpaII fragments which were found to be located within the large HindIl fragment might contain at least parts of the tet gene or of its regulatory region. Therefore, sequence analyses started at those ends of the BclI, ClaI, HinPl, and HpaII inserts which were purportedly within the tet gene. The sequencing strategy is shown in Fig. lb. Analysis of the nucleotide sequence revealed the presence of two open reading frames (ORFs) (Fig. 2). ORF1 encoded a peptide of 20 amino acids (positions 72 to 134) and was

71

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FIG. 2. Nucleotide sequence of a 1,616-bp region of pSTE1 containing the structural tet(L) gene as well as its regulatory region, presented as the noncoding strand. The two putative ribosomal binding sites (SD1 and SD2) as well as three pairs of inverted repeats [(1) and (1'), (2) and (2'), and (3) and (3')] were detected 5' of the tet gene coding region. Another pair of inverted repeats [(4) and (4')] was found 3' of the tet gene coding region. T-he amino acid sequences of the two open reading frames, deduced from the respective nucleotide sequences, are displayed in the single-letter code.

preceded by a potential ribosome binding site 1 (ShineDalgarno sequence 1 [SD1], positions 60 to 65). Three pairs of inverted repeats (Fig. 2) which might play a role in the regulation of tet gene expression were found (Fig. 3). The start codon for ORF2, GTG, was found to be located 3 bp 3' from the SD2 sequence (positions 156 to 164). ORF2 (positions 168 to- 1544) encoded a protein of 458 amino acids. This protein exhibited a mean hydrophobicity index of 0.90. A putative Rho-independent terminator for mRNA transcription was found 9 bp 3' from the translational stop codon. It consisted of two inverted repeated sequences (positions 1554 to 1564 and 1578 to 1587) followed by a set of seven thymine residues (Fig. 4). Comparisons with Tet proteins of other gram-positive bac-

tet(L) IN S. HYICUS

VOL. 36, 1992

583

AG. -4 kcl/mol

(a)

AG. -1.1 kcol/mol

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START tet START LEADER FIG. 3. Probable stem-loop configurations in mRNA transcribed from the tet(L) gene control region. The two putative SD sequences (SD1 and SD2) as well as their free energy of binding to 16S rRNA are indicated. The ORF for the leader peptide is marked by a broad black bar. The start and stop codon of the small ORF as well as the start codon of the tet gene are marked by boxes. The relative stabilities of the stem-loop structures were calculated by the method of Tinocco et al. (52). (a) Possible mRNA secondary structures in the absence of tetracycline; (b) putative mRNA secondary structure in the presence of tetracycline.

teria. The

pSTEl-encoded

Tet

protein corresponded

in size

closely to the plasmid-encoded Tet K proteins previously described for S. aureus. These consisted of 433 amino acids (pT181) (31) and 459 amino acids (pNS1) (34). Tet from pSTE1 was indistinguishable in size from the chromosomally

encoded Tet L protein from Bacillus subtilis (40), the Tet L proteins encoded by the B. cereus plasmid pBC16 (36), and the B. stearothermophilus plasmid pTHT15 (19), each with a size of 458 amino acids. However, transposon-encoded Tet proteins found in S. faecalis (5, 30) and S. mutans (23) as

584

ANTIMICROB. AGENTS CHEMOTHER.

SCHWARZ ET AL.

the Tet proteins originally found in Bacillus spp. than to those encoded by the S. aureus plasmids pT181 and pNS1.

END tot

FIG. 4. Rho-independent terminator for mRNA transcription occurring immediately 3' of the tet gene coding region. The seven consecutive uracil residues which are a characteristic of rho-independent transcriptional terminators are indicated by a black bar.

The stop codon of the tet

gene

is marked by a box.

well as the chromosomal Tet M from S. aureus (33), each with a size of 639 amino acids, were distinctly larger than the pSTE1-encoded Tet. Comparisons revealed that the tet gene from pSTE1 exhibited 99% nucleotide sequence homology and 98% amino acid identity with the tet(L) gene of the B. stearothennophilus plasmid pTHT15. On the basis of this high degree of similarity, the pSTE1-encoded Tcr determinant was assigned to the Tet variants of class L. A distinctly lower homology of 63% in the nucleotide sequences and 60% in the deduced amino acid sequences was observed between Tet L from pSTE1 and Tet K from pT181. An alignment of the predicted amino acid sequences of known Tet proteins from Staphylococcus spp., Streptococcus spp., and Bacillus spp. is shown in Fig. 5. A total of 30 completely conserved amino acids could be observed in all these Tet proteins from gram-positive bacteria. The pSTE1encoded Tet exhibited highest identity to the plasmid-encoded Tet L variants from B. stearothermophilus (98%) and B. cereus (98%) as well as to the chromosomal Tet L from B. subtilis (81%), followed by the pT 181-encoded Tet K (60%) and the pNS1-encoded Tet K (59%), both from S. aureus. However, the pSTE1-encoded Tet shared only minor amino acid identities with the transposon-encoded Tet M proteins from S. faecalis (Tn916, 12%, and TnJS45, 12%), the chromosomal Tet 0 from S. mutans (13%), and the chromosomal Tet M from S. aureus (12%). Phylogeny of Tet variants. A phylogenetic tree was constructed for the predicted amino acid sequences of the 10 Tet variants from gram-positive bacteria. The respective cladogram is shown in Fig. 6. The branching order followed the identity calculations. The numbers along the branches represented relative evolutionary distances calculated according to the values from the distance matrix. The phylogenetic tree suggested the presence of two main groups of Tet proteins. Group 1 consisted of the larger, mostly transposon- or chromosomally encoded Tet variants. Within this group the chromosomally encoded Tet M from S. aureus shared amino acid identities of 97% with Tet M from Tn916 and 92% with Tet M from TnJS45, both originally found in S. faecalis. The second group included the smaller, mostly plasmidencoded Tet proteins from B. cereus, B. stearothermophilus, B. subtilis, S. aureus and S. hyicus. Within this group, the pSTE1-encoded Tet appeared to be more closely related to

DISCUSSION In the present study, we cloned and sequenced the tet gene carried by the Tcr MLU plasmid pSTE1 from S. hyicus. This inducible tet gene differed distinctly from the tet genes previously described for staphylococci. Its regulatory region was located immediately 5' of the tet gene coding region and exhibited 97.3% nucleotide sequence homology to the regulatory region of the inducible tet gene encoded by the plasmid pTHT15 from B. stearothermophilus and 53.7% nucleotide sequence homology to the regulatory region of the inducible tet gene encoded by pT181 from S. aureus. Moreover, the structural elements identified in this regulatory region appeared to be very similar to those previously described for the regulatory regions of other inducible antibiotic resistance genes in staphylococci, such as cat genes specifying chloramphenicol resistance (Cmr) (3, 4, 46, 47) or ern(M) genes specifying MLr (18). All these inducible genes seemed to be regulated via translational attenuation (28). Figure 3 shows two putative forms of folding of mRNA transcribed from the pSTE1 encoded tet gene regulatory region. Although the calculated free energies of the stemloop structures differed slightly, the pSTE1-specific mRNA secondary structures corresponded closely to those previously reported to occur in mRNA from the pTHT15-encoded tet gene regulatory region (19). According to the model of Hoshino et al. (19), an inactive (Fig. 3a) and an active (Fig. 3b) form of mRNA folding were proposed. In the inactive form, translation of the leader peptide as well as ribosome stalling within the small ORF would have no influence on the accessibility of SD2 to ribosomes. SD 2 was still masked in a stem-loop structure (AG = -14.1 kcal/mol). As a result, Tet synthesis might occur not at all or only at a basal level. In the presence of tetracycline, ribosomes were supposed to be stalled during translation of the leader peptide. This might lead to a conformational change of mRNA folding to the thermodynamically more stable stem-loop structure (AG = -16.8 kcal/mol), thereby rendering SD2 accessible to ribosomes and allowing Tet synthesis to occur. The structural tet(L) gene from pSTE1 encoded a protein of 458 amino acids. The start codon was GUG, which had been previously defined to occur as start codon of ,B-lactamase genes from Streptomyces spp. (12, 24) and tet(L) genes from Bacillus spp. (19, 36, 40, 41). Examination of the predicted amino acid sequence of Tet L from pSTE1 revealed an abundance of hydrophobic amino acids. This observation supported the assumption that Tet L from pSTE1 might be a membrane protein, similar to the plasmidencoded Tet K and Tet L proteins in S. aureus and Bacillus spp. (26). Nine nucleotides 3' from the UAA stop codon, a pair of inverted repeats which were supposed to be able to form a stable stem-loop structure on mRNA level could be detected (Fig. 4). The calculated stability of AG = -17.2 kcal/mol (52) indicated that this stem-loop structure might act as a strong terminator. For a better understanding of the structural relationships between Tet L from pSTE1 and the Tet variants isolated from other Staphylococcus spp., Streptococcus spp., and Bacillus spp., we performed an alignment of the predicted Tet amino acid sequences (Fig. 5). The alignment showed that Tet L from pSTE1 differed only by 9 amino acids from Tet L encoded by the plasmid pTHT15 from B. stearothermophilus and by 8 amino acids from Tet L encoded by

tet(L) IN S. 1H1YICUS

VOL. 36, 1992 pNSI pTl8I

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RS L

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585

pTHTl 5 VNTSYSQ.SNLRHNQIL.---IWLCILSFFSVLNENVLNVSLPDIAN-----DFNKPPASTNWVNTAFtKLTFSIG.---TAVYGKt.SDQLGIKRLLLFGI IINCFGS pSTEI VNTSYSQSNLRHNQIL----IWLCILSFFSVLNEHVLNVSLPDIAN. .----DFNKPPASTNWVNTAF'MLTFSIG.---TAVYGIU.SDQ1,G1KRLL1.FG! J NCFGS pBC16 VNTSYSQSNLRHNQIL.---IWLCILSFFSVLNEHVLNVSLPDIAN-----DFNKPPASTNWVNTAFNLTFSIG.---TAVYGKI.SDQt.GIKRLI.LFGI IINCFGS SN 0 NKIINLGILAHVDAGKTTLTESLLYTSGAIAEPGSVDKGTTRTDTMNLERQRGITIQTAVTSFQWEDVKVNI IDTPGIIKDFI,AEVYRSI,SVLDGAVI,LVSAKDGIQAQTR SA N 11I1INIGVLAHVDAGKTTLTESLLYNSGAITELGSVDKGTTRTDNTLLERQRGITIQTGITSFQWENTKVNI IDTPGHNDFLAEIVYRSL.SVI,DGAILLISAKDFVQAQTR Tn916 WKIINIGVLAHVDAGKTTLTESLLYNSGAITELGSVDKGTTRTDNTLLERQRGITIQTGITSFQWENTKVNI IDTPGHMDFI.AEVYRSLSVtLDGAILL,ISAKDGVQAQTR Tnl545 HKIINIGVLAHVDAGKTTLTESLLYNSGAITEtLGSVDRGTTKTDNT(.LERQRGTITIQTAITSFQWKNTKVNI IDTPGHIDFF.AEVYRSI,SVLDGAJIt[ISAKDGVQAQTR

LI--AFIGHINHWFILIFGHL,VQGV--GSAAFPSLIMVVVARNITRKKQGKAFGFTGSIVALGIICLGPSIT---GGI IAHIYI1IWSYLLILPMITI VTJ PFITKVKVPGKSTK LI1--AFIGHNHFF?ILIFGRLVQGV--GSAAFPSLIMVVVARNITRKKQGKAFGFIGSIVALGEGLGPSI ---GGI IAIIYTOIWSYLtLILPMITIVTJPFL.IKVMVPGKSTK 11--GFVGHSFFPILILARFIQGI--GAAAFPALVHVVVARYIPKENRGKAFGL.IGSLVAMGEGVGPAI ---GGMVAHfYIHWSYLLLIPTATI ITVPFLIKLLKKEERIR pTHTl5 VI--GFVGHSFFSLLINARFIQGA--GAMPFPALVN4VVVARYIPKENRGK(AFGLIGSIVAMGEGVGPAI ---GGNIAHYIHWSYLLLIPMITTITTVPFLMKLL.KKEVRIK pNSR pTl8I

BS L

pSTEI pBCI6 SN 0 SA MN Tn916 Tn1545

Vl--GFVGHSFFSLLINARFIQGA--GAAAFPALVNVVVARYIPKENRGKAFGLIGSIVANGEGVCPAI ---GGMIAIIYIIIWSYILLITPI ITITTVPFI,MKI.LKKEVR!K Vl--GFVGlISFFSLLINARFIQGA--GAAAFPALVHVVVARYIPKENRGKAFGL.IGSJVAMGEGVGPAI ---GGMIAIYIYIIWSY.LJLIPMITI ITVPFI.MKI.t.KKIIVRIK ILFIIALQTMKIPTIFFINKIDQEGIDLPHVYRENKAKL,SSRI IVKQKVGQIIPHINVTDNDIXfFQWDTVIMGNDR.LI.EKYMSGKPFKMSEt.EQE.ENRRFQNGTI.FPVYIU(;S LIARNITFIKDNIDSVQIELAIIQVLPMVNT-EWTIEND,.KMGSEIFI-QF FNSFLIG ILFHALRKMGIPTIFFINKIDQNGIDLSTVYQDIKEKLSAEIVIKQKVELYPNVCVTNFTESEQWDTVIEGNDDLLEKYMSGKSLEALEI,EQF.RS RFQNCSL.FPL.YIGS IFARIITFIKDNILTYDKKSEVKKE.INRMFEEWMIGDLEYSK.EL.,QEIFI(SFV[G

. NTLDIVGIVLNMSISIICFMLFTFNYNW1FLILFTIFFVIFIKIIJSRVSNPF'FNPKLIGKNIPFMIG ------L,FSGGLIFSIVAGFISMVPYMMKTJYIIVNVATIG NTLDIVGIVtXSISIICFNLrFTTYNWTFLILFTIFFVIFIKHISRVSNPFINPKLGKNIPFMILG.------LFSGGLIPSIVAGFISNVPYMMKTIYHIVNVATIG GlIIDtNAGIILHSAGIVFFMLFTTSYRFSFI,IISILAFFIFVQHIKIKAQDPFVDPEI,GKNVFFVIG.------TLCGGlJIFGTVAGFVSMVPYiBIKDVFIII.STAAIG pTHTi 5 GHFDIKGI ILNSVGIVFFNLFTTSYSISFLIVSVLSFLIFVKHIRKVTDPFVDPGLGKNIPFMIG.------VLCGGTI FGTVAGFVSMVPYNIHKDVHIQLSTAEIG pSTEI GHFGSI(GIItNSVGIVFFN4LFTTSYSISFLIVSVLSFLIFVKBIIRKVTDPFVDPGLGKNIPFMIG.------VLCGGI IFGTVAGFVSMVPYMMKDVHQtISTAEJG pBCi6 GHFDIKGIILNSVGIVFFNLFTTSYSISFLIVSVLSFLIFVKIIIRKVTDPFVDPGLGKNIPFMIG.------VLCGGI IFGTVAGFVSMVPYNKKDVHQL.STAEIG

pNSI pTl81 BS L

SN 0 SA MN

ANLGRLEISFSTEQECQFIYERRVVISTIIRVRSKKKTMVTGLSDASDVIPDIQNIG KN-INIVTKYSIRPE.GVKFTKQLYR.SV,IRSRSKKKTMTIGICIRYGII.NFK,SIG Tn9SI AK6 -INIEINFSTiGStCNFIYKRRA R.SVIIRXVVFEIVEYSINF.K RYGTILNFK,S1G TnI545AKNS GDL VTKYSIR.SLGVK YERRA tYGIIlRPR EFIIFM,sINFC KYG11,NPK,StG pBS I

NSVIFPGTN.SVIVFGYFGGFLVDRKGSLFVFILGSI.S-ISISFI.TIAFFVEFSM--WLTTFMFIFVMGGI

---------------------

pTI8

NSVIFPGTMSVIVFGYFGGFLVDRKGSLFVFILGSLS-ISISFLTIAFFVEFSM--WLTTFMFIFVNGEL---------------------

BS L

SGI IFPGTMSVI IFGYIGGLLVDR.KGSLYVLTIGSAL-LSSGFLIAAF'FIDAAP--WIMTI IVIFVFGGL--------------------SVI IFPGTMSVI IFGYIGGILVDRRGPLYVLNIGVTF-LSVSFI.TASFLLIET'TS--WFMTI IIVFV1IGGI --------------------SVI IFPGThSVI IFGYIGGILVDRRVPLYALNIGVTIF-LSVSFLTASFI.I.ETTS--WFMTI I VFVLGGL.-------------------SVI IFPGTMSVI IFGYIGGILVDRRGPLYVLNIGVTF-I.SVSFL.TASFLI.ET-TS--WFMTI IIVFVLGGI ----------.----------

pTlITl 5 pSTEI

pBCI6 SM 0

SA M

Tn9I6 TnIi545

EILLPQRKFIENPLPMLQTTIAVKKSEQREILLGALTEISDGDPLI.KYYVIYrTTIIEI II.SFLGNVQMEVICAILEEKYIIVEAEIKF.PTviYMERPLRKAEYTIHIEVPPN TKLLPQRKKIENPHPLLQTTVEPSKPEQREMLLDALLEISDSDPLLRYYVDSTTHEIII.SFLGKVQME.VISALL,QEKYHVEIELKEPTVIYMERPI.KNAEYTTHITEVPPN

TKLLPQRKKIENPHPLLQTTVEPSKPEQREMLLDALLEISDSDPLLRYYVDISTTHIEI IISFI.GKVQMEVISAI,LQEKYHVEIEITEPTVIYMERPL.KNAEYTIHIIEVPPN TKI.LPQRERIENPLPLLQTTVEPSKPQQREMLLDALLEISDSDPLI,RYYVDSATHEII I.SFLGKVQMEVTCALLQEKYiVEI EIKEPTVIYMERPLKKAEYT!HI EVPPN

ILVAMAILIL ----------. SFTKTVISKIVSSSLSEEEVASGMStLLN--FTSFILSEGTGIAIVGGLLSLQLINRKLVLEFINYSSGVYSNIL---.---------------SFTKTVISTVVSSSLKEKEAGAGMISLLN--FTSFL.SEGTGIAIVGGL,LSIGFLDlIRLLPI DVDIISTYLYSNMLILFAGI IVI.------------LFTKTVISTIVSSStLKQQEAGAGKSLLN--FTSFLSEGTGIAIVGGLI.SJPLI.DQRI.PMEVDQSTYI.YSNtL.LLFSGI. IVI ------------SFTKTVISTIVSSSLKQQEAGAGMSLLN--FTSLLSEGTGIAIVGGLLSIPI.L.DPRLLPMEVDQSTYI.YSNLLLLFSGIII ------------.

pNS I pTI81

--- SFTKTVISKIVSSSLSEEEVASGMSLLN--FTSFLSEGTGIAIVGGLLSLQLINRKLVLEFINYSSGVYSN ---

BS L

--------- SFTrKTVJSTIVSSSI.KQQP.AGArMNSII.N--FTSFI,SECTrIAIV.GI.I.I

pTIITI 5 pSTEI

iBC16

SM I) SA MH

TOM1 TnI 545

pNSI p)T 18 1

PI.1DQRI.t.PMV~VDQSTYI.YSNI.I.I.I.FSGT lVI.------------PFWASVGI,SI EPLPIGSGVQYESRVSLGYI,NQSFQNAVIEGVI.YGCP.QGL,Y(WKVTrCKI CFRY;I.YYSPVSTPAI)FRI.I.SPI VI.P.QAI.KKAGTPI.I.LF.PYI.JIF.I YAPI1E

FAILVPP.SMY.SSGLNSQAMGRGE(LGNVDKCKG.YPSPDRLPV.QFKGE,.PLFVAQ PFAIISSLLSMYSVLYNSQAMGRGEG.GNTCIFYIYSVTAFMAIL.VKATLEYSKYAPQR PWSGSALLSVYSVGYNSQAMGIYCGLGN,DKCKG,YPSPFRLPVEVKATLELFIYAPQE ----------------------------CCI.I.TT IVFKRSFKQF ------------

459 13

L.---------------------------CWLVII,NVYKRSRRIIG-------------

458

pT1IT15.--SWLVTI,NVYKII.SQRDF ..-pSTEI ---------------------------SWLVTLNLYKHSQRDF ..----------pBCI6 ---------------------------SWLVTLNVYKIISQRDF -----------YLSRAYNDAPKYCANIVNTQLKNNEVI IIGEIPARCIQIDYRNDI.TFFTNGL,SVCLAEI,KGYQVTTGP.PVCQTBRRLNSRIDKVRYMFNKITr

458 458 458 639 639

YLSRAYNDAPKYCANIVDTQLKNNEVILSGEIPARCIQEYRSDLTFFTNGRSVCLTEI,KGYIIVTTGEPVCQPRRPNSRT DKVRYMFNKTT-

639

BS

SM 0 SA M

Tn9i6 Tn!545

YLSRAYHDAPRYCADIVSTQIKNDEVILKGEIPARCIQEYRNDLTYFTNGQGVCLTELKGYQPAIGKFICQPRRPNSRIDKVRIIMFHIKtA

YL.SRAYNDAPKYCANIVDTQLKNNEVILSGEIPARCIQEYRSDL.TFFTNGRSVCI,TEI,KGYHiVTTGF.PVCQPRRPNSRJDKVRYMFNKIT

6.39

FIG. 5. Alignment of the amino acid sequence of the pSTEl-encoded Tet L with the sequences of Tet variants from Staphylococcus spp., Streptococcus spp., and Bacillus spp. according to the alignment program of Hemn (17). Abbreviations are as follows: pNS1, Tet K encoded by the plasmid pNS1 from S. aureus (34); pT181, Tet K encoded by the plasmid pTl8l from S. aureus (20, 31); BS L, chromosomally encoded Tet L from B. subtilis (40); pTHT15, Tet L encoded by the plasmid pTHT15 from B. stearothermophilus (19); pSTE1, Tet L encoded by the plasmid pSTE1 from S. hyicus; pBC16, Tet L encoded by the plasmid pBC16 from B. cereus (36); SM 0, Chromosomally encoded Tet 0 from S. mutans (23); SA M, chromosomally encoded Tet M from S. aureus (33); Tn916, Tet M encoded by transposon Tn916 from S. faecalis (5); Tn1545, Tet M encoded by transposon Tn 1545 from S. faecalis (30); Asterisks indicate conserved amino acids in all Tet sequences. The Tet L variants encoded by plasmids pTHT15 from B. stearothermophilus (19) and pNS1981 from B. subtilis (41) exhibited the same amino acid sequences; the respective tet(L) genes differed only by 2 base pairs (41).

pBC16 from B. cereus, resulting in 98% amino acid identity between Tet L from pSTE1 and both Bacillus Tet L variants. On the other hand, Tet L from pSTE1 shared distinctly lower amino acid identities of 59 and 60% with the Tet K proteins from the S. aureus Tcr plasmids pNS1 and pT181. Very low amino acid identities of 12 and 13% could be observed between Tet L from pSTE1 and the chromosomally or transposon-encoded Tet M and Tet 0 variants of Streptococcus spp. and S. aureus. The data from this amino acid alignment served for the

construction of a phylogenetic tree of the 10 Tet variants. The cladogram, shown in Fig. 6, demonstrated the presence of two main groups. This subgrouping appeared to be directly related to the structural and biochemical properties of the respective Tet variants. Thus, subgroup 1 consisted of the larger, mostly chromosomally or transposon-encoded Tet variants of the classes M and 0. Members of these two classes also exhibited a substrate spectrum which was not limited to tetracycline but also included the semisynthetic minocycline (Tcr Mnr phenotype). In contrast, subgroup 2

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207

ANTIMICROB. AGENTS CHEMOTHER.

foccais

Tn 1545 TetM

7.faccalis Tn916 Tet M

252

750

S arus Tet M

i!mutans Tat 0

S cereus pBC16 Tet L 77

759

i

pSTEl TetL

Z Bstgiwothermophius pTHT15 TetL

22asutia

Tet L

S.guus pTl8l

Tet K

Saurcus pNSl TetK

FIG. 6. Cladogram of Tet variants from Staphylococcus spp., Streptococcus spp., and Bacillus spp., determined by the method of Hein (17). Branch lengths were determined on the basis of the amino acid alignment; the branch length values indicate relative evolutionary distances.

comprised the smaller, mostly plasmid-encoded Tet variants of classes L and K. All the Tet variants from this subgroup 2 mediated only resistance to tetracycline but not to its semisynthetic derivatives. Both subgroups also differed in their resistance mechanisms. In contrast to Tet M and Tet 0, which specified a cytoplasmatic protein that protects ribosomes from the inhibitory effects of tetracycline (26), Tet L and Tet K mediated a membrane-associated efflux system which prevents tetracycline accumulation within the bacterial cell (26). Hydrophobicity plots of the respective Tet amino acid sequences strongly confirmed these differences

(30). The long evolutionary distances which separated both subgroups in combination with the biochemical and structural properties of their members led to the suggestion that Tet M and Tet 0 had developed from a common ancester, whereas Tet K and Tet L were derivatives of another common ancester. Within subgroup 2, very small evolutionary distances were observed between Tet L from the S. hyicus plasmid pSTE1 and the Tet L variants from the Bacillus plasmids pTHT15 and pBC16, suggesting that they might have diverged more recently. In contrast to those plasmid-encoded Tet L variants, the chromosomal Tet L determinant from B. subtilis appeared to have branched off earlier. The high degrees of homology between these Tet L genes from such evolutionary distant gram-positive bacteria as Staphylococcus spp. and Bacillus spp. implied that these genes obviously had not developed independently from one another. An intergeneric exchange of resistance determinants between Staphylococcus spp. and Bacillus spp. appeared to be more likely. This assumption was supported by the observation that Tet L determinants were preferentially encoded by a family of small Bacillus plasmids. The occurrence of natural plasmid transfer between staphylococci and bacilli had previously been suggested (38). This was supported by the fact that many staphylococcal plasmids were

found to be able to replicate and to express their encoded properties in a Bacillus host (15, 16). Closely related plasmids had also been isolated from S. aureus and soil bacilli (38). The structural relationships between them had been concluded either from restriction map homologies (38) or from nucleotide sequence comparisons (32). Thus, the MLSr plasmid pIM13 from B. subtilis (32) contained a erm methylase gene which was very closely related to the enn(M) and enn(C) genes originally found in MLSr plasmids from S. aureus (18) and S. epidennidis (22). Moreover, ,B-lactamases isolated from Bacillus lichenifornis, B. cereus, and S. aureus exhibited a high degree of amino acid identity (1). These observations indicated that staphylococci might share a certain common pool of resistance determinants with other gram-positive bacteria. In this connection, the Tcr determinant originally isolated from the B. cereus plasmid pBC16 had also been detected by restriction endonuclease mapping on the Tcr plasmid pAMao1 from S. faecalis (37). In this study, we identified a Tcr determinant also very similar to that of pBC16 on the S. hyicus plasmid pSTEl. Thus, the three plasmids pBC16, pAMotl, and pSTE1 isolated from members of the three bacterial genera Bacillus, Streptococcus, and Staphylococcus carried homologous tet(L) genes but differed distinctly in the remaining parts of their restriction maps. This might be explained by intergeneric transfer of resistance determinants. Although many resistance plasmids proved to be unstable in bacterial hosts not closely related to their native hosts (38), a high selective pressure might prevent the bacteria from loss of the newly acquired foreign plasmids and might favor an interplasmidic recombination of the foreign resistance plasmids with those plasmids still resident in the bacterial host. As a result of this substitution of resistance genes and replicons, new resistance plasmids which were well adapted to the respective host might be formed. In the present case, tetracycline had been recommended to be particularly effective in the treatment of piglets with exudative epidermitis and in the control of the causative S. hyicus (13). Thus, the extensive use of tetracycline might have contributed to an increase of Tcr in S. hyicus by enhancing the interspecific transfer of Tet K-encoding pTI81-like Tcr plasmids from other staphylococcal species (44) as well as the intergeneric transfer of tet(L) genes from the ubiquitious bacilli.

ACKNOWLEDGMENTS We thank Detlef Wurkner and Ralf Wahl for help with sequence analysis and alignment programs and Ute Neuschulz and Sabine Grolz-Krug for technical assistance. This study was supported by a project grant of the Justus-LiebigUniversity, Giessen. Marisa Cardoso received a stipend from the German Academic Exchange Service (DAAD). REFERENCES 1. Ambler, R. P. 1975. The amino acid sequence of the Staphylococcus aureus penicillinase. Biochem. J. 151:197-218. 2. Barry, A., and C. Thornsberry. 1985. Susceptibility tests: diffusion test procedures, p. 978-987. In E. H. Lenette, A. Balows, W. H. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 3. Brenner, D. G., and W. V. Shaw. 1985. The use of synthetic oligonucleotides with the universal templates for rapid DNA sequencing: results with staphylococcal replicon pC221. EMBO J. 4:561-568. 4. Bruckner, R., and H. Matzura. 1985. Regulation of the inducible chloramphenicol acetyltransferase gene of the Staphylococcus aureus plasmid pUB112. EMBO J. 4:2295-2300.

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