The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100

The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100 Andreas Tauch,a,b,* Susanne G€ootker,b Alfred P€uuhler,b J€oorn Kalinowski,b and Georg Thierbacha a Degussa AG, Kantstraße 2, D-33790 Halle-K€uunsebeck, Germany b Department of Genetics, University of Bielefeld, P.O. Box 100131, D-33501 Bielefeld, Germany Received 26 November 2001, revised 21 January 2002 Abstract We determined the complete nucleotide sequence of the 27.8-kb R-plasmid pTET3 from Corynebac- terium glutamicum LP-6 which encodes streptomycin, spectinomycin, and tetracycline resistance. The antibiotic resistance determinant of pTET3 comprises an intI1-like gene, which was truncated by the in- sertion sequence IS6100, and the novel aminoglycoside adenyltransferase gene cassette aadA9. The de- duced AADA9 protein showed 61% identity and 71% similarity to AADA6 of integron In51 from Pseudomonas aeruginosa. In addition, pTET3 carries the novel repressor-regulated tetracycline resistance determinant Tet 33 which revealed amino acid sequence homology to group 1 tetracycline efflux systems. The highest level of similarity was observed to the tetracycline efflux protein TetA(Z) from the C. glu- tamicum plasmid pAG1 with 65% identical and 77% similar amino acids. Each antibiotic resistance region of pTET3 is flanked by identical copies of the widespread insertion sequence IS6100 initially identified in Mycobacterium fortuitum. Transposition assays with a cloned copy of IS6100 revealed that this element is transpositionally active in C. glutamicum. These data suggest a central role of IS6100 in the evolutionary history of pTET3 by mediating the cointegrative assembly of resistance gene-carrying DNA seg- ments. � 2002 Elsevier Science (USA). All rights reserved. Keywords: Corynebacterium glutamicum; Plasmid genome; Antibiotic resistance; Class 1 integron; Gene cassette; Tetracycline efflux system; Horizontal gene transfer Plasmid 48 (2002) 117–129 www.academicpress.com *Corresponding author. Fax: +49-0521-106-5626. E-mail address: [email protected] feld.de (A. Tauch). 0147-619X/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved. PII: S0147 -619X(02)00120 -8 1. Introduction Corynebacterium glutamicum is a Gram-posi- tive soil microorganism with a DNA of high G+C content which is widely used by the bio- technological industry for the fermentative pro- duction of numerous metabolites including LL-amino acids. Although intensive work has been carried out on the molecular genetic character- ization of amino acid-producing corynebacteria, only limited information is available on antibiotic resistance determinants in C. glutamicum. In early studies, a streptomycin and spectinomycin resis- tance was identified on the 29-kb R-plasmid pCG4 from C. glutamicum ATCC 31830 (Katsu- mata et al., 1984) and a chloramphenicol resis- tance region was detected on plasmid pXZ10145 from C. glutamicum 1014 (Zheng et al., 1987). The chloramphenicol resistance gene cmr encodes a putative efflux protein and is an integral part of the transposable element Tn45 (Shen et al., 1994). In addition, the 19.8-kb R-plasmid pAG1 from C. glutamicum 22243 was shown to harbor a tetra- cycline resistance determinant (Takeda et al., 1990). Very recently, the complete nucleotide se- quence of pAG1 was determined and the novel tetracycline efflux system Tet Z was described (Tauch et al., 2000). The Tet Z determinant con- sists of two divergently oriented genes encoding tetracycline efflux and repressor proteins and represents the first group 1 tetracycline efflux system found in Gram-positive bacteria (McMurry and Levy, 2000; Chopra and Roberts, 2001). In the present study, a systematic screening of C. glutamicum isolates for the presence of anti- microbial resistances was performed with a set of 23 antibiotics. This screening procedure resulted in the identification of the C. glutamicum R-pla- smid pTET3 which encodes aadA9, a new type of integron gene cassettes, and Tet 33, a new class of tetracycline efflux and repressor proteins. 2. Materials and methods 2.1. Bacterial strains and growth conditions C. glutamicum strains obtained from the American Type Culture Collection (ATCC, Ma- nassas, VA) and used in this study were: ATCC 13032, 13058, 13825, 13868, 13869, 13870, 13744, 13745, 13870, 14017, 14020, 14067, 14068, 14747, 14751, 14752, 14902, 14915, 15243, 15354, 15455, 15990, 17965, 17966, 19223, 19240, 31808, 31830, 31832. In addition, strains C. glutamicum 22220, 22243 (Takeda et al., 1990), and C. glutamicum LP-6 (Sonnen et al., 1991) were included in the study. C. glutamicum strains were routinely grown at 30 �C in LB medium (Sambrook et al., 1989) containing 2 g/l glucose. All cloning experiments were performed in Escherichia coli DH5aMCR (Grant et al., 1990). Recombinant E. coli strains were grown on Antibiotic Medium No. 3 (Oxoid, Wesel, Germany) supplemented with ampicillin ð100 lg=mlÞ and kanamycin ð50 lg=mlÞ, respec- tively. 2.2. Antimicrobial susceptibility screening Antimicrobial susceptibility screening of C. glutamicum isolates was performed by a macro- broth dilution method described by the National Committee of Clinical Laboratory Standards (1997) with the antibiotics amikacin, amoxicillin, ampicillin, cefotaxime, cefuroxime, cephalothin, chloramphenicol, clindamycin, doxycycline, ery- thromycin, fusidic acid, gentamicin, kanamycin, minocycline, neomycin, norfloxacin, novobiocin, oxacillin, penicillin G, rifampicin, spectinomycin, streptomycin, and tetracycline. Antibiotics were purchased from Sigma-Aldrich (Taufkirchen, Germany) and ICN Biomedicals (Eschwege, Germany). Breakpoints for susceptibility were as suggested by Funke et al. (1997). 2.3. Cloning and DNA sequencing of pTET3 To determine the complete nucleotide se- quence of pTET3, plasmid DNA was isolated from C. glutamicum LP-6 by means of a Nucle- oBond AX100 cartridge system (Macherey-Na- gel, D€uuren, Germany). The plasmid preparation was separated on a 0.8% agarose gel and the DNA corresponding to pTET3 was isolated from the gel with the QIAEX II gel extraction kit (Qiagen, Hilden, Germany). Overlapping DNA fragments generated with the restriction endo- nucleases HindIII and XbaI were cloned in E. coli DH5aMCR into compatible sites of pUC19 following standard procedures (Sambrook et al., 1989). The DNA sequence of pTET3 was deter- mined by MWG-Biotech AG (Ebersberg, Ger- many) using a primer walking strategy on the cloned DNA fragments. The complete nucleotide sequence of the R-plasmid pTET3 was deposited in the EMBL database with accession number AJ420072. 118 A. Tauch et al. / Plasmid 48 (2002) 117–129 2.4. Transposition assay with IS6100 in C. glutam- icum ATCC 13032 The insertion sequence IS6100 was cloned as EcoRI-BglII DNA fragment from pCG4 (Katsu- mata et al., 1984) into the mobilizable plasmid pK18mob2 (Tauch et al., 1998) which was di- gested with EcoRI and BamHI. The resulting plasmid pAT6100 (Fig. 4A) was then transferred into the mobilizing donor strain E. coli S17-1 (Simon et al., 1983). Transfer of pAT6100 to C. glutamicum ATCC 13032 was performed by con- jugation as described previously (Sch€aafer et al., 1990) with the exception that the mating mixture was incubated for 16 h at 37 �C. Transconjugants were selected on LB agar containing 25 lg=ml kanamycin and 50 lg=ml nalidixic acid. The fre- quency of kanamycin resistant transconjugants is expressed as the ratio of the total number of se- lected colonies to the number of recipient cells used per mating assay. Chromosomal DNA of selected transconju- gants was isolated as described previously (Tauch et al., 1995a). Blotting and Southern hybridization were performed with the vacuum blotter Vacu- Gene (Amersham Pharamcia, Freiburg, Ger- many) and the DIG DNA Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany). The Southern blot was documented with a Cy- bertech CS1 camera system (Cybertech, Berlin, Germany). Cloning and sequencing of insertion sites were carried out by a plasmid rescue tech- nique (Tauch et al., 1995b). 3. Results and discussion 3.1. Identification and sequence analysis of the R- plasmid pTET3 from C. glutamicum LP-6 A systematic antimicrobial susceptibility screening of C. glutamicum was performed with a collection of 32 isolates and a total of 23 antimi- crobial substances. The investigated set of strains comprises the so-called amino acid-producing corynebacteria which were previously clustered within the species C. glutamicum by taxonomical fatty acid analyses (K€aampfer and Kroppenstedt, 1996). Susceptibility profiling revealed that only three out of the 32 C. glutamicum isolates encode antibiotic resistances, in particular against streptomycin, spectinomycin, and tetracycline. Besides C. glutamicum ATCC 31830 and C. glu- tamicum 22243, which were known to carry the streptomycin–spectinomycin resistance plasmid pCG4 (Katsumata et al., 1984) and the tetracy- cline resistance plasmid pAG1 (Takeda et al., 1990; Tauch et al., 2000), C. glutamicum LP-6 revealed three differences in the susceptibility pattern when compared with the type strain. The minimum inhibitory concentrations (MICs) of the antibiotics streptomycin ð128 lg=mlÞ, spectinomycin ð>1024 lg=mlÞ, and tetracycline ð16 lg=mlÞ were significantly higher in C. glutamicum LP-6 suggesting the presence of re- sistance determinants in this strain. Since strepto- mycin and tetracycline are extensively used in animal husbandry, it is interesting to note that C. glutamicum LP-6 was initially isolated from a pig-manure deodorizing plant (Sonnen et al., 1991). The MICs of the remaining isolates were almost identical to the values determined for the type strain (data not shown) which indicated that antimicro- bial resistance is a rare genetic feature in amino acid-producing corynebacteria in contrast to the multiple resistances found in medically relevant members of the genus (Funke et al., 1997). Since C. glutamicum LP-6 was previously shown to harbor plasmids pGA1 and pGA2 (Sonnen et al., 1991), experiments were performed to localize the putative resistance regions within the genome of this strain. In addition to pGA1 (4.8 kb) and pGA2 (19.2 kb), two large plasmids designated pTET3 (27.8 kb) and pCRY4 (ap- proximately 48 kb) were identified by agarose gel electophoresis. Subsequently, an aliquot of a C. glutamicum LP-6 plasmid preparation containing the four plasmids was transferred to C. glutami- cum ATCC 13032 by electroporation (Liebl et al., 1989). Interestingly, C. glutamicum transformants were selected on LB medium containing 5 lg=ml tetracycline and 250 lg=ml spectinomycin, re- spectively. Reisolation of plasmid DNA from se- lected transformants of both transformation assays and a size determination revealed plasmid bands indistinguishable from pTET3. In addition, the MICs of streptomycin, spectinomycin, and tetracycline were measured in a pTET3-carrying transformant of C. glutamicum ATCC 13032 and found to be identical to the inhibitory concen- trations determined for C. glutamicum LP-6, the original host of pTET3. Therefore, the plasmid transformation assay strongly suggested that the tetracycline and aminoglycoside resistance deter- minants of C. glutamicum LP-6 are located on plasmid pTET3. In conclusion, three antibiotic resistant strains were identified during a system- atic susceptibility screening of C. glutamicum A. Tauch et al. / Plasmid 48 (2002) 117–129 119 isolates and all R-determinants were localized on large plasmids (pAG1, pCG4, and pTET3) indi- cating that the resistances were acquired by hori- zontal gene transfer. To determine the complete nucleotide sequence of plasmid pTET3, overlapping DNA fragments were cloned in pUC19 and sequenced by a primer walking strategy. Computer-assisted assembly of the pTET3 sequence data revealed a plasmid size of 27,856-bp with a mean G+C content of 54.9%. Fig. 1 presents a detailed physical and genetic map of the R-plasmid pTET3 and includes 25 ORFs identified by automated genome interpretation with the database system GenDB version 1.0.5 (University of Bielefeld, Germany). The most sa- lient features of the deduced gene products such as G+C content of the coding regions, protein length, protein size, and the closest relationship found among known protein sequences in data- bases are summarized in Table 1. Besides putative replication and plasmid maintenance functions, six insertion sequences were present on pTET3 including three identical copies of the widespread element IS6100 (Fig. 1). The identified resistance determinants of pTET3 will be discussed in more detail below. 3.2. The streptomycin–spectinomycin resistance region of pTET3 is part of an integron-like structure Annotation of the complete pTET3 plasmid sequence indicated the presence of the genetic components of a class 1 integron (Table 1; Fig. 2A). This genetic organization includes the 30- conserved segment with the qacED1, sulI, and orf5 coding regions and the 50-conserved segment with the intI1 gene and the cassette integration site Fig. 1. Physical and genetic map of the R-plasmid pTET3 from C. glutamicum LP-6. The ORFs deduced from the complete nucleotide sequence of pTET3 are shown by arrows indicating the direction of transcription. Details on the coding regions are summarized in Table 1. The position of insertion sequences (IS) is shown by boxes. 120 A. Tauch et al. / Plasmid 48 (2002) 117–129 Table 1 Coding regions of the Corynebacterium glutamicum R-plasmid pTET3 and closest relationship of the deduced proteins ORF Position Gene G+C content (%) Protein length (amino acids) Protein size (kDa) Closest relationship amino acid identity/similarity/protein/ microorganism GenBank No. ORF1 27,773—264 qacED1 50.0 115 12.3 100%/100% to QacED1 protein; 30-conserved segment of integrons –a ORF2 258 —1097 sulI 61.7 279 30.1 100%/100% to dihydropteroate synthase SulI; 30-conserved segment of integrons –a ORF3 1225—1725 orf5 65.0 166 18.3 100%/100% to conserved ORF5 protein; 30-conserved segment of integrons –a ORF4 2232—2996 tnpA 61.1 254 29.7 100%/100% to IS6100 transposase; Mycobacterium fortuitum X53635 ORF5 5719—3173 ssmT 46.7 848 95.9 19%/29% to XmnI methyltransferase; Xanthomonas campestris U44748 ORF6 7555—6086 repA 54.9 489 54.8 63%/72% to replication protein RepA; C. glutamicum pAG1 AF121000 ORF7 8018—7755 orf7 50.0 87 10 36%/55% to partitioning protein ParB; Corynebacterium jeikeium pK43 AF364477 ORF8 8557—8330 parB 55.3 75 8.2 28%/45% to partitioning protein ParB; C. glutamicum pAG1 AF121000 ORF9 9156—8557 parA 57.5 199 21.0 31%/51% to partitioning protein ParA; C. glutamicum pAG1 AF121000 ORF10 9910—9407 orf10 59.0 167 18.2 – – ORF11 10,067—9687 orf11 59.0 126 13.3 37%/51% to N-terminal region of ParA family protein; Neisseria meningitidis AL162752 ORF12 11,212—10,139 orf12 52.9 357 37.9 – – ORF13 11,447—12,478 res 50.5 343 38.3 94%/96% to resolvase; C. glutamicum pCG4 AF164956 ORF14 13,317—12,607 tnpB 59.3 236 27.2 94%/96% to IS1628 transposase; C. glutamicum pCG4 AF164956 ORF15 13,459—13,815 orf15 41.8 118 13.3 – – 30-end 14,224—13,850 tnpCF 50.1 124 14.8 41%/56% to C-terminal region of IS870 transposase; Agrobacterium tumefaciens Z18270 ORF16 14,345—15,748 tnpC 59.9 467 50.1 25%/42% to IS1676 transposase; Rhodococcus erythropolis AF126281 ORF17 16,379—16,134 orf17 51.2 81 8.7 37%/56% to transcriptional regulator; Neisseria meningitidis AF123569 ORF18 16,480—16,755 orf18 55.1 91 10.0 – – ORF19 17,304—20,798 traA 55.9 1164 127.4 30%/46% to conjugal transfer protein TraA; Rhodococcus equi pREAT701 AP001204 ORF20 20,929—21,693 tnpA 61.1 254 29.7 100%/100% to IS6100 transposase; M. fortuitum X53635 30-end 21,748—22,011 orf26 61.4 87 9.3 – – ORF21 22,901—22,332 tetR(33) 59.1 189 21.0 63%/74% to tetracycline repressor protein TetR(Z);C. glutamicum pAG1 AF121000 ORF22 23,012—24,163 tetA(33) 61.1 383 39.8 65%/77% to tetracycline efflux protein TetA(Z); C. glutamicum pAG1 AF121000 ORF23 24,883—25,647 tnpA 61.1 254 29.7 100%/100% to IS6100 transposase; M. fortuitum X53635 ORF24 26,627—25,464 intI1D1 61.7 387 43.8 100%/100% to N-terminal region of DNA integrase IntI; 50-conserved segment of integrons –a ORF25 26,773—27,609 aadA9 50.4 278 31.0 61%/71% to AADA6 aminoglycoside resistance protein; Pseudomonas aeruginosa In51 AF140629 aDue to the large number of conserved integron sequences no specific GenBank accession number is provided. A . T a u ch et a l. / P la sm id 4 8 ( 2 0 0 2 ) 1 1 7 – 1 2 9 1 2 1 attI1 (Hall et al., 1994). A 123-bp fragment with the characteristic 25-bp inverted repeat sequence from the outer right end of class 1 integrons and a complete copy of the insertion sequence IS6100 were identified adjacent to the 30-conserved seg- ment (Fig. 2A). The 123-bp fragment and the IS6100 insertion site are identical to the nucleotide sequence identified downstream of the 30-con- served segment of integron In4 which is present in Tn1696 (Hall et al., 1994). A unique feature of the Fig. 2. Comparison of the streptomycin–spectinomycin resistance region of pTET3 from C. glutamicum LP-6 with class 1 integron elements. (A) The general gene organization of a class 1 integron structure is shown. Characteristic genetic features are the cassette integration site attI1, the 59-base element (59-be) and the terminal repeat sequences (TR). The approximate position of the integron tandem promoters (P) is indicated by arrows. The insertion sequence IS6100 is shown as box. In addition, the integron structure deduced from the complete sequence of plasmid pCG4 from C. glu- tamicum ATCC 31830 is presented (GenBank AF164956). (B) Global amino acid sequence alignment (Myers and Miller, 1988) between ORF25/AADA8 and the AADA6 protein of integron In51 from P. aeruginosa (Naas et al., 1999). Identical amino acids are marked by asterisks, similar amino acids are shown by dots. Gaps (-) were used to optimize the protein alignment. (C) Comparison between the 59-base elements of the aadA9 and aadA6 gene regions. The length of the 59-base element is indicated by arrows. The core site (GTTRRRY), the inverse core site (RYYYAAC) and the conserved internal regions 2L (GNTCAAGC) and 2R (GCTTANC) are boxed (Stokes et al., 1997). 122 A. Tauch et al. / Plasmid 48 (2002) 117–129 integron-like structure of pTET3 is the truncation of the intI1 gene by a second copy of IS6100 (Fig. 1). The insertion site on pTET3 is different from the IS6100 insertion found in the intI1 gene of Tn610 from Mycobacterium fortuitum (Martin et al., 1990). In the latter element the insertion site is located 43-bp closer to the 50-end of the intI1 coding region indicating independent IS6100 in- sertion events. The genetic organization of the aminoglycoside resistance determinant of pTET3 was further compared with the streptomycin– spectinomycin resistance gene region sequenced on pCG4 from C. glutamicum ATCC 31830 (Fig. 2A). Both resistance determinants revealed com- plete colinearity with the exception of the gene cassette regions. Integron cassettes contain genes, which confer resistances to a range of antimicro- bials, and a recombination site, known as 59-base element (Recchia and Hall, 1995). The integron-like structure of pTET3 contains a gene cassette region (ORF25) which encodes a 31 kDa protein with deduced amino acid sequence homology to streptomycin–spectinomycin resis- tance proteins of the AADA family (Table 1). The AADA enzymes, also termed ANT(300) (9) aminoglycoside resistance proteins, adenylate the 300-hydroxyl position of streptomycin and the 9-hydroxyl group of spectinomycin, thereby in- activating these antibiotics. Global protein align- ments revealed that the ORF25 protein is different from all known integron-encoded AADA proteins (AADA1 to AADA8) displaying only 56–61% amino acid identity (Table 2). The highest amino acid sequence similarity was observed to the AADA6 protein of integron In51 from Pseudo- monas aeruginosa with 61% identical and 71% similar amino acids (Fig. 2B). Like the AADA6 protein from In51, AADA9 carries a C-terminal extension of 16 additional amino acids when compared with the conserved length of AADA proteins (Naas et al., 1999). A global protein alignment of AADA9 with AADA2 from the in- tegron structure of pCG4 from C. glutamicum ATCC 31830 showed 57% identity and 67% sim- ilarity. Sequence analysis of the ORF25 flanking regions revealed the presence of genetic features characteristic of gene cassettes (Recchia and Hall, 1995). These include a DNA sequence with similarity to 59-base elements downstream of ORF25, an inverse core site at the 50-end of the 59-base element and 7-bp sequences indicative of core sites at the ORF25 boundaries (Fig. 2C). The 59-base element of ORF25 has only four nucleotides difference with that of aadA6. The gene cassette present on pTET3 is thus 900-bp in length and contains a 59-base element with a length of 54 nucleotides (Fig. 2C). Conse- quently, the DNA sequence anaylsis revealed that the R-plasmid pTET3 from C. glutamicum LP-6 carries a remnant of a class 1 integron with ORF25 representing a novel aminoglyco- side adenyltransferase gene cassette, designated aadA9. No promoter signals were identified within the aadA9 gene cassette indicating that the integron tandem promoters may be responsible for resis- tance gene expression. Analysis of the common integron promoter region revealed the presence of the weak P1 promoter (TGGACA-N17-TA- AGCT) and the inactive form of the P2 promoter (TTGTTA-N14-TACAGT). Therefore, it is likely that the aadA9 resistance gene is expressed from the weak P1 integron promoter. The aadA9 ex- pression was further investigated in E. coli DH5aMCR with the subclone pTET3-H10 com- prising the pTET3 fragment HindIII24843 to HindIII1 (Fig. 1). The E. coli strain carrying pTET3-H10 was resistant to streptomycin (MIC 128 lg=ml) and spectinomycin (MIC 1024 lg=ml) whereas the E. coli control with pUC19 was sus- ceptible to both antibiotics (MIC 8 lg=ml). The aadA9 gene cassette thus confers a very similar aminoglycoside resistance level to C. glutamicum and E. coli. Class 1 integrons were long believed to exist only in Gram-negative bacteria, but the presence of a short integron remnant comprising the con- served intI1 and sulI (sul3) genes was described for Tn610 fromM. fortuitum (Martin et al., 1990). An aadA1 gene cassette, which has been reported to exist in a range of Gram-negative microorganisms, Table 2 Amino acid sequence similarity of AADA9 to known AADA proteins AADA protein Amino acid identity (%) Amino acid similarity (%) GenBank Accession Number AADA1 57 70 M95287 AADA2 57 67 AF164956 AADA3 57 69 AF047479 AADA4 58 69 Z50802 AADA5 58 70 AF220757 AADA6 61 71 AF140629 AADA7 56 68 AF224733 AADA8 57 68 AF326210 A. Tauch et al. / Plasmid 48 (2002) 117–129 123 was recently detected in Enterococcus faecalis by means of a PCR screening technique (Clark et al., 1999) and a complete integron structure was identified on the C. glutamicum R-plasmid pCG4 (Fig. 2). The only gene cassette of this integron is aadA2 which is also part of the ancestrial P. aeruginosa integron InC and its derivatives (Bissonnette and Roy, 1992; Kasama et al., 1995). Likewise, the novel aminoglycoside resistance gene cassette aadA9 of the C. glutamicum R-plasmid pTET3 is part of a class 1 integron-like structure. The aadA9 resistance gene is thus the first integron gene cassette which is only known in a Gram- positive bacterium. 3.3. The tetracycline resistance region of pTET3 encodes a new class of Tet efflux systems The tetracycline resistance region of pTET3 in- cludes ORF21 and ORF22 which are flanked by IS6100 insertion sequences (Fig. 1). ORF22 en- codes a predominantly hydrophobic protein with a molecular mass of 39.8 kDa. The most abundant amino acids are alanine (14.3%) and leucine (14.3%), followed by glycine (10.3%) and valine (8.8%).A topology prediction (Rost et al., 1995) for the deduced amino acid sequence suggested 12 hy- drophobic protein segments with a minimum length of 18 amino acids that are proposed to be possible transmembrane a-helices (Fig. 3A). Ac- cording to the topology prediction, the N- and C- terminal ends of the ORF22 protein are located in the cytoplasm. BLASTdatabase searches (Altschul et al., 1997) with the ORF22 protein revealed ho- mology to tetracycline efflux proteins of the re- cently defined efflux group 1 (McMurry and Levy, 2000; Chopra and Roberts, 2001). The highest similarity was observed to the TetA(Z) protein from the C. glutamicum R-plasmid pAG1 (Tauch et al., 2000) with 65% identical and 77% similar amino acids. The global amino acid sequence alignment is shown in Fig. 3A. In addition, the conserved amino acid sequence motifs GxLaDrxGr Kx and GX8GX3GPX2GG common to tetracy- cline efflux proteins are present in the cytoplasmic loop connecting the helical segments 2 and 3 and within the helix V domain (McMurry and Levy, 2000; Varela et al., 1995). Replacement of specific amino acid residues within the conserved motifs of the TetA(B) and TetA(C) proteins reduced or totally abolished resistance to tetracycline. The divergently transcribed ORF21 of pTET3 is closely related to tetracycline repressor proteins of group 1 efflux systems (Table 1). A putative helix-turn-helix DNA-binding motif was pre- dicted within the N-terminal half of the protein (Fig. 3B). The ORF21 protein showed 63% iden- tity and 74% similarity to the hypothetical re- pressor protein TetR(Z) of plasmid pAG1 (Fig. 3B) and 48% identity and 63% similarity to the TetR(A) protein of Tn1721 (Allmeier et al., 1992). The intergenic region between ORF21 and ORF22 contains a 30-bp sequence with a 14-bp inverted repeat showing a 23-bp identity to the operator binding sites OL and OR of the TetR(A) protein from Tn1721 (Fig. 3C). Transfer of the R-plasmid pTET3 to C. glu- tamicum ATCC 13032 resulted in an increased tetracycline MIC value of 12 lg=ml when com- pared with the plasmid-free control ðMIC 6 0:5 lg=mlÞ. No minocycline or doxycycline resis- tance was observed in the transformant. In addi- tion, the pTET3-carrying strain was grown in the presence of a subinhibitory tetracycline concen- tration ð0:05 lg=mlÞ to early log-phase cultures before being challenged with higher tetracycline concentrations during a macrobroth dilution assay. This induction experiment increased the tetracycline MIC value to 32 lg=ml in the pTET3- carrying strain whereas the MIC of the plasmid- free control strain was unchanged. These data suggest that the tetracycline resistance determi- nant of pTET3 may be inducible in C. glutami- cum. In addition, a functional assay with the HindIII subclone pTET3-H9 (Fig. 1 HindIII20889 to HindIII24843) was performed in E. coli DH5 aMCR. Plasmid pTET3-H9 conferred a tetracy- cline resistance level of 16 lg=ml providing evi- dence that the resistance gene is also functional in the heterologous host E. coli ðMIC6 0:5 lg=mlÞ. No increase of the MIC values was observed in E. coli upon induction with subinhibitory tetra- cycline concentrations. Global protein alignments of ORF21 and ORF22 with known Tet A and Tet R proteins of group 1 tetracycline efflux systems (McMurry and Levy, 2000; Chopra and Roberts, 2001) revealed a low degree of amino acid sequence identity (Table 3). Therefore,wepropose anew class of tetracycline efflux and repressor proteins. According to the nomenclature for new tetracycline resistance de- terminants (identity 6 80%; Levy et al., 1999) the tetracycline resistance region of the C. glutamicum R-plasmid pTET3 was designated Tet 33 and con- tains the tetA(33) and tetR(33) resistance genes. Tet 33 is the second repressor-regulated resistance de- terminant of group 1 tetracycline efflux systems which was identified in a Gram-positive bacterium. 124 A. Tauch et al. / Plasmid 48 (2002) 117–129 Fig. 3. Comparison of the tetracycline resistance and repressor proteins of pTET3 with homologous proteins from the C. glutamicum plasmid pAG1. (A) Global amino acid sequence alignment between ORF22/TetA(33) and TetA(Z). Identical amino acids are marked by asterisks and similar amino acids are marked by dots. Gaps (-) were introduced by the ALIGN computer program (Myers and Miller, 1988) to optimize the protein alignment. The transmembrane helical segments of the TetA(33) protein are indicated by numbers (I–XII) above the amino acid sequence (Rost et al., 1995). The conserved amino acid sequence motif GxLaDrxGrKx in the loop 2–3 region of the protein (McMurry and Levy, 2000) and the helix V motif GX8GX3GPX2GG (Varela et al., 1995) are specifically marked. (B) Amino acid sequence alignment of ORF21/TetR(33) with TetR(Z). A putative helix-turn-helix DNA-binding motif (Dodd and Egan, 1990) in the N-terminal region of the protein is marked. (C) Comparison between the tetA–tetR intergenic regions of Tet 33 and Tet Z with the tet operator sequences OL (left) and OR (right) present in Tet A of Tn1721 (Allmeier et al., 1992). A 14-bp inverted repeat within the Tet 33 and Tet Z sequences is indicated by arrows. Identical nucleotides are marked by as- terisks. Please note that the OL and OR sequences of Tet A are separated by 19 nucleotides. A. Tauch et al. / Plasmid 48 (2002) 117–129 125 3.4. IS6100 is transpositionally active in C. glutamicum ATCC 13032 An unusual feature of plasmid pTET3 is the presence of three indentical copies of IS6100 flanking the tetracycline and aminoglycoside re- sistance determinants (Fig. 1). This genetic or- ganization implies that IS6100 was involved in plasmid evolution of pTET3. Since IS6100 is a member of the IS6 family of insertion sequences which exclusively integrate by a cointegrative mechanism (Mahillon and Chandler, 1998), the aadA9 and Tet 33 resistance determinants may become part of pTET3 via cointegrative capture. This type of transposition event is consistent with the truncation of the integrase gene intI1 and the hypothetical coding region ORF26 located up- stream of tetR(33) (Fig. 1). To investigate whether IS6100 is an active mobile element in C. glutami- cum, a copy of IS6100 was cloned in the mobil- izable vector pK18mob2 and subsequently applied in transposition assays. The resulting plasmid, termed pAT6100 (Fig. 4A), was transferred to C. glutamicum ATCC 13032 by RP4-mediated high- frequency conjugation using E. coli S17-1 as do- nor (Sch€aafer et al., 1990). Since pAT6100 is unable to replicate in C. glutamicum, a kanamycin resis- tance phenotype can be selected for only upon illegitimate integration or transposition of the vector resistance into the chromosome. Primary selection with kanamycin resulted in the isolation of C. glutamicum transconjugants with a fre- quency of 2:8� 10�6 suggesting the integration of the kanamycin resistance determinant into the chromosome of the recipient strain. The integration of pAT6100 was verified by Southern hybridization using chromosomal DNA from seven randomly selected transconjugants (Fig. 4B). For this purpose, the chromosomal DNA was digested with EcoRI and probed with digoxigenin-labeled pAT6100. The Southern blot clearly indicated that independent and apparently random integration events had occurred in C. glutamicum. The control hybridization (Fig. 4B; lane 2) revealed no DNA sequence homology between plasmid pAT6100 and the C. glutamicum chromosome. In addition, cloning of the seven insertion sites by means of a plasmid rescue technique in E. coli (Tauch et al., 1995b) and subsequent DNA sequence analysis confirmed an IS6100-mediated cointegration of pAT6100 and the presence of an 8-bp target site duplication at the integration sites (data not shown). These data provided experimental evidence that IS6100 is transpositionally active in C. glutamicum and might be responsible for the segmental evolution Fig. 4. Transpositional activity of IS6100 in C. glutamicum ATCC 13032 (A) The genetic map of plasmid pAT6100 is shown. The plasmid consists of the mobilizable (RP4mob) cloning vector pK18mob2 and an IS6100-carrying DNA fragment. The kanamycin resistance marker (Km) can be used for selection. ori E.c.: origin of replication in E. coli. (B) Southern hybridization of chromosomal DNA from seven randomly selected kanamycin resistant transconjugants. The DNA was digested with EcoRI and probed with DIG-labeled pAT6100. Lanes 1 and 10, DIG DNA size marker II (Roche Diagnostics, Mannheim, Germany). Lane 2, C. glutamicum ATCC 13032. Lanes 3–9, C. glutamicum trans- conjugants. 126 A. Tauch et al. / Plasmid 48 (2002) 117–129 of the R-plasmid pTET3. A similar genetic ar- rangement was detected on the R-plasmid pAG1 from C. glutamicum 22243 where plasmid repli- cation and maintenance functions and the tetra- cycline resistance determinant Tet Z not only comprise regions which have significantly different G+C content but are also separated by the in- sertion sequence IS1628 (Tauch et al., 2000). A further integrational ‘‘hot spot’’ was identi- fied around 14-kb on the genetic map of pTET3 where three additional insertion sequences were present (Fig. 1). The pTET3 plasmid segment comprising the hypothetical coding region ORF15 as well as the insertion sequences IS1870 and IS1677 is virtually identical at the nucleotide level to a chromosomal DNA region from the type strain C. glutamicum ATCC 13032 (data not shown). The nucleotide sequence identity ends exactly at the inverted repeat sequences of IS1870 and IS1674 suggesting a genetic exchange between the C. glutamicum chromosome and pTET3. Therefore, it is likely that an ancestor molecule of pTET3was sequently loaded with additional DNA segments of different bacterial origin by means of horizontal mobile elements such as insertion se- quences. This evolutionary ‘‘gene loading’’ re- sulted in the generation of an R-plasmid conferring resistance against streptomycin, spectinomycin, and tetracycline with the aadA9 and Tet 33 genes. Acknowledgments The authors thank L.M. McMurry (Tufts University School of Medicine, Boston, MA) for classifying the tetracycline resistance determinant and R. M. Hall (CSIRO Molecular Science, North Ryde, Australia) for naming the strepto- mycin–spectinomycin resistance gene cassette. This work was supported by Grant BIO4CT960145 from the European Commission. References Allmeier, H., Cresnar, B., Greck, M., Schmitt, R., 1992. Complete nucleotide sequence of Tn1721: Gene organization and a novel gene product with features of a chemotaxis protein. Gene 111, 11–20. Altschul, S.F., Madden, T.L., Sch€aaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. 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Tauch et al. / Plasmid 48 (2002) 117–129 129 The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100 Introduction Materials and methods Bacterial strains and growth conditions Antimicrobial susceptibility screening Cloning and DNA sequencing of pTET3 Transposition assay with IS6100 in C. glutamicum ATCC 13032 Results and discussion Identification and sequence analysis of the R-plasmid pTET3 from C. glutamicum LP-6 The streptomycin-spectinomycin resistance region of pTET3 is part of an integron-like structure The tetracycline resistance region of pTET3 encodes a new class of Tet efflux systems IS6100 is transpositionally active in C. glutamicum ATCC 13032 Acknowledgements References