Jan 17, 1989 - Transconjugant TM4.2321 transferred pGAT400ABglII to Escherichia coli. HB101 at ... By using shuttle vectors such as pGAT400, a chimeric.
JOURNAL OF BACTERIOLOGY, JUlY 1989, p. 3603-3608
Vol. 171, No. 7
0021-9193/89/073603-06$02.00/0
Copyright (C 1989, American Society for Microbiology
Tn4399,
a
Conjugal Mobilizing Transposon of Bacteroides fragilis DAVID W. HECHTt
AND
MICHAEL H. MALAMY*
Department of Molecular Biology and Microbiology, Tuifts University Health Sciences Camputs, 136 Harrison Avenue, Boston, Massachusetts 02111 Received 17 January 1989/Accepted 7 April 1989
Conjugal transposons play an important role in the dissemination of antibiotic resistance determinants in the streptococci and have been postulated to exist in Bacteroides fragilis. To investigate the presence of conjugal transposons in B. fragilis, we employed a Tra- derivative of the transfer factor pBFTM10 contained in the chimeric plasmid pGAT400ABglII. We attempted to restore transferability to this plasmid from a series of transconjugants generated by crossing B. fragilis TMP230 containing the TET transfer factor with B. fragilis TM4000, a standard recipient. Transconjugant TM4.2321 transferred pGAT400ABglII to Escherichia coli HB101 at almost the same frequency as did the Tra+ parental plasmid, pGAT400. Analysis of the transferred plasmids revealed the presence of 9.6 kilobases of additional DNA in every case but at different positions in independent isolates. The presence of this DNA, designated Tn4399, allowed the pGAT400ABgIII derivatives to retransfer from the TM4000 background to B. fragilis or E. coli recipients. DNA hybridization studies demonstrated the presence of one copy of Tn4399 in TMP230 and three copies at new sites in TM4.2321. Tn4399 is a new B. fragilis transposon with unique transfer properties that may play a role in the dissemination of drug resistance genes. It differs from previously described conjugal transposons by its ability to mobilize nonconjugal plasmids in cis.
Many Bacteroides fragilis strains transfer chromosomal antibiotic resistance determinants and autonomous plasmids by a conjugation process to B. fragilis and other Bacteroides species (see references 13 and 17 for recent reviews). Although autonomous transfer factors such as pBF4 and pBFTM10 (=pCP1) have been described in B. fragilis, transfer of resistance markers can also be detected from apparently plasmid-free donor cells (9, 12, 19, 20, 24). The efficiency of transfer of tetracycline resistance from Tetr plasmid-free cells such as TM2000 and TMP230 is increased several orders of magnitude by pretreatment of the donor cells with subinhibitory concentrations of tetracycline (9, 14, 15). Transferability in these Tetr donor cells has been ascribed to a TET element that is presumed to be located in the chromosome (9, 11, 15). The presence of the TET element also leads to increased transfer of autonomous plasmids such as pBFTM10 but only after growth in the presence of tetracycline (9, 17). The nature of the TET element, the mechanism of tetracycline resistance transfer, and the mechanism by which the TET element mobilizes in trans remain open questions. We and others have proposed that the TET element may represent a conjugal transposon like Tn916 of Streptococcus faecalis (2) and that the TET element transfer functions are induced by tetracycline. By using shuttle vectors such as pGAT400, a chimeric plasmid containing pBFTM10 and pDG5 (16; this paper) can be transferred from B. fragilis to Escherichia coli by conjugation. The presence of the TET element in the donor cell increases the frequency of pGAT400 transfer of E. coli after tetracycline pretreatment. Tra- mutants of pGAT400 have been isolated after insertional mutagenesis by TnlOOO (unpublished data) or after deletion of portions of pBFTM10 by in vitro manipulation. Most TM4000 transconjugants containing the TMP230 TET element cannot mobilize these Tra- pGAT400 derivatives.
However, rare Tra+ derivatives of the Tra- plasmids can be isolated from a unique transconjugant of a TMP230 x TM4000 cross; these plasmids all have acquired a 9.6 kilobase (kb) transposable element that we have named Tn4399. In this report, we show that Tn4399 confers transferability on Tra- deletion derivatives and Tra-::Tn1000 mutants of pGAT400 in both B. fragilis x B. fragilis and B. fragilis x E. coli crosses. Thus, Tn4399 represents a new type of conjugal transposon capable of mobilizing Tra- plasmids in cis. MATERIALS AND METHODS
Bacterial strains and media. Characteristics of the bacterial strains used are listed in Table 1. B. fragilis strains were grown on brain heart infusion broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 0.5% hemin and 0.5% yeast extract (BHIS), and the medium was solidified with 1.5% agar when required. Cultures were grown in a Coy anaerobic chamber in an atmosphere of 85% N2-5% CO-10% H2. E. coli strains were grown in L broth or on L agar under aerobic conditions. The following concentrations (micrograms per milliliter) of antibiotics were used for selective media: rifampin, 50; tetracycline, 5; ampicillin (Sigma Chemical Co., St. Louis, Mo.), 200; clindamycin, 6; and trospectomycin (The Upjohn Co., Kalamazoo, Mich.), 10. Resuspension buffer was 8 mM Na2HPO4-2 mM NaH2 P04-0.145 M NaCl, pH 6.9. Plasmid constructions. The shuttle vector pGAT400 (Fig. 1) was constructed by ligating the B. fragilis transfer factor pBFTM10 to the E. coli plasmid pDG5, as previously described for the construction of a similar plasmid, pGAT500 (16). pGAT400 can replicate in both E. coli and B. fragilis. In addition, it contains the Clnr determinant of Tn4400, which is expressed in B. fragilis, and the Ampr determinant of pDG5, which is expressed in E. coli. pGAT400 can transfer at low efficiency from B. fragilis to E. coli, as previously described for pGAT500 (16), and can be efficiently mobilized from E. coli to B. fragilis by the
Corresponding author. t Present address: Loyola University Medical Center, Maywood, IL 60153. *
3603
3604
J . BACTERIOL .
HECHT AND MALAMY TABLE 1. Bacterial strains and plasmids" Source or reference
Relevant phenotype
Strain or plasmid
Bacterial strains B. fragilis Tet' Rif' Tetr Tetr Cln' Trs' Trs' Sm''
TMP230 TM4000 TM4.23 TM4.2321
TM4000(pGAT400ABGIII) TM429 TM420 E. coli HB101 Plasmids pRK231 pGAT400 pGAT400ABGlII pTGD94 pACYC184 pDWH10 pDWH11L pDWH12R
Clinical isolate (11) M. Sebald, Pasteur Institute, Paris, France This paper This paper This paper Spontaneous Trs' mutant of TM4000 Trs' Arg His auxotrophic mutant of TM4000 Maniatis et al. (10)
Cln' Trs'
Rif' Rif' Trs' Rif' Rif' Rif"
Guiney et al. (6) B. ftagilis-E. coli shuttle vector (16; this paper) Tra- deletion of pGAT400 (this paper) Tra- TnlOOO mutant of pGAT400 (this paper) E. coli plasmid used as cloning vector This paper This paper This paper " Abbreviations: Ampr, Clnr, Cmr, Kanr, Rif, Sm', Tet', and Trs', resistance to ampicillin. clindamycin, chloramphenicol, kanamycin, rifampin. streptomycin, tetracycline, and trospectomycin, respectively; Trs\, sensitivity to trospectomycin; Arg, arginine; His, histidine. Kan' Cln' Amp" Tra+ CIn' Amp" TraClnr Amp" TraCm' Tetr Cm'' Cm' Ampr
broad-host-range transfer factor RK231 (an RP4 derivative)
(7).
A Tra- deletion derivative of pGAT400 deficient in transfer from B. fragilis to B. fragilis or to E. coli was created by deleting a series of BglII restriction fragments in pBFTM10. The resulting plasmid, pGAT400ABglII, has lost 4.0 kb and retains a single BglII site (Fig. 1B). TnIOOO insertion mutations of pGAT400 were isolated after mobilization by F' lac as previously described (8). The location of Tn1000 inserted in plasmid pTGD94 (Tra-) is shown in Fig. 1A. Transfer experiments. Filter mating techniques, doublefilter assays, and DNase experiments were carried out as previously described (22). Donor and recipient cultures
were mixed under aerobic conditions in all cases and then incubated either anaerobically or aerobically, as indicated. Transfer frequencies were calculated by dividing the number of antibiotic-resistant transconjugants isolated by the number of input donor cells. Tetracycline induction was achieved by adding 1 pLg of tetracycline per ml to growing B. fragilis strains 2 h before mating. Preparation of plasmid DNA. Plasmid DNA was purified from E. coli and B. fragilis by an alkaline lysis method (1, 10) or after cesium chloride ethidium bromide density gradient ultracentrifugation (3). All restriction endonucleases were obtained from New England BioLabs Inc. (Beverly, Mass.) and were used under the conditions recommended by the supplier. DNA fragTGD94
pRK2 OriT
E
E
A.
H H
B 1B
E
r,%.
v
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v-
-I-
A-_ 'r pDG5 Amp -r%%r
E
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Tn4400 Cln' *Tetr
nRFTM1 pur- I ivi vn
H H
E
HHHE
BH
E
.
E,
25
I
21
I
I
v
1
. v
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Delete this region
E
E
E
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423,12
13
pGAT400ABglII
10 14 H
E
H4IEI
pGAT400ABglII; DNA insertions 1kb FIG. 1. Restriction maps of plasmids pGAT400 and pGAT400ABgll and sites of DNA insertions. (A) Restriction map of pGAT400 showing its components and the site of TnIOOO insertion in pTGD94. (B) Origin of pGAT400ABglIl by deletion of the indicated region in pGAT400, resulting in a plasmid with a unique BglIl site and a Tra- phenotype. (C) Sites of DNA insertions in pGAT400ABgIII ( I ). Numbers indicate individual insertions and correspond to those given in the legend to Fig. 2. Enzyme abbreviations: B, BglIl; E, EcoRI; H, Hindlil.
VOL. 171, 1989
CONJUGAL TRANSPOSON Tn4399
3605
TABLE 2. Transfer of Tetr from TET element-containing strains with and without tetracycline induction Donor strain
Tetracycline induction
TMP230 TMP230 TM4.23 TM4.23 TM4.2315 TM4.2315 TM4.2321 TM4.2321
+
Recipient
+ +
+
Mean frequency of Tet" transfer/ input donor ± SE
TM4000 TM4000 TM429 TM429
8.8 x 10-6
TM4000(pGAT400ABGIII) TM4000(pGAT400ABGIII) TM4000(pGAT400,ABGIII) TM4000(pGAT400ABGlII)
1.5 x
8.5 x
9.2 x 1.3 x
+
4.6 x 10-6 (8)"
