Characterization of the mobilization region of a Bacteroides insertion

JOURNAL OF BACrERIOLOGY, OCt. 1993, p. 6588-6598 0021-9193/93/206588-1 1$02.00/0 Copyright © 1993, American Society for Microbiology

Vol. 175, No. 20

Characterization of the Mobilization Region of a Bacteroides Insertion Element (NBU1) That Is Excised and Transferred by Bacteroides Conjugative Transposons LHING-YEW LI,* NADJA B. SHOEMAKER, AND ABIGAIL A. SALYERS

Department of Microbiology, University of Illinois, Urbana, Illinois 61801 Received 18 February 1993/Accepted 31 July 1993

Many Bacteroides clinical isolates carry large conjugative transposons that, in addition to transferring themselves, excise, circularize, and transfer smaller, unlinked chromosomal DNA segments called NBUs (nonreplicating Bacteroides units). We report the localization and DNA sequence of a region of one of the NBUs, NBU1, that was necessary and sufficient for mobilization by Bacteroides conjugative transposons and by IncP plasmids. The fact that the mobilization region was internal to NBU1 indicates that the circular form of NBU1 is the form that is mobilized. The NBU1 mobilization region contained a single large (1.4-kbp) open reading frame (ORF1), which was designated mob. The oriT was located within a 220-bp region upstream of mob. The deduced amino acid sequence of the mob product had no significant similarity to those of mobilization proteins of well-characterized Escherichia coli group plasmids such as RK2 or of either of the two mobilization proteins of Bacteroides plasmid pBFTM10. There was, however, a high level of similarity between the deduced amino acid sequence of the mob product and that of the product of a Bacteroides vulgatus cryptic open reading frame closely linked to a cefoxitin resistance gene (cfr4).

Species of Bacteroides, a genus of gram-negative obligately anaerobic bacteria, are a major component of the normal microflora of the human colon and can also cause opportunistic infections (23). Many clinical isolates carry large (>60-kbp) conjugative transposons that have been called tetracycline resistance (Tcr) elements because most of them carry a Tcr gene (19, 24, 27). The Tcr elements have an unusual activity. They mediate the excision and circularization of discrete unlinked 10- to 12-kbp segments of chromosomal DNA (32, 38). Since these circle forms do not replicate, they were designated NBUs (nonreplicating Bacteroides units). Two NBUs, NBU1 and NBU2, have been characterized to date (32). They differed in restriction pattern and size but shared some cross-hybridizing DNA, which was located in the interior of each element (30, 32). Many Bacteroides strains contain DNA that cross-hybridizes with NBU1, indicating that carriage of NBU-like elements may be widespread in the Bacteroides group. Previously, we had noted that NBUs were sometimes cotransferred with a Tcr element (la). Thus, it appeared that the Tcr elements could mobilize NBUs as well as excising them from the chromosome. Further evidence that NBUs were mobilizable was obtained when Shoemaker et al. (30) isolated three hybrid plasmids, designated Y5, Y11, and Y17, that consisted of a mobilization-deficient plasmid (pEG920) inserted into different sites on NBU1 (30, 34). Y5 and Y1 1 were mobilized from Bacteroides donors by a Tcr element (Tcr ERL) at a much higher frequency than pEG920 alone. Y5 and Y11 were also mobilized from Escherichia coli donors by the IncP plasmid R751 at a 1,000-fold-higher frequency than pEG920. These results suggested that NBU1 carried a mobilization (mob) region that was recognized both by the Bacteroides Tcr elements and by R751 (34). The third hybrid, Y17, was mobilization deficient, possibly because of insertion of pEG920 into a mobilization gene on NBU1. *

Corresponding author. 6588

We have constructed a vector that is not mobilized by the Tcr elements and have used it to locate the NBU1 mobilization region. We report that a single 1,404-bp open reading frame (ORF) on NBU1 was necessary and sufficient for mobilization by Tcr elements and IncP plasmids and that a 220-bp segment upstream of the start codon probably contains the oriT. Finally, we provide evidence that a cefoxitin resistance gene (cfxA4) found in a clinical isolate of Bacteroides vulgatus (21) is carried on an NBU-like element, indicating that NBUs could be contributing to the spread of antibiotic resistance genes among Bacteroides strains.

MATERIALS AND METHODS

Strains, plasmids, and growth conditions. The strains and plasmids used in this study are listed in Table 1. E. coli DH5aoMCR was used for cloning and as a donor in some mating experiments. E. coli HB101 or EM24NR was used as the recipient in E. coli-E. coli matings. For mating experiments in which a Bacteroides sp. was the donor, derivatives of Bacteroides thetaiotaomicron 5482A were used. This strain was chosen because it contains no known Tcr_type elements or NBUs (27, 32). The Tcr element used to mobilize test plasmids was Tcr ERL, an element originally isolated from Bacteroides fragilis ERL (28, 31). Bacteroides strains were cultivated in prereduced Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extractglucose (TYG) broth or on TYG agar medium in a BBL GasPak jar system (15, 29). Strains of E. coli were grown in Luria broth (LB) or on LB agar plates. Antibiotic concentrations (micrograms per milliliter) were as follows: ampicilin, 100; erythromycin, 100; gentamicin, 200; kanamycin, 100; spectinomycin, 40; streptomycin, 100; tetracycline, 1; thymidine, 100; and trimethoprin, 100. Bacteroides strains harboring plasmids carrying the cefoxitin resistance (Cef) gene (cfxA) were grown in a lower concentration of ampicillin (25 to 50 ,ug/ml) to prevent cell lysis. To test mobilization of plasmids,

MOBILIZATION REGION OF NBU1

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TABLE 1. Bacterial strains and plasmids used in this study Plasmid or strain

Plasmids R751 RP4 pEG920

Tpr Tra+ Tcr Tra+ Apr Tcr (Emr) Mob+/

Y11

Apr Tcr (Emr) Mob'

Y17 Y11-SstI

Y1I-PvuII Y1I-AvaI Y11-PA pFD160R pFD160R-CE

Apr TCr (Emr) Apr TCr (Emr) Apr Tcr (Emr) Apr TCr (Emr) Apr Tcr (Emr) Apr Mob' Apr Mob

pFD288 pFD288-CE

Spr (Emr) Mob' Spr (Emr) Mob

pFD351 pLYL7

Spr (Apr) Mob' Apr (Apr) Mob

pLYLIlS, pLYLIIH

Apr Mob'

pLYL11HR pLYLI1

Apr Mob' Apr (Apr)

pLYLl lEAB

Apr

pLYL20

Apr (Apr)

pLYL21

Cmr

E. coli strains HBI0I EM24NR

DH5tMCR S17-1 B. thetaiotaomicron 5482A

derivatives BT4001 BT4100 BT4104

Description and/or reference

Relevant phenotype"

RecA RecA RecA Mrr RecA-

Mob

Mob' Mob' MobMob-

Strr Strr Nalr Rifr McrA- McrB HsdM- HsdR Tpr Strr

Self-transmissible IncP plasmid (16, 18) Self-transmissible IncP plasmid (7) E. coli-Bacteroides shuttle vector that contains Bacteroides plasmid pB8-51 (28) Hybrid plasmid produced by integration of pEG920 into a site on NBU1 (30) Same as Yt 1 except pEG920 integrated at a different site on NBU1 (30) Deletion of Y11 (this study [Fig. 2]) Deletion of Y11 (this study [Fig. 2]) Deletion of Y11 (this study [Fig. 2]) Deletion of Y11 (this study [Fig. 2]) Bacteroides plasmid pBI143 cloned into pUC19 (36) Deletion of pFD160R that removes the mobilization region on pBI143 (this study [Fig. IA]) Bacteroides ermF gene and RK2 oriT cloned into pI6OR (37) Deletion of pFD288 that removes the mobilization region of pBI143; still mobilizable by RP4 because of oriT of RK2 (this study) Bacteroides cfxA DNA cloned into pFD288 (21) Mobilization-deficient vector (this study [Fig. IB]) 3.2-kbp Clal-PvuII fragment of Yll cloned into pUC19 at the SmaI (S) or Hincll (H) site (this study) Reverse orientation of pLYLIIH (this study) 3.2-kbp ClaI-PvuII fragment of Y 1I cloned into the SmiaI site of pLYL7 (this study) Nested Erase-a-Base deletions into the ClaI-PvuII fragment from Y1 I (this study) 1.9-kbp EcoRl-AfllII fragment of pLYLI IEAB6 cloned into pLYL7 at the SmaI site (this study) 1.9-kbp EcoRI-AfllII fragment of pLYLl 1EAB6 cloned into pACYC184 at the EcoRV site (this study)

Boyer and Roulland-Dussoix (3) Nalr Rif' derivative of EM24 (30) GIBCO/BRL RP4 integrated in the chromosome (35)

Rif' derivative of B. thetaiotaomicron 5482A (33) Thy- Tpr derivative of B. thetaiotaomicron 5482A (33) BT4100 carrying the TCr ERL element (34) ' Phenotypes not in parentheses refer to those expressed in E. coli. Phenotypes in parentheses indicate those expressed in B. thletaiotaomicroni 5482A strains. Abbreviations: Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Nalr, nalidixic acid resistance; Rif, rifampin resistance; Spr, spectinomycin resistance; Strr, streptomycin resistance; TCr, tetracycline resistance; Thy thymidine auxotroph; Tpr, trimethoprim resistance; Mob', mobilized by R751

(Rif) (Thy - Tpr) (Thy- Tpr TCr)

and/or Bacteroides TCr ERL; Mob-, not mobilized by R751 or Bacteroides TCr ERL; Tra+, conjugal plasmid.

cultures of Bacteroides mating donors were grown in TYG plus tetracycline (final concentration, 1 p.g/ml). DNA isolation and analysis. Plasmid DNA was prepared by the Ish-Horowitz modification of the alkaline lysis procedure (17). Restriction endonuclease digestions and T4 ligation reactions were performed according to the instructions of the manufacturer (GIBCO/BRL, Gaithersburg, Md., or New England Biolabs, Inc., Beverly, Mass.). DNA samples were loaded on a 0.8 to 1.2% agarose gel and electrophoresed in 1 x GGB (40 mM Tris, 25 mM sodium acetate, 2 mM EDTA [pH 8.3]) for 2 h at 200 V. A low-melting-point gel (SeaPlaque agarose; FMC BioProducts, Rockland, Maine) was used to purify restriction fragments of interest for plasmid constructions. The Klenow fragment of DNA polymerase I was used to create blunt-ended DNA fragments for ligation. Southern blotting and hybridization were done as described by Maniatis et al. (17). Localization of the region on NBU1 that was necessary for mobilization by IncP plasmids. The initial localization of the

mobilization region on NBU1 was done by constructing deletions of hybrid plasmid Yl 1 that eliminated portions of NBUI and pEG920 and testing them for mobilization from E. coli donors to E. coli recipients by IncP plasmid R751. Deletions were generated by complete or partial restriction digestion of YI1 with each of the following enzymes or combinations: SstI, PvuII, and AvaI. Digested plasmids were religated to generate

YII-SstI, Y11-PvuII, Y11-AvaI, respectively (see Fig. 2). YI1-PA was derived from Y11-PvuII by an AvaI digest. For further localization of the mobilization region, the 3.2-kbp ClaI-PvuII fragment from Y11, which contained 2.5 kbp of NBU1 DNA plus a 0.7-kbp junction DNA from a segment of Tn4400 (that supplied the Bacteroides selectable marker, ermF [22]), was cloned into the SmaI site on pUC19 to generate pLYL1 1S or into the HinclIl site at both orientations to generate pLYLIIH and pLYLIIHR, respectively. The residual 0.7 kbp of Tn4400 DNA and portions of the 2.5-kbp NBU1 DNA segment were removed in Erase-a-Base clones (designated by EAB and a number) which were generated from

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pLYLlIH or pLYLl1S by using the Erase-a-Base kit (Promega, Madison, Wis.). Deletions of Y11 or subclones were transformed into E. coli DH5oxMCR (GIBCO/BRL). To assay for mobilization of these constructs, a triparental mating was done as described previously (39), with E. coli J53(R751) and E. coli DH5oLMCR (carrying the subclone) as donors and E. coli HB1I1 or EM24NR as the recipient. Construction of a mobilization-deficient vector for testing subclones of NBU1 for mobilization in Bacteroides spp. The Bacteroides-E. coli shuttle vector pFD160R (36) was used as the starting plasmid. pFD160R is a hybrid of pUC19 and a small Bacteroides cryptic plasmid, pBI143 (2.7 kbp). pBI143 is known to contain a replication region that works in Bacteroides spp. and a mobilization region that is recognized by R751 and by the Tcr elements (38). Preliminary characterization of the plasmid by Smith (36) had indicated that pBI143 might be a simpler plasmid with a restriction pattern more convenient than those of two other Bacteroides plasmids, pB8-51 and pBFTM 1O, that have been used as bases for shuttle vectors. To locate the mob region on pBI143, a 1.9-kbp Hincll-fragment of pRME19 containing the Tn9O3 kanamycin cartridge (13) was inserted into various restriction sites on pBI143, and the resulting insertional mutants were tested for mobilization from E. coli DH5oLMCR by R751 (Fig. 1A). The mobilization region of pBI143, tentatively identified by these insertional mutagenesis experiments, was eliminated by deleting the ClaI-EcoRI region on pFD16OR, and the resulting construct (pFD160RCE) was found to be completely mobilization deficient in E. coli; i.e., it was no longer mobilized by R751 (< 10 9 transconjugants per recipient). To test whether the deletion in pBI143 abolished mobilization by Tcr elements in Bacteroides spp. but still retained the replication functions of pBI143, it was necessary to introduce a selectable Bacteroides marker in the plasmid and to have some means of introducing the plasmid into Bacteroides spp. To date, attempts to introduce DNA isolated from E. coli into B. thetaiotaomicron 5482A derivatives by electroporation have not been successful, although the same DNA isolated from B. thetaiotaomicron can be reintroduced by electroporation into the same strain at high frequencies (1). Thus, plasmids must be transferred from E. coli to B. thetaiotaomicron by conjugation. To provide mobilization functions that would not interfere with experiments to locate mobilization functions on NBU1, we used the oriT of RK2 (12). This oriT is not recognized either by R751 (11) or by the Bacteroides Tcr elements (33) and thus does not interfere with testing NBU1-mediated mobilization by the Tcr elements or R751. Plasmid pFD288 is pFD160 with a selectable Bacteroides marker (ermF) plus the RK2 oriT (37). The ClaI-EcoRI fragment that contained the mobilization region of pBI143 was removed from the pBI143 portion of pFD288 to construct pFD288-CE. pFD288-CE was mobilized into Bacteroides recipients by IncP plasmid pRP4 (which recognizes the RK2 oril), using the same mating conditions

described previously for E. coli-Bacteroides matings in which R751 was the mobilizing plasmid (39). Although pFD288-CE would have been adequate for the experiments described in this paper, we wanted a test vector that could be used in the future with B. thetaiotaomicron strains that were both Tcr and Emr (e.g., strains that contained elements carrying both Tcr and Emr genes or insertional mutations of the Tcr ERL element constructed with Emr as the selectable marker). Accordingly, the cefoxitin resistance gene cfxA, carried on a BamHI-EcoRI fragment from pFD351 (21), was band extracted and inserted into the unique AatII site on pFD160R-CE to create pLYL7 (Fig. IB). The cfxA gene conferred resistance to both cefoxitin and ampicillin in B. thetaiotaomicron. Two pieces of NBU1 DNA (the 3.2-kbp ClaI-PvuII and the 1.9-kbp piece from bp 491 to 2412) were then cloned into pLYL7 to generate pLYLI I and pLYL20 (Table 1) and tested for mobilization from B. thetaiotaomicron to E. coli by the Bacteroides Tcr ERL element as described previously (39). In trans mobilization to localize the oriT region of NBU1. There are regions of DNA upstream of the NBU1 mob open reading frame that are required for mobilization. To examine whether this is the oriT region, we cloned the 1.9-kbp mobilization-positive NBU1 DNA into pACYC184 (5) to generate pLYL21 and transformed pLYL21, along with various Erasea-Base subclones of the upstream region (which are pUC19based constructs), into S17-1. Mobilization of the potential oriT region could be mediated by a combination of the transfer functions from the IncP RP4 and the cognate NBU1 mob gene in trans. DNA sequence analysis. The DNA sequence of a 2.5-kbp region of NBU1 that was sufficient for mobilization was obtained by the dideoxy-chain termination method with the Sequenase 2.0 kit (U.S. Biochemical, Cleveland, Ohio). The DNA templates were prepared from overlapping Erase-a-Base deletion clones of pLYL1I, pLYLIIH, and pLYLIIS. Both strands were sequenced. DNA sequence analysis was done with GCG package software (Genetics Computer Group, University of Wisconsin) (9). The insertion point of pEG920 into NBUI in the mobilization-deficient hybrid Y17 was first determined to be in the mobilization region by restriction analysis. The precise insertion point was determined by comparing the DNA sequence of the pEG920-NBU1 junction region (28) with the sequence of the NBU1 mobilization region. Nucleotide sequence accession number. The GenBank accession number for the sequence in Fig. 4 is L13840. RESULTS AND DISCUSSION Localization of the NBU1 mob region. Results of the initial localization of NBU1 DNA required for mobilization by R751,

FIG. 1. Construction of pLYL7, the vector used to test mobilization phenotypes of cloned NBU 1 segments by Bacteroides Tcr ERL. (A) Linear map of pBI143 and locations of insertions made with a Knr cassette in the pBI143 segment of pFD160R. The arrows under the insertions indicate the orientation of the cassette. Mobilization phenotypes of the insertional mutants refer to mobilization between E. coli strains by R751. mob-,