production of two proteins encoded by the bacteroides mobilizable

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.00�0 DOI: 10.1128/JB.183.21.6335–6343.2001

Nov. 2001, p. 6335–6343 Vol. 183, No. 21

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Production of Two Proteins Encoded by the Bacteroides MobilizableTransposon NBU1 Correlates with Time-Dependent Accumulation

of the Excised NBU1 Circular FormJUN WANG, GUI-RONG WANG, NADJA B. SHOEMAKER, AND ABIGAIL A. SALYERS*

Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received 4 April 2001/Accepted 8 August 2001

NBU1 is a mobilizable transposon that excises from the Bacteroides chromosome to form a double-strandedcircular transfer intermediate. Excision is triggered by exposure of the bacteria to tetracycline. Accordingly, weexpected that the expression of NBU1 genes would be induced by tetracycline. To test this hypothesis,antibodies that recognized two NBU1-encoded proteins, PrmN1 and MobN1, were used to monitor productionof these proteins. PrmN1 is essential for excision, and MobN1 is essential for transfer of the excised circularform. At first, expression of the genes encoding these two proteins appeared to be regulated by tetracycline,because the proteins were detectable on Western blots only after the cells were exposed to tetracycline.However, when the prmN1 gene and/or the mobN1 gene was cloned on a multicopy plasmid, production of theprotein was constitutive. Initially, we assumed that the constitutive expression was due to loss of a repressorprotein that was encoded by one of the other genes on NBU1. Deletions or insertions in the other genes (orf2and orf3) on NBU1 and various integrated NBU1 derivatives abolished production of PrmN1 and MobN1. Thisis the opposite of what should have happened if one or both of these genes encoded a repressor. A secondpossibility was that when NBU1 excised, it replicated transiently, increasing the gene dosage of prmN1 andmobN1 and thereby producing enough PrmN1 and MobN1 for these proteins to become detectable. In fact, afterthe cells entered late exponential phase the copy number of NBU1 increased to 2 to 3 copies per cell. Productionof PrmN1 and MobN1 showed a similar pattern. Any mutation in NBU1 that decreased or prevented excisionalso prevented elevated production of these two proteins. Our results show that the apparent tetracyclinedependence of the production of PrmN1 and MobN1 is due to a growth phase- or time-dependent increase inthe number of copies of the NBU1 circular form.

Bacteroides species harbor integrated mobilizable elements,called mobilizable transposons (MTns). The first step in mo-bilization of an MTn is excision and circularization, an activitythat requires the trans action of two regulatory proteins, RteAand RteB, which are provided by a coresident conjugativetransposon (CTn). The circular form of the MTn is then mo-bilized by other CTn-encoded transfer proteins to a recipientcell, where it integrates into the chromosome (15, 23). Anexample of an MTn is NBU1, a 10.3-kbp element that is ex-cised and mobilized by CTns of the CTnERL/CTnDOT family.CTnERL and CTnDOT are virtually identical except thatCTnDOT carries an additional 13-kbp region (18, 29). Inte-grated elements that cross-hybridize with NBU1 have beenfound in over half of all Bacteroides strains tested, representingat least 10 different species (28). Thus, excision and transfer ofthese MTns appears to occur commonly in nature.

The rteA and rteB genes that are required to initiate excisionand transfer of NBU1 are part of an operon that also containsthe tetracycline resistance gene tetQ. The proteins encoded byrteA and rteB appear to be members of a two-component reg-ulatory system in which RteA is the sensor and RteB is theresponse regulator (14). Downstream of the tetQ-rteA-rteBoperon is a third regulatory gene, rteC, whose expression iscontrolled by RteA and RteB. RteC controls many CTn exci-

sion and transfer functions but does not appear to be involvedin NBU1 excision (1a, 14, 26). Transcription of the tetQ-rteA-rteB operon is induced by tetracycline (10, 25). This explainswhy exposure of a donor cell to tetracycline is required totrigger excision and transfer of NBU1 (15, 23, 25).

Excision of NBU1 appears to be a complex process involvingseveral NBU-encoded genes as well as the CTn genes rteA andrteB (22). The minimal excision region of NBU1 is indicated inFig. 1. It includes the integrase gene (intN1), four genes ofunknown function (orf2, orf2x, orf3, prmN1), the transfer origin(oriT), and two-thirds of the mobilization gene (mobN1).IntN1, together with the joined ends of NBU1, is necessary andsufficient for integration of the NBU1 circular form. The intN1gene from either the integrated element or a plasmid is ex-pressed constitutively (21). In contrast to excision and mobili-zation, integration does not require the trans action of any ofthe proteins encoded by the CTn. Thus, although intN1 isessential for excision as well as integration (21), it is not re-sponsible for the tetracycline regulation of excision. TheMobN1 protein is required for mobilization of the NBU1 cir-cular form (7, 9). MobN1 nicks at the oriT to initiate transferof the NBU1 circular intermediate through the mating appa-ratus provided by the CTn. Approximately two-thirds of themobN1 gene is required for excision (Fig. 1).

To obtain more information about how NBU1 excision isregulated, it was first necessary to ascertain whether expressionof any of the NBU1 genes is regulated. We focused our atten-tion on two genes required for NBU1 excision and mobiliza-

* Corresponding author. Mailing address: Department of Microbi-ology, 601 S. Goodwin Ave., University of Illinois, Urbana, IL 61801.Phone: (217) 333-7378. Fax: (217) 244-8485. E-mail: [email protected].

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tion, prmN1 and mobN1. There were two reasons for thisdecision. First, the prmN1-oriT-mobN1 region (Fig. 1) is highlyconserved between the two NBUs studied to date, NBU1 andNBU2 (9). Outside this region, there is little sequence similar-ity between the two NBUs (22, 28). Since both NBU1 andNBU2 require RteA and RteB for excision, it seemed likelythat the conserved genes would be the regulated ones if RteBwas activating their expression. Second, previous studies hadshown that a multicopy plasmid carrying prmN1, oriT, andmobN1 (pPOM; Fig. 1) could completely suppress excision ofa wild-type NBU1 when provided in trans with the NBU1 (22).The suppression of NBU1 excision required all of prmN1-oriTand 450 bp of mobN1. Smaller plasmids carrying parts of thisregion (prmN1-oriT, oriT, or mobN1-oriT) did not have anyeffect on excision of NBU1. No other DNA segment from theexcision region had a suppressive effect (22). Thus, PrmN1 andthe N-terminal two-thirds of MobN1 appear to be importantproteins in the excision process. In this report, we show thatcontrary to expectation, both mobN1 and prmN1 are expressedconstitutively (not induced by tetracycline) and that what ini-tially appeared to be regulated expression of these genes wasdue to a time-dependent increase in the copy number of thecircular form of NBU1 after excision from the chromosome.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterial strains and plasmidsused in this study are shown in Table 1. Escherichia coli strain DH5�MCR(Gibco-BRL) was used as a host for plasmid construction. E. coli strainsBL21(DE3) (27) carrying the pLysS inhibitor plasmid (Novagen) and M15 car-rying the pREP4 repressor plasmid (Qiagen) were used for bacterial induction ofthe His6-tagged proteins. The E. coli strains were grown aerobically in Luria-Bertani (LB) broth or plated on LB agar plates at 37°C. The Bacteroides strainslabeled BT (e.g., BT4001 and BT4004) are derivatives of Bacteroides thetaio-taomicron 5482A, also called ATCC 29148 (Virginia Polytechnical InstituteAnaerobe Laboratory, Blacksburg, Va.). The Bacteroides strains are grownanaerobically in prereduced trypticase-yeast extract-glucose (TYG) (6) broth oron TYG agar plates incubated in BBL GasPak jars at 37°C. For tetracyclineinduction of the regulatory operon the Bacteroides strains were grown in TYGbroth or on TYG agar containing 2 �g of tetracycline/ml (20).

Vectors and strain construction. The plasmids used for cloning are de-scribed in Table 1. For insertional disruptions, the DNA fragments of interestwere cloned into either pCQW1 (5) to try to detect transcription or intopGERM (22). These two vectors replicate in and can be mobilized out of E.

coli by IncP plasmids but cannot replicate in B. thetaiotaomicron (see Table1). The resulting vectors were mobilized into BT4104N1-3 (which containedintegrated copies of NBU1 and CTnERL), with selection for the erythromy-cin (10 �g/ml) resistance genes on the plasmids. All insertional disruptionswere checked by Southern blotting to confirm that they had inserted in theexpected NBU1 location. For complementation tests, the NBU1 segment wascloned into pNLY1, which contains a chloramphenicol (15 �g/ml) resistancemarker, or pLYL05 or pLYL7, which carry cfxA, the cefoxitin (20 �g/ml)resistance gene (8, 22). These plasmids are shuttle vectors based on pFD160(24), which replicates both in E. coli and in B. thetaiotaomicron strains. All ofthe NBU1 integrative derivatives containing specific deletions were clonedinto pGERM and were then transferred by conjugation from E. coli donors toBT4004 recipients, where they site-specifically integrated into the 3� end of atRNALeu gene in the chromosomal target site, attBT1-1 (21). The vectorscontaining NBU1 regions used for complementation attempts were trans-ferred by conjugation into various Bacteroides strains containing �pNBU1derivatives or insertion mutants in NBU1 and were tested for excision of theNBU1 as previously described (22).

DNA manipulations. Plasmid DNA was isolated from E. coli and Bacteroidesstrains by the method of Ish-Horowitz as described by Sambrook et al. (16).Restriction digests and ligations were performed essentially as specified by themanufacturer (Bethesda Research Laboratories, Inc., Gaithersburg, Md., or NewEngland Biolabs, Inc., Beverly, Mass.). Chromosomal or total DNA preparationswere made using a modification of the method of Saito and Miura (13) aspreviously described (22).

Southern blots. NBU1 excision assays were conducted as described by Shoe-maker et al. (22). Most of the insertional disruptions and deletions in NBU1genes have been previously described and most were recloned into pGERM forthis study (22). The newly constructed deletions in orf2 and orf2-orf3 are de-scribed in Table 1. The 1.7-kbp HincII fragment of NBU1 containing the joinedends or the attN1 region was used as the probe (21). The fragment used formobN1 detection to follow increased copy number of the NBU1 in a tetracycline-induced culture was the 1-kbp HincII-PvuII 3� end of mobN1. The 0.9-kbp 5� endof the chromosomal starch utilization gene susG (17) was used to probe for asingle copy gene in the same strain. The probes were labeled with fluorescein-dUTP by using random primers as specified in the NEN Life Sciences Renais-sance kit protocol. The Southern blots were developed using a chemiluminescentsubstrate and exposure to film.

Overexpression and purification of mobN1 and prmN1 gene products in E. coli.The pET T7 promoter expression system (Novagen) was used to overexpressN-terminally His6-tagged MobN1 (27). The mobN1 gene was PCR amplifiedfrom B. thetaiotaomicron strain BT4104N1-3, which contains an integratedNBU1, with primers (mobN1-F [bp 8584], AA G AAT TCA TGG CAA CAAAAT CAA BCA TAC AC, and mobN1-R [bp 10,011], TC G AAT TCT ATCATA ATT ACA TTC TGA ATC CT) that contained EcoRI sites (shown in boldletters). The PCR product was first cloned into PCR product cloning vectorpGEM-T (Promega) and the product was sequenced to confirm that there wereno mutations. A 2.4-kbp EcoRI fragment was then isolated and cloned intoexpression vector pET-28a(�) digested with EcoRI. The resulting clone,pETmobN1, was used to transform the expression host strain BL21(DE3) car-rying the inhibitor plasmid pLysS. Kanamycin-resistant transformants (Knr, 50�g/ml) were selected. The T5 promoter-based QIAexpress system (Qiagen) wasused to overexpress C-terminally His6-tagged PrmN1. The full-length 971-bpprmN1 was PCR amplified from B. thetaiotaomicron BT4104N1-3 with the for-ward primer containing an NcoI site (shown in bold) (prmN1-F [bp 7420], CGCAAG A CC ATG GCA ATA GAA GAA) and the reverse primer containing aBglII site (shown in bold) (prmN1-R [bp 8391], AGT GGG AGA TCT CCGAAA GCC GTT TTT). The PCR product was digested with NcoI and BglII andinserted directly into the corresponding sites in pQE60. The resulting clone,pQE60prmN1, was used to transform the expression host strain M15 carryingrepressor plasmid pREP4.

To induce the expression of His-tagged recombinant MobN1 and PrmN1, hoststrains that contained pETmobN1 and pQE60prmN1 were initially grown at37°C in LB broth to an optical density (OD) between 0.5 and 0.7. IPTG (iso-propyl-�-D-thiogalactopyranoside) was then added to the medium to a finalconcentration of 1 mM. The cultures were incubated for an additional 3 to 4 hbefore being harvested. Recombinant MobN1 and PrmN1 were purified withNi-nitrilotriacetic acid metal-affinity columns using a protocol described by themanufacturer (Qiagen).

Generating polyclonal antisera against MobN1 and PrmN1. The proteinswere purified from acrylamide gels and the purified MobN1 and PrmN1 weredialyzed using a microdialyzer (PGC Scientifics). Approximately 1 mg of eachrecombinant protein was used to immunize mice. Ascites fluid was collected,

FIG. 1. Schematic representation of the integrated NBU1. NBU1integrates site specifically into the 3� end of a tRNALeu. The chromo-somal DNA at the junctions of the integrated NBU1 is indicated bydotted lines and the ends of NBU1 are indicated by hatched boxes. Theminimal region of NBU1 required for integration and excision con-tains the left end, including intN1 through two-thirds of the mobN1gene as indicated by the arrow, and the right end from the ScaI site(22). Between PvuII and ScaI, just downstream of mobN1 and high-lighted in gray, are four orfs of unknown functions that are not nec-essary for integration or excision. Below the map of �NBU1 (accessionno. AF238307) is the region containing mobN1, oriT, and prmN1cloned on the plasmid pPOM. This is the region that is nearly identicalto the same region on NBU2 (accession no. AF251288).

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delipified, and stored in saturated ammonium sulfate until needed. Antibodieswere generated by the Immunological Research Center of the University ofIllinois.

Membrane preparations. Both MobN1 and PrmN1 were found primarily inthe membrane fraction of the sonicated cells. Therefore, unless otherwise noted,the membrane fractions were the source of proteins for all experiments. Toobtain the membrane fractions, B. thetaiotaomicron strains containing variousNBU1 derivatives or plasmids were grown anaerobically in 100 ml of prereducedTYG broth to late exponential phase (OD at 650 nm of 0.7 to 0.9) with or withouttetracycline at 2 �g/ml. The cells were harvested at 4°C (10,000 � g for 20 min).The cell pellet was washed once with 20 mM potassium phosphate buffer (pH7.2) and resuspended in 5 ml of the same buffer, and cells were disrupted bysonication. The sonicated samples were spun at 10,000 � g at 4°C for 15 min toremove large cell debris. The membranes were pelleted from the supernatant byultracentrifugation (200,000 � g for 2.5 h at 4°C). The soluble fraction wascollected and saved, and the membrane pellet was thoroughly resuspended in 7ml of the 20 mM phosphate buffer and pelleted again by ultracentrifugationunder the same conditions. The membrane pellet was resuspended in 200 to 300�l of 20 mM potassium phosphate buffer, and the membranes were dispersed bygentle sonication for 15 s. For the soluble fraction, the supernatant from the first

ultracentrifugation step was concentrated using an Amicon Centriprep YM-10system to about 0.5 ml. If no membrane-associated proteins were detected, thesoluble fractions were checked in case there was a localization effect due to theconditions used or the NBU1 derivative being tested. The concentrations of thesoluble and membrane proteins were determined using a Bio-Rad DC ProteinAssay kit.

Immunoblotting of MobN1 and PrmN1. To detect the expression of MobN1and PrmN1, membrane preparations were made from B. thetaiotaomicronBT4104N1-3 containing CTnERL and a wild-type NBU1 and from other BTstrains containing derivatives of NBU1 (Table 1). The membrane fraction wasused because most of the MobN1 and PrmN1 protein fractionated with themembranes. Using a membrane fraction made the assay more sensitive. Samplescontaining 100 �g of protein were loaded in each well. The proteins wereseparated on sodium dodecyl sulfate–10% polyacrylamide gels. Proteins wereelectrotransferred from the gels to a nitrocellulose membrane using a Bio-RadTrans-Blot. The membranes were incubated with polyclonal antibodies, usuallydiluted 500- to 1000-fold in TTBS (20 mM Tris [pH 7.5], 0.2% Tween 20, 0.5 MNaCl) with 1% bovine serum albumin, against MobN1 and/or PrmN1 at roomtemperature for 3 h to overnight. MobN1 and PrmN1 were detected by using aBio-Rad goat anti-mouse horseradish peroxidase Opti-4CN kit and the mem-

TABLE 1. Bacterial strains and plasmids

Bacterial strain or plasmid Relevant characteristicsa,b Description and/or source

B. thetaiotaomicron(BT5482) strains

BT4001 Rifr Spontaneous rifampin-resistant mutant of BT5482BT4004 Rifr Tcr BT4001 transconjugant containing the conjugative transposon CTnERLBT4100 Thy� Tpr Spontaneous thymidine-requiring mutant of BT5482 that also makes the

strain resistant to trimethoprimBT4104 Thy� Tpr Tcr BT4100 transconjugant containing CTnERLBT4100N1-S1 Thy� Tpr BT4100 containing an integrated copy of NBU1 (�NBU1)BT4104N1-3 Thy� Tpr Tcr BT4104 containing �NBU1BT4104�pY5D Thy� Tpr Tcr Emr ExcN1 MobN1� pEG920 integrated into orf3 of NBU1 and the entire pY5D derivative

integrated into BT4104 (22, 23); excision defective but mobilization positive

PlasmidspCQW1 Apr (Emr) uidA reporter (GUS) insertional vector used to detect transcription from

integrated genes (5)pPOM Apr (Cfxr MobN1�) Entire prmN1-oriT-mobN1 region of NBU1 cloned into special oriT-lacking

shuttle vector pLYL7 (22)pLYL20 Apr (Cfxr MobN1�) oriT and mobN1 of NBU1 cloned into pLYL7 (Fig. 1); mobilizable in both

Bacteroides hosts by CTns and in E. coli by IncP plasmids (7)pY5 Apr Tcr MobN1� (Emr MobN1�) Cointegrant of pEG920 and NBU1 that replicates in both Bacteroides and E.

coli hosts; pEG920 is inserted in orf3 of NBU1 (19, 23); mobilized byCTns in Bacteroides strains and IncP plasmids in E. coli

pNLY1 Apr Cmr (Cmr) Chloramphenicol-resistant shuttle vector based on pFD160 (22, 24)pNBS2 (pQA) Apr Cmr (Cmr) pNLY1 containing tetQ-rteA of CTnDOTpNBS4 (pQAB) Apr Cmr (Cmr) pNLY1 containing tetQ-rteA-rteB of CTnDOTpAMS9 (pQABC) Knr Cmr� (Cmr) pNJR24 containing tetQ-rteA-rteB and rteC of CTnDOT (25)pNW17 Apr (Emr ExcN1� MobpB8-51

�) NBU1 from PvuII site through 70% of mobN1, as shown in Fig. 5A, clonedinto a special mobilizable insertional vector, pNV19; integrates and excisesat wild-type levels (22)

pGERM Apr (Emr) Insertional vector containing pUC19, oriT of RK2, and ermG; used toconstruct various NBU1 derivatives, to make insertional disruptions intargeted genes in Bacteroides strains, and to locate possible promoters (22)

pG-Sph18 Apr (Emr ExcN1� MobN1�) pGERM containing the 7.7-kbp SphI fragment of NBU1 from pNW17,which is the minimal clone to excise at wild-type levels (22), and this study

pG-Sph18orf2 Apr (Emr ExcN1�) pG-Sph18 with a 420-bp (bp 4697 to 5117) deletion in orf2 of NBU1 fromthis study

pG-PvuIIOrf3 Apr (Emr ExcN1�) pGERM containing NBU1 SphI to PvuII (all of mobN1) with a 975-bpdeletion (bp 6028 to 7003) within orf3 (22)

pG-Y5Orf2-3 Apr (Emr ExcN1�) PstI-SstI fragment of pY5 that results in a 1.86-kbp deletion of most of orf2,all of orf2X, and most of orf3 (Fig. 1) cloned into the PstI-SstI sites ofpGERM from this study

pG-Sph18prm Apr (Emr ExcN1�) pG-Sph18 with 377-bp deletion within prmN1 (22)

a Abbreviations for antibiotic resistance phenotypes are as follows: Ap, ampicillin; Cfx, cefoxitin; Cm, chloramphenicol; Em, erythromycin; Kn, kanamycin; Rif,rifampin; Tc, tetracycline; and Tp, trimethoprim. Other abbreviations include Thy�, thymidine requiring; Mob, ability to be mobilized (�) or not (�) from oriT-mobN1of NBU1 (MobN1) or from oriT-mob of pB8-51; and ExcN1, detection of the excised circular form of NBU1 (wild-type level [�], low but detectable level [], or notdetectable by Southern blots [�]).

b The plasmid phenotypes shown in parentheses are expressed in Bacteroides hosts and the phenotypes outside the parentheses are expressed in E. coli hosts.

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branes were scanned or photographed. The truncated form of MobN1, calcu-lated to be about 30.6 kDa, could not be detected in either the membrane or theconcentrated soluble protein preparations. In cases where no MobN1 or PrmN1was detected in the membrane fractions, the soluble fraction from the same cellswas concentrated to the same degree as the membrane fraction and probed todetermine whether the proteins were truly missing or were fractionating aber-rantly.

Time course for excision of NBU1 and detection of MobN1. Cultures weregrown in 100-ml bottles containing TYG medium with 2 �g of tetracycline/mladded. The bottles were inoculated with 3 ml of overnight cultures ofBT4104N1-3 or BT4004(pPOM) grown without tetracycline. Five milliliters ofeach overnight culture was used to make total DNA preparations and theremainder of the culture was used for total membrane preparations. The tetra-cycline-induced cultures were incubated at 37°C and the OD at 650 nm wasmonitored. Samples were taken at ODs of 0.3, 0.5, and 0.7 and overnight (ca. 0.8to 0.9), and the times required to reach these densities were noted. At eachgrowth point, 10 to 20 ml of the cultures was used to obtain DNA for the excisionassays by Southern blots and the remaining 90 ml (180 ml for the samples withan OD of 0.3) was used to obtain membranes and soluble fractions for theWestern blot analysis. The estimate of the copy number of NBU1 at the varioustime points was determined by analyzing the Southern blots of the samplesprobed with the mobN1 and susG probes (see above). Serial dilutions of thevarious samples from different time points were included on the Southerns to getthe intensity of the bands in the linear range of the film. The total density of eachband was determined using the Bio-Rad gel documentation and Quantity OneSoftware analysis system. The ratio of the total density of the mobN1 bands tothat of the susG bands for the uninduced BT4104N1-3 sample was normalized to1 and the ratios obtained for all of the other samples were divided by the samefactor.

RESULTS

Location of promoter regions of NBU1 excision genes. Pre-viously, we had shown that there was a promoter upstream ofintN1 and one upstream of mobN1 (9, 22), but it was notknown whether the other NBU1 genes involved in excisionwere organized in an operon. To answer this question, we firsttested cloned regions of the NBU1 excision region for theability to complement excision-deficient mutants to excisionproficiency. None of the clones tested complemented any ofthe mutants. Accordingly, we turned to the alternative strategyillustrated in Fig. 2A. Single crossover disruptions were madethat separated each gene from the one upstream but kept bothgenes intact. If the two genes that were separated from eachother were in the same operon, the insertion should have apolar effect on the downstream gene. Mutant NBU1s withinsertions that separated orf2 from orf3 (bp 5147 to 6028) ororf3 from prmN1 (bp 7030 to 7699) were still able to excise aswell as the wild type (Fig. 2B). Thus, orf3 and prmN1 are inseparate transcriptional units (Fig. 2A, panel 3). SeparatingintN1 from orf2 (bp 4063 to 5154) abolished excision (Fig. 2B),indicating that intN1 and orf2 are in the same transcriptionalunit. Information about the location of promoters was used insubsequent cloning experiments designed to monitor gene ex-pression of prmN1 and mobN1.

FIG. 2. (A) Strategy for determining the approximate location of promoters for various excision genes. The regions containing the 3� end ofthe upstream gene and the 5� end of the downstream gene (panel 1) were cloned into pGERM, a vector that does not replicate in Bacteroides strains(panel 2). When the resultant suicide vectors were transferred into BT4104N1-3, they integrated by homologous recombination into NBU1. Theintegrated vectors separated the two targeted genes by about 5 kbp of vector DNA. If the two genes are in an operon, the insertion will have apolar effect on the downstream gene and will eliminate NBU1 excision. Panel 3 shows a summary of the approximate location of the promotersof genes in the minimal excision region. (B) Southern blots showing the results of the excision assay on the various insertions described in panelA. For panel 1, the DNA from each strain was digested with HincII and the blot was probed with the 1.7-kbp HincII DNA fragment that containsthe joined ends of the NBU1 circular form. The designation �pP2x-3 at the top of the blot indicates the insertion that separated orf2x-orf3 fromorf2. Similarly, the other insertions are labeled with the gene that is downstream from the region where the integration occurred. The arrow to theleft of the blots indicates the position of the 1.7-kbp joined end fragment that is diagnostic for the NBU1 circular form. JL and JR are, respectively,the left and right ends of the copies of NBU1 that are still integrated in the chromosomal site. For panel 2, the DNA was digested with HincIIand KpnI because the intN1�-orf2� fragment used for the �pP2N1 insertion had a small region of identity to the probe. This combination of enzymeswould give a clearer separation of an excision product (indicated by arrow) from the junction- and vector-containing bands.



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Production of PrmN1 and MobN1 from an integrated copyof NBU1. We had found previously that trancriptional fusionsof the upstream regions of prmN1 or mobN1 to uidA did notgive detectable �-glucuronidase activity (21). Thus, we usedantibodies against PrmN1 and MobN1 to detect production ofthe two proteins. Both proteins were detectable on Westernblots of extracts from B. thetaiotaomicron carrying CTnERLand NBU1. The proteins were only detectable in cells that hadbeen stimulated by tetracycline (Fig. 3). Moreover, a plasmidcarrying the tetQ-rteA-rteB operon, pQAB (pNBS4), was ableto replace CTnERL, whereas a plasmid carrying only tetQ-rteA,pQA (pNBS2), was not sufficient. There was also no require-ment for rteC, which is carried on pAMS9.

Gene expression from genes cloned on a plasmid. Althoughexpression of prmN1 and mobN1 appeared to be regulatedwhen produced from NBU1, Western blot analysis of proteinsproduced from a plasmid, pPOM, that carried only prmN1,oriT, and mobN1 revealed that PrmN1 and MobN1 were nowproduced independently of RteAB and of tetracycline induc-tion (Fig. 3). That is, the proteins were produced at the samelevel in a strain that contained only pPOM and had not beenexposed to tetracycline as in a strain that carried both pPOMand CTnERL and had been exposed to tetracycline. Also,PrmN1 was produced from a plasmid that contained onlyprmN1 (pPrm-oriT) and MobN1 was produced from a plasmidthat contained only mobN1 (pLYL20) (data not shown). Theseresults suggested two possible hypotheses. The first hypothesiswas that expression of prmN1 and mobN1 is regulated at thetranscriptional level, but expression of the genes on pPOM wasconstitutive because pPOM did not contain a repressor genethat normally controls these genes. A second hypothesis wasthat excision and circularization of NBU1 had to occur beforethere was detectable production of PrmN1 and MobN1.

If there is a repressor gene on NBU1 that controls prmN1and mobN1 expression, it is likely to be encoded by orf2 or orf3,

because a derivative of NBU1 that contained only the regionstretching from intN1 through mobN1 was capable of excision.To test the hypothesis that a repressor was controlling expres-sion of prmN1 and mobN1, we first tested whether a copy ofNBU1 in the chromosome could affect the production ofMobN1 and PrmN1 from pPOM. The genes on pPOM werestill expressed constitutively. Moreover, deletions in orf2 or inorf3 and a deletion that eliminated both orf2 and orf3 all abol-ished production of both PrmN1 and MobN1 from genes on anintegrated form of NBU1 (Fig. 4).

Excision is required for detectable production of PrmN1and MobN1. It was noteworthy that the plasmid that containedall of NBU1 (pY5; Fig. 4A) produced detectable levels of bothPrmN1 and MobN1 and production was independent of tetra-cycline, similar to that observed for pPOM (data not shown).However, the integrated form of pY5 (�pY5D) produced nodetectable PrmN1 or MobN1 (Fig. 4B). Moreover, any muta-tion in integrated NBU1 that blocked excision was associatedwith no production of PrmN1 and MobN1. These results sup-ported the hypothesis that the amount of the excised circularform might be important for the production of MobN1 andPrmN1. If, after excision, the circular form of NBU1 reached ahigh enough copy number, production of PrmN1 and MobN1,which was not detectable when the NBU1 was present only ina single copy, would become detectable. That is, genes thatwere actually expressed constitutively on integrated NBU1would appear to have tetracycline-induced expression becauseafter NBU1 excision, copies of the NBU1 circular form wouldaccumulate to a high enough number for proteins produced attheir basal level to become detectable.

An examination of cells harvested at intervals after tetracy-cline stimulation produced an unexpected finding: the excisedcircular form did not appear until after 3 h of growth in tetra-cycline, after the cells reached an OD of 0.5 or higher (25).That is, the appearance of the circular form appeared to begrowth phase dependent. We also noted that even though astrong band representing the joined ends of the circular formappeared, the strength of the two bands representing the in-tegrated form (junction bands) did not diminish in abundance(e.g., see Fig. 2B and 5). This is what would be expected ifexcision of NBU1 and amplification of the circular forms bysome kind of replication allowed multiple copies of the NBU1circular form to accumulate while maintaining a copy of theintegrated form (seen as the junction bands). Increased genedosage is not due to large concatemers since we have shownthat in plasmid preparations, the excised form exists as amonomer (20).

If accumulation of the NBU1 circular forms was responsiblefor the increased production of MobN1 and PrmN1, theseproteins should also have a time-dependent rise in abundancethat correlates with the appearance of the excised circularforms. This proved to be the case. The Mob protein appearedat about the same time that the circular form began to becomeprominent (two experiments, shown in Fig. 5A and B). TheSouthern blot for each experiment is shown at the top of thefigure and the Western blot of the samples from the samegrowth points is aligned below the Southern. The Western blotfor MobN1 production from pPOM, done at the same timepoints, is shown in Fig. 5B at the bottom of the panel. MobN1was detectable at all time points at the same level until entry

FIG. 3. Western blot showing the pattern of production of MobN1and PrmN1 from integrated NBU1 and from a plasmid containing thetwo genes. Membrane preparations for the indicated strains wereloaded on a sodium dodecyl sulfate–10% acrylamide gel. The blotcontaining the transferred proteins was probed with antibodies toMobN1 and to PrmN1. The samples are labeled according to thesource of the RteA and RteB proteins in the host strain. The strainswere grown with (�) or without (�) 2 �g of tetracycline (Tc)/ml. Thestrain labeled CTn contains CTnERL and NBU1 (BT4104N1-3). All ofthe other strains were derivatives of BT4100N1S1 (NBU1, no CTn)which contained a plasmid with the indicated CTn regulatory genes:tetQ-rteA on pQA (pNBS2), tetQ-rteA-rteB on pQAB (pNBS4), andtetQ-rteA-rteB and rteC on pQABC (pAMS9). pPOM is a plasmid witha copy number of about 5 per cell that contains prmN1 and mobN1 andis in a strain containing CTnERL which provides RteA and RteB whengrown in tetracycline. Locations of MobN1 (54 kDa) and PrmN1 (36.4kDa) are indicated by arrows at the right.



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into stationary phase, when there was a slight decrease inMobN1 concentration. This control further supports the hy-pothesis that the apparent growth phase regulation of MobN1production is due to increased excision, not necessarily toincreased gene expression. PrmN1 production (not shown)followed the same pattern as MobN1.

To test further the hypothesis that the copy number of thecircular form of NBU1 was increasing in later growth phases,the Southern blot shown in Fig. 5B was stripped and reprobedwith probes for the internal mobN1 gene on NBU1 and forsusG, an unrelated chromosomal marker. The result shown inFig. 5D indicates that the copy number of an internal NBU1gene, mobN1, increased at about the same time that excisionbecame detectable. To estimate the copy number increasemore precisely, a dilution series of the samples shown in Fig.5A and B was made. The total density of each band wasdetermined using a densitometer and was used to calculateratios of the mobN1 band to the susG band for each sample.The ratio in the case of no tetracycline induction ofBT4104N1-3 was normalized to 1 and ratios in the other sam-ples were divided by the same factor to give an estimate of thecopy number of mobN1 relative to susG. The copy number ofmobN1 increased from 1 to 1.5 to 1.7 in the 0.7-OD samplesand to 2.5 to 2.7 in the overnight samples. The ratio of mobN1to susG for pPOM was 4.7 to 5.0 at all of the points in thegrowth curve. The MobN1 concentration associated withpPOM was constant as shown in the bottom panel of Fig. 5B,except for a slight decrease detected in the overnight sample.

This decrease in the concentration of MobN1 (and also ofPrmN1; not shown) was also observed in the Westerns in Fig.5A and B for NBU1 in overnight cultures.

DISCUSSION

When NBU1 or NBU2 is introduced by conjugation into B.thetaiotaomicron, it invariably integrates site specifically intothe recipient’s chromosome. Because of this, it had been as-sumed that NBUs do not replicate in B. thetaiotaomicron. Infact, “NBU” stands for “nonreplicating Bacteroides unit.” Theresults of experiments reported here suggest that this view ofNBUs may need to be modified. Results reported here showthat after excision, copies of the NBU circular form accumu-late to an estimated copy number of 2.5 to 2.7 per cell.Whether this is due to transient replication or to an excisionmethod that produces more than one circle remains to bedetermined. The MobN1 and PrmN1 proteins are only pro-duced at detectable levels at the point when excision begins tooccur. When the prmN1 and mobN1 genes were cloned onto aplasmid having a copy number of 5 per cell such as for pPOM(Fig. 5), expression of the genes was independent of growthphase and tetracycline stimulation. Yet during excision ofNBU1, expression of these genes appeared to be regulatedboth by growth phase and by tetracycline. The topology ofDNA molecules has been shown to influence transcription ofgenes (2–4). Possibly, transient changes in the supercoiling of

FIG. 4. (A) Integrated deletion derivatives of NBU1 and NBU1 plasmid clones tested for production of PrmN1 and MobN1. At the top is aschematic of the integrated NBU1 with the open reading frames not necessary for excision between PvuII and SphI indicated by “//.” Thederivatives of NBU1 with the plasmid (�p) designation were replicated in E. coli and integrated in B. thetaiotaomicron. An asterisk by the nameindicates that this construct has the entire mobN1 gene. The other constructs have only two-thirds of mobN1 (to the arrow indicating the end ofthe minimal excising region and depicted by a vertical dotted line) and do not produce any MobN1 detectable with our antibody. Several of theconstructs were made to contain the minimal excising region and as a result they are missing the region downstream of mobN1, extending to theSphI site near the right end of NBU1. The deletions downstream of the end of the minimal region are shown as gray filled rectangles. The openboxes indicate the deletions in the genes within the region essential for excision. At the bottom of the figure are the plasmids used to detect theproduction of PrmN1 and/or MobN1. pY5 is the replicative form of the �Y5D shown in the top section. To the right of the drawing are twocolumns summarizing the results of Western blot detection of MobN1 and PrmN1. �C, the protein was produced constitutively and did not requireinduction of the cells with tetracycline. (B) Western blots on which the results listed as “�” and “�” for some of the integrated derivatives of NBU1shown in panel A are based. Membrane proteins (100 �g) were loaded for each sample. The Western blot was developed for both MobN1 andPrmN1 and the expected position for each of the two proteins is indicated on the right. The predicted location for the 30.6-kDa truncated formof MobN1 (MobN1�) is indicated by an arrow near the bottom of the blot. This truncated form of MobN1, if produced, was not detectable by ourantibodies in total membranes or in soluble fractions (not shown). �Tc, cultures grown without tetracycline induction.



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the circular form of NBU1 that occur during the excisionprocess could trigger increased expression of NBU1 genes.

Although excision seems to be required to cause maximalexpression of prmN1 and mobN1, the concentration of protein

is not completely correlated with the amount of excised circu-lar form. As seen in Fig. 5A and B, the level of MobN1 riseswhen the circular form first begins to reach detectable levels,and then the amount of MobN1 decreases as the cells enter

FIG. 5. Correlation of the appearance of MobN1 and the appearance of the excised circular form of NBU1 following induction withtetracycline. (A) The top half of the panel is a Southern blot showing the assay for the joined ends of the excised circular form of NBU1. The DNAfrom the culture grown to the indicated OD following tetracycline (Tc) induction was digested with HincII and the blot was probed with the 1.7-kbpHincII joined-ends fragment of NBU1. The lower strip is a Western blot of membrane preparations from cells taken at the same time point as forthe Southern blot (see Materials and Methods). The lanes are labeled with the OD of the culture from which the DNA and total membraneproteins were obtained. (B) Results shown here are from a duplicate experiment of the one shown in panel A. It is provided to show that therewas some variation in the OD at which the circular form of NBU1 and MobN1 appeared. Also, in this experiment, a strain containing pPOM wasalso included in parallel. As can be seen from the second Western blot strip at the bottom of the panel, MobN1 from pPOM was expressed at alltime points in this strain. (C) Growth curve for the samples shown in panel A, with arrows indicating the points at which the samples were taken.(D) The Southern blot shown in panel B was stripped and reprobed with a probe that detects mobN1 and thus the concentration of NBU1 DNA(integrated or circular form) in the cell and a probe that detects an unrelated chromosomal gene, susG. This panel shows that the concentrationof NBU1 DNA increases at the time when the circular form of NBU1 appears and accumulates in stationary phase. Copy numbers for mobN1relative to susG were determined by band density determinations for both NBU1 and pPOM for the same samples shown in panels A and B ona separate Southern blot (not shown).



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stationary phase, even though the amount of NBU1 circularform increases or remains constant. This may be due to insta-bility of the MobN1 or PrmN1 proteins or to a decrease inprotein synthesis as cells enter stationary phase, because whenthese proteins are produced from the genes carried on a plas-mid (pPOM), the level of the proteins in the cell remainsconstant in the exponential growth phases but decreases rela-tive to the plasmid copy number in the stationary phase (Fig.5B).

The increase in copy number of the circular form of NBU1could be explained if excision involves more than one step: onestep produces the excised circular form, and then in a secondstep the circular form accumulates so there is more than onecopy per cell. Having multiple circular forms per cell couldincrease the probability that the circular form would be nickedby MobN1 and transferred by conjugation. According to thishypothesis, there is a burst of PrmN1 and MobN1 productionduring the first step but expression of these genes ceases ordecreases during the second step as the cells go into stationaryphase. The need for increased MobN1 after excision is clear; itis required for mobilization of the circular form from the donorto a recipient. This explanation does not apply to PrmN1,which appears not to be necessary for mobilization (7). PrmN1might contribute, however, to the step in which the number ofcopies of the circular form increases. Indirect evidence sup-porting this hypothesis is that a mutation in prmN1 (�pY11D)that abolished excision as detected by Southern blot still al-lowed some low-level mobilization of the integrated NBU1 toa recipient similar to that observed for �pY5D in orf3 (22, 23).This suggests that some excision was occurring even thoughaccumulation of the circular form to the level necessary fordetection on a Southern blot was not occurring.

The accumulation of multiple copies of the circular formmay not be true replication, in the sense that it involves aplasmid-like replication origin, but could be due to a replica-tive mode of excision. In the past, it was assumed that NBU1excision is not a replicative process because it is possible todetect the site from which NBU1 had excised by PCR ampli-fication. Further work may reveal that NBU1 excision does infact occur by a replicative mechanism under some conditions.

Another surprising finding was that none of the NBU1 ex-cision genes tested was able to complement excision-deficientmutants in trans. From the insertional disruption data andfrom Western blot data, it is clear that the cloned segmentscontained a promoter region, and in the case of prmN1 andmobN1, were producing the protein encoded by the gene. Inthe case of mobN1, we had shown previously that the genecould complement mobilization in trans. The fact that it couldnot complement a mutant of NBU1 to excision proficiency intrans suggests that for excision the genes are cis acting.

Conjugal transfer of DNA is a form of replication becausethe transferred single-stranded copy of the element and thesingle-stranded copy that remains behind are both copied torecreate the double-stranded circular form. This is not theexplanation for the increase in copy number of the NBU1circular form, because the phenomenon occurs in cells thathave only the tetQ-rteA-rteB portion of the CTn but not theregion that contains transfer genes. If, as our hypothesis sug-gests, excision occurs in two phases, with the excision eventfollowed by a rise in copy number of the circular form, there

should be one or more NBU1 genes involved in the secondstage. As mentioned previously, prmN1 could be such a gene.The existence of a second phase in the excision process couldexplain why so many genes are required for excision. Mostexcision systems consist of an integrase gene and one othergene. This is true of the lambdoid phages (1), the gram-positiveCTn Tn916 (11, 12), and the Bacteroides CTn CTnDOT (1a).Additional proteins may be involved, but these are host factorssuch as IHF that aid in bending DNA to form the synapticexcision complex. At this point, we suggest an alternative hy-pothesis, that NBU1 excision can occur by a conservative pro-cess under some conditions and by a replicative process underother conditions. Further studies will be needed to determinewhich hypothesis is correct.

ACKNOWLEDGMENT

This work was supported by grant number AI 22383 from the Na-tional Institutes of Health.

REFERENCES

1. Abremski, K., and S. Gottesman. 1981. Site specific recombination: Xis-dependent excisive recombination of bacteriophage lambda. J. Mol. Biol.153:67–78.

1a.Cheng, Q., Y. Sutanto, N. B. Shoemaker, J. F. Garnder, and A. A. Salyers.2001. Identification of genes required for the excision of CTnDOT, a Bac-teroides conjugative transposon. Mol. Microbiol. 41:625–632.

2. Dorman, C. J. 1995. 1995 Flemming lecture. DNA topology and the globalcontrol of bacterial gene expression: implications for the regulation of viru-lence gene expression. Microbiology 141:1271–1280.

3. Dorman, C. J. 1991. DNA supercoiling and environmental regulation ofgene expression in pathogenic bacteria. Infect. Immun. 59:745–749.

4. Dorman, C. J. 1996. Flexible response: DNA supercoiling, transcription andbacterial adaptation to environmental stress. Trends Microbiol. 4:214–216.

5. Feldhaus, M. J., V. Hwa, Q. Cheng, and A. A. Salyers. 1991. Use of anEscherichia coli beta-glucuronidase gene as a reporter gene for investigationof Bacteroides promoters. J. Bacteriol. 173:4540–4543.

6. Holdeman, L. V., and W. E. C. Moore. 1975. Anaerobe laboratory manual,4th ed. Virginia Polytechnical Institute and State University, Blacksburg, Va.

7. Li, L. Y., N. B. Shoemaker, and A. A. Salyers. 1993. Characterization of themobilization region of a Bacteroides insertion element (NBU1) that is ex-cised and transferred by Bacteroides conjugative transposons. J. Bacteriol.175:6588–6598.

8. Li, L. Y., N. B. Shoemaker, and A. A. Salyers. 1995. Location and charac-teristics of the transfer region of a Bacteroides conjugative transposon andregulation of transfer genes. J. Bacteriol. 177:4992–4999.

9. Li, L. Y., N. B. Shoemaker, G. R. Wang, S. P. Cole, M. K. Hashimoto, J.Wang, and A. A. Salyers. 1995. The mobilization regions of two integratedBacteroides elements, NBU1 and NBU2, have only a single mobilizationprotein and may be on a cassette. J. Bacteriol. 177:3940–3945.

10. Nikolich, M. P., N. B. Shoemaker, and A. A. Salyers. 1992. A Bacteroidestetracycline resistance gene represents a new class of ribosome protectiontetracycline resistance. Antimicrob. Agents Chemother. 36:1005–1012.

11. Rudy, C., K. L. Taylor, D. Hinerfeld, J. R. Scott, and G. Churchward. 1997.Excision of a conjugative transposon in vitro by the Int and Xis proteins ofTn916. Nucleic Acids Res. 25:4061–4066.

12. Rudy, C. K., J. R. Scott, and G. Churchward. 1997. DNA binding by the Xisprotein of the conjugative transposon Tn916. J. Bacteriol. 179:2567–2572.

13. Saito, H., and K. I. Miura. 1963. Preparation of transforming deoxy-ribonu-cleic acid by phenol treatment. Biochim. Biophys. Acta 72:619–629.

14. Salyers, A. A., N. B. Shoemaker, and A. M. Stevens. 1995. Tetracyclineregulation of conjugal transfer genes, p. 393–400. In J. A. Hoch and T. J.Silhavy (ed.), Two-component signal transduction. American Society forMicrobiology, Washington, D.C.

15. Salyers, A. A., N. B. Shoemaker, A. M. Stevens, and L. Y. Li. 1995. Conju-gative transposons: an unusual and diverse set of integrated gene transferelements. Microbiol. Rev. 59:579–590.

16. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor,N.Y.

17. Shipman, J. A., K. H. Cho, H. A. Siegel, and A. A. Salyers. 1999. Physiolog-ical characterization of SusG, an outer membrane protein essential for starchutilization by Bacteroides thetaiotaomicron. J. Bacteriol. 181:7206–7211.

18. Shoemaker, N. B., R. D. Barber, and A. A. Salyers. 1989. Cloning andcharacterization of a Bacteroides conjugal tetracycline-erythromycin resis-tance element by using a shuttle cosmid vector. J. Bacteriol. 171:1294–1302.

19. Shoemaker, N. B., L. Y. Li, and A. A. Salyers. 1994. An unusual type of



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Dow

nloaded from

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cointegrate formation between a Bacteroides plasmid and the excised circularform of an integrated element (NBU1). Plasmid 32:312–317.

20. Shoemaker, N. B., and A. A. Salyers. 1988. Tetracycline-dependent appear-ance of plasmidlike forms in Bacteroides uniformis 0061 mediated by conjugalBacteroides tetracycline resistance elements. J. Bacteriol. 170:1651–1657.

21. Shoemaker, N. B., G. R. Wang, and A. A. Salyers. 1996. The Bacteroidesmobilizable insertion element, NBU1, integrates into the 3� end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrasefamily. J. Bacteriol. 178:3594–3600.

22. Shoemaker, N. B., G. R. Wang, and A. A. Salyers. 2000. Multiple geneproducts and sequences required for excision of the mobilizable integratedBacteroides element NBU1. J. Bacteriol. 182:928–936.

23. Shoemaker, N. B., G. R. Wang, A. M. Stevens, and A. A. Salyers. 1993.Excision, transfer, and integration of NBU1, a mobilizable site-selectiveinsertion element. J. Bacteriol. 175:6578–6587.

24. Smith, C. J. 1987. Nucleotide sequence analysis of Tn4551: use of ermFS

operon fusions to detect promoter activity in Bacteroides fragilis. J. Bacteriol.169:4589–4596.

25. Stevens, A. M., J. M. Sanders, N. B. Shoemaker, and A. A. Salyers. 1992.Genes involved in production of plasmidlike forms by a Bacteroides conjugalchromosomal element share amino acid homology with two-component reg-ulatory systems. J. Bacteriol. 174:2935–2942.

26. Stevens, A. M., N. B. Shoemaker, L. Y. Li, and A. A. Salyers. 1993. Tetra-cycline regulation of genes on Bacteroides conjugative transposons. J. Bac-teriol. 175:6134–6141.

27. Studier, F. W. 1991. Use for bacteriophage T7 lysozyme to improve aninducible T7 expression system. J. Mol. Biol. 219:37–44.

28. Wang, J., N. B. Shoemaker, G. R. Wang, and A. A. Salyers. 2000. Charac-terization of a Bacteroides mobilizable transposon, NBU2, which carries afunctional lincomycin resistance gene. J. Bacteriol. 182:3559–3571.

29. Whittle, G., B. D. Hund, N. B. Shoemaker, and A. A. Salyers. 2001. Char-acterization of the 13-kilobase ermF region of the Bacteroides conjugativetransposon CTnDOT. Appl. Environ. Microbiol. 67:3488–3495.



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production of two proteins encoded by the bacteroides mobilizable

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