Characterization of the integrase of NBU1, a Bacteroides mobilizable transposon

NBU1 is a Bacteroides mobilizable transposon (MTn) that is integrated within the host chromosome and requires CTnDOT functions for its excision and transfer into a new host. The NBU1 integrase IntN1 has been classified as a tyrosine recombinase based on the presence of conserved residues. We created alanine mutants of the residues R291, K314, H393, R396, H419 and the conserved substitution Y429F and tested them for integration efficiency. The results suggest that these residues in IntN1 are important for integration, and Y429 could be the catalytic nucleophile. We employed suicide substrates and partially purified IntN1 to determine the positions of IntN1 cleavage within the 14 bp common core region that is identical in both NBU1 att sites. We show that IntN1 makes 7 bp staggered cuts on the top and bottom strands. From previous mutational analysis of the att sites, we show that two specific mutations near the site of bottom strand cleavage within this 7 bp region increased integration, and mutations of the two bases near top strand cleavage site had no effect on integration. These results indicate that IntN1 lacks the strict requirement for homology between the recombining sites seen with other tyrosine recombinases. We also show that phosphorothioate substitutions at the cleavage site and 1 bp downstream inhibited cleavage by IntN1. This differs from other studied tyrosine recombinases where inhibition occurs by substitutions at the cleavage site only.     

The Bacteroides mobilizable insertion element, NBU1, integrates into the 3' end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrase family.

NBU1 is a 10.3-kbp integrated Bacteroides element that can be induced to excise from the chromosome and can be mobilized to a recipient by trans-acting functions provided by certain Bacteroides conjugative transposons. The NBU1 transfer intermediate is a covalently closed circle, which is presumed to be the form that integrates into the recipient genome. We report here that a 2.4-kbp segment of NBU1 was all that was required for site-specific integration into the chromosome of Bacteroides thetaiotaomicron 5482. This 2.4-kbp region included the joined ends of the NBU1 circular form (attN1) and a single open reading frame, intN1, which encoded the integrase. Previously, we had found that NBU1 integrates preferentially into a single site in B. thetaiotaomicron 5482. We have now shown that the NBU1 target site is located at the 3' end of a Leu-tRNA gene. The NBU1 integrase gene, intN1, was sequenced. The predicted protein had little overall amino acid sequence similarity to any proteins in the databases but had limited carboxy-terminal similarity to the integrases of lambdoid phages and to the integrases of the gram-positive conjugative transposons Tn916 and Tn1545. We also report that the intN1 gene is expressed constitutively.     

The mobilization regions of two integrated Bacteroides elements, NBU1 and NBU2, have only a single mobilization protein and may be on a cassette.

Bacteroides conjugative transposons can act in trans to excise, circularize, and transfer unlinked integrated elements called NBUs (for nonreplicating Bacteroides units). Previously, we localized and sequenced the mobilization region of one NBU, NBU1, and showed that this mobilization region was recognized by the IncP plasmids RP4 and R751, as well as by the Bacteroides conjugative transposons. We report here that the single mobilization protein carried by NBU1 appears to be a bifunctional protein that binds to the oriT region and catalyzes the nicking reaction that initiates the transfer process. We have also localized and sequenced the mobilization region of a second NBU, NBU2. The NBU2 mobilization region was 86 to 90% identical at the DNA sequence to the oriT-mob region of NBU1. The high sequence similarity between NBU1 and NBU2 ended abruptly after the stop codon of the mob gene and about 1 kbp upstream of the oriT region, indicating that the oriT-mob regions of NBU1 and NBU2 may be on some sort of cassette. A region on NBU1 and NBU2 which lies immediately upstream of the oriT region had 66% sequence identity to a region upstream of the oriT region on a mobilizable transposon, Tn4399, an element that had previously appeared to be completely unrelated to the NBUs.     

Excision, transfer, and integration of NBU1, a mobilizable site-selective insertion element.

The Bacteroides species harbor a family of conjugative transposons called tetracycline resistance elements (Tcr elements) that transfer themselves from the chromosome of a donor to the chromosome of a recipient, mobilize coresident plasmids, and also mediate the excision and circularization of members of a family of 10- to 12-kbp insertion elements which share a small region of DNA homology and are called NBUs (for nonreplicating Bacteroides units). The NBUs are sometimes cotransferred with Tcr elements, and it was postulated previously that the excised circular forms of the NBUs were plasmidlike forms and were transferred like plasmids and then integrated into the recipient chromosome. We used chimeric plasmids containing one of the NBUs, NBU1, and a Bacteroides-Escherichia coli shuttle vector to show that this hypothesis is probably correct. NBU1 contained a region that allowed mobilization by both the Tcr elements and IncP plasmids, and we used these conjugal elements to allow us to estimate the frequencies of excision, mobilization, and integration of NBU1 in Bacteroides hosts to be approximately 10(-2), 10(-5) to 10(-4), and 10(-2), respectively. Although functions on the Tcr elements were required for the excision-circularization and mobilization of NBU1, no Tcr element functions were required for integration into the recipient chromosome. Analysis of the DNA sequences at the integration region of the circular form of NBU1, the primary insertion site in the Bacteroides thetaiotaomicron 5482 chromosome, and the resultant NBU1-chromosome junctions showed that NBU1 appeared to integrate into the primary insertion site by recombining within an identical 14-bp sequence present on both NBU1 and the target, thus leaving a copy of the 14-bp sequence at both junctions. The apparent integration mechanism and the target selection of NBU1 were different from those of both XBU4422, the only member of the conjugal Tcr elements for which these sequences are known, and Tn4399, a mobilizable Bacteroides transposon. The NBUs appear to be a distinct type of mobilizable insertion element.     

Characterization and DNA sequence of the mobilization region of pLV22a from Bacteroides fragilis.

A 4.2-kb plasmid (pLV22a) native to Bacteroides fragilis LV22 became fused to a transfer-deficient Bacteroides spp.-Escherichia coli shuttle vector by an inverse transposition event, resulting in a transferrable phenotype. The transfer phenotype was attributable to pLV22a, which was also capable of mobilization within E. coli when coresident with the IncP beta R751 plasmid. Transposon mutagenesis with Tn1000 localized the mobilization region to a 1.5-kb DNA segment in pLV22a. The mobilization region has been sequenced, and five open reading frames have been identified. Mutants carrying disruptions in any of the three genes designated mbpA, mbpB, and mbpC and coding for deduced products of 11.3, 30.4, and 17.1 kDa, respectively, cannot be mobilized when coresident with R751. Mutations in all three genes can be complemented in the presence of the respective wild-type genes, indicating that the products of mbpA, mbpB, and mbpC have roles in the mobilization process and function in trans. The deduced 30.4-kDa MbpB protein contains a 14-amino-acid conserved motif that is also found in the DNA relaxases of a variety of conjugal and mobilizable plasmids and the conjugative transposon Tn4399. Deletion analysis and complementation experiments have localized a cis-acting region of pLV22a within mbpA.     

Characterization of the 13-kilobase ermF region of the Bacteroides conjugative transposon CTnDOT

The conjugative transposon CTnDOT is virtually identical over most of its length to another conjugative transposon, CTnERL, except that CTnDOT carries an ermF gene that is not found on CTnERL. In this report, we show that the region containing ermF appears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although the ermF region contains genes also carried on Bacteroides transposons Tn4351 and Tn4551, it does not contain the IS4351 element which is found on these transposons. In CTnDOT, insertion of the ermF region occurred near a stem-loop structure at the end of orf2, an open reading frame located immediately downstream of the integrase (int) gene of CTnDOT, and in a region known to be important for excision of CTnERL and CTnDOT. The chimera that comprises the ermF region can apparently no longer excise and circularize, but it contains a functional mobilization region related to that described for the Bacteroides MTn Tn4399. Analysis of 19 independent Bacteroides isolates showed that the ermF region is located in the same position in all of the strains analyzed and that the compositions of the ermF region are almost identical in these strains. Therefore, it appears that CTnDOT-like elements present in community and clinical isolates of Bacteroides were derived from a common ancestor and proliferated in the diverse Bacteroides population.     

Production of two proteins encoded by the Bacteroides mobilizable transposon NBU1 correlates with time-dependent accumulation of the excised NBu1 circular form

NBU1 is a mobilizable transposon that excises from the Bacteroides chromosome to form a double-stranded circular transfer intermediate. Excision is triggered by exposure of the bacteria to tetracycline. Accordingly, we expected 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 production of these proteins. PrmN1 is essential for excision, and MobN1 is essential for transfer of the excised circular form. 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 the protein was constitutive. Initially, we assumed that the constitutive expression was due to loss of a repressor protein that was encoded by one of the other genes on NBU1. Deletions or insertions in the other genes (orf2 and orf3) on NBU1 and various integrated NBU1 derivatives abolished production of PrmN1 and MobN1. This is the opposite of what should have happened if one or both of these genes encoded a repressor. A second possibility was that when NBU1 excised, it replicated transiently, increasing the gene dosage of prmN1 and mobN1 and thereby producing enough PrmN1 and MobN1 for these proteins to become detectable. In fact, after the cells entered late exponential phase the copy number of NBU1 increased to 2 to 3 copies per cell. Production of PrmN1 and MobN1 showed a similar pattern. Any mutation in NBU1 that decreased or prevented excision also prevented elevated production of these two proteins. Our results show that the apparent tetracycline dependence of the production of PrmN1 and MobN1 is due to a growth phase- or time-dependent increase in the number of copies of the NBU1 circular form.     

Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1

NBU1 is an integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision-required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision. The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRS(HIII)) inhibited excision when provided in trans on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized.     

A gene product related to Tral is required for the mobilization of Bacteroides mobilizable transposons and plasmids

The antibiotic-resistance transposon Tn4555 from Bacteroides can be transferred between strains by conjugation. The transposon is not self-transmissible and must be mobilized by resident chromosomal tetracy-cline-resistance elements. In the present report, the mechanism of transfer was examined at the genetic level by deletion analysis and nucleotide sequencing of clones that conferred a transmissible phenotype on a non-mobilizable plasmid. The results suggested that the product of mobA_Tn was required for mobilization and it worked in concert with a cis-acting oriT-like sequence. This mechanism was compared with the mobilization system of a cryptic Bacteroides plasmid, pBl143, and the two systems were found to share a common transfer strategy. The mobA gene products from both genetic elements were related and they had limited homology to the broad group of mobilization proteins (relaxases) typified by Tral of RP4. Phylogenetic analysis of MobA and several other mobilization proteins from commensal gastrointestinal tract organisms suggested that they formed a new subgroup of the Tral superfamily. The mobilization regions of both Tn4555 and pBl143 were located on discrete segments of DNA within the parent genetic element. These segments were delineated by regions of secondary structure, suggesting that they could be defined mobilization cassettes.     

NBU1, a mobilizable site-specific integrated element from Bacteroides spp., can integrate nonspecifically in Escherichia coli.

NBU1 is an integrated Bacteroides element that can he mobilized from Bacteroides donors to Bacteroides recipients. Previous studies have shown that a plasmid carrying the internal mobilization region of NBU1 could be transferred by conjugation from Bacteroides thetaiotaomicron to Escherichia coli. In this report, we show that NBU1 can integrate in E. coli. Whereas integration of NBU1 in B. thetaiotaomicron is site specific, integration of NBU1 in E. coli was relatively random, and the insertion frequency of NBU1 into the E. coli chromosome was 100 to 1,000 times lower than the frequency of integration in B. thetaiotaomicron. The frequency of NBU1 integration in E. coli could be increased about 10- to 70-fold, to a value close to that seen with B. thetaiotaomicron, if the primary integration site from B. thetaiotaomicron, BT1-1, was provided on a plasmid in the E. coli recipient or the NBU1 integrase gene, intN1, was provided on a high-copy-number plasmid to increase the amount of integrase available in the recipient. When the primary integration site was available in the recipient, NBU1 integrated site specifically in E. coli. Our results show that NBUs have a very broad host range and are capable of moving from Bacteroides spp. to distantly related species such as E. coli. Moreover, sequence analysis of NBU1 integration sites provided by integration events in E. coli has helped to identify some regions of the NBU1 attachment site that may play a role in the integration process.     

Tn5253, the pneumococcal omega (cat tet) BM6001 element, is a composite structure of two conjugative transposons, Tn5251 and Tn5252.

Tn5253, carrying tetracycline and chloramphenicol resistance determinants, is a 65.5-kb conjugative transposon originally detected in the chromosome of Streptococcus pneumoniae BM6001. We have identified an 18-kb segment of DNA carrying the tet determinant within Tn5253 to be an independent conjugative transposon when removed from the context of the larger element. In vivo deletion of this DNA segment, now termed Tn5251, from within Tn5253 did not affect the conjugative transposition properties of the remaining sequences. Thus, Tn5253 is a composite element of two conjugative structures: Tn5252, constituting the sequences beyond Tn5251 within Tn5253, and Tn5251. The transfer properties of Tn5252 and Tn5251 suggest that these may belong to two different classes of mobile elements even though they were initially found associated. The notion that a tet-carrying transposon like Tn5251 may have been the ancestral element in the evolution of the larger streptococcal conjugative transposons must be reevaluated in the light of present observations.     

Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030.

The self-transmissible plasmid pTR2030 mobilized nonconjugative heterologous cloning vectors pGK12 (Cmr Emr) and pSA3 (Emr) at frequencies of 10(-5) to 10(-6) per input donor. Transconjugants harbored a 51- or 58-kilobase (kb) plasmid not found in the parental strains that cotransferred at high frequency with Cmr Emr and pTR2030-encoded phage resistance (Hsp+) in second-round matings (10(-1) per input donor). Restriction endonuclease mapping and DNA-DNA hybridization identified the 51- to 58-kb plasmids as pTR2030::vector cointegrates. Examination of four cointegrates indicated that pGK12 and pSA3 had inserted within two locations on pTR2030. Resolution of the cointegrates generated vector derivatives containing a 0.8-kb insert of pTR2030 DNA. Restriction analyses of several resolution plasmids indicated that the 0.8-kb element had inserted into various positions within pGK12 and pSA3 and in certain cases had inactivated the Cmr or Emr marker of pGK12. A conjugative mobilization assay demonstrated that the 0.8-kb element, designated IS946, mediated transpositional recombination. Nucleotide sequence determination identified IS946 as an 808-base-pair (bp) insertion sequence sharing ca. 96% homology with lactococcal insertion sequence ISS1. IS946 differed by 27 and 31 bp from ISS1S and ISS1T, respectively, and in 2 of 226 amino acids in the deduced sequence of the putative transposase. IS946 has perfect 18-bp terminal inverted repeats, identical to ISS1, and similarly generated 8-bp direct repeats of the target site upon insertion.     

Characterization of a new type of Bacteroides conjugative transposon, Tcr Emr 7853.

Results of previous investigations suggested that the conjugative transposons found in human colonic Bacteroides species were all members of a closely related family of elements, exemplified by Tcr Emr DOT. We have now found a new type of conjugative transposon, Tcr Emr 7853, that does not belong to this family. Tcr Emr 7853 has approximately the same size as the Tcr Emr DOT-type elements (70 to 80 kbp) and also carries genes encoding resistance to tetracycline (Tcr) and erythromycin (Emr); however, it differs from previously described conjugative transposons in a number of ways. Its transfer is not regulated by tetracycline and its transfer genes are not controlled by the regulatory genes rteA and rteB, which are found on Tcr Emr DOT and related conjugative transposons. Its ends do not cross-hybridize with the ends of Tcr Emr DOT-type conjugative transposons, and the Emr gene it carries does not cross-hybridize with ermF, the Emr gene found on all previously studied Bacteroides conjugative transposons. There is only one region with high sequence similarity between Tcr Emr 7853 and previously characterized elements, the region that contains the Tcr gene, tetQ. This sequence similarity ends 145 bp upstream of the start codon and 288 bp downstream from the stop codon. A 2-kbp region upstream of tetQ on Tcr Emr 7853 cross-hybridized with four additional EcoRV fragments of Bacteroides thetaiotaomicron 7853 DNA other than the one that contained tetQ. These additional cross-hybridizing bands were not part of Tcr Emr 7853, but one of them cotransferred with Tcr Emr 7853 in some matings. Thus, at least one of the additional cross-hybridizing bands may be associated with another conjugative element or with an element that is mobilized by Tcr Emr 7853. DNA that cross-hybridized with the upstream region was found in one clinical isolate of Bacteroides ovatus and four Tcr isolates of Prevotella ruminicola.     

Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element.

Bacteroides-Escherichia coli shuttle vectors containing a nonmobilizable pBR322 derivative and either pBFTM10 (pDP1, pCG30) or pB8-51 (pEG920) were mobilized by IncP plasmid R751 or pRK231 (an ampicillin-sensitive derivative of RK2) between E. coli strains and from E. coli to Bacteroides recipients. IncI alpha R64 drd-ll transferred these vectors 1,000 times less efficiently than did the IncP plasmids. pDP1, pCG30, and pEG920 could be mobilized from B. uniformis donors to both E. coli and Bacteroides recipients by a conjugative Bacteroides Tcr (Tcr ERL) element which was originally found in a clinical Bacteroides fragilis strain (B. fragilis ERL). However, the shuttle vector pE5-2, which contains pB8-51 cloned in a restriction site that prevents its mobilization by IncP or IncI alpha plasmids, also was not mobilized at detectable frequencies from Bacteroides donors by the Tcr ERL element. The mobilization frequencies of pCG30, pDP1, and pEG920 by the Tcr ERL element in B. uniformis donors to E. coli recipients was about the same as those to isogenic B. uniformis recipients. Transfer of the shuttle vectors from B. uniformis donors to E. coli occurred at the same frequencies when the matings were done aerobically or anaerobically. Growth of the B. uniformis donors in tetracycline (1 microgram/ml) prior to conjugation increased the mobilization frequencies of the vectors to both E. coli and Bacteroides recipients 50 to 100 times.     

Location and characteristics of the transfer region of a Bacteroides conjugative transposon and regulation of transfer genes.

Many Bacteroides clinical isolates contain large conjugative transposons, which excise from the genome of a donor and transfer themselves to a recipient by a process that requires cell-to-cell contact. It has been suggested that the transfer intermediate of the conjugative transposons is a covalently closed circle, which is transferred by the same type of rolling circle mechanism used by conjugative plasmids, but the transfer origin of a conjugative transposon has not previously been localized and characterized. We have now identified the transfer origin (oriT) region of one of the Bacteroides conjugative transposons, TcrEmr DOT, and have shown that it is located near the middle of the conjugative transposon. We have also identified a 16-kbp region of the conjugal transposon which is necessary and sufficient for conjugal transfer of the element and which is located near the oriT. This same region proved to be sufficient for mobilization of coresident plasmids and unlinked integrated elements as well as for self-transfer, indicating that all of these activities are mediated by the same transfer system. Previously, we had reported that disruption of a gene, rteC, abolished self-transfer of the element. rteC is one of a set of rte genes that appears to mediate tetracycline induction of transfer activities of the conjugative transposons. On the basis of these and other data, we had proposed that RteC activated expression of transfer genes. We have now found, however, that when the transfer region of TcrEmr DOT was cloned as a plasmid that did not contain rteC and the plasmid (pLYL72) was tested for transfer out of a Bacteroides strain that did not have a copy of rteC in the chromosome, the plasmid was self-transmissible without tetracycline induction. This and other findings suggest that RteC is not an activator transfer genes but is stimulating transfer in some other way.     

The unstable wDZL mutation of Drosophila is caused by a 13 kilobase insertion that is imprecisely excised in phenotypic revertants

We have analyzed the lesion in wDZL, a genetically unstable mutant allele of the eye color locus, white, of Drosophila melanogaster. We have cloned the DNA of the white locus region of flies carrying the wDZL allele and find a 13 kilobase insertion not present in the wild-type at the corresponding location. In 12 independent cases examined, reversion to a wild-type eye color phenotype correlates with the excision of a portion of this 13 kilobase insertion, indicating that the insertion is the cause of the mutation. The portion of the insertion that is excised in these eye color revertants is heterogeneous in size but appears to include the central 6 kilobases of the insertion in all cases. Many of these eye color revertants continue to undergo mutation at the white locus, indicating that the residual portion of the insertion in these revertants is sufficient to promote mutations.     

Transfer region of a Bacteroides conjugative transposon contains regulatory as well as structural genes.

Conjugative transposons (CTns) are integrated elements that excise themselves from the chromosome to form a circular transfer intermediate that is transferred by conjugation to a recipient. In an earlier paper, the excision step was shown to be regulated by tetracycline and to be dependent on the regulatory gene, rteC. In this paper, we report that genes involved in conjugal transfer are also regulated by tetracycline but that regulation is more complex. Genes contained within a 20-kbp region that is sufficient for conjugal transfer were disrupted by single crossover integration events. Most of the disruptions abolished transfer of the CTn. None of them abolished excision. Antibodies to two of the proteins encoded in this region (TraG and TraN) were obtained and used to show that production of these proteins was dependent on tetracycline stimulation. Both TraG and TraN were membrane proteins. A surprising finding was that a disruption in the gene traQ increased transfer of CTnERL over 100-fold. Thus, TraQ may be a repressor protein that controls expression of transfer genes. If so, TraQ is not the only protein that controls expression of transfer genes because production of TraG and TraN in the traQ disruption mutant was still dependent on tetracycline stimulation.     

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.