Bacteroides fragilis transfer factor Tn5520: the smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli

Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400DeltaBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520 mobilizes plasmids in both B. fragilis and Escherichia coli at high frequency and also transposes in E. coli.     

Isolation and characterization of cLV25, a Bacteroides fragilis chromosomal transfer factor resembling multiple Bacteroides sp. mobilizable transposons

Horizontal DNA transfer contributes significantly to the dissemination of antibiotic resistance genes in Bacteroides fragilis. To further our understanding of DNA transfer in B. fragilis, we isolated and characterized a new transfer factor, cLV25. cLV25 was isolated from B. fragilis LV25 by its capture on the nonmobilizable Escherichia coli-Bacteroides shuttle vector pGAT400DeltaBglII. Similar to other Bacteroides sp. transfer factors, cLV25 was mobilized in E. coli by the conjugative plasmid R751. Using Tn1000 mutagenesis and deletion analysis of cLV25, two mobilization genes, bmgA and bmgB, were identified, whose predicted proteins have similarity to DNA relaxases and mobilization proteins, respectively. In particular, BmgA and BmgB were homologous to MocA and MocB, respectively, the two mobilization proteins of the B. fragilis mobilizable transposon Tn4399. A cis-acting origin of transfer (oriT) was localized to a 353-bp region that included nearly all of the intergenic region between bmgB and orf22 and overlapped with the 3' end of orf22. This oriT contained a putative nic site sequence but showed no significant similarity to the oriT regions of other transfer factors, including Tn4399. Despite the lack of sequence similarity between the oriTs of cLV25 and Tn4399, a mutation in the cLV25 putative DNA relaxase, bmgA, was partially complemented by Tn4399. In addition to the functional cross-reaction with Tn4399, a second distinguishing feature of cLV25 is that predicted proteins have similarity to proteins encoded not only by Tn4399 but by several Bacteroides sp. transfer factors, including NBU1, NBU2, CTnDOT, Tn4555, and Tn5520.     

Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis.

Conjugal transposons play an important role in the dissemination of antibiotic resistance determinants in the streptococci and have been postulated to exist in Bacteroides fragilis. To investigate the presence of conjugal transposons in B. fragilis, we employed a Tra- derivative of the transfer factor pBFTM10 contained in the chimeric plasmid pGAT400 delta BglII. We attempted to restore transferability to this plasmid from a series of transconjugants generated by crossing B. fragilis TMP230 containing the TET transfer factor with B. fragilis TM4000, a standard recipient. Transconjugant TM4.2321 transferred pGAT400 delta BglII to Escherichia coli HB101 at almost the same frequency as did the Tra+ parental plasmid, pGAT400. Analysis of the transferred plasmids revealed the presence of 9.6 kilobases of additional DNA in every case but at different positions in independent isolates. The presence of this DNA, designated Tn4399, allowed the pGAT400 delta BglII derivatives to retransfer from the TM4000 background to B. fragilis or E. coli recipients. DNA hybridization studies demonstrated the presence of one copy of Tn4399 in TMP230 and three copies at new sites in TM4.2321. Tn4399 is a new B. fragilis transposon with unique transfer properties that may play a role in the dissemination of drug resistance genes. It differs from previously described conjugal transposons by its ability to mobilize nonconjugal plasmids in cis.     

Heterologous expression of the Bacteroides ruminicola xylanase gene in Bacteroides fragilis and Bacteroides uniformis

A cloned xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 was transferred by conjugation into the colonic species Bacteroides fragilis and Bacteroides uniformis by using the Escherichia coli-Bacteroides shuttle vector pVAL-1. The cloned gene was expressed in both species, and xylanase specific activity in crude extracts was found to be at least 1400-fold greater than that found in the B. ruminicola strain. Analysis of crude extract proteins from the recombinant B. fragilis by SDS-PAGE demonstrated a new 60,000 molecular weight protein. The xylanase activity expressed in both E. coli and B. fragilis was capable of degrading xylan to xylooligosaccharides in vitro. This is the first demonstration that colonic Bacteroides species can express a gene from a ruminal Bacteroides species.     

Screening for the presence of the insertion sequence IS1 in the genus Bacteroides

Abstract A specific DNA probe, containing a conserved region of the insertion sequence IS1, was hybridised to dot blots of total genomic DNA from 2 oral and 5 intestinal Bacteroides spp. Using Escherichia coli K12 as a positive control and Pseudomonas aeruginosa as a negative control, DNA homologous to the probe could not be detected in Bacteroides corporis, Bacteroides intermedius, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron or 2 strains of Bacteroides fragilis . The total DNA included plasmid DNA of 30.2, 42.7 and 42.7 MDa from B. fragilis, B. intermedius and B. corporis , respectively.
IS1 is commonly found in members of the Enterobacteriaceae, and it was concluded that the 2 groups of bacteria are not closely related.     

Facilitated transfer of IncP beta R751 derivatives from the chromosome of Bacteroides uniformis to Escherichia coli recipients by a conjugative Bacteroides tetracycline resistance element.

The broad-host-range IncP beta plasmid R751 can mobilize itself from Escherichia coli to Bacteroides spp, but it is not maintained in Bacteroides spp. If R751 carries the Bacteroides transposon Tn4351, it can be integrated into the Bacteroides chromosome. Previously we showed that R751, integrated in the chromosome of Bacteroides uniformis, cannot mobilize itself out of B. uniformis into E. coli or isogenic B. uniformis strains. In this report, we showed that if the Bacteroides conjugative tetracycline resistance element Tcr ERL was coresident with the R751 insertion in B. uniformis, derivatives of R751 were transferred to E. coli, where they were recovered as plasmids. The most common derivatives were R751::Tn4351 and R751::IS4351, but some strains transferred R751 derivatives, containing additional DNA segments ranging in size from 10 to 23 kilobases. These DNA inserts cross-hybridized with chromosomal DNA from B. uniformis which did not carry the Tcr ERL element. Therefore, the inserts appeared to be segments of the wild-type B. uniformis chromosome and were not associated with the Tcr ERL element. The transfer of integrated R751 from B. uniformis was independent of the RecA phenotype of the E. coli recipients and did not appear to be due to transfer of B. uniformis chromosomal DNA, followed by RecA-dependent recombination between homologous IS4351 sequences to form the resultant R751 plasmid derivatives. Consistent with this, no transfer of Tn4351 (associated with the cointegrated R751) from B. uniformis donors to isogenic B. uniformis recipients was detected (< 10(-8)). Our data support the hypothesis that R751 excises from the B. uniformis chromosome by recombination involving flanking Tn4351 or IS4351 sequences and forms nonreplicating circles. The mobilization of these circular forms out of B. uniformis to E.coli is then facilitated by the Tcr ERL 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.     

Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression

Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments. pDK3 was used to test the expression in B. fragilis of five foreign tetracycline resistance (TcR) genes. The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes.     

Genetic transfer systems in Bacteroides: cloning and mapping of the transferable tetracycline-resistance locus

Conjugation systems that transfer antibiotic resistance in the absence of detectable plasmids are common in Bacteroides, but the mechanism of transfer is poorly understood. We found that linked transfer of tetracycline (TcR) and clindamycin (ClR) resistance by Bacteroides fragilis strain 1126 is induced by growth in either Tc or Cl. We cloned the transferable TcR locus as a 13 kb fragment on the shuttle vector pPH6 in Escherichia coli and showed that this region expresses TcR in Bacteroides but not E. coli. The TcR gene was mapped to a 3 kb region and the ClR gene was shown not to be present in the 13 kb insert. Homologous TcR genes are found in B. fragilis V479 and 1792. Using pulsed-field electrophoresis, the transferable TcR gene was shown to be physically associated with high molecular-weight DNA, suggesting that it is located on the chromosome. A new TcR shuttle vector, pPH7 delta 1.1, was constructed to facilitate use of this selective marker in Bacteroides genetics.     

Conjugal transfer of antibiotic resistance factors in Bacteroides fragilis: the btgA and btgB genes of plasmid pBFTM10 are required for its transfer from Bacteroides fragilis and for its mobilization by IncP beta plasmid R751 in Escherichia coli.

Transferable plasmids play an important role in the dissemination of clindamycin-erythromycin resistance in Bacteroides fragilis. We previously described the isolation and properties of pBFTM10, a 14.9-kb ClnR transfer factor from B. fragilis TMP10. We also reported the isolation of a transfer-deficient deletion derivative of pBFTM10 contained in the B. fragilis-Escherichia coli shuttle vector pGAT400. In the present study we used pGAT400 and a similar shuttle vector, pGAT550, to characterize and sequence a region of pBFTM10 required for its transfer from B. fragilis to B. fragilis or E. coli recipients and for its mobilization by the broad-host-range plasmid R751 from E. coli donors to E. coli recipients. Deletion of certain BglII restriction fragments from pBFTM10 resulted in partial or complete loss of transfer ability. Tn1000 insertions into this same region also resulted in altered transfer properties. We used the sites of Tn1000 insertions to determine the DNA sequence of the transfer region. Two potential open reading frames encoding proteins of 23.2 and 33.8 kDa, corresponding to two genes, btgA or btgB, were identified in the sequence. Tn1000 insertions within btgA or btgB or deletion of all or portions of btgA or btgB resulted in either a transfer deficiency or greatly reduced transfer from B. fragilis donors and alterations in mobilization by R751 in E. coli. A potential oriT sequence showing similarity in organization to the oriT regions of the IncP plasmids was also detected. Thus, pBFTM10 encodes and requires at least two proteins necessary for efficient transfer from B. fragilis. These same functions are expressed in E. coli and are required for mobilization by R751.     

Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells.     

Pathogenicity of Bacteroides fragilis group in rat intra-abdominal abscesses.

Attempts were made to study the pathogenicity of some strains of Bacteroides fragilis group in the rat intra-abdominal abscess model. Multiple intraabdominal abscesses were produced in 50 to 70% of animals when an inoculum containing 10(9) CFU/ml of any of the five species of Bacteroides fragilis group was injected. Rising homologous antibody titers determined by indirect fluorescent antibody test were observed till the 3rd week when tested last, indirectly confirming the multiplication of the organisms as also evident by viable count of bacteria in the abscesses. In some cases in addition to inoculated organisms some intestinal bacteria like Escherichia coli, Proteus mirabilis and Streptococcus spp. were also recovered from the abscess pus. Studies with the electron microscope showed presence of capsular polysaccharide only in Bacteroides fragilis and Bacteroides thetaiotaomicron. It was doubtful in Bacteroides distasonis and absent in Bacteroides ovatus and Bacteroides vulgatus, suggesting that virulence factor beside the capsular polysaccharide may be playing a role. Further studies are required to investigate the virulence factor responsible for the pathogenicity of noncapsulated species.     

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.     

Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.

Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.     

Cloning and expression in Escherichia coli of a recA-like gene from Bacteroides fragilis

The recombinant plasmid pHG100, containing a 5.2-kb DNA fragment from Bacteroides fragilis, complemented defects in homologous recombination, DNA repair and prophage induction to various levels in an Escherichia coli recA mutant strain. There was no DNA homology between the cloned B. fragilis recA-like gene and E. coli chromosomal DNA. pHG100 produced two proteins with Mr of approx. 39,000 and 37,000 which cross-reacted with antibodies raised against E. coli RecA protein. The production of these proteins was not increased after UV induction. The cloned B. fragilis recA-like gene product did not enhance the production of native but defective E. coli RecA protein after UV irradiation.     

Plasmid transformation of Bacteroides spp. by electroporation

Transformation of Bacteroides spp. with a variety of plasmid DNAs was accomplished using electroporation. The standard transformation assay system used to deduce the optimal electroporation parameters employed a 50-to 100-fold concentrated cell suspension of mid-logarithmic phase Bacteroides fragilis strain 638 and the 5.4-kb clindamycin resistance (Ccr) vector, pBI191. A variety of electroporation buffers were used successfully in transformation experiments but of these, 1 mM MgCl2 in 10% glycerol was superior. The incorporation of MgCl2 was essential for optimum viability prior to electroporation and for optimum transformation. Transformants were routinely obtained using 5-ms pulses over a range of field strengths from 5 to 12.5 kV/cm, with a maximum of greater than 10(6) micrograms-1 DNA at 12.5 kV/cm. The number of transformants increased linearly with respect to DNA concentration over the range 0.01-2 micrograms tested. Recovery of transformants required an expression period of up to 2.5 h following exposure to the electric field. This period, however, was dependent on the antibiotic resistance marker used for selection of transformants, with a significantly shorter incubation required when chloramphenicol rather than clindamycin was used in the selective medium. The effect of the DNA source on transformation was tested using the shuttle vector pFD288. Plasmid DNA isolated from Bacteroides uniformis, Bacteroides ovatus, or Bacteroides thetaiotaomicron transformed B. fragilis 638 at frequencies 7.5- to 12.5-fold less than those observed for controls with homologous DNA. Further reductions were seen with Escherichia coli purified pFD288, which transformed at 1000-fold lower frequencies. Finally, using homologous pFD288 or pBI191 isolated from strain 638, several strains of B. fragilis, B. uniformis, and B. ovatus were transformed successfully without modification of the standard assay system. Two strains each of B. thetaiotaomicron and Bacteroides ruminicola were not transformed using the methods described here.     

Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1.95 +/- 0.05 x 10(6) for plasmids pBY1 and pBY2, respectively. Plasmid DNA purified by cesium chloride-ethidium bromide gradient centrifugation was used to transform a restriction- and modification-negative strain of Escherichia coli. Penicillin G- and tetracycline-resistant transformants were screened for the presence of plasmid DNA. A plasmid band corresponding to a molecular weight of 1.95 x 10(6) was present in all transformants tested. Curing experiments demonstrated that the plasmid, referred to as pBY22 when present in transformants, was responsible for penicillin G and tetracycline resistance. Plasmid pBY22 was mobilized and transferred to other E. coli strains by plasmid R1drd-19. Stability of pBY22 was examined in different E. coli strains and was shown to be stably maintained in both restriction-negative and restriction-positive strains. Unexpectedly, pBY2 and pBY22 were resistant to digestion by 12 different restriction endonucleases.     

Discerning the Role of Bacteroides fragilis in Celiac Disease Pathogenesis

Celiac disease (CD) is associated with intestinal dysbiosis, which can theoretically lead to dysfunctions in host-microbe interactions and contribute to the disease. In the present study, possible differences in Bacteroides spp. and their pathogenic features between CD patients and controls were investigated. Bacteroides clones (n = 274) were isolated, identified, and screened for the presence of the virulence genes (bft and mpII) coding for metalloproteases. The proteolytic activity of selected Bacteroides fragilis strains was evaluated by zymography and, after gastrointestinal digestion of gliadin, by high-pressure liquid chromatography/electrospray ionization/tandem mass spectrometry. The effects of B. fragilis strains on Caco-2 cell culture permeability and inflammatory response to digested gliadin were determined. B. fragilis was more frequently identified in CD patients than in healthy controls, in contrast to Bacteroides ovatus. B. fragilis clones carrying virulence genes coding for metalloproteases were more abundant in CD patients than in controls. B. fragilis strains, representing the isolated clones and carrying metalloprotease genes, showed gelatinase activity and exerted the strongest adverse effects on the integrity of the Caco-2 cell monolayer. All B. fragilis strains also showed gliadin-hydrolyzing activity, and some of them generated immunogenic peptides that preserved or increased inflammatory cytokine production (tumor necrosis factor alpha) and showed increased ability to permeate through Caco-2 cell cultures. These findings suggest that increased abundance of B. fragilis strains with metalloprotease activities could play a role in CD pathogenesis, although further in vivo studies are required to support this hypothesis.     

Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4.

Prevotella ruminicola (formerly Bacteroides ruminicola) is an anaerobic, gram-negative, polysaccharide-degrading bacterium which is found in the rumina of cattle. Since P. ruminicola is thought to make an important contribution to digestion of plant material in rumina, the ability to alter this strain genetically might help improve the efficiency of rumen fermentation. However, previously there has been no way to introduce foreign DNA into P. ruminicola strains. In this study we transferred a shuttle vector, pRDB5, from the colonic species Bacteroides uniformis to P. ruminicola B(1)4. The transfer frequency was 10(-6) to 10(-7) per recipient. pRDB5 contains sequences from pBR328, a cryptic colonic Bacteroides plasmid pB8-51, and a colonic Bacteroides tetracycline resistance (Tcr) gene. pRDB5 was mobilized out of B. uniformis by a self-transmissible Bacteroides chromosomal element designated Tcr Emr 12256. pRDB5 replicated in Escherichia coli as well as in Bacteroides spp. and was also mobilized from E. coli to B. uniformis by using IncP plasmid R751. However, direct transfer from E. coli to P. ruminicola B(1)4 was not detected. Thus, to introduce cloned DNA into P. ruminicola B(1)4, it was necessary first to mobilize the plasmid from E. coli to B. uniformis and then to mobilize the plasmid from B. uniformis to P. ruminicola B(1)4.     

Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4.

Prevotella ruminicola (formerly Bacteroides ruminicola) is an anaerobic, gram-negative, polysaccharide-degrading bacterium which is found in the rumina of cattle. Since P. ruminicola is thought to make an important contribution to digestion of plant material in rumina, the ability to alter this strain genetically might help improve the efficiency of rumen fermentation. However, previously there has been no way to introduce foreign DNA into P. ruminicola strains. In this study we transferred a shuttle vector, pRDB5, from the colonic species Bacteroides uniformis to P. ruminicola B(1)4. The transfer frequency was 10(-6) to 10(-7) per recipient. pRDB5 contains sequences from pBR328, a cryptic colonic Bacteroides plasmid pB8-51, and a colonic Bacteroides tetracycline resistance (Tcr) gene. pRDB5 was mobilized out of B. uniformis by a self-transmissible Bacteroides chromosomal element designated Tcr Emr 12256. pRDB5 replicated in Escherichia coli as well as in Bacteroides spp. and was also mobilized from E. coli to B. uniformis by using IncP plasmid R751. However, direct transfer from E. coli to P. ruminicola B(1)4 was not detected. Thus, to introduce cloned DNA into P. ruminicola B(1)4, it was necessary first to mobilize the plasmid from E. coli to B. uniformis and then to mobilize the plasmid from B. uniformis to P. ruminicola B(1)4.