Comparison of the transposon-like structures encoding clindamycin resistance in Bacteroides R-plasmids

The R-plasmids pBF4, pBFTM10, and pBI136 encode transmissible clindamycin resistance (Ccr) in Bacteroides spp. These plasmids are distinct replicons but the regions implicated in Ccr share some homology and appear to have a transposon-like structure. To better understand the mechanism of dissemination and to locate the Ccr determinant(s), the genetic and structural properties of the Ccr regions of each plasmid were compared and contrasted. For this work a single EcoRI restriction fragment containing the Ccr region from each plasmid was cloned into pBR322 in Escherichia coli. Results of restriction mapping and heteroduplex experiments showed that the pBF4 EcoRI-D and pBFTM10 EcoRI-B fragments shared more than 90% base sequence homology but that the EcoRI-C fragment of pBI136 had diverged significantly. The pBI136 fragment also did not confer tetracycline resistance in E. coli as shown for the pBF4 EcoRI-D fragment (D.G. Guiney, P. Hasegawa, and C. E. Davis, 1984, Plasmid 11, 248-252). Heteroduplex experiments showed that the pBI136 EcoRI-C and pBF4 EcoRI-D fragments shared a 1.2-kb region of homology attributed to a directly repeated sequence which bounds the Ccr region. Southern hybridization studies indicated that an additional 0.85 kb of the pBI136 EcoRI-C fragment was homologous to the EcoRI-D fragment of pBF4. This region was characterized by its sequential restriction endonuclease sites for HindIII, AvaII, and DdeI, and it is proposed that the Ccr gene(s) resides in this area.     

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.     

Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant.

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.     

Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region.

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.     

Characterization and mapping of regions encoding clindamycin resistance, tetracycline resistance, and a replication function on the Bacteroides R plasmid pCP1.

The Bacteroides drug resistance plasmid pCP1 encodes clindamycin resistance (Clr) and a cryptic tetracycline resistance (Tcr) determinant that is expressed in Escherichia coli cells grown aerobically, but not anaerobically, and is not expressed phenotypically in Bacteroides spp. Localization of genetic functions on pCP1 was facilitated by the construction of hybrid shuttle plasmids containing portions of pCP1 ligated to pDG5, a pBR322 derivative carrying the RK2 transfer origin. pDP1 delta 4 is a BglII deletion derivative of pCP1 linked to pDG5 and can be maintained in both E. coli and Bacteroides fragilis. By using Tn5 mutagenesis and subcloning, we localized the Clr and Tcr regions on the EcoRI B fragment between the 1.2-kilobase direct repeats of pCP1. The Clr and Tcr determinants are distinct and appear to be transcribed separately. Control of the Tcr phenotype is unusual in that expression is constitutive and is enhanced by a region encompassing the adjacent direct repeat. In addition, a region of pCP1 required for replication in Bacteroides spp. has been identified in the neighboring EcoRI A fragment.     

Homology between clindamycin resistance plasmids in Bacteroides

Two different species of clindamycin-resistant Bacteroides were isolated from the same infection. One isolate contained a single 15-kb plasmid (pCP1) which encoded transferable clindamycin resistance. pCP1 appears similar to the Bacteroides clindamycin resistance plasmid pBFTM10 isolated independently by F.P. Tally, D.R. Snydman, M.J. Shimell, and M.H. Malamy (1982, J. Bacteriol. 151, 686-691). The second strain had a 10-kb plasmid (pCP2) but did not transfer resistance. DNA hybridization studies revealed that pCP1 shares a 5-kb region of homology with the B. fragilis R plasmid pBF4 studied by R.A. Welch and F.L. Macrina (1981, J. Bacteriol. 145, 867-872). This region in both plasmids was shown to be bounded by homologous direct repeats and contains the putative clindamycin resistance determinant. pCP1 and pCP2 were found to share extensive homology but sequences homologous to the clindamycin resistance region were missing from pCP2 and found instead in the whole cell DNA of the host strain. These results identify a transposon-like structure on Bacteroides clindamycin resistance plasmids.     

A site-specific DNA inversion in Bacteroides plasmid pBF4 is influenced by the presence of the conjugal tetracycline resistance element.

pBF4 is a 42-kb R plasmid from Bacteroides fragilis which transfers clindamycin resistance (Clr) independently of the chromosomal tetracycline resistance (Tcr) transfer element. We have found that this plasmid exists in two nonequimolar conformations, A and B. These forms differ by an inversion of approximately 11.5 kb which does not involve the repeated DNA sequences previously mapped on the plasmid. The presence of chromosomal tetracycline resistance conjugal elements influences the relative amounts of the two conformations: induction with tetracycline shifts the dominant form from B to A.     

Cloning and analysis of the Clostridium perfringens tetracycline resistance plasmid, pCW3

Clostridium perfringens strain CW92 carries pCW3, a conjugative 47-kb plasmid that confers inducible resistance to tetracycline. The plasmid was examined by restriction endonuclease analysis and by cloning each of the five ClaI fragments of pCW3 in Escherichia coli, using pBR322. Analysis of the recombinant plasmids allowed the deduction of a detailed restriction map of pCW3. The tetracycline resistance determinant of pCW3 was mapped by examining the phenotype of recombinant E. coli clones derived from the cloning, into pUC vector plasmids, of EcoRI fragments from pCW3. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed EcoRI fragments which together encompass a 4-kb region of pCW3. Deletion experiments showed that the tetracycline resistance gene, and/or its control regions, contained internal EcoRI and SphI sites. E. coli strains that carried recombinant plasmids with only the 4-kb region were found to express tetracycline resistance constitutively. In contrast, recombinant plasmids harboring a 10.5-kb ClaI fragment of pCW3, that included the 4-kb region, coded for an inducible tetracycline resistance phenotype. The existence of a negatively regulated resistance gene, similar to that proposed for several other bacteria is postulated.     

Mobilization of Bacteroides plasmids by Bacteroides conjugal elements.

A 4.2-kilobase cryptic Bacteroides plasmid, pB8-51, is found in several colonic Bacteroides species. To determine whether pB8-51 is mobilized by any of the known Bacteroides conjugal elements, we constructed an Escherichia coli-Bacteroides shuttle vector, pVAL-1, which contains pB8-51. We constructed Bacteroides uniformis 0061 derivatives which carry pVAL-1 and various Bacteroides conjugal elements. The Bacteroides conjugal elements tested were six conjugal tetracycline resistance (Tcr) elements (which appear to be chromosomal), i.e., Tcr ERL, Tcr V479, Tcr Emr ERL, Tcr Emr 12256, Tcr Emr DOT, and Tcr Emr CEST, and the conjugal erythromycin resistance (Emr) plasmid pBF4. These Tcr conjugal elements have not been extensively characterized, except for Tcr ERL. All six Tcr elements tested mobilized pVAL-1 at high frequency (10(-3) to 10(-5)) from one Bacteroides strain to another or from a Bacteroides strain to E. coli. Pregrowth of the donors (containing one of the Tcr elements and pVAL-1) in 1 microgram of tetracycline per ml enhanced the transfer of pVAL-1 by 20- to 10,000-fold, depending on which Tcr element was present in the donor. An Ems derivative of pBF4 (pBF4 delta E2) mobilized pVAL-1 from one Bacteroides strain to another at a frequency of 10(-4) but did not mobilize pVAL-1 from a Bacteroides strain to E. coli as efficiently. Thus the Tcr conjugal elements and pBF4 recognize a mobilization region on pB8-51.     

Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome.

The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.     

Large transmissible clindamycin resistance plasmid in Bacteroides ovatus.

Bacteroides ovatus IB106 contained two plasmids, pBI106 (46 kilobases) and pBI136 (82 kilobases). Transmissible clindamycin-erythromycin resistance (Ccr) was mediated by pBI136 , whose Ccr determinant was closely related to the determinant on the Bacteroides R plasmids pBF4 and pBFTM10 . Hybridization studies showed that pBI106 was not involved in Ccr transfer, but it shared extensive homology to pBF4 with the exception of the pBF4 region implicated in Ccr.     

Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. Both enzymes have very similar biochemical properties. To determine whether both enzymes are required for growth on chondroitin sulfate, we constructed a Bacteroides suicide vector, pE3-1, and used it to create an insertional mutation that interrupts the chondroitin lyase II gene of Bacteroides thetaiotaomicron. pE3-1 contains a 4.4-kilobase cryptic B. eggerthii plasmid (pB8-51), the Escherichia coli cloning vector pBR328, and the EcoRI D fragment from the conjugative B. fragilis plasmid pBF4. A 0.8-kilobase fragment from the center of the B. thetaiotaomicron chondroitin lyase II gene was inserted in pE3-1 to create pEG817. Although, pEG817 is stably maintained in E. coli and can be mobilized into B. thetaiotaomicron by the IncP plasmid R751, pEG817 is not maintained as a plasmid in Bacteroides spp. When pEG817 was mobilized into B. thetaiotaomicron, with selection for a drug marker on pEG817, transconjugants were obtained which had pEG817 inserted into the chondroitin lyase II gene. Western blot analysis was used to confirm that intact chondroitin lyase II is not produced in the mutant. The mutant was able to utilize chondroitin sulfate as a sole source of carbon, although no active chondroitin lyase II was produced. Thus chondroitin lyase I alone appears to be sufficient for growth on chondroitin sulfate. The mutant also had some minor changes in its outer membrane protein profile. However, there was no evidence that any of the major chondroitin sulfate-associated polypeptides in the outer membrane were affected by the insertion in the chondroitin lyase II gene.     

Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli

Bacteroides fragilis TMP10, which is clindamycin-erythromycin resistant (Clnr) and tetracycline resistant (Tetr), contains several plasmids and is capable of transferring drug resistance markers to suitable recipients. We were able to separate a 14.6-kilobase self-transmissible Clnr plasmid, pBFTM10, from the other plasmids of TMP10 in a tetracycline-sensitive recipient strain, B. fragilis TM4000. All Clnr transconjugants acquired an unaltered pBFTM10 and became plasmid donor strains. Transfer is proposed to occur by conjugation since it required to cell-to-cell contact of filter matings and was insensitive to DNase, but sensitive to chloroform treatment of donor cells. The efficiency of transfer of pBFTM10 in a Tets background (TM4003) was not affected by pretreatment of donor cells with clindamycin. A spontaneously occurring Clns derivative, pBFTM10 delta 1, suffered a deletion of DNA, which included a 4.4-kilobase EcoRI fragment. A complex interaction between the autonomous plasmid pBFTM10 and a tetracycline transfer element also present in strain TMP10 was observed since pretreatment of this donor with tetracycline or clindamycin resulted in a marked increase in transfer of both tetracycline and clindamycin resistance.     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.     

Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli.

Transfer factor pBFTM10, isolated from the obligate anaerobic bacterium Bacteroides fragilis, carries a clindamycin resistance determinant which we have suggested is part of a transposable element. DNA homologous to this determinant is found in many Clnr Bacteroides isolates, either in the chromosome or on plasmids. We have now established that Ccr resides on a transposon, Tn4400. In addition to the Ccr determinant that functions under anaerobic conditions in B. fragilis, Tn4400 also carries a determinant for tetracycline resistance (Tcr) which only functions in Escherichia coli under aerobic conditions. The presence of Tn4400 on pBFTM10 does not confer tetracycline resistance on B. fragilis cells containing it. DNA from pBFTM10 was cloned in E. coli, with pDG5 as the cloning vector, to form pGAT500. Using a mobilization assay involving pGAT500 and an F factor derivative, pOX38, we determined that a 5.6-kilobase region of pBFTM10 DNA was capable of mediating replicon fusion and transposition. Most of the mobilization products resulted from inverse transposition reactions, while some were the result of true cointegrate formation. Analysis of the cointegrate molecules showed that three were formed by the action of one of the ends of Tn4400 (IS4400), and one was formed by the action of the whole element (Tn4400). The cointegrate molecule carrying intact copies of Tn4400 at the junction of the two plasmids could resolve to yield an unaltered donor plasmid (pGAT500) and a conjugal plasmid containing a copy of Tn4400 or a copy of one insertion sequence element (pOX38::Tn4400 or pOX38::IS4400). Thus, Tn4400 is a compound transposon containing active insertion sequence elements as directly repeated sequences at its ends.     

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.     

Two independent conjugal transfer systems operating in Bacteroides fragilis V479-1.

Bacteroides fragilis V479-1 (also designated strain 92) has previously been shown to contain a conjugative plasmid, pBF4, that specifies resistance to clindamycin (Cc). A report of inducible tetracycline (Tc) resistance in this strain suggested that this phenotype was also plasmid associated (G. Privitera et al., Nature [London] 278:657-659, 1979) and prompted further investigation. Mating experiments with V469-1 and a Cc-sensitive derivative of V479-1, V598, showed that Tc resistance transfer occurred by a conjugation-like event which was insensitive to DNase, was not mediated by donor culture cell-free filtrates, and required cell-to-cell contact. Results from transfer experiments with V479-1 indicated that Tc and Cc resistance determinants were not linked and segregated independently in matings. Progeny recovered from matings with the V479-1 or V598 donor strain were able to transfer the Tc resistance marker in secondary crosses. Tc resistance transfer from V479-1 or V598 was greatly stimulated by pregrowth in the presence of Tc but not Cc. pBF4-mediated Cc resistance transfer was not affected by pregrowth in the presence of Cc or Tc. Filter blot DNA hybridization studies revealed that pBF4 sequences were not involved in either the Tc resistant phenotype or its associated conjugal transfer properties. The Tc resistance transfer element was not associated with pBF4 or any other extrachromosomal DNA element.     

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.     

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.