Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.     

A newEscherichia coli: Bacteroides shuttle vector,pRRI207, based on theBacteroides ruminicola plasmid replicon pRRI2

A 3.4kb cryptic plasmid, pRRI2, was isolated fromBacteroides ruminicola 223/M2/7 and used as the basis for a newBacteroides/Escherichia shuttle vector. Constructs were made incorporating pRRI2, aBacteroides erythromycin/clindamycin resistance marker and theE. coli pUC8-derived vector pHG165. One of these, pRRI207 (11 kb), was capable of introduction into strains belonging to four different species ofBacteroides (B. uniformis, B. distasonis, B. thetaiotaomicron, orB. ruminicola) either by conjugal transfer fromE. coli or by electrotransformation. pRRI207 carries several unique restriction sites derived from the pUC8 multiple cloning site. Only one of sixB. ruminicola strains tested was used successfully as a recipient for pRRI207, indicating that further modifications to transfer procedures or marker genes may be needed for wider application in this species.     

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.     

Cloning, sequencing and complementation analysis of the recA gene from Prevotella ruminicola

Abstract Degenerate PCR primers based on conserved RecA protein regions were used to amplify a portion of recE from Prevotella ruminicola strain 23, which was used as a probe to isolate the full-length recA gene from the P. ruminicola genomic library. The P. ruminicola recA gene encoded a protein of 340 amino acids with a molecular mass of 36.81 kDa. P. ruminicola RecA was highly similar to other RecA proteins and most closely resembled that of Bacteroides fragilis (75% identity). It alleviated the methyl methanesulfonate and mitomycin C sensitivities of Escherichia coli recA mutants, but did not restore the resistance to UV-light irradiation. Mitomycin C treatment of otherwise isogenic E. coli strains showed a higher level of prophage induction in a recA harboring lysogen.     

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.     

Introduction of the transposon Tn919 into Lactobacillus curvatus Lc2-c

Frequencies of greater than 10(5) transformants per microgram DNA were achieved in Bacteroides ruminicola F101 by electroporation of cells under anaerobic conditions, using the 19.5 kbp tetracycline resistance plasmid pRRI4. Similar procedures gave frequences of 10(6) erythromycin resistant transformants per microgram DNA with the shuttle plasmid pDP1 (19 kbp) in Bacteroides uniformis. Transformation of B. uniformis occurred at a far lower frequency (10(3) micrograms) when pDP1 DNA was derived from E. coli rather than B. uniformis.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola.

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Epidemiological characteristics of infections caused by Bacteroides, Prevotella and Fusobacterium species: a prospective observational study

In order to investigate differences among infections due to Gram-negative anaerobic bacteria (Bacteroides, Prevotella and Fusobacterium spp.), clinical, epidemiological, and microbiological data were collected and evaluated from 206 anaerobic infections. The most frequently isolated species was Bacteroides fragilis. The majority of the cases were intra-abdominal infections (49%) followed by skin and soft tissue infections (24.7%). Logistic regression analysis showed that Bacteroides spp. strains were more often isolated from intra-abdominal infections (p?=?0.002), whereas Prevotella spp. were isolated more frequently from cases with shorter duration of hospitalization (p?=?0.026), and less frequently from bloodstream infections (p?=?0.049). In addition, Bacteroides spp. were associated with coinfection due to Enterobacteriaceae species (p?=?0.007), whereas Prevotella spp. were associated with coinfection due to Staphylococcus spp. (p?=?0.002). Patients with an infection due to B.?fragilis, were more frequently admitted in a general surgical ward (p?=?0.017), or have been treated with a 2nd generation cephalosporin before anaerobic infection onset (p?=?0.05). Total mortality was 10.9% and was associated with bacteremia (p?=?0.026), and hematological (p?=?0.028), or solid organ malignancy (p?=?0.007). Metronidazole resistance was detected only among Prevotella spp. (16.2%) and B. fragilis group (0.8%) isolates. In conclusion, this study indicated differences between infections due to the most frequently isolated Gram-negative anaerobic species, differences that may affect the design and implementation of empirical antimicrobial chemotherapy guidelines.     

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.     

Construction of new shuttle plasmid vectors for Escherichia coli-Bacteroides transgeneric cloning

Abstract An Escherichia coli-Bacteroides shuttle vehicle (pKBF367-1) was constructed by combining the pBR322 derivative pKC7 (5.9 kb) with [1] a 4.6 kb cryptic plasmid from Bacteroides fragilis ; and [2] the 4.2 kb Eco RI-B fragment of the B. fragilis plasmid pBFTM10. This latter component allowed selection of clindamycin-resistant transconjugants upon helper plasmid-mediated transfer to a recipient strain of Bacteroides distasonis . To improve the potential of pKBF367-1 (14.7 kb) as cloning vector, successive deletions generated derivatives of 12.8, 10.5 and 9.3 kb, which were still able to replicate in B. distasonis 419. These bifunctional vectors were successfully employed to introduce transposon Tn 501 (Hg^r) into B. distasonis 419, but expression of mercury resistance was not observed. This plasmid vehicles series may be useful for cloning Bacteroides genes in E. coli and studying their expression in a heterologous Bacteroides strain.     

Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.     

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.     

Peptidases of the rumen bacterium, Prevotella ruminicola

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested.Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.     

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.     

Sequencing the gene for an imipenem-cefoxitin-hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus beta-lactamase II.

Using a newly constructed Bacteroides fragilis-Escherichia coli cloning shuttle vector, pJST61, we have cloned the cefoxitin (FOX)-imipenem (IMP) resistance determinant from B. fragilis TAL2480. FOX-IMP resistance in this strain results from the production of a periplasmic, Zn2(+)-containing beta-lactamase which hydrolyzes carbapenems and cephamycins and whose activity is resistant to clavulanic acid but sensitive to Zn2(+)-binding reagents, including EDTA. The pJST61 vector permits efficient library construction in E. coli and allows for the transfer of the library to B. fragilis recipients for the screening or selection of specific phenotypes. The library clone containing the FOX-IMP resistance gene was detected after transfer to B. fragilis TM4000 (Fox-Imps) selecting for Foxr. One of the isolates carrying plasmid pJST241 is resistant to FOX and IMP and synthesizes a periplasmic protein with substrate and inhibitor properties identical to those of strain TAL2480. On the basis of deletion analysis, Tn1000 insertion mutations, and DNA sequencing, we have defined the 747-base cfiA (FOX-IMP resistance) gene within the 3.6-kilobase cloned insert in pJST241. The cfiA gene contains an open reading frame that could code for a precursor protein of 249 amino acids and with a molecular mass of 27,260 daltons. A potential signal sequence has been identified at the N terminus of this protein; cleavage within this sequence would result in a protein of 231 amino acids with a molecular mass of 25,249 daltons. The CfiA protein shows remarkable similarities to the exported, Zn2(+)-requiring, type II beta-lactamase Blm proteins from Bacillus cereus 569/H and 5/B/6. Although overall amino acid identity is only 32%, the Zn ligand-binding His and Cys residues are precisely conserved and the amino acids in the vicinity of these sites show strong similarities (greater than 80%) when the CfiA and Blm proteins are compared.     

Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4.

A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B(1)4 was reconstructed by adding a cellulose binding domain from a Thermomonospora fusca cellulase and was conjugally transferred from Escherichia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW). pTC-COW was specifically constructed to facilitate conjugal transfer of vectors from B. uniformis donors to P. ruminicola recipients. B. uniformis transconjugants containing CMCase constructs cloned into pTC-COW expressed Cmr, but they did not produce the reconstructed CMCase until a xylanase promoter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC). The xylanase promoter allowed the B. uniformis transconjugants to produce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells. Although the reconstructed CMCase alone did not allow B. uniformis to grow on acid-swollen cellulose, rapid growth was observed when two exocellulases were added to the culture supernatant. Under these conditions, the reconstructed CMCase permitted faster growth than the wild-type CMCase. The frequency of transfer of pTC-XRCMC from B. uniformis to P. ruminicola B(1)4 was increased 100-fold when strictly anaerobic conditions, nitrocelluose filters (cell immobilization), and more stringent selections were employed. Although the P. ruminicola B(1)4 (pTC-XRCMC) transconjugates expressed Tcr and had DNA that hybridized with a probe to the shuttle vector, these transconjugants did not produce detectable levels of the reconstructed CMCase even when xylan was the carbon source. On the basis of these results, it appears that not all of the promoters recognized by B. uniformis and P. ruminicola 23 are functional in P. ruminicola B(1)4. However, the results with B. uniformis suggest that the introduction of a P. ruminicola B(1)4 promoter should allow expression of the reconstructed CMCase in P. ruminicola B(1)4.