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.     

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.     

Characterization of an outer membrane mannanase from Bacteroides ovatus

Bacteroides ovatus utilizes guar gum, a high-molecular-weight branched galactomannanan, as a sole source of carbohydrate. No extracellular activity was detectable. Approximately 30% of the total cell-associated mannanase activity partitioned with cell membranes. When inner and outer membranes of B. ovatus were separated on sucrose gradients, the mannanase activity was associated mainly with fractions containing outer membranes. Enzyme activity was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or by Triton X-100 at a detergent-to-protein ratio of 1:1. The enzyme was stable for only 4 h at 37 degrees C and for 50 to 60 h at 4 degrees C. Analysis of the products of the CHAPS-solubilized mannanase on Bio-Gel A-5M and Bio-Gel P-10 gel filtration columns indicated that the enzyme breaks guar gum into high-molecular-weight fragments. The CHAPS-solubilized mannanase was partially purified by chromatography on a FPLC Mono Q column. The partially purified mannanase preparation contained three major polypeptides (Mr 94,500, 61,000, and 43,000) and several minor ones. High mannanase activity was seen only when B. ovatus was grown on guar gum. Cross-absorbed antiserum detected two other guar gum-associated outer membrane proteins: a CHAPS-extractable 49,000-dalton polypeptide and a 120,000-dalton polypeptide that was not solubilized by CHAPS. Neither of these polypeptides was detectable in the partially purified mannanase preparation. These results indicate that there are at least two guar gum-associated outer membrane polypeptides other than the mannanase.     

Characterization of a Metal-independent CAZy Family 6 Glycosyltransferase from Bacteroides ovatus

The myriad functions of complex carbohydrates include modulating interactions between bacteria and their eukaryotic hosts. In humans and other vertebrates, variations in the activity of glycosyltransferases of CAZy family 6 generate antigenic variation between individuals and species that facilitates resistance to pathogens. The well characterized vertebrate glycosyltransferases of this family are multidomain membrane proteins with C-terminal catalytic domains. Genes for proteins homologous with their catalytic domains are found in at least nine species of anaerobic commensal bacteria and a cyanophage. Although the bacterial proteins are strikingly similar in sequence to the catalytic domains of their eukaryotic relatives, a metal-binding Asp-X-Asp sequence, present in a wide array of metal ion-dependent glycosyltransferases, is replaced by Asn-X-Asn. We have cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that is linked to inflammatory bowel disease. Functional characterization shows it to be a metal-independent glycosyltransferase with a 200-fold preference for UDP-GalNAc as substrate relative to UDP-Gal. It efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A and may participate in the synthesis of the bacterial O-antigen. The kinetics for GalNAc transfer to 2′-fucosyl lactose are characteristic of a sequential mechanism, as observed previously for this family. Mutational studies indicate that despite the lack of a metal cofactor, there are pronounced similarities in structure-function relationships between the bacterial and vertebrate family 6 glycosyltransferases. These two groups appear to provide an example of horizontal gene transfer involving vertebrates and prokaryotes.The structures of complex glycans are determined by the specificities of the glycosyltransferases (GTs)² that catalyze their biosynthesis. GTs fall into two groups that differ in mechanism, based on whether the anomeric configuration of the donor substrate (α for most UDP-sugars) is retained or inverted in the product (1–3). They are classified into 90 different families in the CAZy data base based on sequence similarities (4, 5), but the majority of those that have been structurally characterized fall into one of two fold types, designated GT-A and GT-B (2). The retaining GTs of CAZy family 6 (GT6) have a GT-A fold and catalyze the transfer of either galactose or GalNAc into an α-linkage with the 3-OH group of β-linked galactose or GalNAc. GT6 includes the histo-blood group A and B GTs (GTA and GTB), the α-galactosyltransferase (α3GT) that catalyzes the synthesis of the xenoantigen or α-gal epitope, Forssman glycolipid synthase, isogloboside 3 synthase, and their homologues from other vertebrates (6). GT6 enzymes from vertebrates are type-2 membrane proteins with N-terminal cytosolic domains, a transmembrane helix, a spacer, and a C-terminal catalytic domain (6). Crystallographic studies of recombinant catalytic domains of GTA, GTB, and α3GT have provided detailed information about their interactions with substrates, metal cofactor, and inhibitors (7–9). Most GT-A fold GTs, including those in the GT6 family, require divalent metal ions, such as Mn²⁺, for catalytic activity; their metal dependence is linked to a shared DXD sequence motif. Residues of this motif interact with the metal ion and both the ribose and phosphates of the donor substrate to produce an appropriate substrate orientation and conformation for catalysis and to stabilize the UDP leaving group (3, 7–10).Mammalian members of GT6 are responsible for variations in glycan structures between different species and individuals as the result of selective enzyme inactivation in certain species (α3GT, Forssman glycolipid synthase, and isogloboside 3 synthase) or the inheritance of multiple alleles at one locus that encode enzymes with different substrate specificity (GTA and GTB) or are inactive (11–14). The presence of circulating antibodies against glycan structures that are subject to interspecies and individual variability has been linked to resistance to pathogens that also carry the glycans; these antibodies are thought to arise from exposure to potential pathogens, including enveloped viruses and bacteria that carry structurally similar glycans (11).In addition to the well characterized enzymes discussed previously, atypical members of the GT6 family have been identified in mammals that have sequence changes in highly conserved regions of the active site, including the DXD motif (6). However, no glycosyltransferase activity was detected in recombinant forms of two of these, and their functions are unclear (6). Although GT6 members are widely distributed among vertebrates, no homologues have been found in other eukaryotes (6). However, GT6 members have been identified in several bacterial species (15–17). GT6 enzymes from Escherichia coli O86, and Helicobacter mustelae that appear to function in the biosynthesis of the lipopolysaccharide O-antigen have been cloned and expressed by Wang and co-workers (16, 17) and found to have specificities similar to those of human GTB and GTA, respectively. These enzymes have been applied in the enzymatic synthesis of oligosaccharides. Other homologues are encoded by Hemophilus somnus, Psychroacter sp., PRwf-1 (15), Francisella philomiragia, and three Bacteroides species, Bacteroides ovatus, Bacteroides caccae, and Bacteroides stercoris, as well as a cyanophage, PSSM-2 (15). Genes for other homologues from unidentified species are present in the marine metagenome (18, 19) and human gut metagenome (20, 21). The phage and bacterial enzymes are substantially truncated at the N terminus relative to the catalytic domains of vertebrate GT6 representatives and are smaller than the reported minimal functional unit of a primate α3GT (22). When bacterial and vertebrate GT6 amino acid sequences are aligned (Fig. 1 and supplemental Figs. S1 and S2), it can be seen that the metal-binding DXD of the eukaryotic GTs is replaced by NXN (where X is Ala, Gly, or Ser) in the bacterial homologues. The cyanophage GT6 member and related proteins in the marine metagenome, however, retain the DXD motif. This conspicuous difference in the bacterial proteins is particularly interesting, since, in the mammalian enzymes, the aspartates of the DXD and adjacent residues are crucial for catalytic activity (10, 23).Open in a separate windowFIGURE 1.An alignment of selected bacterial, cyanophage and mammalian GT6 amino acid sequences. Abbreviations and Interpro sequence IDs (in parentheses) are as follows. HuA, human histo-blood group A synthase (A1EAJ6); Bova, bovine α1,3-galactosyltransferase ({"type":"entrez-protein","attrs":{"text":"P14769","term_id":"120970","term_text":"P14769"}}P14769); PSSM2, cyanophage PSSM-2 ({"type":"entrez-protein","attrs":{"text":"Q58M87","term_id":"75096044","term_text":"Q58M87"}}Q58M87); Bs, B. stercoris (B0NSM3); Bo1, B. ovatus GT1 (A7LVT2); Bo2, B. ovatus GT2 (A7M0P3); Bc, B. caccae (A5ZC71). The boxed regions in the alignment identify regions that have been shown to be involved in interactions with substrates and cofactor and in catalysis in bovine α1,3-galactosyltransferase and histo-blood group A and B enzymes. These are labeled (below) as follows. A, interactions with uracil; B, interactions with the galactose moiety of UDP-Gal; C, interactions with Mn²⁺, phosphates, and galactose; D, interactions with acceptor substrate; E, interactions with Gal or GalNAc of donor substrate; F, interactions with monosaccharide of donor substrate and acceptor and catalysis; The arrow (above) denotes the intron/exon boundary in vertebrate GT6s, and the asterisks indicate the residues in BoGT6a that were subjected to mutagenesis.B. ovatus is a Gram-negative commensal bacterium that inhabits the distal mammalian gut and has been implicated in the pathology of inflammatory bowel disease in humans (24). The B. ovatus genome contains two genes that encode GT6 representatives (Fig. 1). We selected one of these for initial investigation, and designate it BoGT6a (family 6 glycosyltransferase 1 of Bacteroides). The gene for this protein was amplified by PCR and cloned and expressed in His-tagged form in E. coli BL21(DE3). Assays with a variety of substrates show that its substrate specificity is similar to that of human GTA. Previous studies of the activities of bacterial enzymes were conducted in the presence of Mn²⁺ (16, 17), but we find that the B. ovatus enzyme does not require divalent metal ions for activity and is fully active in EDTA. Despite this striking difference, BoGT6a is similar to its metal-dependent relatives in catalytic properties; also, the effects of amino acid substitutions for residues corresponding to several that act in substrate binding and catalysis in vertebrate GT6 glycosyltransferases suggest that they have similar structure-function relationships. These results indicate that the metal cofactor is not a conserved feature in the GT6 family. They also raise questions about the catalytic mechanism of prokaryotic GT6 members and the evolutionary relationship between bacterial, phage, and vertebrate enzymes.     

Identification and DNA sequence of the mobilization region of the 5-nitroimidazole resistance plasmid pIP421 from Bacteroides fragilis.

The nucleotide sequence of the DNA mobilization region of the 5-nitroimidazole resistance plasmid pIP421, from strain BF-F239 of Bacteroides fragilis, was determined. It contains a putative origin of transfer (oriT) including three sets of inverted repeats and two sequences reminiscent of specific integration host factor binding sites. The product of the mobilization gene mob421 (42.2 kDa) is a member of the Bacteroides mobilization protein family, which includes the MobA of pBI143, NBUs, and Tn4555. Sequence similarity suggests that it has both oriT binding and nicking activities. The transfer frequency of pIP421 in a B. fragilis donor strain possessing a Tc(r) or Tc(r) Em(r)-like conjugative transposon was significantly enhanced by tetracycline. Moreover, the mobilization region of pIP421 confers the ability to be mobilized from Escherichia coli by an IncP plasmid.     

The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region.

Genes coding for three xylan-degrading activities, xylanase, xylosidase, and arabinosidase, were simultaneously cloned from the colonic anaerobic organism Bacteriodes ovatus. The genes for the three enzymes were located on a 3.8-kilobase EcoRI genomic insert and were cloned by using plasmid pUC18. All three activities were expressed in Escherichia coli JM83, and all were cell associated. Expression of the xylanase gene was independent from expression of the xylosidase and arabinosidase genes, whereas expression of the latter two genes appeared to be coordinated. Restriction endonuclease analysis of the arabinosidase and xylosidase genes and partial purification of these enzyme activities from E. coli suggested that these activities were catalyzed by a bifunctional protein or two proteins of very similar molecular weight. All three enzyme activities were regulated in B. ovatus in response to the carbon source used for growth. This is the first report of the cloning and expression of B. ovatus genes.     

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.     

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.     

Purification and characterization of a cell-associated, soluble mannanase from Bacteroides ovatus.

Bacteroides ovatus, a human colonic anaerobe, utilizes the galactomannan guar gum as a sole source of carbohydrate. Previously, we found that none of the galactomannan-degrading enzymes were extracellular, and we characterized an outer membrane mannanase which hydrolyzes the backbone of guar gum to produce large fragments. We report here the purification and characterization of a second mannanase from B. ovatus. This enzyme is cell-associated and soluble. Using ion-exchange chromatography, gel filtration, and chromatofocusing steps, we have purified the soluble mannanase to apparent homogeneity. The enzyme has a native molecular weight of 190,000 and a monomeric molecular weight of 61,000. It is distinct from the membrane mannanase not only with respect to cellular location but also with respect to stability and isoelectric point (pI of 6.9 for the membrane mannanase and pI of 4.8 for the soluble mannanase). The soluble mannanase, like the membrane mannanase, hydrolyzed guar gum to produce large fragments rather than monosaccharides. However, if galactosyl side chains were removed from the galactomannan fragments by alpha-galactosidase, both the soluble mannanase and the membrane mannanase could degrade guar gum to monosaccharides. Thus either or both of these two enzymes, working together with alpha-galactosidase, appear to be sufficient for the breakdown of guar gum to the level of monosaccharides.     

Intracellular glycosidases of human colon Bacteroides ovatus B4-11.

Activity of various glycosidases in the intracellular enzyme fraction of Bacteroides ovatus B4-11 was investigated. During 120 h of incubation at 37 degrees C, ca. 30% of the crude hemicellulose was hydrolyzed by an intracellular enzyme fraction of strain B4-11. Xylose was the major sugar released from crude hemicellulose. Glycosidases (alpha-1,6-glucosidase, alpha-1,4-glucosidase, beta-1,4-glucosidase, and beta-1,4-xylosidase) were induced in B. ovatus B4-11 by crude hemicellulose and heteroxylan. When B. ovatus B4-11 was grown on either crude hemicellulose or heteroxylan, the predominant enzyme in the intracellular enzyme fraction was beta-1,4-xylosidase.     

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.     

Tetracycline-inducible transfer of tetracycline resistance in Bacteroides fragilis in the absence of detectable plasmid DNA.

Tetracycline resistance of three Bacteroides fragilis strains was shown to be inducible by subinhibitory concentrations of tetracycline. Tetracycline resistance markers could be transferred to another B. fragilis strain by filter mating. The transferability was inducible by subinhibitory concentrations of tetracycline and did not take place in the absence of tetracycline. The optimum concentration of tetracycline for induction of transfer was about 2 microgram/ml. The transfer was shown to be a conjugation-like process requiring cell-to-cell contact between donor and recipient. Screening of parental donor strains for the presence of plasmid DNA did not demonstrate any detectable plasmids in two of the strains. A 3.0-megadalton plasmid, designated pBY5, was present in the third donor strain. Mobilization of pBY5 by another plasmid (pBF4) showed that pBY5 did not carry the genes responsible for tetracycline resistance. It appears that the genes responsible for resistance to tetracycline as well as those responsible for conjugal transfer may be carried on the chromosome in all three donor strains.     

Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.     

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.     

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 drug resistance plasmid NTP16

A functional and physical analysis of the multicopy plasmid NTP16 is presented. The plasmid-encoded drug resistance determinants are located, as are regions encoding the origin of replication, incompatibility functions, copy number determinants, and mobility functions. It is demonstrated that NTP16 probably arose from the closely related plasmid NTP1 by the acquisition of a novel kanamycin resistance transposon, Tn4352, followed by deletion of some NTP1 sequences. The incompatibility behavior of NTP16 derivatives indicates a system of control rather more complex than that which operates in ColE1. In addition to the RNA I/primer RNA system, the production of a further trans-acting product is demonstrated and its site of action located. A series of derivative plasmids have been created which may prove useful as vectors for genetic engineering.     

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.     

Polyethylene glycol-facilitated transformation of Bacteroides fragilis with plasmid DNA.

A method for the transformation of Bacteroides fragilis with plasmid DNA was developed by using the clindamycin resistance plasmid pBFTM10 as the source of transforming DNA. The method was technically simple to perform and resulted in an average of 4.2 X 10(3) transformants per microgram of pBFTM10 added. A method for the preparation of frozen competent cells is also described.