Isolation of Bacteroides fragilis mutants with in vivo growth defects by using Tn4400', a modified Tn4400 transposition system, and a new screening method.

A modified version of the Bacteroides fragilis transposon Tn4400, designated Tn4400', enabling rapid isolation and analysis of B. fragilis mutants has been constructed. To identify potential virulence factors, Tn4400'-generated mutants were screened by a new method; this resulted in the isolation of 21 mutant strains with impaired growth characteristics on tissue culture monolayers but normal growth in rich medium anaerobically.

Identification and cloning of genes from Porphyromonas gingivalis after mutagenesis with a modified Tn4400 transposon from Bacteroides fragilis.

Porphyromonas gingivalis is a gram-negative, black-pigmented, oral anaerobe strongly associated with adult periodontitis. Previous transposon mutagenesis studies with this organism were based on the Bacteroides transposon Tn4351. Characterization of Tn4351-disrupted genes by cloning has not been an efficient way to analyze large numbers of mutants and is further complicated by the high rate of cointegration of the suicide delivery vector containing Tn4351. In this study, we mutagenized P. gingivalis with a modified version of the Bacteroides fragilis transposon Tn4400. Plasmid pYT646B carrying the transposon was mobilized from Escherichia coli to P. gingivalis ATCC 33277 by conjugation. Both normal and inverse transposition frequencies were similar (3 x 10(-8)). However, the inverse transposon (Tn4400') contains a pBR322 replicon and a beta-lactamase gene; thus, the cloning of disrupted genomic DNAs from inverse transposition mutants was easily accomplished after ligation of genomic fragments and transformation into E. coli. Thousands of transconjugants could be obtained in a single mating experiment, and inverse transposition was random as demonstrated by Southern hybridization. By this procedure the disrupted genes from P. gingivalis pleiotropic mutants were quickly cloned, sequenced, and identified.

Characterization of the Batl (Bacteroides aerotolerance) operon in Bacteroides fragilis: isolation of a B. fragilis mutant with reduced aerotolerance and impaired growth in in vivo model systems.

YT135.2.8, a Tn4400' insertion mutant of Bacteroides fragilis strain TM4000, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) cell monolayers and is outcompeted by wild-type strains in mixed infections. YT135.2.8 also shows defects in the rat granuloma pouch model system in monoculture and is completely outcompeted by the wild-type strain in a mixed infection. In addition, this mutant shows defects in a new model system consisting of CHO suspension cell columns. All of these defects may be explained by the finding that YT135.2.8 shows decreased tolerance to exposure to atmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur-containing reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non-sulphur reducing agent Tris-(2-carboxylethyl)phosphine (TCEP). The defects in YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chromosome. DNA sequencing of this fragment and of the regions flanking the Tn4400' insertion in the B. fragilis chromosome revealed the presence of five open reading frames, corresponding to genes bat (Bacteroides aerotolerance) A, B, C, D, E, which form the Batl operon; Tn4400' inserted within batD. All of the hypothetical proteins possess one or more membrane-spanning domains. BatA and BatB show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and BatE contain 2-4 repeated sequences similar to the tetratricopeptide repeats (TPRs) seen in many eukaryotic proteins. The function of TPR sequences in protein interactions in other systems leads to the suggestion that the Bat proteins form a complex. The Batl complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragilis cell.

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains.

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.

Requirements for strand- and site-specific cleavage within the oriT region of Tn4399, a mobilizing transposon from Bacteroides fragilis.

Replicons that contain Tn4399, a conjugal mobilizing transposon isolated from Bacteroides fragilis, can be mobilized in the presence of broad-host-range IncP plasmids RP4 and R751 in Escherichia coli to B. fragilis or E. coli recipients (C. G. Murphy and M. H. Malamy, J. Bacteriol. 175:5814-5823, 1993). To identify the initial DNA processing events involved in Tn4399-mediated mobilization in E. coli, plasmid DNA from pCGM328 (a pUC7 vector that contains the mobilization region of Tn4399) was isolated from donor cells following the release of plasmid DNA from the relaxation complex. Site- and strand-specific cleavage within the oriT region of Tn4399 was detected by denaturing gel electrophoresis and Southern hybridization analysis of this DNA in the presence or absence of IncP plasmids. Mutations in either mocA or mocB, two genes which are encoded by Tn4399 and are required for mobilization, significantly decrease the amount of specifically nicked DNA detected. These results suggest roles for the MocA and MocB gene products in specific processing of Tn4399-containing plasmid DNA prior to mobilization. By isolation of the nicked strand and primer extension of this template, we mapped the precise 5' end of the single-stranded cleavage reaction. The nucleotide position of nicTn4399 is adjacent to two sets of inverted repeats, a genetic arrangement similar to those of previously characterized oriT regions. Two site-directed mutations which remove nicTn4399 (oriT delta 1 and oriT delta 2) cannot be mobilized to recipients when they are present in trans along with functional MocA and MocB proteins and an IncP mobilizing plasmid; they are cis-dominant loss-of-function mutations.

A role for Bacteroides fragilis neuraminidase in bacterial growth in two model systems.

Two Bacteroides fragilis neuraminidase-deficient mutants were used to study the role of neuraminidase activity in growth of B. fragilis in tissue culture monolayers (CHO cells) and in the in vivo rat granuloma pouch. The nanH structural gene for neuraminidase was cloned from B. fragilis TM4000 and was used to create two isogenic strains with chromosomal disruptions at the nanH gene. B. fragilis VRC404 contains an insertion flanked by disrupted copies of the nanH gene, and B. fragilis VRC426 contains a deletion of a significant portion of nanH coding sequences. The insertion mutant VRC404 is capable of reverting to nanH+. It grew as well as the wild type in CHO monolayers. However, between 48 and 72 h after infection, the bacterial population was enriched with nanH+ bacterial cells (10 to 20%). In the rat pouch 48 h after infection, more than 90% of the population sampled had become nanH+. The deletion mutant VRC426 showed a severe growth defect in the rat pouch model. In addition, VRC426 was efficiently outgrown by the wild type in competition experiments, even when the mutant was present at 10 times the number of wild-type cells at the time of infection. A common characteristic of both model systems is a drastic decrease in the free glucose concentration 16 to 24 h postinfection. We suggest that neuraminidase activity may be required for B. fragilis to grow to maximal levels in the tissue culture and rat pouch systems by making other carbon sources available after glucose levels are reduced.

Characterization of a "mobilization cassette" in transposon Tn4399 from Bacteroides fragilis.

Derivatives of nonconjugal plasmids that carry Tn4399, a transposon isolated from Bacteroides fragilis, can be mobilized for transfer by the broad-host-range IncP plasmids pRK231 or R751 in Escherichia coli. To characterize regions of Tn4399 involved in mobilization, we have isolated and analyzed subcloned fragments of Tn4399 in E. coli, as well as mutations within the element. We have identified a "mobilization cassette" within a 2.8-kb region of Tn4399 which, when cloned into mobilization-deficient plasmids, allows these plasmids to be mobilized in trans by the IncP plasmids pRK231 and R751. The 2.8-kb region has been sequenced, and several open reading frames have been identified. Mutants defective in two genes, designated mocA and mocB, coding for deduced products of 36.4 and 16.4 kDa, respectively, cannot be mobilized by either IncP plasmid; these mutants can be complemented in the presence of the respective wild-type genes in trans. This suggests that the putative MocA and MocB proteins have a role in the mobilization process. The 36.4-kDa MocA protein contains a 14-amino-acid sequence which is closely related to a highly conserved motif within DNA relaxases encoded by a wide variety of conjugal or mobilizable plasmids. Subcloning experiments also lead to the localization of an oriT region within a 199-bp fragment, internal to the mobilization cassette.

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 and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli.

We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type of NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda pR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and Tn1000 mutagenesis, the NANase structural gene and control region that functions in B. fragilis were localized to a 1.5- to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tn1000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.

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.

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.

Characterization of the termini and transposition products of Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis.

We have isolated a 9.6-kilobase conjugal transposon, Tn4399 from Bacteroides fragilis, that is capable of mobilizing nonconjugal plasmids in cis. Here we characterize the ends of the transposon, its target-site requirements, and the products of transposition into the B. fragilis chromosome and two sets of B. fragilis-Escherichia coli shuttle vectors. With the exception of an additional cytosine residue in the left end, there are perfect 13-base-pair (bp) inverted repeats at the ends of Tn4399. Insertion of Tn4399 resulted in a 3-bp target-site repeat in 8 out of 12 independent transpositions and showed a high insertion-site specificity. A remarkable feature of Tn4399 insertions is the presence of an additional 5 bp located between the right inverted repeat and the target-site repeat. Four sequence variations of the 5 bp were found, with absolute conservation at positions 1, 2, and 5. Only two of the variations were present in junction fragments of all three copies of Tn4399 contained in the chromosome of the original donor strain, B. fragilis TM4.2321. Tn4399 appears to represent a new type of conjugal transposon. In contrast to Tn916 and Tn1545, described in streptococci, Tn4399 creates a target-site repeat and contains an additional 5 bp at the right end only, between the transposon and the target sequence. In addition, Tn4399 can mobilize plasmids in cis.

Permeability to beta-lactams in Bacteroides fragilis.

Bacteroides fragilis strains TAL2480 and TAL3636 were used to assess outer membrane permeability to various beta-lactam compounds. These strains were chosen because they possess beta-lactamases capable of hydrolysing all commonly employed beta-lactams except monobactams. The beta-lactamases are located in the periplasmic space and could be released by sonication and osmotic shock. Permeability was calculated by the method of Zimmerman & Rosselet (1977, Antimicrobial Agents and Chemotherapy 12, 368-72). The rank order of permeative ability (from fastest to slowest) was as follows: cephaloridine, imipenem, cefotaxime, cefoxitin, cefoperazone, nitrocefin, cephalothin, and latamoxef. Our results showed that ionic charge, hydrophobicity, and molecular weight influenced beta-lactam drug uptake by B. fragilis. These results are similar to findings for Escherichia coli, where increased negative charge and increased molecular weight are associated with decreased drug uptake. However, unlike E. coli, increased drug hydrophobicity, for a given charge and molecular weight of the drug, was associated with increased uptake by B. fragilis.

Cryptic tetracycline resistance determinant (class F) from Bacteroides fragilis mediates resistance in Escherichia coli by actively reducing tetracycline accumulation.

Escherichia coli bearing a cryptic tetracycline resistance determinant from Bacteroides fragilis expressed low-level constitutive resistance to tetracycline under aerobic, but not anaerobic, growth conditions and accumulated less tetracycline aerobically than did isogenic susceptible cells. This decreased uptake was energy dependent and reversible by increased concentrations of tetracycline, suggesting a saturable carrier-mediated active efflux mechanism. Decreased uptake was not seen when the cells were grown and assayed anaerobically. Other tetracycline resistance determinants (classes A to E) isolated from gram-negative enteric bacteria expressed resistance and generated active efflux of tetracycline under anaerobic as well as aerobic conditions. When the Bacteroides determinant was placed in the same cell with any of the class A to E tetracycline resistance determinants, there was an increase in resistance under aerobic conditions of as much as 48% more than was projected by adding the resistances expressed by the determinants individually. In cells bearing the class A determinant together with the Bacteroides determinant, saturation of the active efflux system required over twofold more exogenous tetracycline than did cells bearing the class A determinant alone. We have designated this new tetracycline resistance determinant class F.

Beta-lactamase-mediated imipenem resistance in Bacteroides fragilis.

Imipenem has excellent antimicrobial activity owing in part to beta-lactamase stability. We found that only 2 of over 350 Bacteroides fragilis group clinical isolates were resistant to imipenem, with an MIC of more than 16 micrograms/ml. These two isolates from the Tufts Anaerobe Laboratory (TAL) were resistant to all other beta-lactam agents tested. The organisms were able to inactivate imipenem in broth cultures and contained similar beta-lactamases that were able to hydrolyze carbapenems, cephamycins, cephalosporins, and penicillins. The molecular sizes of the beta-lactamases in TAL2480 and TAL3636 were estimated to be 44,000 daltons. The novel beta-lactamase contained Zn2+ as a cofactor. An additional factor contributing to resistance was determined. The outer membranes of these two organisms were found to limit free diffusion of the drugs into the periplasm. This novel beta-lactamase, associated with a barrier to drug permeation, resulted in high-grade beta-lactam drug resistance.

Transfer of beta-lactamase-associated cefoxitin resistance in Bacteroides fragilis.

A cefoxitin-resistant Bacteroides fragilis isolate, TAL 4170, which inactivates cefoxitin, was able to transfer beta-lactamase-mediated cefoxitin resistance to a susceptible B. fragilis recipient. Cefoxitin-resistant transconjugants acquired a new beta-lactamase with a pI of 8.1 and were able to inactivate cefoxitin and retransfer cefoxitin resistance. No plasmids were detected in the donor or transconjugants.

Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution.

We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor. Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII-BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region. We constructed a series of pMB080 deletions in vitro using exonucleases S1 and Bal31. Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction. Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system. The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids. We have determined the precise location of these recombination sites within oriV1 by DNA sequencing. These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base-pair inverted repeats found to be important for oriV1-oriV1 interactions. We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements. Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site-specific recombination process.

Mutational and in vivo methylation analysis of F-factor PifC protein binding to the pif operator and the region containing the primary origin of mini-F replication.

We have used in vivo methods to identify multiple DNA-binding sites for the negatively autoregulated mini-F replication factor PifC. Sequence analysis of pif operator constitutive mutants, isolated as insensitive to repression by PifC, establishes the structure of pifO. This site contains a 17-base-pair (bp) region of dyad symmetry with 7-bp perfect inverted repeats separated by 3 bp. In vivo DNA methylation studies with dimethyl sulfate show that the reactivity of five of six guanine residues in the pifO region is altered in the presence of PifC protein. In addition, there are several sites of PifC-dependent methylation enhancement and protection upstream of pifO within repeated sequences bearing homology to pifO. The significance of the repeated PifC binding sequences and their relationship to the primary origin of mini-F replication (oriV1) are discussed.

Resistance factors in anaerobic bacteria.

Resistance transfer factors have been described in both Bacteroides and clostridia. The clindamycin (Cln) resistance transfer factors from the Bacteroides fragilis group of organisms have been best studied, including our own plasmid pBFTM10. The clindamycin resistance determinant (Cln X) of pBFTM10 can be detected in 90% of Cln resistant Bacteroides isolated from dispersed geographical areas. This determinant can be located in the chromosome and on plasmids. Recent studies from our laboratory have shown that the Cln X genes of pBFTM 10 are carried on a compound transposon, Tn4400. Bacteroides plasmids have been cloned in Escherichia coli and shuttle vectors have been developed that allow transfers of DNA from E. coli back to B. fragilis, using the broad host range plasmid RK2 to supply essential conjugation functions. We have shown that shuttle vectors containing pBFTM 10 can be retransferred from B. fragilis back to E. coli. In addition, a tetracycline transfer element from B. fragilis strain TM230 is able to promote high frequency conjugation between B. fragilis and E. coli. The results of these investigations indicate that Bacteroides has efficient mechanisms to exchange genetic material and that genetic exchange can occur between Bacteroides and E. coli, which exist in intimate contact in the human colon.