Tetracycline-dependent appearance of plasmidlike forms in Bacteroides uniformis 0061 mediated by conjugal Bacteroides tetracycline resistance elements.

Some human colonic Bacteroides strains carry conjugal tetracycline resistance (Tcr) elements, which are thought to be chromosomal. We have found that some of these Tcr elements can mediate the appearance of plasmidlike forms in Bacteroides uniformis 0061. When B. uniformis 0061, containing a conjugal Tcr element designated Tcr ERL, was grown in medium containing tetracycline (1 microgram/ml), two circular DNA forms were found in the alkaline plasmid preparations: NBU1 (10.3 +/- 0.5 kilobases) and NBU2 (11.5 +/- 0.5 kilobases). Restriction analysis of NBU1 and NBU2 showed that they were not identical, although Southern blot analysis indicated that they did contain some region(s) of homology. Results of Southern blot analysis also demonstrated that both NBU1 and NBU2 were normally integrated in the chromosome of B. uniformis or in some undetected large plasmid. Although we were unable to determine the exact structure and location of the integrated forms of NBU1 and NBU2 in B. uniformis, they appear to be in close proximity to each other. Neither NBU1 or NBU2 could be detected as a plasmidlike form in cells exposed to UV light, thymidine starvation, mitomycin C, or autoclaved chlortetracycline (50 micrograms/ml). Four conjugal Tcr elements other than the Tcr ERL element were able to mediate the appearance of NBU1 alone, and two Tcr elements did not mediate the excision of either NBU1 or NBU2. Three strains from different Bacteroides species contained some DNA sequences which had homology to NBU1 and NBU2.     

A cryptic 65-kilobase-pair transposonlike element isolated from Bacteroides uniformis has homology with Bacteroides conjugal tetracycline resistance elements.

A 65-kilobase-pair element, XBU4422, which has some transposonlike characteristics but carries no known antibiotic resistance genes, has been isolated from Bacteroides uniformis 0061. XBU4422 was trapped on Bacteroides-Escherichia coli shuttle vectors during experiments in which one of the conjugal Bacteroides tetracycline resistance (Tcr) elements was being used to mobilize the shuttle vectors to Bacteroides recipients. Results of Southern hybridization experiments showed that XBU4422 is normally integrated in the B. uniformis 0061 chromosome and is found only in some strains. Insertion of XBU4422 in the shuttle vectors was site specific and orientation specific. Nonmobilizable vectors that had acquired XBU4422 became transmissible and could be transferred to Bacteroides or E. coli recipients. In B. uniformis transconjugants, the XBU4422 insertion in the vectors was usually intact, but XBU4422 was always lost in matings with E. coli, Bacteroides thetaiotaomicron, or B. ovatus. The loss of XBU4422 did not visibly alter the vector; in the case of E. coli, the loss of the insertion appeared to be RecA dependent. Although XBU4422 carried no antibiotic resistances, it shared regions of homology with six conjugal Bacteroides Tcr elements; this homology was strongest with the ends of XBU4422. Using a strain of B. thetaiotaomicron that contains no XBU4422-hybridizing sequences, we showed that the ends of XBU4422 were probably reacting with the ends of the Tcr elements. These results provide the first direct evidence that the Tcr elements, like XBU4422, are integrated in the chromosome and that insertion of the least some Tcr elements, such as TcrEmr DOT, is relatively site specific.     

Transfer of conjugal elements in oral black-pigmented Bacteroides (Prevotella) spp. involves DNA rearrangements.

Conjugal genetic elements in isolates of oral black-pigmented Bacteroides denticola (Prevotella denticola) and B. intermedius (P. intermedia) transfer tetracycline and penicillin resistance in the absence of plasmids. Transverse alternating-field electrophoresis of restricted chromosomal DNAs from transconjugants revealed arrangements indicating that transfer and insertion can involve more than one 60-kb copy of the elements and occurs at strongly preferred sites in the recipient chromosome.     

Inhibition of conjugal transfer of R plasmids by nitrofurans

Nifurzide is a nitrofuran with antibacterial activity. As nitrofurans have been reported to interact with DNA, we tested the ability of nifurzide to inhibit plasmid transfer. Inhibition of plasmid transfer between Escherichia coli strains was obtained for ten plasmids belonging to nine incompatibility groups. The same effect was observed when plasmid RP4 was harboured in six different members of the Enterobacteriaceae. Inhibition depended on the reduction of the -NO2 group of nifurzide and was obtained with four other nitrofuran derivatives.     

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.     

Detection of conjugal transfer systems in oral, black-pigmented Bacteroides spp.

Oral, black-pigmented Bacteroides spp. are important pathogens in oral anaerobic infections and dental disease. We detected conjugation systems in isolates of Bacteroides denticola and Bacteroides intermedius that transferred tetracycline resistance (Tetr) and penicillin resistance to Bacteroides buccae and to Bacteroides fragilis, an intestinal Bacteroides species. A cloned Tetr gene from B. fragilis hybridized to the transferable Tetr locus in the oral strains, indicating that genetic exchange occurs between these two groups of anaerobes.     

Mobilization of the Proteus morganii chromosome by R plasmids

Mutants of Pseudomonas aeruginosa PAC1 which could grow on L-threonine were isolated. These mutants, like the parent strain, synthesized a biosynthetic threonine deaminase, but its apparent Km value for threonine was higher than that of the enzyme from strain PAC1. These mutants also synthesized an inducible NAD-dependent threonine dehydrogenase, which was not present in the parent strain. No threonine aldolase activity could be detected. The results suggest that the threonine deaminase with lowered affinity for L-threonine, together with L-threonine dehydrogenase, enabled these mutants to utilize L-threonine as the sole source of carbon for growth.     

Mobilization of nonconjugative antibiotic resistance plasmids in Haemophilus ducreyi.

A clinical isolate of Haemophilus ducreyi was found to harbor three plasmids: a 23.5-megadalton (Mdal) phenotypically cryptic plasmid, a 7.0-Mdal ampicillin resistance plasmid, and a 4.0-Mdal sulfonamide resistance plasmid. The two smaller plasmids were transferable by conjugation to Haemophilus recipients, but only if the donor cell harbored the 23.5-Mdal plasmid as well, indicating that this large plasmid had mobilizing capabilities. Transfer was also possible to Escherichia coli recipients. Haemophilus influenzae transconjugants which had acquired both the 23.5-Mdal plasmid and one of the R-plasmids could subsequently retransfer the R-plasmid to other Haemophilus recipients at higher frequencies. A derivative of the 23.5 Mdal plasmid was isolated which was shown by restriction endonuclease analysis to contain an ampicillin resistance transposon and to have retained its conjugative ability.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains.

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector.

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.     

Inter- and intraspecies conjugal transfer of different plasmids in bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

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.     

Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation.

Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3, between strains of B. thuringiensis subsp. israelensis were examined. Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h. Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006. However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B. thuringiensis subsp. israelensis. A specific protease-sensitive coaggregation between strains of B. thuringiensis subsp. israelensis was found to be unambiguously correlated with plasmid transfer. Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies. Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution. Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr- that is cells involved in aggregation formation is presented. Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr- cell. The aggregation phenotype was found to be transferred with high frequency (approximately 100%) in broth matings, and the appearance of Agr- isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid. No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.     

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.     

Insertion elements on lactococcal proteinase plasmids.

DNA segments of 809 and 808 nucleotides, with 18-base-pair terminal inverted repeats, are present on the proteinase plasmids pWV05 from Lactococcus lactis subsp. cremoris Wg2 and pSK111 from L. lactis subsp. cremoris SK11, respectively. These DNA segments are highly similar: 77% identical nucleotides and both contain an open reading frame that can encode a protein of 226 amino acids. Furthermore, both DNA segments are located downstream of the proteinase maturation gene prtM, but they differ individually in their orientation with respect to the prtM gene. On the basis of the striking similarity between ISS1, an 808-base-pair insertion sequence (IS) from L. lactis subsp. lactis ML3 lactose plasmid pSK08, and the DNA segments of pWV05 and pSK111, we propose that these DNA segments comprise IS elements. The IS elements from strains Wg2 and SK11 were named ISS1W and ISS1N, respectively. On pWV05, ISS1W is flanked on one side by only part of a second IS element, indicating that pWV05 evolved as a deletion derivative of a precursor plasmid that carried at least two IS elements.     

Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element.

Bacteroides-Escherichia coli shuttle vectors containing a nonmobilizable pBR322 derivative and either pBFTM10 (pDP1, pCG30) or pB8-51 (pEG920) were mobilized by IncP plasmid R751 or pRK231 (an ampicillin-sensitive derivative of RK2) between E. coli strains and from E. coli to Bacteroides recipients. IncI alpha R64 drd-ll transferred these vectors 1,000 times less efficiently than did the IncP plasmids. pDP1, pCG30, and pEG920 could be mobilized from B. uniformis donors to both E. coli and Bacteroides recipients by a conjugative Bacteroides Tcr (Tcr ERL) element which was originally found in a clinical Bacteroides fragilis strain (B. fragilis ERL). However, the shuttle vector pE5-2, which contains pB8-51 cloned in a restriction site that prevents its mobilization by IncP or IncI alpha plasmids, also was not mobilized at detectable frequencies from Bacteroides donors by the Tcr ERL element. The mobilization frequencies of pCG30, pDP1, and pEG920 by the Tcr ERL element in B. uniformis donors to E. coli recipients was about the same as those to isogenic B. uniformis recipients. Transfer of the shuttle vectors from B. uniformis donors to E. coli occurred at the same frequencies when the matings were done aerobically or anaerobically. Growth of the B. uniformis donors in tetracycline (1 microgram/ml) prior to conjugation increased the mobilization frequencies of the vectors to both E. coli and Bacteroides recipients 50 to 100 times.     

Antibiotic resistance patterns and plasmids of ruminal strains of Bacteroides ruminicola and Bacteroides multiacidus

Summary Plasmids were recovered and are described from three ruminal Bacteroides strains — 23, 223/M2/7 and 46/5(2). Although all three were originally isolated as Bacteroides ruminicola, 46/5(2) is shown here to be a strain of Bacteroides multiacidus, a species not previously described from the rumen. An 11.7 kbp plasmid present in strain 46/5(2) gave the same digestion pattern with Sal I and Sma I as a plasmid in B. multiacidus subgroup b type strain P208-58. Strains 46/5(2) and P208-58 both showed resistance to tetracycline, as did B. ruminicola strain 223/M2/7. B. multiacidus P208-58, and, to a lesser degree, B. ruminicola 23, showed resistance to ampicillin. Four other strains of B. ruminicola and one of B. multiacidus in which plasmids were not detected were sensitive to both antibiotics. It has still to be established whether these resistance traits are plasmid or chromosomally coded.     

Insertion and excision of Bacteroides conjugative chromosomal elements.

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.