A Conjugative Transfer System for the Rumen Bacterium, Butyrivibrio fibrisolvens, Based on Tn916-Mediated Transfer of the Staphylococcus aureus Plasmid pUB110

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.     

Analysis of the Requirement for a pUB110 mob Region during Tn916-Dependent Mobilization

Tn916-dependent mobilization of nonconjugative plasmids pUB110 and its derivative pUB110Deltam was compared. Deleting a 787-bp fragment from the pUB110 mob region created plasmid pUB110Deltam. Deletion of the mob region of pUB110 rendered the plasmid nontransferable by the conjugative plasmids of Bacillus thuringiensis subsp. israelensis. During matings between Bacillus subtilis (Tn916) and B. thuringiensis subsp. israelensis, however, Tn916-dependent mobilization of plasmids pUB110 and pUB110Deltam was observed at a frequency of approximately 2 x 10(-6) transconjugants per donor. The results show that Tn916-mediated conjugal transfer of plasmids is a mob-independent event. Jaworski and Clewell (J. Bacteriol 177; 6644-6651) recently demonstrated the presence of an IncP-like nicking site in the oriT of Tn916. These data suggest that a IncP-like nickling site is essential for Tn916-mediated plasmid transfer.     

The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis

Summary The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon, Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.Deceased, 7/89. O. Torres and R. Korman contributed equally to this work     

Tn916-dependent conjugal transfer of PC194 and PUB110 from Bacillus subtilis into Bacillus thuringiensis subsp. israelensis

The Staphylococcus aureus plasmids pC194 and pUB110 were introduced into Bacillus thuringiensis subsp. israelensis by using the Streptococcus faecalis transposon Tn916 as a mobilizing agent. Plasmid transfer occurred only when B. thuringiensis subsp. israelensis was mated with a B. subtilis donor that contained both pC194 and pUB110 and Tn916; plasmid transfer was not observed in the absence of the transposon. B. thuringiensis transconjugants resistant to chloramphenicol (Cmr) and tetracycline (Tetr) were detected at a frequency of 1.96 x 10(-6) per recipient cell, whereas the Tetr phenotype, but not the Cmr, was observed at a frequency of 1.09 x 10(-4). The converse, Cmr but not Tetr, was observed at a frequency of 2.94 X 10(-5). The transfer of pUB110 from B. subtilis to B. thuringiensis subsp. israelensis was observed at a frequency of 3.0 x 10(-6) per recipient cell but concomitant transfer of pUB110 and Tn916 was not observed. Mobilization of plasmid pE194 was not observed under these conditions. Transconjugants were detected in filter matings only, not in broth. The Tn916 phenotype was maintained during serial passage of B. thuringiensis without selection, whereas the pC194 phenotype was not. Unlike pC194, however, pUB110 remained stable in B. thuringiensis during several passages through nonselective medium. Southern hybridization analysis demonstrated that Tn916 had inserted into several different sites on the B. thuringiensis chromosome and that pC194 and pUB110 were maintained as an autonomous plasmid.     

Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum

The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred at high frequency (10^-2–10^-3 per recipient, selecting for tetracycline resistance) to the Gram-positive anaerobe Clostridium acetobutylicum. Selection in the presence of two antibiotics (tetracycline and erythromycin) with the plasmids pAM 180 and pINY1275 yielded only low numbers of transconjugants (10^-8 per recipient). Matings were done by combining liquid and filter mating procedures under anaerobic conditions. No plasmid DNA could be detected in the transconjugants selected on a minimal medium in the presence of tetracycline. DNA-DNA hybridization experiments with restricted chromosomal DNA using biotinylated pAM120::Tn916 as probe revealed the presence of homologous sequences in the transconjugants but not in Clostridium acetobutylicum wild type. The transconjugants were used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec. diacetylactis. In both cases tetracycline-resistant strains were found. Transfer frequencies in these experiments were less than 10^-7 per recipient.     

Conjugal transfer of enterococcal transposons in Bacillus megaterium

The conjugative enterococcal transposons Tn916 and Tn919 were introduced into Bacillus megaterium by a filtermating technique. The transfer frequencies obtained ranged from 1.3×10^-6 to 6.6×10^-7. The transposons integrated stably into the B. megaterium chromosome. Tn916 could generate auxotrophs and was transferred from B. megaterium Tn916 transconjugants to other species.     

Introduction of the Streptococcus faecalis transposon Tn916 into Bacillus thuringiensis subsp. israelensis

The conjugative Streptococcus faecalis transposon Tn916 was introduced into Bacillus thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline (Tetr) were detected at a frequency of approximately 7.0 X 10(-7) per recipient cell during filter matings, whereas transfer of Tn916 was not observed in broth matings. The Tetr phenotype in subsp. israelensis was stable in the absence of antibiotic selection. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome but insertion into plasmid DNA was not observed. Movement of Tn916 was demonstrated when Tetr B. thuringiensis transconjugants were mated with isogenic recipients. Southern hybridizations, however, showed that the resulting Tetr isolates contained Tn916 junction fragments that were nearly identical to the donor, suggesting that this movement resulted from transfer of chromosomal DNA from donor to recipient or from a fusion of mating cells, rather than conjugative transposition of the Tn element.     

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10^–9 to 9.8×10^–5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10^–9 to 3.7×10^–7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.     

Development of High-Frequency Delivery System for Transposon Tn919 in Lactic Streptococci: Random Insertion in Streptococcus lactis subsp. diacetylactis 18-16

The conjugative transposon Tn919, originally isolated in Streptococcus sanguis FC1, is capable of low-frequency transfer (10⁻⁷ and 10⁻⁸ per recipient) on membrane filters to a wide number of streptococcal recipients including the industrially important lactic streptococci. The introduction of pMG600 (Lac⁺ Lax⁻; a lactose plasmid capable of conjugative transfer at high frequencies and which, in certain hosts, confers an unusual clumping phenotype) into a Streptococcus lactis CH919 donor, generating S. lactis CH001, resulted in a significant improvement in the transfer frequency of Tn919 to S. lactis CK50 (1.25 × 10⁻⁴ per recipient). In addition, these matings could be performed on agar surfaces, allowing the recovery of a greater number of recipients than with filter matings. Tn919 also transferred at high frequency to S. lactis subsp. diacetylactis 18-16S but not to Streptococcus cremoris strains. Insertion in 18-16S transconjugants generated from filter matings with an S. lactis CH919 donor was random, occurring at different sites on the chromosome and also in plasmid DNA. Thus, the conditions necessary for the practical exploitation of Tn919 in the targeting and cloning of genes from a member of the lactic streptococci, namely, high-frequency delivery and random insertion in host DNA, were achieved.     

Multiple copies of functional, Tet(M)-encoding Tn916-like elements in a clinical Enterococcus faecium isolate

Tn916 and similar elements are very common in clinical enterococcal isolates, and are responsible for transmission of a variety of resistance determinants. It is commonly assumed that clinical strains carrying Tn916 have a single copy, although the actual number of copies in clinical isolates has never been systematically studied. We report a clinical isolate of Enterococcus faecium in which three distinct and excision-proficient copies of Tn916-like elements are present in the genome. All of the elements contain tet(M) genes, at least one of which confers resistance to tetracycline and minocycline. Two elements (Tn6085a, Tn6085b) are indistinguishable, containing an inserted 2758 bp Group II intron at the start of open reading frame Tn916ORF_06. The third (Tn6084) also contains the intron, but also has an ISEfa11 integrated upstream of tet(M). All three copies are able to excise from plasmid vectors when cloned in E. coli, and at least two of the elements can transfer to an E. faecium recipient strain. These data indicate that nearly identical Tn916-like elements encoding Tet(M)-mediated tetracycline/minocycline resistance can coexist in clinical E. faecium isolates.     

Insertion of Tn916 into Bacillus pumilus plasmid pMGD302 and evidence for plasmid transfer by conjugation.

As part of an effort to develop systems for genetic analysis of strains of Bacillus pumilus which are being used as a microbial hay preservative, we introduced the conjugative Enterococcus faecalis transposon Tn916 into B. pumilus ATCC 1 and two naturally occurring hay isolates of B. pumilus. B. pumilus transconjugants resistant to tetracycline were detected at a frequency of approximately 6.5 x 10(-7) per recipient after filter mating with E. faecalis CG110. Southern hybridization confirmed the insertion of Tn916 into several different sites in the B. pumilus chromosome. Transfer of Tn916 also was observed between strains of B. pumilus in filter matings, and one donor strain transferred tetracycline resistance to recipients in broth matings at high frequency (up to 3.4 x 10(-5) per recipient). Transfer from this donor strain in broth matings was DNase-resistant and was not mediated by culture filtrates. Transconjugants from these broth matings contained derivatives of a cryptic plasmid (pMGD302, approx 60 kb) from the donor strain with Tn916 inserted at various sites. The plasmids containing Tn916 insertions transferred to a B. pumilus recipient strain at frequencies of approx 5 x 10(-6) per recipient. This evidence suggests that pMGD302 can transfer by a process resembling conjugation between strains of B. pumilus.     

Marking of a Sym plasmid of Rhizobium with Tn7

Summary Transposon Tn7 was inserted into wide host range plasmid pSUP202 and used as a suicide plasmid vehicle for transposon mutagenesis in Rhizobium leguminosarum. Tn7 is transposed with high frequency into the self-transmissible plasmid pJB5JI without affecting the transfer, nodulation and nitrogen fixation functions. Tn7 transposition provides a useful tool for marking symbiotic plasmids.     

Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus

Broad host range conjugating transposon Tn916 has been introduced into the extreme thermophile Thermus by transposon transformation and transposition into the Bacillus subtilis chromosome followed by broth mating with Thermus aquaticus ATCC27634. Tetracycline resistant Thermus transconjugants were obtained at a frequency of 1.4 X 10(-7) per donor and 1.2 X 10(-7) per recipient. Transposon transfer from Thermus to Bacillus subtilis was also demonstrated in similar broth matings. Transfer characteristics were consistent with the conjugation mechanism described for Tn916 in mesophiles.     

Plasmid Transfer between Bacillus thuringiensis subsp. israelensis Strains in Laboratory Culture, River Water, and Dipteran Larvae

Plasmid transfer between strains of Bacillus thuringiensis subsp. israelensis was studied under a range of environmentally relevant laboratory conditions in vitro, in river water, and in mosquito larvae. Mobilization of pBC16 was detected in vitro at a range of temperatures, pH values, and available water conditions, and the maximum transfer ratio was 10⁻³ transconjugant per recipient under optimal conditions. Transfer of conjugative plasmid pXO16Tn5401 was also detected under this range of conditions. However, a maximum transfer ratio of 1.0 transconjugant per recipient was attained, and every recipient became a transconjugant. In river water, transfer of pBC16 was not detected, probably as a result of the low transfer frequency for this plasmid and the formation of spores by the introduced donor and recipient strains. In contrast, transfer of plasmid pXO16Tn5401 was detected in water, but at a lower transfer ratio (ca. 10⁻² transconjugant per donor). The number of transconjugants increased over the first 7 days, probably as a result of new transfer events between cells, since growth of both donor and recipient cells in water was not detected. Mobilization of pBC16 was not detected in killed mosquito larvae, but transfer of plasmid pXO16::Tn5401 was evident, with a maximum rate of 10⁻³ transconjugant per donor. The reduced transfer rate in insects compared to broth cultures may be accounted for by competition from the background bacterial population present in the mosquito gut and diet or by the maintenance of a large population of B. thuringiensis spores in the insects.     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

Behaviour of the transposons Tn5 and Tn7 in Xanthomonas campestris pv. campestris

Summary Xanthomonas campestris pv. campestris was tested for its ability to maintain various plasmids after they had been transferred by conjugation from Escherichia coli donors. Broad host-range plasmids belonging to incompatibility groups P and Q could be maintained but X. campestris was unable to support replication of narrow host-range ColE1, pACYC184 and pBR325 replicons. Delivery systems based on E. coli donors of suicide plasmids and on X. campestris Hfrs were used to introduce Tn7 and Tn5 into X. campestris. Tn7 insertions were recovered at high frequency while Tn5 transposed at low frequency. Three auxotrophic Tn5 insertions were isolated but transposition of Tn7 into the X. campestris genome did not generate any auxotrophs. DNA hybridization analysis showed that Tn7 had inserted into the same hot spot(s) in all cases tested.     

Characteristics of Tn5307 exchange and intergeneric transfer of genes associated with nisin production

Transfer of the Lactococcus lactis 11454 nisin-sucrose conjugative transposon, Tn5307, was investigated to develop a methodology for conjugation of this element to other lactic acid bacteria. Tn5307 exchange was sensitive to temperature and pH but was not affected by protease or amylase treatments to donor cells. Moreover, conjugation studies demonstrated that the direct-plate method could be employed to rapidly identify LM2301 transconjugants able to transfer Tn5307 at least ten times more efficiently than 11454. Intergeneric transfer of nisin and sucrose genes between L. lactis and a dairy Enterococcus sp. was also investigated. Erythromycin-resistant Enterococcus sp. recipients were developed by electro-transformation with pGK13 or by conjugal introduction of the broad-host-range plasmid pAM\1. Matings between L. lactis 11454 and an Enterococcus sp. recipient that contained pAM\1 yielded sucrose-positive, nisin-immune transconjugants at a frequency of 2.3 × 10^–9 transconjugants per donor cfu. Agar-overlay assays for nisin production revealed that enterococcal transconjugants did not produce the bacteriocin, but DNA·DNA hybridization with a nisA-specific probe demonstrated that these bacteria had acquired the nisin structural gene.     

Replicon fusion mediated by a single-ended derivative of transposon Tn1721

Summary Tn17221K, a derivative of transposon Tn1721 lacking one terminal inverted repeat (IR) and conferring kanamycin resistance, promotes transposition of the resistance marker to a target replicon at about 100-fold lower frequency than the wild-type element. A study involving restriction analysis of 16 independent Tn17221K-mediated events led to the following results: (i) Tn17221K mediates fusions of the donor (pRU506) and target (RSF1010) replicons; the fused entities are non-permuted. (ii) Tn17221K promotes insertions of donor DNA at many different sites in the target replicon. (iii) The analyzed fusion plasmids contain the entire target and various lengths of donor DNA. Eleven products contain the entire donor plasmid plus a duplication of the IR (class A), whereas five products contain only portions adjacent to the single IR (class B). (iv) In each case the two replicons are joined at (or very close to) the single IR. The second junction is located shortly beyond the duplicated IR in class A and at different sites within the donor plasmid in class B. These results are interpreted in terms of asymmetric replicative transposition.