Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola

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

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola.

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

Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required.

Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient. Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer. Two distinct models have been generally considered for the mechanism of retrotransfer. In the two-way conduction model, no transfer of the conjugative plasmid is required. The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions. In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor. We have tested these models by the construction of a mutant of the self-transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer. Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E. K.Ayres, V. J. Thomson, G. Merino, D. Balderes, and D. H. Figurski, J. Mol. Biol. 230:174-185, 1993). The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type. Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer. Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient. The results prove that retrotransfer occurs by two sequential DNA transfer events.     

Effect of growth phase and parental cell survival in river water on plasmid transfer between Escherichia coli strains.

We evaluated the transfer to and from Escherichia coli of endogenously isolated plasmid material from the River Butrón during the growth of three donor strains and two recipient strains as well as after the survival of these parental cells in river water. Transfer frequency varied greatly during the growth of donor cells, with minimum values in the exponential phase; frequency remained constant, however, during the growth of recipient strains. After survival in river water, donor cells lost their ability for plasmid transfer before any other physiological variations in the cells caused by environmental stress were detected. Under the same conditions and during equal periods, however, no variation in the ability of recipient cells to receive and express plasmid material was observed.     

Conjugative transfer of tetracycline resistance in rumen streptococcal strains

In 11% of testedStreptococcus bovis strains a conjugative transfer of tetracycline resistance was observed when mating experiments were carried out on membrane filters. The recipient strain used wasS. bovis BM114 with chromosomal resistance to rifampicin. In addition, in two strains tetracycline resistance was transferred also to recipient strainEnterococcus faecium AL6. The transfer frequencies were in the range of 10⁻⁶ to 10⁻³. The donor strains were screened for the presence of plasmids and one up to four bands of plasmid DNA in all tested strains were revealed. In spite of that isolation of plasmid DNA was successful only in 53/4/114 transconjugants. Transconjugant 32/114 contained amylase activity which was higher than in the donor strain.     

Plasmid DNA transfer in Mycobacterium smegmatis involves novel DNA rearrangements in the recipient, which can be exploited for molecular genetic studies

The establishment of molecular genetic techniques is essential for development of new treatments for mycobacterial infections. To this end, we recently described a novel DNA transfer process that occurs in the model mycobacterial organism Mycobacterium smegmatis. This transfer system is most like conjugal DNA transfer in that it requires two viable parents, is DNAse resistant and occurs between distinct donor and recipient strains. Cis-acting sequences called bom, which confer transferability, are distinct from the prototypical oriT sites of conjugative plasmids, as they occur at multiple locations in the chromosome and require RecA in the recipient to mediate plasmid recircularization. Here, we show that a plasmid containing two of these bom regions can undergo several fates in the recipient cell, each of which require recipient recombination functions. The products of plasmid transfer that we observed provide further insights toward a model for DNA transfer. Furthermore, we have taken advantage of the recombination events that occur in the recipient to develop simple procedures for capturing, or replacing specific segments of the recipient chromosome. To demonstrate the potential of the system, we describe the capture and deletion of 25 kb of the M. smegmatis chromosome, and targeted-allele exchange of the recipient recB and recD genes. Using these transfer-mediated rearrangements, we demonstrate that homology with the recipient chromosome and RecB, but not RecD, are essential for DNA transfer.     

Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid.

The Pseudomonas putida TOL plasmid pWW0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). Transconjugants are recipient cells that have received DNA from donor cells, whereas retrotransconjugants are donor bacteria that have received DNA from a recipient. The TOL plasmid pWW0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrated into the chromosome of other P. putida strains, a process that appears to involve a single conjugational event. The rate of retrotransfer (as well as of direct transfer) of the chromosomal marker is influenced by the location of the kanamycin marker on the chromosome and ranges from 10(-3) to less than 10(-8) retrotransconjugants per donor (transconjugants per recipient). The mobilized DNA is incorporated into the chromosome of the retrotransconjugants (transconjugants) in a process that seems to occur through recombination of highly homologous flanking regions. No interspecific mobilization of the chromosomal marker in matings involving P. putida and the closely related Pseudomonas fluorescens, which belongs to rRNA group I, was observed.     

Hfr-mediated conjugative transfer of pBR322 vector carrying the chromosomal DNA of Escherichia coli

Hybrid plasmids consisting of a non-mobilized plasmid, pBR322, and a segment of chromosomal DNA of Escherichia coli could be transferred from an Hfr donor to recipient cells by a bacterial mating. When the chromosomal DNA in the plasmid corresponded to the early transfer region of the Hfr, the frequency of the transfer was high. The recA function of both donor and recipient cells was required in the transfer. The physical association of the hybrid plasmid with the transferring Hfr chromosome through the homologous sequences may mediate the transfer of the non-mobilized plasmid. This phenomenon is useful for the determination of the chromosomal location of an unidentified fragment cloned in a non-mobilized plasmid.     

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.     

Mobilization of phase I plasmid in Shigella sonnei and stability of its inheritance in rough strains of Shigella and Escherichia

The plasmid pSS120, determining the synthesis of species specific I phase antigen of Shigella sonnei is mobilized for genetic transfer into E. coli K12 recipient cells with the frequency 12-41%. The frequency depends on the type of mobilized plasmid and recipient strain. The I phase antigen is normally expressed in II phase recipient cells and in E. coli cells. During mobilization pSS120 forms cointegrates representing a recombinant of mobilizing and mobilized plasmids DNA. The study of pSS120 inheritance stability has shown the plasmid to be unstable during culturing of bacteria and to be partially lost from the parent Shigella sonnei strains as well as from the "hybrid" transconjugants obtained. The 60 Md plasmid present in the donor strains of Shigella sonnei is prone to structural fragmentation particularly expressed in Shigella sonnei/E. coli hybrids.     

Transferable lincosamide-macrolide resistance in Bacteroides.

Inter- and intraspecies transfer of resistance to clindamycin, lincomycin, and erythromycin in the strict anaerobe, Bacteroides, is described. This lincosamide-macrolide resistance was found to be specified by a 27 × 10⁶-dalton plasmid, designated pBF4, originally identified in a clinical Bacteroides fragilis isolate. Transfer of this plasmid to a strain of Bacteroides uniformis was demonstrated to occur by a deoxyribonuclease insensitive process which required cell-to-cell contact. Chloroform sterilized donor cell supernatants or filtrates of donor cells did not mediate resistance transfer. Transfer of the antibiotic resistance and pBF4 plasmid deoxyribonucleic acid (DNA) were always coincident. Drug resistant progeny recovered from such matings were able to transfer the pBF4 plasmid and its associated resistance markers to a suitable B.fragilis recipient strain. Compared to interspecies matings, resistance transfer was 100- to 1000-fold greater between isogenic donor and recipient strains, suggesting the possibility of a host controlled restriction-modification system.     

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.     

Genetic functions and cell–cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis

Pheromone-inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1-5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell-cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.     

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 pXO16::Tn5401 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 pXO16::Tn5401 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.     

Efficiency of the transfer of a pSAM2-derivative plasmid between two strains of Streptomyces lividans in conditions ranging from agar slants to non-sterile soil microcosms

Abstract: The aim of this work was to determine the efficiency of the conjugative plasmid pTS130 to transfer in various environmental conditions between two strains of Streptomyces lividans . This plasmid is a derivative of the conjugative and integrative plasmid pSAM2 isolated originally from Streptomyces ambofaciens and capable of transfer to a large range of bacteria. Our results demonstrate the high frequency of the conjugation mechanism since more than 60% of the recipient cells developed on agar slants harbored the plasmid pTS130 (as evidenced by Southern hybridization with a pSAM2 derivative plasmid probe). When donor and recipient strains were inoculated into sterile and non-sterile soil microcosms, transconjugants were detected after two days of incubation in both cases. However, the number of donor, recipient and transconjugant cells were established at a lower level in the non-sterile soil than in the sterile soil experiments. Moreover, nutrient amendment of the sterile soil was found to increase the population levels of parental strains and transfer frequencies both significantly and simultaneously. On the other hand, modifying water potential of the soil microcosms did not result in affecting the establishment of the Streptomyces lividans cells or the transfer rate.     

Bacterial conjugation between pseudomonads in the rhizosphere of wheat

Abstract Transfer of plasmid RP4 between introduced pseudomonads was studied in rhizosphere and non-rhizosphere soil of wheat, in soil chambers and in culture tubes. In both experiments, the presence of growing wheat roots stimulated the occurrence of plasmid transfers in the soil. The plasmid transfer frequencies in rhizosphere soil in the soil chambers were consistently higher than those in rhizosphere soil in the culture tubes, indicating an influence of the experimental set-up.
In the soil chambers, both the survival of introduced donor and recipient strains and the plasmid transfer frequencies decreased drastically at increasing distances from the roots. In addition, plasmid transfer frequencies were influenced by the inoculum densities of both donor and recipient strains; higher frequencies were observed in soil that was initially inoculated with higher cell numbers.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Stimulation in dnaB(ts) Donors by Minicells

R64-11(+) donor cells that are thermosensitive for vegetative DNA replication will synthesize DNA at the restrictive temperature when recipient minicells are present. This is conjugal DNA replication because it is R64-11 DNA that is being synthesized and there is no DNA synthesis if minicells that cannot be recipients of R64-11 DNA are used. The plasmid DNA present in the donor cells before mating is transferred to recipient minicells within the first 20 min of mating, but additional copies of plasmid DNA synthesized during the mating continue to be transferred for at least 90 min. However, the transfer of R64-11 DNA to minicells is not continuous because the plasmid DNA in minicells is the size of one R64-11 molecule or smaller, and there are delays between the rounds of plasmid transfer. DNA is synthesized in minicells during conjugation, but this DNA has a molecular weight much smaller than that of R64-11. Thus, recipient minicells are defective and are not able to complete the synthesis of a DNA strand complementary to the single-stranded R64-11 DNA received from the donor cell.     

Plasmid dynamics in a model soil column

Continuous-flow column reactors were used to study the dynamics of plasmid exchange in a structured, thermodynamically open system containing either Enterobacter cloacae or Pseudomonas cepacia , both carrying the transmissible recombinant plasmid R388::Tn1721. Plasmid transfer rates were higher in vermiculite and sterile soil columns supplied with nutrient solution than those in sterile and non-sterile soil columns without input of nutrient solution. For both species, donor and recipient strains took about 5 days to reach their maximum densities in effluents from the columns supplied with nutrient solution. After about 8 day s the donor and transconjugant populations of P. cepacia in the effluent solution decreased exponentially, whereas E. cloacae donor, recipient and transconjugant strains maintained steady-state concentrations. The difference between plasmid stability in the two species may have significant consequences in terms of releasing plasmid-bearing genetically modified microorganisms into the natural environment. The plasmid is persistent in E. cloacae in non-sterile soil even though its transfer to the marked recipient in non-sterile soil was minimal.     

Conjugation mapping of Pseudomonas mendocina bacteria

Donor strains of the Hfr type were isolated using plasmid pRK2013 with transposons Tn10 and Tn5 as a chromosome-mobilizing factor. The isolated strains were shown to promote transfer of donor chromosome from different origins in different directions during isogenic matings of Pseudomonas mendocina bacteria. The created collection of donors and polyauxotrophic recipient bacteria permitted mapping 26 genetic determinants on the bacterial chromosome and identifying the genome of these microorganisms as a circular DNA molecule.     

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 (p19 pUB110) 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.