Plasmids spread very fast in heterogeneous bacterial communities

Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes.     

Conjugative DNA synthesis: R1162 and the question of rolling-circle replication

Strand-replacement synthesis during conjugative mating has been characterized by introducing into donor cells R1162 plasmid DNA containing a base-pair mismatch. Conjugative synthesis in donors occurs in the absence of vegetative plasmid replication, but with a lag between rounds of transfer, and with most strands being initiated at the normal site within the replicative origin. These characteristics argue against the idea that multiple plasmid copies are generated for successive rounds of transfer by rolling-circle replication. However, the R1162 relaxase protein can process molecules containing multiple transfer origins in the manner expected for the conversion of single-strand multimers, generated by rolling-circle replication, to unit-length molecules. This capability appears to be the result of a secondary cleavage reaction carried out by the protein. The possibility is raised that the processing of molecules with more than one origin of transfer might be a repair mechanism directed against adventitious DNA synthesis during transfer.     

Effect of Genomic Location on Horizontal Transfer of a Recombinant Gene Cassette Between Pseudomonas Strains in the Rhizosphere and Spermosphere of Barley Seedlings

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 × 10⁻³ transconjugants/(donors × recipients)^1/2 in the rhizosphere and 4.57 × 10⁻² transconjugants/(donors × recipients)^1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 × 10⁻³ transconjugants/(donors × recipients)^1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Received: 21 July 2000 / Accepted: 21 August 2000     

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.     

Chromosomally integrated conjugative plasmids are common in antibiotic-resistant Haemophilus influenzae.

Twenty-three highly antibiotic-resistant strains of Haemophilus influenzae and two of Haemophilus parainfluenzae without detectable large plasmids were examined for conjugative transfer of their resistance to H. influenzae strain Rd or to other strains. Very inefficient transfer was observed for 18 H. influenzae strains and 1 H. parainfluenzae strain. All H. influenzae transcipients carried a large plasmid, and they were in turn efficient donors of their resistances in standard conjugation crosses with isogenic recipients. This was not seen for the H. parainfluenzae transcipients. It is concluded that most of the original antibiotic-resistant cultures carried an integrated conjugative R plasmid which had been excised in a few cells in each population. It was these cells which transferred resistance in the primary crosses.     

Investigating the impact of bisphosphonates and structurally related compounds on bacteria containing conjugative plasmids

Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in Escherichia coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli cells. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation.     

Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri

Most Vibrio fischeri strains isolated from the Euprymna scolopes light organ carry plasmids, often including both a large (>40kb) plasmid, and one or more small (<12kb) plasmids. The large plasmids share homology with pES100, which is the lone plasmid in V. fischeri type strain ES114. pES100 appears to encode a conjugative system similar to that on plasmid R721. The small plasmids lack extensive similarity to pES100, but they almost always occur in cells that also harbor a large plasmid resembling pES100. We found that many or all of these small plasmids share homology with pES213, a plasmid in strain ES213. We determined the 5501-bp pES213 sequence and generated selectable antibiotic resistance encoding pES213 derivatives, which enabled us to examine replication, retention, and transfer in V. fischeri. An 863-bp fragment of pES213 with features characteristic of theta-type replicons conferred replication without requiring any pES213 open reading frame (ORF). We estimated that pES213 derivatives were maintained at 9.4 copies per genome, which corresponds well with a model of random plasmid segregation to daughter cells and the approximately 10(-4) per generation frequency of plasmid loss. pES213 derivatives mobilized between V. fischeri strains at frequencies up to approximately 10(-4) in culture and in the host, apparently by employing the pES100 conjugative apparatus. pES213 carries two homologs of the putative pES100 origin of transfer (oriT), and V. fischeri strains lacking the pES100 conjugative relaxase, including a relaxase mutant, failed to serve as donors for transmission of pES213 derivatives. In other systems, genes directing conjugative transfer can function in trans to oriT, so it was noteworthy that ORFs adjacent to oriT, VFB51 in pES100 and traYZ in pES213, enhanced transfer 100- to 1000-fold when provided in cis. We also identified and disrupted the V. fischeri recA gene. RecA was not required for stable pES213 replication but surprisingly was required in donors for efficient transfer of pES213 derivatives. These studies provide an explanation for the prevalence and co-occurrence of pES100- and pES213-type plasmids, illuminate novel elements of pES213 mobilization, and provide the foundation for new genetic tools in V. fischeri.     

Vibrio cholerae conjugative plasmid pSJ15 contains transposable prophage dVcA1

Evidence is presented that defective prophage dVcA1 in Vibrio cholerae strain 162 was transposed to the hybrid P::Tn1 plasmid pSJ5. Properties of the resulting conjugative plasmid, pSJ15, indicated that bacteriophage VcA1, like coliphage Mu, can insert at many sites. By analogy with other Hfr-like donors, the high-frequency, polarized chromosomal transfer mediated by plasmid pSJ15 in strain 162 appeared to depend on plasmid integration through the homologous dVcA1 sequences in both replicons. When strain 162(pSJ15) donors were mated to the nonlysogenic El Tor strain RJ1, many potential ampicillin-resistant transconjugants were zygotically induced. However, surviving transconjugants (i) were immune to phage VcA1, (ii) cotransferred immunity and ampicillin resistance to nonlysogenic recipients, and (iii) did not preferentially transfer any chromosomal markers. Recombinant plasmids that transferred wild-type VcA1 prophages were readily isolated from strain RJ1 (VcA1+) lysogens that contained plasmid pSJ15. Physical measurements revealed that plasmid pSJ15 and the recombinant plasmids were about one VcA1 genome (22 to 24 megadaltons) larger than the 51-megadalton pSJ5 plasmid. Similar Hfr-like donors were constructed by introducing plasmid pSJ15 into different strain RJ1 (VcA1+) lysogens. Transfer properties of these donors indicated that the VcA1 prophage was integrated at several sites in the strain RJ1 chromosome.     

Plasmid transfer in Haemophilus influenzae.

Twenty-nine strains of Haemophilus influenzae highly resistant to ampicillin, chloramphenicol, or tetracycline were examined for the presence of plasmids. Agarose gel electrophoresis of ethanol-precipitated cell extracts revealed large plasmids in 11 strains, of which 7 were conjugative. Plasmid transfer by conjugation between isogenic strains was quite efficient, but transfer between different serotypes was nearly always much more inefficient. Type I or II restriction enzymes do not appear to be barriers to this transfer. Encapsulated cells can be both efficient donors and recipients. Small plasmids were seen in three strains, but only two of the three are resistance factors (RSF0885, pUB703). Thus, in 17 isolates antibiotic resistance genes are believed to be located in the bacterial chromosome. Most of these resistances could be transferred by genetic transformation into the widely used Rd strain. In some cases transfer of chromosomal resistance into conjugative plasmids was observed in both rec+ and rec host cells. Since transfer by conjugation seems to be the more efficient process, it is puzzling that in the majority of the 29 isolates studied resistance genes appeared to be in the chromosome.     

Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.     

Induction and loss of Ti plasmid conjugative competence in response to the acyl-homoserine lactone quorum-sensing signal

Conjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of TraR and its signal N-(3-oxo-octanoyl)-L-homoserine lactone. This system is, in turn, controlled by the conjugative opines produced by crown gall tumors induced on plants by the bacteria. Using nonpolar traI mutants, we examined the kinetics of induction of conjugative transfer in response to exogenous acyl-homoserine lactone. In the absence of the antiactivator TraM, onset of induction of transfer requires about 30 min, 15 to 20 min of which is needed for expression and construction of the conjugative apparatus. TraM delays the onset of conjugation by 30 min. While the rate of development of conjugative competence was not significantly affected by levels of TraR, maximum efficiencies of transfer were correlated with amounts of the activator in the donors. Donors harboring Ti plasmids lacking TraM were fully induced by the quormone at concentrations as low as 100 pM. TraM raised the concentration of signal required for maximum activity to 1 nM. Donors grown in batch culture retained conjugative competence following signal removal, even when in stationary phase. However, donors kept in balanced growth rapidly lost transfer ability following signal removal. Loss of transfer was mirrored by a decrease in levels of active TraR. Decreases in TraR activity and conjugative competence could be accounted for by dilution associated with cell division, suggesting that while induction of Ti plasmid conjugation is an active process, the cells lack a mechanism for disassembling the conjugative apparatus when signals become limiting.     

The diversity of conjugative relaxases and its application in plasmid classification

Bacterial conjugation is an efficient and sophisticated mechanism of DNA transfer among bacteria. While mobilizable plasmids only encode a minimal MOB machinery that allows them to be transported by other plasmids, conjugative plasmids encode a complete set of transfer genes (MOB+T4SS). The only essential ingredient of the MOB machinery is the relaxase, the protein that initiates and terminates conjugative DNA processing. In this review we compared the sequences and properties of the relaxase proteins contained in gene sequence databases. Proteins were arranged in families and phylogenetic trees constructed from the family alignments. This allowed the classification of conjugative transfer systems in six MOB families: MOB_F, MOB_H, MOB_Q, MOB_C, MOB_P and MOB_V . The main characteristics of each family were reviewed. The phylogenetic relationships of the coupling proteins were also analysed and resulted in phylogenies congruent to those of the cognate relaxases. We propose that the sequences of plasmid relaxases can be used for plasmid classification. We hope our effort will provide researchers with a useful tool for further mining and analysing the plasmid universe both experimentally and in silico .     

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.     

Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron

The Ll.LtrB group II intron from the low-G+C gram-positive bacterium Lactococcus lactis was the first bacterial group II intron shown to splice and mobilize in vivo. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids.     

Broad host range gene transfer: plasmids and conjugative transposons

Abstract Conjugation is the primary route of broad host range DNA transfer between different genera of bacteria. Plasmids are the most familiar conjugative elements, but there are also self-transmissible integrated elements called conjugative transposons. Conjugative transposons have been found in many genera of gram-positive bacteria, in mycoplasmas and in gram negative bacteria such as Bacteriodes spp. and Moraxella spp., and they have a very broad host range. The best-studied conjugative transposons are: the ones related to Tn 916 , a 16 kb conjugative transposon found originally in Gram-positive bacteria; Tn 5276 , a 70 kb conjugative transposon from Lactococcus lactis ; and a group of large (> 70 kb) conjugative transposons found in Bacteroides spp. Transfer of conjugative transposons takes place in three steps: excision to form a circular intermediate, transfer of one strand of the circular intermediate to a recipient, and integration into the recipient genome. Some conjugative transposons integrate almost randomly, whereas other integrate site-specifically. Conjugative transposons not only transfer themselves but also mobilize co-resident plasmids, either by providing transfer functions in trans or by inserting themselves into the plasmid. In addition, the conjugative transposons found in Bacteroides spp. can excise and mobilize unlinked integrated elements, called NBUs. Transfer of many of the Bacteroides conjugative transposons is regulated by tetracycline, whereas transfer of Tn 916 and other conjugative transposons appears to be constitutive. The conjugative transposons are clearly widespread in clinical isolates, but their distribution in environmental isolates remains to be determined.     

The BlcC (AttM) Lactonase of Agrobacterium tumefaciens Does Not Quench the Quorum-Sensing System That Regulates Ti Plasmid Conjugative Transfer

The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil.

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.