Donor/recipient interactions affecting plasmid transfer amongStreptomyces species: A conjugative plasmid will mobilize nontransferable plasmids in soil

Streptomyces parvulus was used as the recipient for plasmid pIJ303 and pIJ211, two conjugative plasmids derived from the self-transmissible plasmid pIJ101. One of the resulting transconjugantS. parvulus strains containing plasmid pIJ303 was used withS. lividans to evaluate the effects of the host strain on the frequency of pIJ303 transfer betweenStreptomyces species. Only 30% ofS. parvulus cells acquired plasmid pIJ303 in crosses in whichS. lividans was the donor, whereas 100% ofS. lividans cells acquired the plasmid whenS. parvulus was the donor. This indicates that the frequency of transfer of the conjugative plasmid was determined by the recipient. The other resulting transconjugantS. parvulus strain containing plasmid pIJ211 was evaluated for its ability to mobilize the nonconjugative plasmid pIJ702 fromS. lividans, on agar and in sterile soil. AfterS. lividans containing pIJ702 was crossed on agar and in sterile soil withS. parvulus containing pIJ211, recombinantS. parvulus colonies carrying pIJ702 and expressing pigments characteristic of both species were recovered, from both agar and soil. Although a large percentage ofS. parvulus transconjugants lost pIJ211 during incubation in soil, the mobilization of pIJ702 fromS. lividans intoS. parvulus still occurred. Plasmid integration into the chromosome of the donor and the transconjugant was evaluated by Southern blot hybridization. Hybridization of plasmid pIJ303, with chromosomal DNA fromS. lividans andS. parvulus transconjugants, using biotinylated DNA, indicated that no integration had occurred. Genetic exchange betweenStreptomyces species also occurred in a liquid medium. The finding of plasmid mobilization in soil is significant. It demonstrates that genetic exchange in the environment can occur between released genetically engineeredStreptomyces species and nativeStreptomyces species that contain conjugative plasmids.Paper of the Idaho Agricultural Experiment Station.     

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.     

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.Subject terms: Antibiotics, Microbial ecology, Phylogenomics     

Growth and Survival of Streptomycete Inoculants and Extent of Plasmid Transfer in Sterile and Nonsterile Soil

The growth and survival of strains of Streptomyces lividans and S. violaceolatus in sterile and nonsterile soil was investigated by using inoculated soil microcosms run as batch systems. It was evident that, after an initial short mycelial growth phase of 2 to 3 days, sporulation occurred and inoculants survived as spores. The transfer of a high-copy-number, self-transmissible plasmid, pIJ673, was detected by using intra- and interspecific crosses. The initial detection of transconjugants correlated with the development of the mycelial state of the inoculants (as confirmed by scanning electron microscopy) after 2 days of incubation. Subsequent spread of the plasmid was attributed to spread within existing mycelium followed by sporulation. In natural soil, inoculant numbers remained constant or declined, but plasmid transfer was readily detected.     

Interspecific Transfer of Streptomyces Giant Linear Plasmids in Sterile Amended Soil Microcosms

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30°C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 × 10⁻⁴ and 3.6 × 10⁻⁵ CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.     

Transfer of the Pheromone-Inducible Plasmid pCF10 among Enterococcus faecalis Microorganisms Colonizing the Intestine of Mini-Pigs

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10⁶ and 10⁷ CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10⁶ CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10³ and 10⁴ CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.     

Establishment of New Genetic Traits in a Microbial Biofilm Community

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacI^q gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.     

Transfer of the Pea Symbiotic Plasmid pJB5JI in Nonsterile Soil

Transfer of the pea (Pisum sativum L.) symbiotic plasmid pJB5JI between strains of rhizobia was examined in sterile and nonsterile silt loam soil. Sinorhizobium fredii USDA 201 and HH003 were used as plasmid donors, and symbiotic plasmid-cured Rhizobium leguminosarum 6015 was used as the recipient. The plasmid was carried but not expressed in S. fredii strains, whereas transfer of the plasmid to R. leguminosarum 6015 rendered the recipient capable of nodulating pea plants. Confirmation of plasmid transfer was obtained by acquisition of plasmid-encoded antibiotic resistance genes, nodulation of pea plants, and plasmid profiles. Plasmid transfer in nonsterile soil occurred at frequencies of up to 10⁻⁴ per recipient and appeared to be highest at soil temperatures and soil moisture levels optimal for rhizobial growth. Conjugation frequencies were usually higher in sterile soil than in nonsterile soil. In nonsterile soil, transconjugants were recovered only with strain USDA 201 as the plasmid donor. Increasing the inoculum levels of donor and recipient strains up to 10⁹ cells g of soil⁻¹ increased the number of transconjugants; peak plasmid transfer frequencies, however, were found at the lower inoculum level of 10⁷ cells g of soil⁻¹. Plasmid transfer frequencies were raised in the presence of the pea rhizosphere or by additions of plant material. Transconjugants formed by the USDA 201(pJB5JI) × 6015 mating in soil formed effective nodules on peas.     

Analysis of plasmid pMZ1 from Micromonospora zionensis

Plasmid pMZ1, isolated from Micromonospora zionensis, was also able to replicate by the rolling circle mechanism in Micromonospora melanosporea and Streptomyces lividans. Southern hybridisation experiments with probe prepared from pMZ1 and immobilised M. zionensis DNA fragments separated on pulsed-field gel electrophoresis, indicated that the plasmid is present in the progenitor strain in both integrated and autonomous states. Thiostrepton resistant derivatives of pMZ1 plasmid, pMZS25 and pMZS34, were used to study conjugal transfer in M. melanosporea and S. lividans. A 3.4 kb NcoI-MluI fragment from pMZ1 cloned in pIJ702 (plasmid pIJNM3) was shown to be sufficient to promote plasmid transfer and pock formation in S. lividans.     

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.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPα plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 ± 0.2) × 10⁻² S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPα plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPα plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained. The text was submitted by the authors in English.     

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.     

Effect of Temperature on the Activities of Cytochrome c Oxidase and Respiration in Cassava Root Mitochondria

Cosmid pR4Cl is a derivative of multicopy plasmid pIJ365 which has an insertion of the cos (cohesive end site) region of actinophage R4 [T. Morino, H. Takahashi and H. Saito, Mol. Gen. Genet., 198, 228 (1985)]. When the donor R4 phage was propagated in S. lividans carrying the plasmid, the phage lysate contained transducing particles which encapsulated head-to-tail concatemers of the plasmid DNA. These particles could mediate transfer of the plasmid at a high frequency. We examined conditions that gave a maximum transduction frequency of cosmid pR4Cl. Conditions which depress R4 phage propagation, such as incubation of recipient S. parvulus at a high temperature, improved the frequency. Obviously such conditions minimized the lethal effect of viable phage propogation. The highest transduction frequency obtained so far was around 3 × 10^-3 transductants per infected phage when S. lividans was used as the recipient. This was about 30 per cent of the cosmid transducing particles estimated from the cosmid DNA content in the transducing lysate. The significance of cosmid transduction for gene manipulation in Streptomyces strains is also discussed.     

In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities

Gene transfer of the conjugative plasmid pBF1 from Pseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385–390, 1998). pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacI^q). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp. Transfer to recipient strains lacking the repressor results in expression of gfp. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of the gfp gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from P. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 × 10⁻⁶ to 2.2 × 10⁻⁴ transconjugants per recipient were recorded after 3 days of incubation.     

Estimating the rate of plasmid transfer: an end-point method

We describe a method for determining the rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities and the growth rate in exponential phase of the mating culture. The formula for estimating the plasmid transfer rate, gamma, was derived from a mathematical model describing cell growth and plasmid transfer in batch culture. Computer simulations were used to explore the sensitivity of this method to the realities of bacterial life, such as growth rate differences, plasmid segregation and transitory derepression of pilus synthesis. As predicted by the theory, mating experiments with the plasmid R1 in Escherichia coli K12 demonstrated that the estimate gamma is unaffected by cell density, donor:recipient ratio and mating time. Unlike previous techniques, our method allows us to investigate the effect of environmental factors on plasmid transfer rates when these factors also influence population growth rates. To illustrate this, we examined the effect of temperature on the rate of plasmid transfer.     

Survival of,and plasmid transfer between modified Enterobacter cloacae strains under long-term starvation conditions in a continuous-flow system

Continuous-flow, packed-bed column reactors, which provide an experimental model of a soil profile, were used to investigate survival of, and plasmid transfer between, strains of Enterobacter cloacae. When columns, inoculated with nutrient-sufficient donor and recipient strains, were provided with a minimal salts medium with no added carbon source, transconjugant cells appeared in their effluents. During the first few days of such experiments, the concentration of cells in the effluent declined but then the donor population stabilized, while the recipient and transconjugant populations continued to decrease. The results indicate that the amount of nutrient required to maintain and transfer plasmids is very low. No transconjugants were observed in the effluent from columns inoculated with pre-starved donor and recipient strains.     

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere.

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.     

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

pBLI is a conjugative linear extrachromosomal element of 43 kb previously isolated after interspecific mating between Streptomyces bambergiensis and S. lividans. Cloning experiments using the non-conjugative, circular Streptomyces vector pIJ702 allowed the identification of a 5.74 kb region from pBL1 which facilitates plasmid transfer. Insertion and deletion mutagenesis, gene disruptions, and sequence data suggest that at least five previously unknown genes of pBL1 are required for efficient plasmid transfer and its regulation.     

Expression of a Thermomonospora fusca Cellulase Gene in Streptomyces lividans and Bacillus subtilis

A cellulase gene from Thermomonospora fusca coding for endocellulase E₅ was introduced into Streptomyces lividans by using shuttle plasmids that can replicate in either S. lividans or Escherichia coli. Plasmid DNA isolated from E. coli was used to transform S. lividans, selecting for thiostrepton resistance. The transformants expressed and excreted the endocellulase, but the ability to produce the endocellulase was unstable. This instability was shown to result from deletion of the endocellulase gene from the plasmid. Plasmid DNA prepared from a culture in which plasmid modification had occurred was used to transform E. coli, selecting for Amp⁺ cells, and all of the transformants were cellulase positive, showing that pBR322 and T. fusca DNA were deleted together. When a plasmid was constructed containing only T. fusca DNA in plasmid pIJ702, the transformants were more stable, and the level of endocellulase activity produced in the culture supernatant after growth on 0.2% glucose was close to the level produced by T. fusca cultures grown on 0.2% cellulose. About 50% of the total protein in the culture supernatant of the S. lividans transformant was endocellulase E₅. The enzyme produced by the S. lividans transformant was identical to pure T. fusca E₅ in its electrophoretic mobility and was completely inhibited by antiserum to E₅. Shuttle plasmids containing the E₅ gene that could replicate in Bacillus subtilis and E. coli were also constructed and used to transform B. subtilis. Again there was extensive deletion of the plasmid DNA during transformation and growth in B. subtilis. There was no evidence of E₅ activity, even in those B. subtilis transformants that retained the E₅ gene.