Biotic and abiotic factors affecting plasmid transfer in Escherichia coli strains.

The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.     

Conjugal TOL transfer from Pseudomonas putida to Pseudomonas aeruginosa: effects of restriction proficiency, toxicant exposure, cell density ratios, and conjugation detection method on observed transfer efficiencies

The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp. was investigated by examinations of filter matings. A Pseudomonas putida KT2442-derived strain carrying a gfp-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10(-3) for PAO1162N and 2 x 10(1) for PAO2002N) and total cell densities (10(5) cells/mm(2) for PAO1162N and 10(6) cells/mm(2) for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10(-7) (events/mm(2))(-1) for PAO1162N and 10(-11) (events/mm(2))(-1) for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (P < 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.     

Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa: Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies

The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp. was investigated by examinations of filter matings. A Pseudomonas putida KT2442-derived strain carrying a gfp-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10⁻³ for PAO1162N and 2 × 10¹ for PAO2002N) and total cell densities (10⁵ cells/mm² for PAO1162N and 10⁶ cells/mm² for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10⁻⁷ (events/mm²)⁻¹ for PAO1162N and 10⁻¹¹ (events/mm²)⁻¹ for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (P < 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.     

Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501.

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.     

Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10^–1 to 1.9×10^–7. Highest numbers of transconjugants, 1.5×10⁷ colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 10³ colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.     

Transfer and properties of some natural and suicide replicons in Pasteurella multocida

We tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013--a narrow-host-range plasmid with the broad-host-range IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1011, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.     

Measuring genetic stability in bacteria of potential use in genetic engineering.

Four commonly used conjugation techniques, colony cross streak (CCS), broth mating (BM), combined spread plate (CSP), and membrane filtration (MF), were compared with each other regarding reliability, sensitivity, and complexity in evaluating the transfer of conjugative plasmids. Five plasmids representing several incompatibility groups plus a variety of laboratory and environmental isolates were used as mating pairs. The suitability of each method was evaluated for use in a routine assessment of the genetic stability of genetically engineered microorganisms. By the CSP and MF techniques with laboratory strains such as Escherichia coli and Pseudomonas species as recipients, transconjugants were usually produced in 100% of the mating trials. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% of some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the CSP and MF techniques compared with the BM and CCS techniques were not statistically significant at the P less than or equal to 0.05 level. Occasionally, certain mating pairs consistently produced transconjugants by CCS or BM but not by CSP or MF. Since any single conjugation mating technique is not completely reliable in detecting transconjugants, we have developed a combined mating technique which integrates the CCS, CSP, BM, and MF methods as a single procedure to assess the mobility of plasmid DNA of genetically engineered microorganisms.     

Measuring genetic stability in bacteria of potential use in genetic engineering

Four commonly used conjugation techniques, colony cross streak (CCS), broth mating (BM), combined spread plate (CSP), and membrane filtration (MF), were compared with each other regarding reliability, sensitivity, and complexity in evaluating the transfer of conjugative plasmids. Five plasmids representing several incompatibility groups plus a variety of laboratory and environmental isolates were used as mating pairs. The suitability of each method was evaluated for use in a routine assessment of the genetic stability of genetically engineered microorganisms. By the CSP and MF techniques with laboratory strains such as Escherichia coli and Pseudomonas species as recipients, transconjugants were usually produced in 100% of the mating trials. However, when environmental strains isolated from plants and soil were used as recipients, transconjugants were detected in 100% of some crosses and in as little as 30% in other crosses depending on the plasmid and recipient used. In general, differences in the percentage of successful matings between the CSP and MF techniques compared with the BM and CCS techniques were not statistically significant at the P less than or equal to 0.05 level. Occasionally, certain mating pairs consistently produced transconjugants by CCS or BM but not by CSP or MF. Since any single conjugation mating technique is not completely reliable in detecting transconjugants, we have developed a combined mating technique which integrates the CCS, CSP, BM, and MF methods as a single procedure to assess the mobility of plasmid DNA of genetically engineered microorganisms.     

Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus

Mutants defective in chemolithoautotrophic growth (Aut-) have been isolated from Alcaligenes eutrophus strains H16, N9A, G27, and TF93. Spontaneous Aut- mutants were obtained only with strain TF93. Mutants of the other strains were selected after conventional mutagenesis or treatment with mitomycin. Most of the mutants, including the spontaneous Aut- strains, lacked hydrogenase activity (Hox-) but possessed the ability to fix carbon dioxide (Cfx+). Agar mating of A. eutrophus H16 with Hox- mutants of the various strains resulted in transconjugants which had recovered the ability to grow autotrophically and to express activity of hydrogenase as examined by enzymatic and immunochemical analysis. Transfer of hydrogen-oxidizing ability occurred in the absence of a mobilizing plasmid such as Rp4. The transfer frequency was particularly high (ca. 10(-2) per donor) when the spontaneous Hox- mutants of strain TF93 were used as recipients. These strains proved to be plasmid free, whereas donors, transconjugants, and the mutagen-treated Hox- mutants contained a large plasmid (molecular weight, 270 +/- 10 X 10(6) revealed by agarose gel electrophoresis. The results allow the conclusion that A. eutrophus H16 harbors a self-transmissible plasmid designated pHG1, which carries information for hydrogen-oxidizing ability.     

The TOL plasmid is naturally derepressed for transfer

Pseudomonas putida mt-2, formerly known as Pseudomonas arvilla mt-2, which carries the wild-type TOL plasmid, and P. putida strain AC37 carrying TOL, were completely lysed by the pilus-adsorbing plasmid-specific bacteriophages PR4 and PRD1. Pseudomonas putida strain PpS388, also harbouring the plasmid, was not lysed. In a P. putida mt-2 host, TOL transferred 18-fold better on a surface (2.5 X 10(-1) transconjugants per donor h-1) than in liquid; when P. putida PpS388 was the host, however, a frequency of only 2.3 X 10(-4) transconjugants per donor h-1 was obtained. Thus, TOL was derepressed for transfer in P. putida mt-2 and P. putida AC37, but not in P. putida PpS388. Electron microscopy revealed that TOL determined thick (8.5-10 nm diameter) flexible pili in large numbers, suggesting constitutive expression in its derepressed state.     

Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin

These studies represent the first systematic survey of the incidence of conjugative antibiotic resistance in Clostridium perfringens. Ninety-two antibiotic-resistant porcine strains were examined to see if they could donate their antibiotic-resistance determinants to sensitive recipient strains. Fifteen of the 89 tetracycline-resistant strains transferred their tetracycline resistance in mixed-plate mating experiments but no transfer of macrolide-lincosamide resistance was detected. The efficiencies of transfer of tetracycline resistance varied from 1.3 X 10(-3) to 1.9 X 10(-6) transconjugants per donor cell. Significantly higher transfer efficiencies were observed when both the donor and recipient strains were derivatives of strain CW 362. These values ranged from 3.7 X 10(-1) to 4.6 X 10(-2) transconjugants per donor cell. This high frequency transfer system should prove invaluable for further genetic studies on this microorganism.     

Plasmid associated with diplococcin production in Streptococcus.

The ability to produce diplococcin (Dip+) was transferred by conjugation from Streptococcus cremoris 346 to two plasmid-free S. cremoris recipients at a high frequency (10(-1) per donor). Dip+ transconjugants from each mating gained a 54-megadalton plasmid. Spontaneous loss of this plasmid restored the Dip- phenotype.     

Plasmid associated with diplococcin production in Streptococcus

The ability to produce diplococcin (Dip+) was transferred by conjugation from Streptococcus cremoris 346 to two plasmid-free S. cremoris recipients at a high frequency (10(-1) per donor). Dip+ transconjugants from each mating gained a 54-megadalton plasmid. Spontaneous loss of this plasmid restored the Dip- phenotype.     

Genetic and physiological analysis of conjugation in Streptococcus faecalis.

In an effort to elucidate the mechanisms of conjugal plasmid transfer in Streptococcus faecalis, a genetic analysis of the sex pheromone-dependent tetracycline resistance plasmid pCF-10 was initiated. Rare transconjugants obtained from short matings with wild-type donors not exposed to sex pheromones were screened for increased donor potential in a subsequent mating. From this screening, a mutant plasmid, designated pCF-11, whose transfer functions are expressed in the absence of pheromone induction was isolated. Cells carrying pCF-11 spontaneously clump when grown in broth culture but do not excrete sex pheromones active against wild-type donors. In the course of initial experiments, it was observed that physiological conditions could affect plasmid transfer frequency. Therefore, a set of standardized optimal mating conditions was defined. The experiments carried out to determine these conditions revealed that a transient increase in transfer frequency of about 2 order of magnitude occurred in early-exponential-phase donor cells. This peak of activity is independent of sex pheromone response, since it was observed with induced or uninduced donor cells carrying either pCF-11 or pCF-10.     

Plasmid donor affects host range of promiscuous IncP-1beta plasmid pB10 in an activated-sludge microbial community

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI(q). The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the alpha- and gamma-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.     

Major outer membrane proteins of Prochlorothrix hollandica (Prochloraceae)

Abstract Rhizobium sp. isolated from Lablab purpureus utilized catechol as sole carbon and energy source, a property which is plasmid encoded. The heat curable (39–41°C) plasmid, designated as pAMG1, was transferred to cat⁻ strains of Rhizobium sp. with a transfer frequency of 2.6 × 10⁻⁶ transconjugants/donor cell.     

Detection and Characterization of Plasmid pJP4 Transfer to Indigenous Soil Bacteria

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10⁷ and 10⁸ transconjugants g of dry soil⁻¹ for samples supplemented with 500 and 1,000 μg of 2,4-D g of dry soil⁻¹, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2,4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.