Effect of Plant Species on the Kinetics of Conjugal Transfer in the Rhizosphere and Relation to Bacterial Metabolic Activity

Conjugal transfer of a derivative of the RP4 plasmid between Pseudomonas fluorescens AS12 and Serratia plymuthica RF7 was compared in the rhizosphere of pea, wheat, and barley and related to the metabolic activity of the bacteria. To obtain a reliable measure of transfer, which allowed comparison of results between experiments, mathematical mass-action models were used to determine plasmid intrinsic kinetic coefficients. The data showed that not only were the rhizospheres highly conducive of transfer, with rates up to six orders of magnitude higher than in bulk soil, but differences between rhizospheres were also observed. Highest intrinsic kinetic coefficients were found in the pea rhizosphere (1.1-4.1 x 10-11), followed by the barley rhizosphere (2.4-7.2 x 10-12) and the wheat rhizosphere (2.2-2.9 x 10-13). It was further shown that the metabolic activity of the cells in the rhizosphere of the three plants was not significantly different, and that activity and transfer were not correlated. Thus, the data demonstrated species specific rhizosphere effects on the conjugal transfer process that could not be attributed to different metabolic activities of the bacteria.     

Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among gram-positive and gram-negative bacteria indigenous to the barley rhizosphere

The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 x 10(-2) +/- 3.84 x 10(-2). This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.     

Effect of Parental Growth on Dynamics of Conjugative Plasmid Transfer in the Pea Spermosphere

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.     

Monitoring the conjugal transfer of plasmid RP4 in activated sludge and in situ identification of the transconjugants

A GFPmut3b-tagged derivative of broad host-range plasmid RP4 was used to monitor the conjugative transfer of the plasmid from a Pseudomonas putida donor strain to indigenous bacteria in activated sludge. Transfer frequencies were determined to be in the range of 4 x 10(-6) to 1 x 10(-5) transconjugants per recipient. In situ hybridisation with fluorescently labeled, rRNA-targeted oligonucleotides was used to phylogenetically affiliate the bacteria that had received the plasmid.     

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.     

Transfer of plasmid RP4 in the spermosphere and rhizosphere of barley seedling

Transfer of plasmid RP4 to indigenous bacteria in bulk soil could only be detected in soil with nutrient amendment. Lack of physiological active donor and recipient cells was apparently one of the limiting factors in un-amended bulk soil. Plasmid transfer was detected both in the spermosphere and rhizosphere of barley seedlings. Transfer occured from seed coated donor bacteria (i) to introduced recipient bacteria and (ii) to indigenous bacteria present in soil. Plasmid transfer was also detected from donor bacteria introduced to the soil to seed coated recipient bacteria. Transfer efficiencies in the rhizosphere were significantly below the transfer efficiencies obtained in the spermosphere. The transfer efficiencies detected in the barley spermosphere were among the highest reported from any natural environment.     

Plasmid R68.45 and S-a transduction by the temperate bacteriophage 59 of Erwinia carotovora 268

Temperature bacteriophage 59 of Erwinia carotovera 268 had transduced extrachromosomal DNA: plasmids of R68.45 and S-a. Before plasmid transduction experiments the suitable donor strains of indicator culture Erwinia horticola 450 harbouring R68.45 and S-a were created. The frequency of plasmid R68.45 transfer from Pseudomonas putida to E. horticola 450-8 by conjugation was equal to 5 x 10(-8) per a donor cell and in the case of S-a--from E. coli C600 for the same recipient cells--was 2 x 10(-6). Bacteriophage 59 has transduced only separate markers of plasmid R68.45, since plasmid S-a is probably transduced by the phage as an intact unit.     

The specific growth rate of Pseudomonas putida PAW1 influences the conjugal transfer rate of the TOL plasmid.

The kinetics of the conjugal transfer of a TOL plasmid were investigated by using Pseudomonas putida PAW1 as the donor strain and P. aeruginosa PAO 1162 as the recipient strain. Short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. The experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. This method allowed analysis of the mating experiments by a single intrinsic kinetic parameter for conjugal plasmid transfer. Further results indicated that the specific growth rate of the donor strain antecedent to the mating experiment had a strong impact on the measured intrinsic plasmid transfer rate coefficient, which ranged from 1 x 10(-14) to 5 x 10(-13) ml per cell per min. Preliminary analysis suggested that the transfer rates of the TOL plasmid are large enough to maintain the TOL plasmid in a dense microbial community without selective pressures.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

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.     

Gene transfer and plasmid instability within pilot-scale sewage filter beds and the invertebrates that live in them

The environmental plasmid pQKH6 was transferred conjugatively between strains of Pseudomonas putida at mean frequencies of up to 8.4x10(-4) within pilot-scale sewage filter beds. This frequency was 10-fold higher than that reported previously for this environment and was probably due to seasonal temperature changes. Many (45%) of the plasmids isolated subsequently from the filter beds had restriction fragment length polymorphism (RFLP) profiles that differed from that expected for pQKH6. RFLP analysis revealed structural rearrangements occurring within a particularly restriction-site-rich region of the plasmid. Although no evidence was obtained showing the indigenous invertebrate populations within the filter beds to influence the rate of gene transfer, pQKH6 was transferred with frequencies of up to 1.6x10(-2) within the guts of the filter-bed-dwelling Sylvicola fenestralis larvae during laboratory experiments. This transfer was strongly influenced by donor to recipient ratios. Laboratory experiments also showed that Serratia fonticola survived better within invertebrate guts than P. putida. This evidence, along with experiments showing that S. fonticola could participate in pQKH6 transfer within filter-bed biofilm, identify this bacterium as a better model than P. putida for examining the effect of invertebrates on gene 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.     

Rhizosphere competitiveness of trichloroethylene-degrading, poplar-colonizing recombinant bacteria

Indigenous bacteria from poplar tree (Populus canadensis var. eugenei 'Imperial Carolina') and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome. The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 x 10(5) to 23 x 10(5) CFU/cm of root). In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% +/- 12% of all rhizosphere bacteria after 28 days (0.2 x 10(5) to 31 x 10(5) CFU/cm of root). Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp. strain PsK recombinants) retained the ability to express TOM for 29 days as 100% +/- 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively.     

Occurrence, transfer and mobilization in epilithic strains of Acinetobacter of mercury-resistance plasmids capable of transformation

A 7.8 kb plasmid (pQM17) encoding mercury resistance was isolated from two epilithic strains of Acinetobacter calcoaceticus. The plasmid had a broad host range when mobilized by RP1, transferring into Pseudomonas aeruginosa, P. putida, P. fluorescens, Escherichia coli, Proteus vulgaris and Chromobacterium sp. with frequencies ranging from 5.3 x 10(-9) to 4.6 x 10(-4) per recipient. The plasmid could be transferred into A. calcoaceticus BD413 using intact cells of donor and recipient bacteria (i.e. natural transformation) and there was a broad temperature optimum (14-37 degrees C) for transformation. Transformation was as efficient in liquid matings as on plates but there was no effect of pH in the range 5.6-7.9. Maximum transformation frequencies were obtained after 24 h on agar plates containing 3.5-10 g C 1-1 with donor to recipient ratios ranging from 6 to 415.     

Integration of R91-5::Tn501 into the Pseudomonas putida PPN chromosome and genetic circularity of the chromosomal map

Derivatives of the Pseudomonas aeruginosa plasmid R91-5, loaded with the transposon Tn501, were transferred to P. putida PPN. Over 90% of exconjugants, which arose at a frequency of ca. 10(-6) per donor cell, exhibited high-frequency (greater than 10(-2) per donor cell) polarized transfer of chromosomal markers. In one instance it was demonstrated by transduction that the plasmid had been inserted into a gene required for serine biosynthesis. The integrated nature of the plasmid in this and other P. putida (R91-5::Tn501) derivatives was supported by the failure to detect covalently closed circular DNA in these strains. The transfer origins of six different Hfr donors have been characterized genetically, and time-of-entry kinetics obtained from interrupted matings have enabled the construction of a circular genetic map 103 min in length and containing 35 markers. The genetic map of P. putida PPN shows significant differences in marker order to that of P. aeruginosa PAO.     

Root carbon inputs to the rhizosphere stimulate extracellular enzyme activity and increase nitrogen availability in temperate forest soils

The exudation of carbon (C) by tree roots stimulates microbial activity and the production of extracellular enzymes in the rhizosphere. Here, we investigated whether the strength of rhizosphere processes differed between temperate forest trees that vary in soil organic matter (SOM) chemistry and associate with either ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi. We measured rates of root exudation, microbial and extracellular enzyme activity, and nitrogen (N) availability in samples of rhizosphere and bulk soil influenced by four temperate forest tree species (i.e., to estimate a rhizosphere effect). Although not significantly different between species, root exudation ranged from 0.36 to 1.10 g C m^?2 day^?1, representing a small but important transfer of C to rhizosphere microbes. The magnitude of the rhizosphere effects could not be easily characterized by mycorrhizal associations or SOM chemistry. Ash had the lowest rhizosphere effects and beech had the highest rhizosphere effects, representing one AM and one ECM species, respectively. Hemlock and sugar maple had equivalent rhizosphere effects on enzyme activity. However, the form of N produced in the rhizosphere varied with mycorrhizal association. Enhanced enzyme activity primarily increased amino acid availability in ECM rhizospheres and increased inorganic N availability in AM rhizospheres. These results show that the exudation of C by roots can enhance extracellular enzyme activity and soil-N cycling. This work suggests that global changes that alter belowground C allocation have the potential to impact the form and amount of N to support primary production in ECM and AM stands.     

Conjugal transfer at natural population densities in a microcosm simulating an estuarine environment

Abstract Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. Donor cells were added at a concentration similar to the natural abundance of bacteria in the water column (10⁶ cells ml⁻¹). Transfer was not detected in any of the test microcosms (calculated limit of detection of 10⁻⁷ and 10⁻⁴ transconjugants donor⁻¹ in water column and sediment, respectively), with the exception of transfer to an isogenic recipient (added at 10⁵ cells ml⁻¹) in sediments of controls that had been inoculated with both donors and recipients. The same plasmid was transferred with high efficiencies (10⁻¹ to 10⁻³) to a variety of recipients in filter and broth matings. These results suggest that if conjugal gene transfer occurred, it was at efficiencies that were not detectable in estuarine microcosms simulating natural population densities.     

Field and soil microcosm studies on the survival and conjugation of a Pseudomonas putida strain bearing a recombinant plasmid, pADPTel

Pseudomonas putida CR30RNS (pADPTel) is an antibiotic-resistant strain with a recombinant plasmid that confers resistance to tellurite and the ability to catabolize atrazine. The survival of this strain as well as its ability to transfer genes for atrazine degradation and tellurite resistance to indigenous soil bacteria were tested in both fallow soil and canola (Brassica napus) rhizosphere by the use of parallel field and laboratory releases. Culturable CR30RNS (pADPTel) were enumerated in field and microcosm soils at 7- to 14-day intervals over 49 d. Strain CR30RNS (pADPTel) survived for up to 7 weeks in microcosm soils at a density of 10(4) CFU/g soil, whereas in field soils the population declined to 10(3) CFU/g soil by the fourth week. In contrast, when CR30RNS (pADPTel) was introduced into the soil as a seed coating of canola (B. napus 'Karoo'), the bacterium established at higher cell densities in the rhizosphere (10(6)-10(5) CFU/g fresh root mass), with no subsequent decrease in numbers. The presence of selective pressure (i.e., atrazine) had no significant effect on the survival of CR30RNS (pADPTel) in either field or microcosm soils. One year postinoculation field sites were examined for the presence of CR30RNS (pADPTel) and no evidence of culturable parental cells was observed when samples were plated onto selective media. However, the atzC and telAB gene segments were amplified from the field soils at that time. Under laboratory conditions, indigenous soil bacteria were capable of receiving and expressing the engineered plasmid construct at frequencies ranging from 1 to 10(-3) transconjugants per donor. However, no plasmid transfer to indigenous soil bacteria was detected in the field or microcosm soils regardless of the presence of canola rhizosphere and (or) the application of atrazine. Our results show that the survival and population size of P. putida CR30RNS (pADPTel) might be sufficient for degradation of environmental pollutants but that the transfer frequency was too low to be detected under the conditions of this study.     

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.