Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Manure Enhances Plasmid Mobilization and Survival of Pseudomonas putida Introduced into Field Soil

We report a field study on plasmid mobilization in an agricultural soil. The influence of pig manure on the mobilization of the IncQ plasmid pIE723 by indigenous plasmids or by the IncP(alpha) plasmid pGP527 into the recipient Pseudomonas putida UWC1 (Rif(supr) Nal(supr)) was studied in field soil. Six plots were prepared in duplicate, three of which were treated with manure prior to inoculation of the donor and recipient strains. As a donor strain, either Escherichia coli J53(pIE723) or E. coli 600(pIE723, pGP527) was used. Putative transconjugants obtained on a selective medium were confirmed by DNA hybridization and PCR. Plasmid mobilization by indigenous mobilizing plasmids was observed on two occasions in manured soil. Manuring of soil significantly enhanced the frequency of pIE723 mobilization by pGP527, since mobilization frequencies into P. putida UWC1 were at least 10-fold higher in manured soil than in nonmanured soil. Enhanced numbers of P. putida UWC1 transconjugant and recipient colonies could be observed in manured soil throughout the 79-day field test. Transfer of pIE723 or pG527 into indigenous soil or rhizosphere bacteria could not be detected when indigenous bacteria isolated by selective cultivation were screened for the presence of these plasmids by DNA hybridization. Furthermore, the presence of IncN-, IncP-, or IncQ-specific sequences was confirmed in total community DNA extracted directly from the manured or nonmanured soil by PCR. IncW plasmids were detectable only in manured soil, indicating entry of these plasmids into soil via manure.     

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.     

Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere

Abstract: A molecular method for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere was developed. The system consisted of polymerase chain reaction (PCR) amplification of part of the variable V1 to V4 regions of the 16S ribosomal RNA gene, followed by hybridization with a specific oligonucleotide probe homologous to part of the intervening region. In vitro specificity tests showed that the detection system worked specifically for P. azotofixans strains, and did not detect other Paenibacillus species or species of other bacterial genera. Vegetative cells of a rifampicin resistant P. azotofixans derivative were trackable in Flevo silt loam (FSL) soil in 24 h experiments using both selective plating and most probable number (MPN)-PCR combined with probing, and plate counts parallelled MPN-PCR estimations of numbers of specific targets. MPN-PCR allowed for the detection of down to 10² introduced cells per g of dry soil. Introduced P. azotofixans spores did not form colonies on selective plates, but were detectable via PCR. The P. azotofixans populations introduced into the silt loam soil suffered a slow decline of the detectable plate count over a period of 14 days. MPN-PCR revealed a similar decline of the number of specific DNA targets. Greater numbers of targets were found in wheat rhizosphere from Flevo silt loam soil, and these numbers persisted throughout the experiment. Soil drying resulted in enhanced persistence of the target sequences, whereas in a constantly moist soil the numbers of target sequences declined. Rewetting of dried soil resulted in declining target sequence numbers. The MPN-PCR detection method is adequate to assess the impact of stress conditions affecting P. azotofixans in FSL and probably other soils, since it abolishes the need for culturing or specific markers and is direct and unambiguous due to its high specificity.     

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.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.     

Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the γ subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Co^r, Zn^r, and Cd^r), Alcaligenes eutrophus (a member of the β subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.     

Role of the intraspecific competition in the regulation of Agrobacterium tumefaciens transconjugant population level in soil experiments

Abstract In microcosms of sterilized soil simultaneously inoculated with Pseudomonas aeruginosa carrying the plasmid R68-45 and the plasmid-free Agrobacterium tumefaciens , transconjugants were detectable after two days of incubation and their number remained constant thereafter. The growth of a transconjugant strain was monitored in sterile soil. When mixed together with the parental strains at high inoculum or when the soil was previously colonized by the donor, the transconjugant was able to grow. If the recipient was the first soil colonizer, the challenging population of transconjugant remained stable at its initial level. We demonstrated the possible role of intraspecific competition in the limitation of transconjugant numbers.     

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.     

Detection of plasmid RP4 transfer in soil and rhizosphere,and the occurrence of homology to RP4 in soil bacteria

Plasmid RP4 transfer between introduced pseudomonads was studied in non-rhizosphere and rhizosphere soil. The addition of nutrients to the non-rhizosphere soil stimulated plasmid transfers between introduced donor and recipient cells, and no transfer was detected in nonamended soil. Transfer was also detected in soil in a model rhizosphere, but not in corresponding non-rhizosphere soil. Colony hybridization with whole plasmid RP4 DNA as a probe was employed to detect transfers to indigenous organisms in soil. Although transfers to introduced recipient cells were easily detected in parallel controls, no indigenous organisms were identified that had received RP4. Background levels of soil organisms with the RP4 resistance pattern were considerable, and about 10% of these populations contained DNA sequences with homology to RP4. However, no plasmids could be detected in any of 20 isolates, nor was resistance transfer to aPseudomonas fluorescens recipient detected in filter matings.     

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.     

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.     

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.     

Detection and enumeration of viable but non-culturable transconjugants of Escherichia coli during the survival of recipient cells in river water

Viable but non-culturable transconjugant cells were detected by a modification of the direct viable count (DVC) method. This modification involved the addition of parental antimicrobial markers (kanamycin and streptomycin) to the elongation medium in order to promote selective elongation of the transconjugant cells. Presence of viable, other than culturable, transconjugants was demonstrated in matings with parental cells from TSB culture as well as with recipient cells from survival in river water (under illuminated and non-illuminated systems). In matings with a recipient strain from illuminated systems, culturable transconjugants were not detected after the third day of recipient cell survival. In spite of this, viable transconjugants were detected in numbers that exceeded 10⁵cells ml⁻¹. These results clearly show that a fraction of non-culturable recipient cells is able to receive and express plasmids by conjugation processes and form viable but non-culturable transconjugant cells.     

A microcosm for measuring survival and conjugation of genetically engineered bacteria in rhizosphere environments

A microcosm is described to evaluate and measure bacterial conjugation in the rhizosphere of barley and radish with strains ofPseudomonas cepacia. The purpose was to describe a standard method useful for evaluating the propensity of genetically engineered microorganisms (GEMs) to transfer DNA to recipient bacteria. Results demonstrated the formation of transconjugants from the rhizosphere of each plant 24 h after inoculation. Transconjugant populations peaked at 1.8 × 10² colony forming units (CFU)/g root and associated soil in barley and 2.0×10² CFU/g root and associated soil in radish; they then declined over the next five days of the experiment. No significant differences were found in the survival of transconjugant populations monitored from the two plant species. The microcosm was also used to document the formation of false positive transconjugants, which resulted from donor and recipientP. cepacia mating on the surface of selective agar plates instead of in microcosms. Transconjugants resulting from such plate mating occurred in substantial numbers during the first 5 days of the experiment but declined to undetectable numbers by day 7. The use of nalidixic acid was investigated to determine the magnitude of plate mating. The number of transconjugants detected from radish rhizosphere was reduced by two orders of magnitude by including nalidixic acid in the plating medium; this indicated that 99% of the transconjugants were a result of plate mating.     

Transfer of plasmid RP4 between pseudomonads after introduction into soil; influence of spatial and temporal aspects of inoculation

Abstract Transfer of plasmid RP4 between introduced strains of Pseudomonas fluorescens was studied in 2 soils, Ede loamy sand and Guelph loam, in non-rhizosphere and rhizosphere soil using soil chambers and microcosm systems. Short-term organism survival was generally at high levels (> 10⁶/g dry soil), in both soils, whereas long-term survival was poorer, particularly in the loamy sand. Amendment of this soil with bentonite clay improved bacterial survival. Plasmid transfer between donor and recipient strains freshly introduced into separate portions of Ede loamy sand, which were subsequently mixed, was only detected in the vicinity of growing wheat roots, suggesting roots stimulate bacterial migration and/or growth. However, no transfer was detected between resident donor and recipient cell populations (introduced 48 days previously), due to poor organism survival. Plasmid transfer was detected in the rhizosphere between established, resident donor cell populations, and newly-introduced recipients, and vice-versa, in both soils. These data suggested that plant roots enhance the frequency of bacterial matings not only between organisms present in the same niches, but also between organisms from different niches, or in different conditions of stress, probably by stimulating bacterial migration and/or growth, or by providing additional surfaces for cell-to-cell contact.     

Intraspecific competition as a regulating factor limiting the number of transconjugants in soil

A laboratory study was carried out to determine survival of transconjugant cells ofPseudomonas fluorescens intro duced into sterile soil. The transconjugant survived significantly better when it was the only strain inoculated into the soil; when introduced into soil pre-colonized by the recipient strain, the transconjugant was undetectable. These results indicate that intraspecific competition is a regulating factor limiting the number of transconjugants in soil.     

Growth and survival of cowpea rhizobia in bauxitic silt loam and sandy clay loam soils

Survival of 4 cowpea Rhizobium strains, IRC291, MI-50A, JRW3 and JRC29, in two soil types (bauxitic silt loam and sandy clay loam) undergoing drying at 30°C and 37°C was examined. While all strains except JRW3 showed a general pattern of increase in their numbers during the first 3 weeks in sterile soils, none of the strains showed any increase in their population in non-sterile soils. Cowpea rhizobia showed better survival in non-sterile bauxitic silt loam than in clay loam soils at 30°C. However, the long-term survival (examined up to 6 months) of rhizobia in both soils was poor at 37°C as compared to 30°C. We also found that cowpea rhizobia survived better in soils undergoing drying than in moist soils at 30°C. Our results suggest that (a) cowpea rhizobia survived better in bauxitic silt loam than in clay loam soil and (b) the low indigenous cowpea rhizobial population in Jamaican soils may be due to their poor long-term survival and weak saprophytic competence.