Quantification and Modeling of Plasmid Mobilization on Seeds and Roots

Mobilization frequencies of the nonconjugative plasmid pMON5003 were quantified using Escherichia coli TB1(pRK2013) as donor of a helper plasmid, E. coli M182 (pMON5003) as donor of the nonconjugative plasmid, and Pseudomonas fluorescens as recipient. Initial mating experiments were conducted in nutrient and minimal salts media and pea seed exudates. Mobilization rates were higher during early stationary growth of donors, helpers, and recipients. Numbers of transconjugants were higher in biparental matings when donors contained both conjugative and nonconjugative plasmids, versus tri-parental matings. A mathematical model was developed to predict a nonconjugative plasmid transfer rate parameter (δ), estimating the proportion of conjugative matings in which a plasmid is mobilized. Values of δ ranged from 8 × 10⁻³ to 7.9 × 10⁻¹. Transfer frequencies for pMON5003 from E. coli to P. fluorescens on pea seeds and roots were determined. Transconjugants (P. fluorescens 2-79 (pMON5003)) were isolated from seeds, roots, and soil, but mobilization frequencies were lower than in liquid media.     

Inability of Some <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> Strains to Act as Recipients of Plasmid pRAS1 in Conjugal Transfer Experiments

Plasmids belonging to the IncU incompatibility group are mobile genetic elements isolated frequently from Aeromonas spp. These plasmids share structural and functional characteristics and often carry Class-1 integrons bearing antibiotic resistance genes. In this work the ability of two IncU plasmids, pAr-32 and pRAS1 to establish in different A. hydrophila strains after conjugal transfer was studied. In vitro transfer frequencies on solid surface ranged from 10⁻¹ to 10⁻⁶ for pAr-32 and from 10⁻³ to 10⁻⁵ for pRAS1. While carrying out these experiments we detected four strains unable to acquire plasmid pRAS1, indicating that the genetic background of recipients affects the establishment of the plasmid. We explored the possible reasons why these strains failed to yield transconjugants after mating experiments using A. salmonicida 718 as a donor. Factors included donor cell recognition, incompatibility, surface exclusion and restriction of incoming DNA. We found that none of these factors could explain the refractivity of non-receptive A. hydrophila strains to yield transconjugants. Although we do not know the reasons of this refractivity, we may speculate that these isolates lack a product necessary to replicate or stabilize plasmid pRAS1. Alternatively, these strains could contain a product that impedes plasmid establishment.     

Exclusion in IncI-type Escherichia coli conjugations: the stage of conjugation at which exclusion operates

The stage at which exclusion operates in matings between donors belonging to the I-type incompatibility group (IncI) was investigated. Mating between Escherichia coli cells harbouring the I-type plasmid R144 and E. coli cells harbouring the R144-derived recombinant plasmid pRAH308, which causes a hundredfold exclusion, was performed on a membrane filter to test whether mating aggregate formation was disturbed. Besides, level and kinetics of the formation of mating aggregates in mixtures of R144⁺ donor cells and recipient cells carrying plasmid pRAH308 (exclusion-proficient) was compared with the aggregate formation in mixtures of the donor cells and exclusion-deficient recipient cells. Results from these experiments revealed that the exclusion by pRAH308 does not operate at the level of aggregate formation, but acts at the stage of DNA transfer. The exclusion phenomenon by the recombinant plasmid pRAH308 appeared to be representative for exclusion caused by plasmid R144, since essentially identical results were obtained if plasmid R144 was used as exclusion-determining factor.     

Heterotrophic bacteria of the freshwater neuston and their ability to act as plasmid recipients under nutrient deprived conditions

Significantly higher numbers of Gram-negative heterotrophic bacteria were present at the air-water interface (neston) of freshwater lakes than in the bulk water. Neuston bacteria were distinguished as a population distinct from bacteria in the bulk water by a higher incidence of pigmented colony types and significantly greater levels of multiple resistance to antibiotics and heavy metals. The incidence of plasmids in 236 neuston and 229 bulk water strains were similar (14 and 16.2%, respectively). Nine of 168 plasmid-free strains and 2 of 14 plasmid carrying strains, isolated from both bulk water and neuston, acted as recipients of plasmid R68.45 in plate matings with aPseudomonas aeruginosa donor strain PAO4032 at 21°C, but at frequencies below that of matings with a restriction-minus recipient strain ofP. aeruginosa, strain PAO1168. In a model system composed of nutrient-free synthetic lake water, plasmid R68.45 was shown to transfer betweenP. aeruginosa strains at frequencies between 10⁻³ and 10⁻⁵. Transconjugants were detected about 100 times more frequently at the interface than in the bulk water, which in part reflected a greater enrichment of the donor at this site. None of the aquatic isolates were able to act as recipients of plasmid R68.45 in this model system with strain PAO4032 as donor. The results suggest that under nutrient deprived conditions, the spread of plasmid R68.45 and similar plasmids by lateral transfer into this particular aquatic population would be a rare event.     

The requirements for conjugal DNA synthesis in the donor strain during flac transfer.

Although neither rifampicin nor spectinomycin had any effect on the frequency of Flac transfer by a sensitive donor, rifampicin but not spectinomycin prevented donor conjugal DNA synthesis as measured in matings between a dnaB donor and a tdk recipient. An untranslated RNA species is therefore probably required for this synthesis, although transfer took place even in its absence. Donor conjugal DNA synthesis was abolished in a dnaE donor, showing that DNA polymerase III is responsible for this process; again, plasmid DNA transfer was not affected.Flac mutants lacking the F pilus gave neither donor conjugal DNA synthesis nor plasmid DNA transfer, probably because they could not receive a “mating signal” to activate the transfer process. The products of traI and traM were also required both for donor conjugal DNA synthesis and for physical transfer of plasmid DNA, probably being involved in the conversion of covalently closed circular plasmid DNA into the open circular form that is the substrate for the independent although normally simultaneous synthesis and transfer steps. In contrast, donor conjugal DNA synthesis took place at a normal rate in both piliated traG and traN mutants, and at a reduced rate in traD mutants, although in no case was there physical transfer of plasmid DNA. These gene products are therefore required for DNA transfer to the recipient, and in addition, the absence of the traD product may hinder DNA synthesis.Based upon these results, a scheme for the processing of DNA during conjugation is presented.     

Conjugative transfer of tetracycline resistance in rumen streptococcal strains

In 11% of testedStreptococcus bovis strains a conjugative transfer of tetracycline resistance was observed when mating experiments were carried out on membrane filters. The recipient strain used wasS. bovis BM114 with chromosomal resistance to rifampicin. In addition, in two strains tetracycline resistance was transferred also to recipient strainEnterococcus faecium AL6. The transfer frequencies were in the range of 10⁻⁶ to 10⁻³. The donor strains were screened for the presence of plasmids and one up to four bands of plasmid DNA in all tested strains were revealed. In spite of that isolation of plasmid DNA was successful only in 53/4/114 transconjugants. Transconjugant 32/114 contained amylase activity which was higher than in the donor strain.     

Long-term survival of and plasmid stability inPseudomonas andKlebsiella species and appearance of nonculturable cells in agricultural drainage water

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with³²P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.     

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.     

Transfer and expression of a hydrocarbon-degrading plasmid pHCL from Pseudomonas putida to marine bacteria

Transfer of a catabolic plasmid from Pseudomonas putida to indigenous marine bacteria and obligate halophilic bacteria was carried out under both in vitro and in situ conditions. The marine recipients, which could not otherwise grow on hydrocarbon substrates, were able to degrade them after the horizontal transfer of the catabolic plasmid from P. putida. Mating conducted on nutrient plates yielded comparatively more transconjugants than in broth mating under laboratory conditions (10⁶ c.f.u./ml). The transconjugants stably maintained the plasmid when they were maintained in seawater amended with selective pressure (antibiotics/Hg (25 g/l) even after 30 days, whereas under non-selective conditions they progressively lost the plasmid after 24 days. The expression of the plasmid in the marine recipients was investigated by gas chromatographic analysis. The overall objective of this study is to evolve a novel strategy for bioremediation of oil spills and the results of the present study suggest that the present approach would offer a better solution for the removal of harmful substances from the environment by avoiding serious interference with the microbial flora of the ecosystem.     

Capacity of aquatic bacteria to act as recipients of plasmid DNA.

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.     

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.     

Capacity of aquatic bacteria to act as recipients of plasmid DNA

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.     

Plasmid-Mediated Gene Transfer Between Insect-Resident Bacteria, Enterobacter cloacae, and Plant-Epiphytic Bacteria, Erwinia herbicola, in Guts of Silkworm Larvae

Five strains of Enterobacter cloacae isolated from several species of plants and insects were able to grow in the guts of silkworm larvae. A much larger population of Ent. cloacae strains was detected in the insect guts and feces collected 3 and 6 days than in samples collected 1 day after feeding artificial diets contaminating these bacteria. Furthermore, insect-origin strains of Ent. cloacae were mated with a donor strain, epiphytic Erwinia herbicola, harboring RSF1010 and pBPW1::Tn7 plasmids in the insect guts by introducing these bacteria through separate artificial diets administered at different times. A number of transconjugants, Ent. cloacae strains which had acquired RSF1010 plasmid, were detected from guts and fecal samples at transfer frequencies of 10⁻² to 10⁻³ per recipient. Thus, gene transfer between epiphytic Er. herbicola and insect-resident Ent. cloacae strains in the insect guts was confirmed. These findings may provide significant information about the role of ′′in insecta mating' in the evolution of these bacteria. Received: 16 June 1998 / Accepted: 7 July 1998     

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.     

Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: Role of different regions within the zebra fish intestinal tract,and impact on fish intestinal microbiota

The aqueous environment is one of many reservoirs of antibiotic resistance genes (ARGs). Fish, as important aquatic animals which possess ideal intestinal niches for bacteria to grow and multiply, may ingest antibiotic resistance bacteria from aqueous environment. The fish gut would be a suitable environment for conjugal gene transfer including those encoding antibiotic resistance. However, little is known in relation to the impact of ingested ARGs or antibiotic resistance bacteria (ARB) on gut microbiota. Here, we applied the cultivation method, qPCR, nuclear molecular genetic marker and 16S rDNA amplicon sequencing technologies to develop a plasmid‐mediated ARG transfer model of zebrafish. Furthermore, we aimed to investigate the dissemination of ARGs in microbial communities of zebrafish guts after donors carrying self‐transferring plasmids that encode ARGs were introduced in aquaria. On average, 15% of faecal bacteria obtained ARGs through RP4‐mediated conjugal transfer. The hindgut was the most important intestinal region supporting ARG dissemination, with concentrations of donor and transconjugant cells almost 25 times higher than those of other intestinal segments. Furthermore, in the hindgut where conjugal transfer occurred most actively, there was remarkable upregulation of the mRNA expression of the RP4 plasmid regulatory genes, trbBp and trfAp. Exogenous bacteria seem to alter bacterial communities by increasing Escherichia and Bacteroides species, while decreasing Aeromonas compared with control groups. We identified the composition of transconjugants and abundance of both cultivable and uncultivable bacteria (the latter accounted for 90.4%–97.2% of total transconjugants). Our study suggests that aquatic animal guts contribute to the spread of ARGs in water environments.     

Growth of Amoebae and Flagellates on Bacteria Deposited on Filters

Abstract Artificial bacterial biofilms were formed by making microwave-irradiated, dual-radioisotope-labelled Vibrio bacteria adhere to 0.4 μm pore size filters with albumin. The rate of release of ³H from thymidine label in these bacteria into the surrounding seawater when protozoa were incubated with the biofilm indicated the predator's grazing rate, and the rate of accumulation of ¹⁴C in the predators from leucine label in the bacteria indicated the assimilation rate of the protozoa. The amoeba Vanella septentrionalis consumed about 60% of the available bacteria between the 5th and 15th days of incubation with a gross growth efficiency of 22 ± 6%, compared with about 75% consumption at 29 ± 8% efficiency for the surface-feeding flagellate Caecitellus parvulus, and about 55% consumption at 16 ± 5% efficiency for the suspension-feeding flagellate Pteridomonas danica. As a result of their grazing and metabolism these protozoa regenerated about 70–85% of the nutrients present in their food and released these nutrients in the immediate vicinity of the bacterial biofilm. The biomass of the amoeba Vanella was calculated to be 166 pg protein cell⁻¹ during maximum growth and 93 pg protein cell⁻¹ in the stationary phase. Received: 3 August 1998; Accepted: 20 November 1998     

Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation

Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMβ1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 × 10³ transformants per μg of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene into pAT187. Mating of E. coli containing pKV12 with A. woodii resulted in transfer frequencies of 3 × 10^-6 to 7 × 10^-6 per donor or recipient.     

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.     

High-frequency conjugal transfer of a gonococcal penicillinase plasmid.

Gonococci containing a 24 X 10(6)-dalton conjugal plasmid were able to mobilize for transfer a smaller, non-self-transmissible penicillinase (Pcr) plasmid with high frequency under appropriate conditions. In some strains, over 10% of donor colony-forming units transferred the Pcr plasmid in a mating of less than 2 h, which suggests that the conjugal system was naturally derepressed. Colony-opacity variants containing different quantities of an approximately 28,000-dalton outer membrane protein were altered in their ability to act as conjugal donors and recipients. Maximal transfer of the Pcr plasmid was observed between transparent donors and recipients, lacking appreciable amounts of the 28,000-dalton protein. Under conditions of high-frequency Pcr plasmid mobilization, no conjugal mobilization of chromosomal markers could be discerned.     

A Highly Selectable and Highly Transferable Ti Plasmid to Study Conjugal Host Range and Ti Plasmid Dissemination in Complex Ecosystems

A conjugal donor system, ST2, was constructed to study the conjugal dissemination of a Ti plasmid to wild-type recipient bacteria in vitro and in situ. The system consisted of a polyauxotrophic derivative of C58 harboring a hyperconjugative and highly selectable Ti plasmid, pSTiEGK, which was constructed by inserting a multiple antibiotic resistance cassette in the traM-mcpA region of pTiC58accR. ST2 transfers pSTiEGK constitutively at frequencies up to 10^–1 to plasmidless Agrobacterium recipients. The host range of pSTiEGK includes all the known genomic species of Agrobacterium, indigenous soil agrobacteria and some Rhizobium and Phyllobacterium spp. All transconjugants became pathogenic upon acquisition of the Ti plasmid and were also able to transfer pSTiEGK by conjugation. This host range was indistinguishable from that of its wild-type parent pTiC58, and therefore pSTiEGK constitute a valid proxy to study the dissemination of Ti plasmids directly in the environment. Transconjugants can be selected on a combination of four antibiotics, which efficiently prevents the growth of the indigenous microbiota present in complex environments. The transfer of pSTiEGK to members of the genus Agrobacterium was affected primarily by the plasmid content of the recipient strain (10³- to 10⁵-fold reduction), e.g., the presence of incompatible plasmids. As a consequence, a species should be considered permissive to Ti transfer whenever one permissive isolate is found.