Conjugal transfer of lactose-fermenting ability fromStreptococcus lactis C2 toLeuconostoc cremoris CAF7 yieldsLeuconostoc that ferment lactose and produce diacetyl

Summary Conjugation between lactose-fermenting (Lac⁺)Streptococcus lactis C2 and Lac^– Leuconostoc cremoris CAF7 was performed. The frequency of Lac⁺ transfer was 1.5 · 10^–2 per donor cell. Lac⁺ Leuconostoc transconjugants could ferment lactose significantly faster than wild-type cells. When grown in litmus milk fortified with 0.2% yeast extract, Lac⁺ transconjugants reached pH 4.68 within 24 h at 30°C and produced diacetyl. The identity of the transconjugants asLeuconostoc derivatives was confirmed by their resistance to phage c2 and to vancomycin (>500 g/ml), and by growth on selective medium containing azide. Plasmid profiles of 10 transconjugants showed two unique patterns. A novel enlarged plasmid was found. Southern blot hybridization revealed some homology with the 30 Md Lac⁺ plasmid of donor, recipient and the transconjugants, as well as with some of the remaining plasmids of the donor.Technical Paper No. 7953, Oregon Agricultural Experiment Station.     

Cocolonization of the Rhizosphere by Pathogenic Agrobacterium Strains and Nonpathogenic Strains K84 and K1026, Used for Crown Gall Biocontrol

The crown gall biocontrol agent strain K84 and three mutants derived from it, K1026 (Tra⁻ deletion mutant of pAgK84), K84 Agr⁻ (lacking pAgK84), and K1143 (lacking pAgK84 and pNoc), significantly reduced gall formation caused by two pathogenic strains resistant to agrocin 84 in peach × almond seedlings planted in infested soil. Cocolonization of roots by pathogenic and nonpathogenic strains was observed in these biocontrol experiments under field conditions. In spite of the efficient biocontrol observed, average populations consisting of 10² and 10⁶ pathogenic agrobacteria per g of root were found 8 months after planting. The total numbers of pathogenic bacteria on roots were similar for plants treated with the biocontrol strains and for the untreated plants. Strain K84 and the genetically engineered organism K1026 survived at a level of 10⁶ agrocin 84-producing bacteria per g of root. The population size of genetically engineered strain K1026 was not significantly different than the population size of wild-type strain K84 8 months after root inoculation. Strains K84 and K1026 controlled two pathogens resistant to agrocin 84 without reducing the total number of pathogenic bacteria in the root system. In addition, this study shows that some biological control activity of strain K84 against agrocin 84-resistant pathogens is independent of plasmids pAgK84 and pNoc.     

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.     

Functional Comparison of Conjugative Transposons Tn916and Tn925

During interspecies matings betweenBacillus subtilisandBacillus thuringiensissubsp.israelensis,transfer of conjugative transposon Tn916was detected at a frequency of 1.1 × 10⁻⁴transconjugants per donor. Tn916-dependent transfer of plasmids pC194 and pE194 was detected at frequencies of 1.4 × 10⁻⁵and 3.2 × 10⁻⁷transconjugants per donor, respectively. Similar frequencies were obtained during parallel matings with otherwise isogenic strains that contain Tn925instead of Tn916. Tn916- or Tn925-dependent transfer of plasmids pC194 or pUB110 from the recipient to the donor (retrotransfer) was not observed during inter- or intraspecies matings. Transposon-mediated plasmid transfer by Tn916and Tn925is a Rec independent event. Thus, the data from studies in which otherwise isogenic donor and recipient strains were used indicated that Tn916and Tn925are, from a functional point of view, much more similar than previously suggested.     

Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes

Agrobacterium rhizogenes strain 15834, which incites hairy root disease in plants, harbors three large plasmids: pAr15834a (107 x 10(6) daltons), pAr15834b (154 x 10(6) daltons), and pAr15834c (258 x 10(6) daltons). Kanamycin-resistant transconjugants were selected in a cross of kanamycin-resistant derivate of strain 15834 and an avirulent recipient. The transconjugants belonging to one class were virulent and contained all three donor plasmids. These transconjugants also acquired sensitivity to the bacteriocin agrocin 84. The loss of plasmids from virulent transconjugants during growth at 37 degrees C indicated that virulence genes reside on pAr15834b, whereas agrocin 84 sensitivity genes reside on pAr15834a. The pathology induced by the virulent transconjugants containing only pAr15834b was identical to that produced by the wild-type strain of A. rhizogenes. Restriction endonuclease fragment analysis of plasmids from the transconjugants and the donor revealed that pAr15834c is a cointegrate of pAr15834a and pAr15834b. Kanamycin-resistant transconjugants belonging to a second class were avirulent and contained an altered form of pAr15834b. Strain 15834 can utilize octopine. However, this trait was not detected in any of the transconjugants. Octopine is not synthesized by infected plant tissue.     

Bacterial conjugation betweenEscherichia coli andPseudomonas spp. donor and recipient cells in soil

Summary Experiments conducted in microcosms containing loam soil samples inoculated with eitherE. coli orPseudomonas spp. donor and recipient cells showed that bacterial cells survived and conjugated over a 24-h incubation period.E. coli transconjugants were detected 6 h after donor and recipient strains were introduced into sterile soil samples. In non-sterile soil samples, transconjugants were detected between 8 and 24 h incubation.Pseudomonas transconjugants were recovered from sterile soil samples between 6 and 12 h after their introduction and as early as 2 h in non-sterile soil. The results show that genetic interactions occur in non-sterile soil in relatively short periods of time at relatively high transfer frequencies (10^–3 to 10^–4). Studies on genetic interactions in soil are becoming necessary in risk assessment/environmental impact studies prior to the release of genetically engineered or modified organisms into uncontained environments.     

Comparison of yoghurt, heat treated yoghurt, milk and lactose effects on plasmid dissemination in gnotobiotic mice

The effect of yoghurt, heat-treated fermented milk, milk and lactose solution intake on plasmid transfer and establishment of the resulting transconjugants in the digestive tract of mice colonised with human faecal flora were examined. Yoghurt lowered the population level of transconjugants more efficiently than heat-treated fermented milk (–2 log and –1 log respectively) and indicated a beneficial effect of viable bacteria. On the other hand consumption of milk drastically inhibited the establishment of transconjugants, which were below the detection threshold of 10² UFC per g of faeces. We were not able to recover transconjugants from faecal samples with lactose supplementation, indicating a possible inhibition of plasmid transfer. Since the yoghurt, heat-treated fermented milk, milk and lactose solution contained approximately the same lactose concentration it is fair to speculate that lactose may contribute to the inhibiting effects of the various supplementations. The inhibitions described were not associated with other intestinal parameters like the intestinal transit time, the population levels of the recipient, or the donor and total anaerobic microflora. It is evident that other parameters need to be investigated such as the composition of the endogenous microflora and metabolic products.     

Agrobacterium plasmids encode structurally and functionally different loci for catabolism of agrocinopine-type opines

Summary Agrobacterium tumefaciens strains C58, T37, K827 and J73, A. rhizogenes strains A4 and 15834, and A. radiobacter strain K299 were all susceptible to agrocin 84 and this sensitivity was enhanced in each case by addition of agrocinopines A and B. Analysis of transconjugants showed that sensitivity of strain A4 to agrocin 84 was encoded by pArA4a and not by the rhizogenic plasmid, pRiA4. The acc region of the A. tumefaciens nopaline-type Ti plasmid pTiC58, contained on the recombinant plasmid pTHH206, hybridized strongly to restriction fragments of plasmids from strains T37, K827, J73 and K299. Hybridizing fragment patterns generated with BamHI and EcoRI were identical among the four Ti plasmids while pAtK299 showed restriction fragment length polymorphisms at acc with the two enzymes. At moderate stringency, the pTiC58 acc region hybridized weakly to a single restriction fragment from the Ar plasmid of A. rhizogenes strain A4, but not to pTiBo542, which encodes catabolism of the closely related opines agrocinopines C and D. Plasmid pAtK84b of A. radiobacter strain K84 is induced for conjugal transfer by agrocinopines A and B. However, no hybridization was detected between this plasmid and acc from pTiC58 under conditions of moderate stringency. Like pTiC58, pAtK84b conferred transport of agrocinopines A and B on its host bacteria despite the absence of detectable sequence homology with the pTiC58-derived acc probe. However, unlike pTiC58, pAtK84b failed to confer sensitivity to or uptake of agrocin 84 on its bacterial host. These results indicate that at least four distinguishable systems exist for catabolism of the two agrocinopine opine families with the prototype locus, exemplified by acc from pTiC58, being strongly conserved among nopaline-type Ti plasmids.     

Low-Frequency Horizontal Transfer of an Element Containing the Chlorocatechol Degradation Genes from Pseudomonas sp. Strain B13 to Pseudomonas putida F1 and to Indigenous Bacteria in Laboratory-Scale Activated-Sludge Microcosms

The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes (clc) from Pseudomonas sp. strain B13 were investigated in activated-sludge microcosms. When the clc genes were transferred into an appropriate recipient bacterium such as Pseudomonas putida F1, a new metabolic pathway for chlorobenzene degradation was formed by complementation which could be selected for by the addition of mono- or 1,4-dichlorobenzene (CB). Under optimized conditions with direct donor-recipient filter matings, very low transfer frequencies were observed (approximately 3.5 × 10⁻⁸ per donor per 24 h). In contrast, in matings on agar plate surfaces, transconjugants started to appear after 8 to 10 days, and their numbers then increased during prolonged continuous incubation with CB. In activated-sludge microcosms, CB-degrading (CB⁺) transconjugants of strain F1 which had acquired the clc genes were detected but only when strain B13 cell densities of more than 10⁵ CFU/ml could be maintained by the addition of its specific growth substrate, 3-chlorobenzoate (3CBA). The CB⁺ transconjugants reached final cell densities of between 10² and 10³ CFU/ml. When strain B13 was inoculated separately (without the designated recipient strain F1) into an activated-sludge microcosm, CB⁺ transconjugants could not be detected. However, in this case a new 3CBA-degrading strain appeared which had acquired the clc genes from strain B13. The effects of selective substrates on the survival and growth of and gene transfer between bacteria degrading aromatic pollutants in a wastewater ecosystem are discussed.     

A Conjugative Transfer System for the Rumen Bacterium, Butyrivibrio fibrisolvens, Based on Tn916-Mediated Transfer of the Staphylococcus aureus Plasmid pUB110

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.     

Characteristics of Tn5307 exchange and intergeneric transfer of genes associated with nisin production

Transfer of the Lactococcus lactis 11454 nisin-sucrose conjugative transposon, Tn5307, was investigated to develop a methodology for conjugation of this element to other lactic acid bacteria. Tn5307 exchange was sensitive to temperature and pH but was not affected by protease or amylase treatments to donor cells. Moreover, conjugation studies demonstrated that the direct-plate method could be employed to rapidly identify LM2301 transconjugants able to transfer Tn5307 at least ten times more efficiently than 11454. Intergeneric transfer of nisin and sucrose genes between L. lactis and a dairy Enterococcus sp. was also investigated. Erythromycin-resistant Enterococcus sp. recipients were developed by electro-transformation with pGK13 or by conjugal introduction of the broad-host-range plasmid pAM\1. Matings between L. lactis 11454 and an Enterococcus sp. recipient that contained pAM\1 yielded sucrose-positive, nisin-immune transconjugants at a frequency of 2.3 × 10^–9 transconjugants per donor cfu. Agar-overlay assays for nisin production revealed that enterococcal transconjugants did not produce the bacteriocin, but DNA·DNA hybridization with a nisA-specific probe demonstrated that these bacteria had acquired the nisin structural gene.     

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.     

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.     

Mercury resistance determined by a self-transmissible plasmid inBacillus cereus 5

Summary Inducible mercuric reductase activity inBacillus cereus 5 was plasmid-encoded. Plasmid analysis revealed three plasmids with molecular masses of 2.6, 5.2 and 130 MDa. A mating system permitted transfer of the resistance determinant among strains ofB. cereus andB. thuringiensis. Transfer of mercury resistance fromB. cereus 5 toB. cereus 569 andB. thuringiensis occurred during mixed culture incubation on agar surfaces. The 130-MDa plasmid (pGB130) was responsible for transfer; frequencies ranged from 10^–5 to 10^–4.B. cereus 569 transconjugants inheriting pGB130 were also effective donors. High transfer frequencies and the finding that cell-free filtrates of donor cultures were ineffective in mediating transfer suggested mercury-resistance transfer was not phage-mediated. Transfer was also insensitive to DNase activity. Further evidence that pGB130 DNA carried the mercury-resistance determinant was transformation ofB. cereus 569 by electroporation with pGB130 DNA isolated fromB. cereus 5 and a mercury-resistantB. cereus 569 transconjugant. Mercury-resistant transconjugants and transformants exhibited mercuric reductase activity. Plasmid pGB130 also conferred resistance to phenylmercuric acetate.     

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.     

Use of plasmid pULB113 (RP4::Mini-Mu) to construct a genomic map ofAeromonas hydrophila

Plasmid pULB113 (RP4::Mini-Mu) promoted homologous gene transfer inAeromonas hydrophila; transfer of chromosomal markers occurred at frequencies of between 10^–3 and 10^–4 per donor cell regardless of the marker selected; this indicated chromosome transfer from multiple origins. With a variety of amino acid biosynthetic markers, a single circular map of this bacterium was constructed.     

Effect of Bacterial Distribution and Activity on Conjugal Gene Transfer on the Phylloplane of the Bush Bean (Phaseolus vulgaris)

Conjugal plasmid transfer was examined on the phylloplane of bean (Phaseolus vulgaris) and related to the spatial distribution pattern and metabolic activity of the bacteria. The donor (Pseudomonas putida KT2442) harbored a derivative of the TOL plasmid, which conferred kanamycin resistance and had the gfp gene inserted downstream of a lac promoter. A chromosomal insertion of lacI^q prevented expression of the gfp gene. The recipient (P. putida KT2440) had a chromosomal tetracycline resistance marker. Thus, transconjugants could be enumerated by plating and visualized in situ as green fluorescent cells. Sterile bean seedlings were inoculated with donors and recipients at densities of approximately 10⁵ cells per cm². To manipulate the density and metabolic activity (measured by incorporation of [³H]leucine) of the inoculated bacteria, plants were grown at various relative humidities (RH). At 100% RH, the transconjugants reached a density of 3 × 10³ CFU/cm², corresponding to about one-third of the recipient population. At 25% RH, numbers of transconjugants were below the detection limit. Immediately after inoculation onto the leaves, the per-cell metabolic activity of the inocula increased by up to eight times (100% RH), followed by a decrease to the initial level after 96 h. The metabolic activity of the bacteria was not rate limiting for conjugation, and no correlation between the two parameters was observed. Apparently, leaf exudates insured that the activity of the bacteria was above a threshold value for transfer to occur. Transconjugants were primarily observed in junctures between epidermal cells and in substomatal cavities. The distribution of the transconjugants was similar to the distribution of indigenous bacteria on nonsterile leaves. Compared to polycarbonate filters, with cell densities equal to the overall density on the leaves, transfer ratios on leaves were up to 30 times higher. Thus, aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on transfer.