Conjugational transfer of the nodulation-conferring plasmid pWZ2 in Rhizobium trifolii

Summary When the nodulating Rhizobium trifolii strain 24Vio^r containing plasmid RP4 was conjugated with the non-nodulating R. trifolii mutant strain 24Str^rNod^-35, plasmid RP4 was transferred at a frequency 10^-3–10^-4. Two out of nearly three thousand tested transconjugants which contained plasmid RP4 had acquired the ability to form nodules on clovers. Molecular studies of the DNA of both these nodulating transconjugants showed the presence of plasmid RP4 and another plasmid which was not found in the original recipient strain. The size of this second plasmid corresponded to that of the plasmid pWZ2, the elimination of which was correlated with irreversible loss of the nodulating ability of R. trifolii strain 24 (Zurkowski and Lorkiewicz 1979). Plasmid RP4 was eliminated from cells by ethidium bromide, without the loss of nodulating properties. The nodulation capacity, however, was eliminated from transconjugants after incubation of bacteria at elevated temperature. Non-nodulating clones obtained after such incubation did not contain the plasmid pWZ2. The results indicate that the plasmid pWZ2 is a necessary element for induction of nodules by R. trifolii, and that it can be mobilized by plasmid RP4.     

Natural Transformation-Mediated Transfer of Erythromycin Resistance in Campylobacter coli Strains from Turkeys and Swine

Erythromycin resistance in Campylobacter coli from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli strains from turkeys than in swine-derived strains (P < 0.01). The frequency of transformation to erythromycin resistance was 10⁻⁵ to 10⁻⁶ for turkey-derived strains but 10⁻⁷ or less for C. coli from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 μg/ml), as did the C. coli donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 μg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals.     

R-Plasmid Transfer to and from Escherichia coli Strains Isolated from Human Fecal Samples

Strains of Escherichia coli recently isolated from human feces were examined for the frequency with which they accept an R factor (R1) from a derepressed fi⁺ strain of E. coli K-12 and transfer it to fecal and laboratory strains. Colicins produced by some of the isolates rapidly killed the other half of the mating pair; therefore, conjugation was conducted by a membrane filtration procedure whereby this effect was minimized. The majority of fecal E. coli isolates accepted the R factor at lower frequencies than K-12 F⁻, varying from 10⁻² per donor cell to undetectable levels. The frequencies with which certain fecal recipients received the R-plasmid were increased when its R⁺ transconjugant was either cured of the R1-plasmid and remated with the fi⁺ strain or backcrossed into the parental strain. The former suggests the loss of an incompatibility plasmid, and the latter suggests the modification of the R1-plasmid deoxyribonucleic acid (DNA). In general, the fecal R⁺E. coli transconjugants were less effective donors for K-12 F⁻ and heterologous fecal strains than was the fi⁺ K-12 strain, whereas the single strain of Citrobacter freundii examined was generally more competent. Passage of the R1-plasmid to strains of salmonellae reached mating frequencies of 10⁻¹ per donor cell when the recipient was a Salmonella typhi previously cured of its resident R-plasmid. However, two recently isolated strains of Salmonella accepted the R1-plasmid from E. coli K-12 R⁺ or the R⁺E. coli transconjugants at frequencies of 5 × 10⁻⁷ or less.     

Intergeneric mobilization of Rhizobium nif genes to Agrobacterium and Klebsiella.

Summary The P-1 incompatibility group plasmid RP1 transfers itself from Escherichia coli J53 to the clover endosymbiont bacterium Rhizobium trifolii strain T1 at low frequency in agar surface matings. R. trifolii T1 R-plasmid recipients display a phenotype identical to the wild-type parent strain in all respects except RP1 antibiotic resistances, allowing straightforward donor counterselection and differentiation of excojugants in further intergeneric plasmid transfer experiments. Hence RP1 can readily transfer itself intergenerically from R. trifolii T1 to the related plant pathogenic organism Agrobacterium tumefaciens and to a strain of the free-living diazotroph Klebsiella pneumoniae.Using R. trifolii T1 (RP1) as donor and as recipient LBA 4006, an avirulent strain of A. tumefaciens lacking the tumour-inducing (Ti) plasmid, selection was made for intergeneric transfer of the R-plasmid and its potential as vector of nitrogen-fixation genes evaluated by subsequent indirect screening. Exconjugant Agrobacteria were obtained which carried RP1 resistance markers and, given specific physiological conditions, would reduce acetylene under air. This is the first report of expression of nif genes in a hybrid strain of A. tumefaciens and is of interest since the Ti plasmid of this organism has been suggested as a natural vector for the introduction of these genes into plants. Plasmid RP1 also cotransferred Rhizobium nif genes to KP52, a strain of K. pneumoniae M5al, with deletion by phage eduction of the chromosomal genes for histidine biosynthesis, one of the nif regulatory genes (nif A), the gene for molybdenum cofactor (nif B) and for an electron transport protein of the nitrogen-fixation pathway (nif F). KP52 exconjugants carried RP1 drug resistances and reduced acetylene under anaerobic conditions.     

Response of RP1+ and RP1− strains ofEscherichia coli to antibacterial agents and transfer of resistance toPseudomonas aeruginosa

The sensitivity of strains ofEscherichia coli, with and without the RP1 R-factor, to antibiotics and other antibacterial agents has been studied. RP1⁺ strains ofE. coli were resistant to kanamycin, carbenicillin, and tetracycline, resistance to the first two antibiotics being produced by destruction of the drugs. This resistance could be transferred to two strains ofPseudomonas aeruginosa. The parent strain ofE. coli UB 1005, its two mutant strains (DC2 and DC3), and two of the strains with the RP1 R-factor showed a similar order of sensitivity to phenylmercuric nitrate, chlorhexidine, thiomersal, and mercuric chloride.E. coli strains DC2 and DC2 (RP1⁺) were the most sensitive to benzalkonium chloride and cetrimide. RP1⁺ strains were more resistant than RP1⁻ strains to lysozyme-ethylenediaminetetraacetic acid, but treatment of the former strains with acriflavine rendered the cells more sensitive to the lytic system. There was no evidence thatP. aeruginosa (RP1⁺) strains possessed increased resistance to polymyxin or to disinfectants, although they became somewhat less sensitive to lysozyme-ethylenediaminetetraacetic acid.     

Transfer of IncP Plasmids to Extremely Acidophilic Thiobacillus thiooxidans

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10^-5 to 10^-7 per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 × 10^-3 per recipient.     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

A Fast and Practical Yeast Transformation Method Mediated by Escherichia coli Based on a Trans-Kingdom Conjugal Transfer System: Just Mix Two Cultures and Wait One Hour

Trans-kingdom conjugation is a phenomenon by which DNA is transferred into a eukaryotic cell by a bacterial conjugal transfer system. Improvement in this method to facilitate the rapid co-cultivation of donor bacterial and recipient eukaryotic cell cultures could make it the simplest transformation method, requiring neither isolation of vector DNA nor preparation of competent recipient cells. To evaluate this potential advantage of trans-kingdom conjugation, we examined this simple transformation method using vector combinations, helper plasmids, and recipient Saccharomyces cerevisiae strains. Mixing donor Escherichia coli and recipient S. cerevisiae overnight cultures (50 μL each) consistently yielded on the order of 10¹ transformants using the popular experimental strain BY4742 derived from S288c and a shuttle vector for trans-kingdom conjugation. Transformation efficiency increased to the order of 10² using a high receptivity trans-kingdom conjugation strain. In addition, either increasing the amount of donor cells or pretreating the recipient cells with thiols such as dithiothreitol improved the transformation efficiency by one order of magnitude. This simple trans-kingdom conjugation-mediated transformation method could be used as a practical yeast transformation method upon enrichment of available vectors and donor E. coli strains.     

Transformation of an R-factor from Pseudomonas aeruginosa into Rhizobium trifolii

Summary The transfer by bacterial transformation of an R-factor giving resistance to kanamycin, ampicillin and chloramphenicol from Pseudomonas aeruginosa into Rhizobium trifolii, strain T1 is described.     

Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis

Either NO₃⁻ (16 millimolar) or NH₄⁺ (1 millimolar) completely inhibited infection and nodulation of white clover seedlings (Trifoliin repens) inoculated with Rhizobium trifolii. The binding of R. trifolii to root hairs and the immunologically detectable levels of the plant lectin, trifoliin, on the root hair surface had parallel declining slopes as the concentration of either NO₃⁻ or NH₄⁺ was increased in the rooting medium. This supports the role of trifoliin in binding R. trifolii to clover root hairs. Agglutination of R. trifolii by trifoliin from seeds was not inhibited by these levels of NO₃⁻ or NH₄⁺. The results suggest that these fixed N ions may play important roles in regulating an early recognition process in the Rhizobium-clover symbiosis, namely the accumulation of high numbers of infective R. trifolii cells on clover root hairs.     

Conjugative Plasmid Transfer between Bacteria under Simulated Marine Oligotrophic Conditions

Marine Vibrio S14 strains and an Escherichia coli strain were starved in artificial seawater (NSS) with no added carbon, nitrogen, or phosphorus. The broad-host-range plasmid RP1 was transferred between the starving S14 strains and also from the E. coli donor to the S14 recipient under oligotrophic conditions, in which mixtures of donor and recipient cells were held on Nuclepore filters either floated on NSS or held such that NSS flowed through the filter. Transconjugants were obtained from S14 donors and recipients starved for at least 15 days before being mixed together for conjugation, whereas transconjugants were recovered from the E. coli donor and S14 recipient for up to 3 days of prestarvation, but not after 5 days. Transconjugants were obtained when there were as few as about 10⁵ and 10⁴ cells of starving S14 donors and recipients, respectively, per ml held on the filters. Starved donor and recipient mixtures incubated at 4 or 26°C, as well as those allowed to mate for 2, 5, or 24 h, all yielded numbers of transconjugants which were not significantly (P > 0.05) different.     

Photoreactivation, Excision, and Strand-Rejoining Repair in R Factor-Containing Minicells of Escherchia coli K-12

Chromosomeless “minicells” are formed by misplaced cell fissions near the polar extremities of an Escherichia coli K-12 mutant strain. Resistance (R)-factor deoxyribonucleic acid (DNA) can be introduced into minicells by segregation from an R⁺ (R64-11) derivative of the original mutant. We have assessed the ability of R⁺ minicells to correct defects produced in their plasmid DNA by ultraviolet (UV) and gamma radiations. Minicells harboring plasmid DNA, in comparison with their repair-proficient minicell-producing parents, possess (i) an equal competence to rejoin single-strand breaks induced in DNA by gamma rays, (ii) a reduced capacity for the photoenzymatic repair of UV-induced pyrimidine dimers, and (iii) a total inability to excise dimers, apparently owing to a deficiency in UV-specific endonuclease activity responsible for mediating the initial incision step in excision repair. Assuming that the DNA repair properties of R⁺ minicells reflect the concentration of repair enzymes located in the plasmid-containing polar caps of entire cells, these findings suggest that: (i) the enzymes responsible for rejoining single-strand breaks are distributed throughout the cell; (ii) photoreactivating enzyme molecules tend to be concentrated near bacterial DNA and to a lesser extent near plasmid DNA; and (iii) UV-specific endonuclease molecules are primarily confined to the central region of the E. coli cell and, thus, seldom segregate with R-factor DNA into minicells.     

Chromosome transfer and R-prime formation by an RP4::mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis

We have introduced into the wide host range conjugative plasmid RP4, a mini-Mu derivative which was known to be able to transpose spontaneously in E. coli K-12, and to induce in such a host several kinds of chromosomal rearrangements including replicon fusions. Unlike RP4, RP4::mini-Mu can mediate the transfer of the host chromosome to a recipient bacterium and generate R primes at high frequencies (10^?4 for the transfer of a given marker, 10^?5 for the formation of R primes carrying a given marker). Two such RP4::mini-Mu plasmids were introduced into one Salmonella typhimurium strain, one Klebsiella pneumoniae strain, and one Proteus mirabilis strain. Each of these three strains were mated with an E. coli K-12 recipient and transconjugants carrying R primes were recovered in all three cases at frequencies ranging from 5 × 10^?6 to 10^?7.     

Efficient Transformation System for Propionibacterium freudenreichii Based on a Novel Vector

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (≥10⁸ colonies per μg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10⁵ transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10⁴ transformants per μg of plasmid DNA for strain ATCC 27405 and ~1 × 10³ transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ~50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10⁵/μg of DNA to 10⁷/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.     

Horizontal transfer of antibiotic resistance genes in a membrane bioreactor

Growing attention has been paid to the dissemination of antibiotic resistance genes (ARGs) in wastewater microbial communities. The application of membrane bioreactors (MBRs) in wastewater treatment is becoming increasingly widespread. We hypothesized that the transfer of ARGs among bacteria could occur in MBRs, which combine a high density of bacterial cells, biofilms, and antibiotic resistance bacteria or ARGs. In this study, the transfer discipline and dissemination of the RP4 plasmid in MBRs were investigated by the counting plate method, the MIDI microorganism identification system, and quantitative polymerase chain reaction (qPCR) techniques. The results showed that the average transfer frequency of the RP4 plasmid from the donor strain to cultivable bacteria in activated sludge was 2.76 × 10⁻⁵ per recipient, which was greater than the transfer frequency in wastewater and bacterial sludge reported previously. In addition, many bacterial species in the activated sludge had received RP4 by horizontal transfer, while the genera of Shewanella spp., Photobacterium spp., Pseudomonas spp., Proteus spp., and Vibrio spp. were more likely to acquire this plasmid. Interestingly, the abundance of the RP4 plasmid in total DNA remained at high levels and relatively stable at 10⁴ copies/mg of biosolids, suggesting that ARGs were transferred from donor strains to activated sludge bacteria in our study. Thus, the presence of ARGs in sewage sludge poses a potential health threat.     

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae Cell Lysates

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp^r gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per μg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.     

Isolation of Lightning-Competent Soil Bacteria

Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl₂) or an electrical (electroporation) method. However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil. In this paper, we report on the isolation of two “lightning-competent” soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tc^r, Sp^r, Sm^r). The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E. coli DH10B and Pseudomonas fluorescens C7R12. The electrotransformation frequencies measured reached 10⁻³ to 10⁻⁴ by electroporation and 10⁻⁴ to 10⁻⁵ by simulated lightning, while no transformation was observed in the absence of electrical current. Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp. strains.     

Different pathways of choline metabolism in two choline-independent strains of Streptococcus pneumoniae and their impact on virulence

The two recently characterized Streptococcus pneumoniae strains—R6Chi and R6Cho⁻—that have lost the unique auxotrophic requirement of this bacterial species for choline differ in their mechanisms of choline independence. In strain R6Chi the mechanism is caused by a point mutation in tacF, a gene that is part of the pneumococcal lic2 operon, which is essential for growth and survival of the bacteria. Cultures of lic2 mutants of the encapsulated strain D39Chi growing in choline-containing medium formed long chains, did not autolyze, had no choline in their cell wall, and were completely avirulent in the mouse intraperitoneal model. In contrast, while the Cho⁻ strain carried a complete pneumococcal lic2 operon and had no mutations in the tacF gene, deletion of the entire lic2 operon had no effect on the growth or phenotype of strain Cho⁻. These observations suggest that the biochemical functions normally dependent on determinants of the pneumococcal lic2 operon may also be carried out in strain Cho⁻ by a second set of genetic elements imported from Streptococcus oralis, the choline-independent streptococcal strain that served as the DNA donor in the heterologous transformation event that produced strain R6Cho⁻. The identification in R6Cho⁻ of a large (20-kb) S. oralis DNA insert carrying both tacF and licD genes confirms this prediction and suggests that these heterologous elements may represent a “backup” system capable of catalyzing P-choline incorporation and export of teichoic acid chains under conditions in which the native lic2 operon is not functional.