Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria

Abstract We have developed a vector strategy that allows transfer of plasmid DNA by conjugation from Escherichia coli to various Gram-positive bacteria in which transformation via natural competence has not been demonstrated. The prototype vector constructed, pAT187, contains the origins of replication of pBR322 and of the broad host range streptococcal plasmid pAMβ1, a kanamycin resistance gene known to be expressed in both Gram-negative and Gram-positive bacteria, and the origin of transfer of the IncP plasmid RK2. This shuttle plasmid can be mobilised efficiently by the self-transferable IncP plasmid pRK212.1 co-resident in the E. coli donors, and was successfully transferred by filter matings at frequencies of 2 × 10⁻⁸ to 5 × 10⁻⁷ to Enterococcus faecalis, Streptococcus lactis, Streptococcus agalactiae, Bacillus thuringiensis, Listeria monocytogenes and Staphylococcus aureus .     

Transfer of conjugative plasmid pAMβ1 from Lactococcus lactis to mouse intestinal bacteria

Conjugal transfer of plasmid pAMβ1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 × 10⁻³ and with lower frequencies to other species. In vivo , using gastric intubation with the pAMβ1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis . However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 × 10⁻³, in mice whose anus was artificially closed after gastric intubation with pAMβ1-bearing Lactococcus lactis . These results demonstrate clearly that pAMβ1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.     

Conjugative transfer of the transposon Tn919 to lactic acid bacteria

Abstract The streptococcal transposon Tn 919 was transferred from Streptococcus faecalis GF590 to selected Group N Streptococcus strains and to one strain each of Lactobacillus plantarum and Leuconostoc cremoris , using the filter mating method. An S. lactis MG1363 Rif^r Tc^r transconjugant also acted as a donor, but was less efficient than GF590. Frequencies of transfer varied between 4.0 × 10⁻⁸ and 5.29 × 10⁻⁵ per recipient. Further analysis of S. lactis MG1363 Sm^r Tc^r transconjugants showed that insertion of Tn 919 into the chromosome was site-specific.     

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Heterogeneric conjugal transfer of the pheromone-responsive plasmid pIP964 (IncHlyI) of Enterococcus faecalis in the apparent absence of pheromone induction

Abstract Erythromycin-resistant derivatives of the pheromone-responsive plasmid pIP964 from Enterococcus faecalis were constructed to study its host range. This was done by inserting the integrative vector pAT112 and the related replicon pTCR1 harboring oriR of the broad host range plasmid pAMβ1 into the hemolysin-bacteriocin operon of pIP964, to give pTCR2 and pTCR3, respectively. Plasmid pTCR2 was transferred by filter matings from E. faecalis to Enterococcus faecium and Listeria monocytogenes at frequencies of 2×10⁻⁷ and 5×10⁻⁷ per donor, respectively, in the apparent absence of pheromone induction and cellular aggregation. In these hosts, pTCR2 remained intact as a self-replicating element and maintained its transfer capabilities. Plasmid pTCR3, but not pTCR2, was transferred at similar frequencies from E. faecalis to Lactococcus lactis and Streptococcus agalactiae . Thus, the transfer system of pIP964 possesses a broader host-range than its replication system.     

Plasmid transfer between Pseudomonas spp. within epilithic films in a rotating disc microcosm

Abstract A microcosm using rotating slate discs in a chemostat was used to study bacterial population dynamics and genetic interactions in river epilithon. Populations of all introduced donor and recipient Pseudomonas spp. decreased with time but all the bacteria survived better on the slate discs than in the liquid phase. Conjugal transfer of an epilithic plasmid encoding mercury resistance (pQM1) occured with transfer frequencies of 1.4 × 10⁻⁶ to 3.6 × 10⁻³ per recipient, which were about 100-fold lower than in standard membrane filter mating experiments.     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

Conjugal transfer of the determinants for bacteriocin (lacticin 481) production and immunity in Lactococcus lactis subsp. lactis CNRZ 481

Abstract The lacticin 481-producer (Lct⁺), L. lactis subsp. lactis (L. lactis ) CNRZ 481 harbours 5 plasmids of 6.5, 7.5, 20, 37 and 69 kb. Novobiocin treatment of L. lactis 481 led to the appearance of lacticin 481 deficient variants which had all lost the 69 kb plasmid. Conjugal transfer of the lacticin 481 structural gene ( lct ) into the plasmid free strain L. lactis IL1441 yielded Lct⁺ transconjugants at a 10⁻⁴ frequency, which carried a plasmid with an apparent size of 120–130 kb. Southern hybridization analyses showed that the lct gene was located on the 69 kb plasmid in L. lactis 481 and on the 120–130 kb plasmid in the transconjugants. The lct gene was in higher copy number in transconjugants than in the parental strain resulting in two-fold higher lacticin 481 production in the former strain.     

Transfer and maintenance of IncP and IncW group plasmids into and between extra-slow-growing Bradyrhizobium japonicum strains

Abstract IncP group plasmid pRL180 was conjugally transferred from Agrobacterium tumefaciens LBA928 into extra-slow-growing (ESG) Bradyrhizobium japonicum strains and between ESG strains, RJ17W and RJ12S. pRL180 was integrated into the chromosome of RJ12S, RJ17W and RJ19FY. ESG strains efficiently transferred pRL180 into Escherichia coli at about a 3 × 10⁻⁵ frequency. IncW group plasmid pTY97 was transferred in intergeneric matings from E. coli into ESG strains at a high frequency of 2.5 × 10⁻³; between RJ17W and RJ12S transfer was about 5.6 × 10⁻⁴. pTY97 was maintained as an R' plasmid in RJ12S. The R' plasmid was resolved upon transfer into E. coli C where only pTY97 was autonomously replicated.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10⁻¹⁴ (standard error (S.E.) 1.25 × 10⁻¹⁵) ml·cell⁻¹min⁻¹ and 3.32 × 10⁻¹³ (S.E. 4.42 × 10⁻¹⁴) ml·cell⁻¹min⁻¹, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10⁻⁶ to 8.3 10⁻⁶ min⁻¹, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.     

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

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

In vitro and in situ conjugal transfer of an R-plasmid among enterobacteriaceae species isolated from meat samples

Abstract An R-plasmid donor strain of Escherichia coli isolated from a meat sample was mated with potential bacterial recipients belonging to the family Enterobacteriaceae isolated from ground beef and chicken samples. Nine different strains having different plasmid profiles were used as recipients in broth conjugation experiments. The recipients were identified as Enterobacter cloacae, Hafnia alvei, E. coli, Klebsiella pneumoniae and K. oxytoca . Of 1250 ampicillin resistant, tetracycline sensitive colonies tested, the incidence of recipients was estimated to be 3% (in ground beef) and 11% (in chicken) of the bacteria population. Two of the recipients, E. coli and K. Oxytoca also behaved as donors and transferred their R-plasmids to a laboratory recipient strain of E. coli K12-711. In vitro R-plasmid transfer frequencies varied within a wide range, from 10⁻² to 10⁻⁷ among recipients. Generally, frequencies of plasmid transfer were highest at 30°C and declined with decreasing temperature. Three of the recipient isolates, E. cloacae, H. alvei and E. coli displayed transfer of R-plasmids at 10°C in broth matings. Similar trends in R-plasmid transfer frequencies also were observed under in situ mating conditions in raw ground beef and pasteurized milk samples.     

Mobilization of a recombinant nonconjugative plasmid at the interface between wastewater and the marine coastal environment

Abstract The ability of aquatic bacteria isolated from habitats around the outlet of treated wastewater in a coastal marine ecosystem to mobilize the nonconjugative recombinant plasmid pCE328 was studied. A total of 208 strains were screened for their large plasmid content; 51 strains carried at least one large plasmid. Of these, 6 strains from wastewater and 8 from the marine environment were able to mobilize pCE328. Mobilizing strains were isolated from all habitats, and the majority belonged to the genus Aeromonas . The frequencies of mobilization in plate mating experiments ranged from 2 × 10⁻⁷ to 4.4 × 10⁻⁵ per donor at 15°C and 20°C. Mobilization occurred at lower frequencies in microcosm experiments. The results suggest that recombinant DNA released at such interfaces may be transferred rapidly to the autochtonous populations through several bacterial species.     

Plasmid transfer in natural soil: a case by case study with nitrogen-fixing Enterobacter

Abstract In strains of nitrogen-fixing Enterobacter agglomerans , isolated from the rhizosphere of cereals, the nif genes are located on large plasmids. Plasmid pEA9 (200 kb) is self-transmissible between closely related strains. To collect data on possible uncontrolled gene spread, for planned releases of such bacteria, plasmid pEA9 was labelled with transposons (Tn 1725 and Tn 5 ) and used in mating experiments between homologous Enterobacter strains with soil as substrate. The soil was from a plot into which an actual release was being planned. In the majority of experiments it was not sterilized.
Survival and plasmid transfer is described, as are variations in temperature, time, moisture, pH and soil packing. Further experiments were with or without added energy sources, and with or without plant roots. Under standard conditions (22°C, pH 5.2, 15.5% moisture, loose soil, 2 × 10⁷ inoculated donor and recipient cells each per g soil, 3 days incubation) sterilized soil gave low rates of plasmid transfer (10⁻⁶ per donor) but non-sterilized soil gave none. Adding Luria broth or sucrose to non-sterilized soil elicited strong cell propagation, together with plasmid transfer (optimum after incubation for 1 day: 10⁻⁴ exconjugants per donor). No transfer could be registered in the presence of wheat seedling roots for periods up to 5 weeks.     

Diversity and stability of plasmid transfer in isolates from a single field population of Rhizobium leguminosarum bv. viciae

Abstract Isolates of R. leguminosarum bv. viciae from pea and lentil nodules taken at one field site in France were tested in the laboratory for their ability to donate and receive plasmids by conjugation. Five isolates of 20 tested as donors were found to be capable of donating a plasmid which restored the ability to nodulate V. sativa to an isolate which had spontaneously lost this ability. Of 16 isolates tested as recipients all were found to be competent to receive one or more Tn5-labelled test plasmids at a frequency that varied widely (10⁻⁹− 10⁻³ per recipient) dependent upon both the recipient and the plasmid transferred. Three distinct plasmids carrying genes essential for symbiotic functions (pSym) were consistently shown to be transferred at a lower frequency than a cryptic plasmid. Collectively, these results indicate a significant potential for plasmid transfer within the natural soil population. During this work, several independent derivatives were obtained which contained two bv. viciae pSym. These plasmids usually appeared to be compatible together in cells ex planta, but the one acquired in matings was apparently frequently lost (10⁻² per cell) in nodules of V. sativa . Hybrid derivatives containing bv. viciae and bv. phaseoli pSym, apparently retained both plasmids in nodules when P. vulgaris was the host plant but lost the bv. phaseoli pSym at high frequency (4 × 10⁻¹ per cell) in nodules of V. sativa . Structural rearrangements among the plasmids of these transconjugants were also detected in cells recovered from nodules.     

Isolation of chromosomal mutations of Lactococcus lactis subsp. lactis biovar. diacetylactis 18-16 after introduction of Tn919

Abstract: The conjugative transposon Tn 919 was introduced at high frequency to L. lactis subsp. lactis biovar. diacetylactis 18-16 and transconjugants were screened for mutations in two chromosomally located genotypes; citrate metabolism and maltose utilization. A citrate negative mutant, lacking citritase activity, was isolated at a frequency of 1.18 × 10⁻⁴. The mutant, 18-16C5, contained a single copy of Tn 919 in a chromosomal location. A junction fragment of Tn 919 ::18-16C5 chromosomal DNA was cloned in Escherichia coli . Mutations in maltose metabolism were detected at a frequency of 4.0 × 10⁻⁴. No mutants were detected when Tn 919 was not introduced. Reversion to a Mal⁺ phenotype occurred at high frequency, but was not due to Tn 919 transposition.     

Transformation of group A streptococci by electroporation

Abstract The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10³ to 4 × 10⁴ per μg of DNA. The method was also used to introduce an integrative plasmid and transformants were obtained, albeit at a somewhat lower frequency (2 × 10²). Some of the plasmids used in this study are derivatives of the Lactococcus lactis subsp. cremoris Wg2 plasmid pWV01. These broad host range vectors replicate in Gram-positives as well as Gram-negatives (viz. Escherichia coli ). Here we show that they also replicate in S. pyogenes and S. sanguis .     

Conjugation and heterologous gene expression in Gluconobacter oxydans ssp. suboxydans

Abstract: Conjugal transfer of a series of incompatibility group P and Q plasmids has been studied in the acetic acid bacterium, Gluconobacter oxydans ssp. suboxydans . Transfer frequencies for the IncP/Q vectors ranged from 10⁻⁵−10⁻⁹ exconjugants per recipient cell. It was found in the case of the IncP vector, pRK290, that Bgl II insert constructs displayed increased conjugal transfer frequencies over pRK290 per se, the parent plasmid. A gentamycin-resistant encoding pRK290 vector which was constructed offers considerable potential as a versatile gene delivery system for Gluconobacter . The lactose transposon, Tn951, was used as a model to examine heterologous gene expression in G. oxydans ssp. suboxydans . The expression level of Tn951 encoded β-galactosidase in this strain was found to be less than 5% of that found in the parent Escherichia coli strain, JC3272.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.     

Antimicrobial resistance and in vitro gene transfer in bacteria isolated from the ulcers of EUS-affected fish in India

Aims:  The occurrence of drug resistance and plasmid-mediated transferability was investigated in 15 Aeromonas isolates collected from the ulcers of epizootic ulcerative syndrome (EUS)-affected fishes Katla ( Catla catla ), Mrigel ( Cirrhinus mrigala ) and Punti ( Puntius sp.).
Methods and Results:  Disc diffusion assay showed that all the strains were resistant to ampicillin and sensitive to streptomycin. Of the 15 isolates examined, 93·3% isolates were resistant to erythromycin, sulfadiazine and novobiocin, while 66% were resistant to rifampin and 20% to chloramphenicol. All isolates harboured plasmids with sizes ranging from 64 to 23 kbp with a 23-kbp plasmid in common. Plasmids from 11 Aeromonas strains were transferred to Escherichia coli DH5α recipient strain along with the transfer of ampicillin, erythromycin and chloramphenicol resistance determinants with frequencies ranging from 7·0 × 10⁻⁶ to 1·8 × 10⁻⁵ transconjugants per recipient cell.
Conclusions:  The resistance to ampicillin, erythromycin, sulfadiazine, novobiocin and chloramphenicol is prevalent among the bacteria isolated from EUS-affected fish, and resistant determinants of some of these antibiotics have been transferred to the bacteria of other origin.
Significance and Impact of the Study:  The emergence of antibiotic resistance bacteria and gene transfer in vitr o suggests that antibiotics should be used more cautiously to treat Aeromonas infections in aquaculture.