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.     

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.     

Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

Abstract The role of helper elements in the mobilisation of pBR recombinant plasmids ( tra ⁻, mob ⁻, ori T⁺ and tra ⁻, mob ⁻, ori T⁻) from genetically engineered Escherichia coli K12 strains to other K12-strains and to wild-type E. coli strains of human faecal origin was examined. Transfer experiments were done in the digestive tract of axenic (germ free) and gnotobiotic mice, associated with human faecal flora, HFF. The kinetics of implantation of donors, recipients and transconjugants were determined. Mobilisation of ori T⁺ pBR-type plasmids, by trans-complementation with the products of tra and mob genes was obtained with E. coli K12, in the digestive tract of axenic mice and the resulting transconjugants became established together with the recipient and donor strains. Such mobilisation was only observed sporadically with one E. coli of human origin in axenic mice, but did not occur in gnotobiotic HFF mice. The E. coli strains of human origin were able to promote transfer of an ori T⁻ pBR-type plasmid in vitro but not in axenic or gnotobiotic mice. Transconjugants of wild-type strains obtained in in vitro mating experiments and inoculated into gnotobiotic HFF mice were eliminated as rapidly as the recombinant K12 strains. This work indicates that ≥ 50% of wild-type E. coli strains were able to promote transfer of pBR ori T⁻ plasmids in vitro.     

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 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 .     

Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Conjugal plasmid transfer between lactococci on solid surface matings and during cheese making

Abstract The concept of deliberate use of genetically enginereed microorganisms in dairy products requires a clear understanding of their behaviour and of the dissemination of introduced DNA in these strains. Thus, transfer of a self-transmissible plasmid and a non-self-transmissible but mobilizable plasmid from an engineered strain of Lactococcus lactis subsp. lactis IL 1403 to wild-type strains of L. lactis subsp. lactis and subsp. cremoris of technological interest was studied on standard solid surface matings and in cheese during manufacture. On solid surface matings, transfer of the conjugative plasmid occurred at frequencies ranging from < 2.3 × 10⁻⁹ to 2.8 × 10⁻⁴. Mobilization of the non-conjugative plasmid was observed at a lower frequency (ca. 10⁻⁵) in only one recipient which was then selected along with another recipient strain (presenting intermediate transfer frequencies) for making Camembert cheese. During cheese making, only the transfer of the self-transmissible plasmid was observed. It occurred in the early stages of manufacturing. The transfer frequencies were 7.0 × 10⁻⁸ or 7.6 × 10⁻¹ 1, depending upon the recipient strain. These were about 3 to 4 orders of magnitude lower than on solid surface matings. Mobilization of the non-conjugative plasmid was never detected in cheese.     

Major outer membrane proteins of Prochlorothrix hollandica (Prochloraceae)

Abstract Rhizobium sp. isolated from Lablab purpureus utilized catechol as sole carbon and energy source, a property which is plasmid encoded. The heat curable (39–41°C) plasmid, designated as pAMG1, was transferred to cat⁻ strains of Rhizobium sp. with a transfer frequency of 2.6 × 10⁻⁶ transconjugants/donor cell.     

Transfer of broad host-range plasmids to sulphate-reducing bacteria

Abstract The broad-host-range, IncQ, plasmid R300B (Sm, Su) has been stably transferred to two strains of sulphate-reducing bacteria ( Desulfovibrio sp. 8301 and Desulfovibrio desulfuricans 8312), using the IncP1 transfer system of the helper plasmid pRK2013 and cocultivation of sulphate-reducing bacteria with facultative anaerobes in media provided with sulphate and nitrate ions as electron acceptors. R300B was transferred at a frequency of 10⁻² to 1 per acceptor cell. The Sm^R marker was expressed in both sulphate-reducing bacteria strains while the Su^R was expressed only in strain 8301. R300B can also be transferred back to E. coli strains provided with IncP1 plasmids taking advantage of the retrotransfer ability of these plasmids. This occurs at a frequency up to 10⁻⁴ by recipient E. coli cell.     

Emission rate of amino acids and ammonia and their role in olfactory preference behaviour of juvenile Arctic charr, Salvelinus alpinus (L.)

The behaviour of juvenile Arctic charr, Salvelinus alpinus (L.) , to an abrupt concentration step of L-amino acids, L-alanine and ammonium chloride was studied by fluviarium technique. The emission rates of these substances were studied. Juvenile Arctic charr emit 8.0 × 10⁻⁴ mol total ammonia-N kg⁻¹ h⁻¹ and 3.3 × I0⁻⁵ mol amino acids kg⁻¹ h⁻¹. In behaviour tests the charr avoided 5.6x 10⁻⁶and 5.6 × 10⁻⁷ M ammonium chloride. The 17 L-amino acid mixture, ranked as observed in the analysis of emission, was avoided at 4.6 × 10⁻⁷ M, while 100 times dilution of this value gave neither avoidance nor attraction. The charr avoided L-alanine tested alone at the concentration of 4.6 × 10 ⁻⁷ M. Anosmic charr showed neither avoidance nor attraction to the mixture of 17 amino acids tested at 4.6 × 10⁻⁷ M. The results indicate that ammonia as well as emitted amino acids are not responsible for the olfactory mediated attraction to conspecific odour shown earlier in Arctic charr. On the contrary, these substances may have a negative effect by reducing the strength of attraction.     

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.     

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 .     

Permeability of the Chara cell membrane for ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol

The permeability of internodal cells of Chara australis R. Brown for polyol molecules was determined by using a turgor balance to measure the increase in the osmotic pressure of an internodal cell incubated in artificial pond water containing one of the polyol compounds tested. The permeabilities for ethylene glycol, glycerol, meso -erythritol, xylitol and mannitol were (4. 39 ± 0. 52) × 10⁻⁹, (1. 49 ± 0. 40) × 10¹⁰, (4. 92 ± 0. 27) × 10⁻¹⁰ (9. 9 ± 3. 4) × 10¹¹ and (7. 6 ± 4. 8) × 10⁻¹² m s⁻³, respectively. The permeability for glycerol was slightly smaller than that for meso -erythritol, whose molecular weight is larger than that of glycerol in this homologous series: but the reason for this is not clear.     

Induction of a wheat Em promoter by ABA and optically pure ABA analogs in white spruce (Picea glauca) protoplasts

In white spruce ( Picea glauca ) protoplasts, abscisic acid (ABA) and optically pure ABA analogs induced expression of a reporter gene under regulation of a wheat ABA-responsive promoter. A fusion of a 650 bp promoter fragment from the wheat Em gene promoter and the Escherichia coli uidA sequence encoding β -glucuronidase (GUS) was linked in the plasmid pBM 113Kp. Expression of the Em-uidA fusion varied among 6 white spruce genotypes. Protoplasts from 4-day-old embryogenic suspension cultures gave the highest GUS activity relative 10 other stages in the 7-day growth cycle of suspension cultures. Racemic ABA [R.S-(±)-ABA] induced a significant increase of protoplast GUS activity over background at a concentration of 1 × 10⁻⁵ M , but maximum GUS activity was found at 1 × 10⁻³ M , ABA stereochemistry had a significant effect on gene expression. The natural isomer of ABA [S-(+)-ABA] was an effective inducer at a concentration as low as 1 × 10⁻⁷ M , but a concentration of greater than 1 × 10⁻⁴ M was required for induction by [R-(—)-ABA]. Moreover, analogs with the same configuration at C-l¹ as that of natural ABA were more effective for induction of expression from the Em-uidA . insert at 1 × 10⁻⁴ M than were their enamiomers. Plasnud pBI511. carrying the chloramphenicol acety] transferase (CAT) gene driven by the constitutively expressed, tandemly duplicated cauliflower mosaic virus 35S promoter, was co-electroporated with pBM113Kp for monitoring Ihe influence of addition of exogenous ABA or ABA analogs on heterologous gene expression in protoplasts. CAT activity was not significantly affected by the presence or absence of ABA or the analogs used.     

Selection of strains cured of the Rhizobium leguminosarum Sym-plasmid pRL1JI by using small bacteriocin

Abstract Fast growing Rhizobia are usually insensitive to a low M _r (small) bacteriocin. Introduction of the Sym-plasmid pRL1JI into these strains results in sensitivity towards small bacteriocin. Of such strains, small bacteriocin insensitive mutants (Sbs⁻) were selected.
A high percentage of these Sbs⁻ mutants appeared to be cured of pRL1JI. This selection of cured strains was feasible as well for the wild-type plasmid pRL1JI, as for a variety of transposon marked derivatives in several bacterial backgrounds.     

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.     

A light intensity threshold for schooling in the Atlantic mackerel, Scomber scombrus

The schooling behaviour of Atlantic mackerel was studied in a large tank at different light intensities in the range 12.6–1.8 × 10⁻¹⁰μEs⁻¹ m⁻². Variable light intensity was produced by accurately controlling the current to a green light-emitting diode (LED) 3 m above the experimental tank. Under high light levels (1.8 × 10⁻⁶μEs⁻¹ m⁻²) mackerel always formed a single school, whereas at lower levels (1.8 × 10⁻⁸μEs⁻¹ m⁻²) they swam as individuals. At light levels down to 1.0 × 10⁻⁶μEs⁻¹ m⁻² the mean nearest neighbour distance in a school remained relatively constant (0.3–0.9 body lengths), and individual mackerel swam along a path which deviated from the position of their nearest neighbours by less than 14°. As light dropped below 1.8 × 10⁻⁷μEs⁻¹ m⁻², both nearest neighbour distance and heading angle between nearest neighbours increased, with mean values of 1–1.8 body lengths and 23–92°, respectively, at 1.8 × 10⁻⁹μEs⁻¹ m⁻². The results are discussed in terms of ambient light conditions in the sea.     

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.     

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.     

A demonstration of bacterial conjugation within the alimentary canal of Rhabditis nematodes

Abstract: Rhabditis nematodes fed a diet of Escherichia coli defecate viable undigested bacteria. These bacteria retain phenotypic characteristics, including those encoded on plasmids. Nematodes can survive a 2-min surface sterilization with 2% chlorine bleach; internalized bacteria also survive this treatment and are released in the nematode wastes. Bacteria alone or on the surface of dead nematodes are unable to survive incubation with this solution. There were 3.2 × 10⁵ viable bacteria per nematode, indicating that sufficient bacteria were present for gene transfer. Transconjugants ( lac ⁻ nal ^R str ^R cm ^R) were recovered in the nematode fecal material following a protocol where nematodes were initially fed a plasmidless lac ⁻ nal ^R str ^S cm ^S E. coli and then, after surface sterilization, a lac ⁺ nal ^S E. coli plasmid donor containing the conjugative R100JA ( str ^R cm ^R) plasmid. The presence of plasmids in the transconjugants was confirmed by gel electrophoresis. The occurrence of conjugation in the gut was confirmed by dissection of individual surface-sterilized nematodes and isolation of transconjugants.