Conjugal transfer of R plasmids to and from Enterobacteriaceae isolated from sewage.

The potential of the transfer of natural plasmids between sewage strains has been studied. In vitro transfer was conducted at 37 degrees C in tryptone soya broth and sterile raw sewage as mating media. In situ transfer was carried out in sterile raw sewage within membrane diffusion chambers at 10.6 degrees C. When the recipient was a laboratory strain of Escherichia coli K-12, the in situ frequency values were significantly lower (P less than 0.001) than those obtained in vitro for the same mating pair. When the laboratory recipient was replaced with recipients from the same sewage source, frequency values decreased progressively from the optimum conditions to the most adverse. However, in situ frequency values were higher than those for the same donors mated with a laboratory recipient.     

Conjugal transfer of R plasmids to and from Enterobacteriaceae isolated from sewage

A. FERNANDEZ-ASTORGA, A. FERNANDEZ DE ARANGUIZ, M. POCINO, A. UMARAN AND R. CISTERNA. 1992. The potential of the transfer of natural plasmids between sewage strains has been studied. In vitro transfer was conducted at 37°C in tryptone soya broth and sterile raw sewage as mating media. In situ transfer was carried out in sterile raw sewage within membrane diffusion chambers at 10.6°C. When the recipient was a laboratory strain of Escherichia coli K-12, the in situ frequency values were significantly lower ( P < 0.001) than those obtained in vitro for the same mating pair. When the laboratory recipient was replaced with recipients from the same sewage source, frequency values decreased progressively from the optimum conditions to the most adverse. However, in situ frequency values were higher than those for the same donors mated with a laboratory recipient.     

Influence of salts and temperature on the transfer of mercury resistance from a marine pseudomonad to Escherichia coli

Thirty-one strains of marine bacteria were examined for their ability to transfer mercury resistance to Escherichia coli in complex media; eight strains were able to transfer their resistance marker, with frequencies ranging from 10(-3) to 10(-8). Frequencies generally increased with the increase of the mating period. Additional mating experiments were carried out with one strain, belonging to the pseudomonads, to estimate the influence of temperature, salinity, and time on the conjugal transfer frequency of mercury resistance markers. The higher frequencies occurred at 30 degrees C, in a salt medium (37%), after 24 h of mating.     

Influence of salts and temperature on the transfer of mercury resistance from a marine pseudomonad to Escherichia coli.

Thirty-one strains of marine bacteria were examined for their ability to transfer mercury resistance to Escherichia coli in complex media; eight strains were able to transfer their resistance marker, with frequencies ranging from 10(-3) to 10(-8). Frequencies generally increased with the increase of the mating period. Additional mating experiments were carried out with one strain, belonging to the pseudomonads, to estimate the influence of temperature, salinity, and time on the conjugal transfer frequency of mercury resistance markers. The higher frequencies occurred at 30 degrees C, in a salt medium (37%), after 24 h of mating.     

The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis

Summary The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon, Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.Deceased, 7/89. O. Torres and R. Korman contributed equally to this work     

Utilization of a thermosensitive episome bearing transposon TN10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi

A thermosensitive episome bearing the transposon Tn10, F(Ts)::Tn10 Lac+, has been successfully transferred from Escherichia coli to several wild strains of the enterobacteria Erwinia carotovora subsp. chrysanthemi, which are pathogenic on Saintpaulia ionantha. In one of these strains, all of the characters controlled by this episome (Lac+, Tetr, Tra+) were expressed, and its replication was stopped at 40 degrees C and above. At 30 degrees C, the episome was easily transferred between strains derived from E. carotovora subsp. chrysanthemi 3937j and to E coli. Hfr donor strains were obtained from a F' strain of 3937j by selecting clones which grew at 40 degrees C on plates containing tetracycline. One of these strains, Hfrq, was examined in more detail: the characters Lac+ and Tetr were stabilized and did not segregate higher than its parental F' strain. The mating was most efficient at 37 degrees C on a membrane. Hfrq transferred its chromosome to recipient strains at high frequency and in a polarized fashion, as evidenced by the gradient of transfer frequencies, the kinetics of marker entry (in interrupted mating experiments), and the analysis of linkage between different markers. The chromosome of Hfrq was most probably transferred in the following sequence: origin...met...xyl...arg...ile...leu...thr...cys...pan...ura...gal...trp...his. ..pur... Moreover, this genetic transfer system proved to be efficient in strain construction.     

Efficient Transfer of the Pheromone-Independent Enterococcus faecium Plasmid pMG1 (Gmr) (65.1 Kilobases) to Enterococcus Strains during Broth Mating

Plasmid pMG1 (65.1 kb) was isolated from a gentamicin-resistant Enterococcus faecium clinical isolate and was found to encode gentamicin resistance. EcoRI restriction of pMG1 produced five fragments, A through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. The clockwise order of the fragments was ACDEB. pMG1 transferred at high frequency to Enterococcus strains in broth mating. pMG1 transferred between Enterococcus faecalis strains, between E. faecium strains, and between E. faecium and E. faecalis strains at a frequency of approximately 10⁻⁴ per donor cell after 3 h of mating. The pMG1 transfers were not induced by the exposure of the donor cell to culture filtrates of plasmid-free E. faecalis FA2-2 or an E. faecium strain. Mating aggregates were not observed by the naked eye during broth mating. Small mating aggregates of several cells in the broth matings were observed by microscopy, while no aggregates of donor cells which had been exposed to a culture filtrate of E. faecalis FA2-2 or an E. faecium strain were observed, even by microscopy. pMG1 DNA did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. These results indicate that there is another efficient transfer system in the conjugative plasmids of Enterococcus and that this system is different from the pheromone-induced transfer system of E. faecalis plasmids.     

Inheritance of nocardiophage phi EC in matings of nocardial lysogens.

Lysogens of Nocardia erythropolis were mated with nonlysogenic strains to study the inheritance of the phi EC prophage. Crosses between lysogenic strains of the Mat-Ce mating type and nonlysogenic Mat-cE strains produced Mat-cE lysogens at a recovery rate of 17%, whereas recombination frequencies between chromosomal traits were about 2.3 x 10(-5). Crosses of lysogenic Mat-cE mating types with nonlysogenic Mat-Ce produced Mat-Ce lysogens at a recovery rate of 19%, whereas recombinants for chromosomal traits were recovered at only 1.8 x 10(-5). Crosses of homologous mating types, lysogenic Mat-Ce with nonlysogenic Mat-Ce or lysogenic Mat-cE with nonlysogenic Mat-cE, failed to transfer the prophage. It was concluded that the phi EC prophage exists as a plasmid and can be transferred at high frequencies with patterns of transfer controlled like typical nocardial fertility. Evidence that the prophage may also exist as an integrated element was observed from recombination analyses.     

Chromosome Transfer from F-lac+ Strains of Escherichia coli K-12 Mutant at recA, recB, or recC

The frequency of chromosome transfer from various recombination-deficient F-lac(+) donor strains was estimated by standardizing the yield of conjugants receiving a male chromosomal marker against the level of episome transfer in the mating mixture. The efficiency of chromosome transfer from newly formed F-lac(+) cells carrying recB21 or recC22 was more than 50% of the wild-type value, although it was about 10 and 20%, respectively, if the male cell lines had become established. In contrast, recA13 donors transmitted the chromosome with less than 10(-4) of the normal frequency. If chromosome transfer from F-lac(+) strains reflects the cutting and subsequent joining of homologous single strands of episomal and chromosomal deoxyribonucleic acid by recombination, these results imply that the completed unions are not made in recA cells, but can be effected with more than 50% of normal efficiency in newly formed partial diploids mutant at either recB or recC. Thus, the defective stage in recA mutants may precede strand joining, whereas the deficiency in recB or recC cells may involve a later step in recombinant formation.     

Conjugational transfer of genes determining plant virulence in Erwinia amylovora.

A stable virulent donor strain (EA 178R1-99) of Erwinia amylovora can transfer, by conjugation during a 3-h mating period, the gene or genes which determine(s) plant virulence to avirulent recipient strains (EA178-M64S1 and EA178-M173S1) of Escherichia amylovora. The virulence of over 200 recombinant clones was tested; they all were as virulent on immature Bartlett pear fruits (and, in the smaller series of strains tested, also, on Pyracantha twigs) as was the parent donor strain. Although the avirulent recipeint strains are amino acid auxotrophs, addition of the required amino acids to the inocula in plant virulence trials does not of itself restore virulence. Two small series of prototrophic revertant clones were selected from the auxotrophic avirulent recipient strains; only nine of the 21 prototrophic revertant clones regained virulence, whereas the other 12 prototrophic revertant clones remained avirulent, again suggesting a lack of parallelism between nutritional status and virulence in this system. Preliminary interrupted mating trials, carried out at 15-min intervals over 3 h, show that ser is transferred during the first 15 min, that pro starts entering at about 75 min (and with a higher frequency later), and that lac (originating from an integrated Escherichia coli F'lac) enters toward the end of the 3-h mating period and at a reduced frequency compared to the other markers. The gene or genes which determine(s) plant virulence in this Escherichia amylovora donor strain appear(s) to be transferred readily and seemingly completely to recipient strains during the first 15 min of a 3-h mating period. Exposure of the virulent donor strain to acridine orange or ethidium bromide does not result in loss of virulence, suggesting (but, of course, not proving conclusively) that the determinant(s) of virulence in Escherichia amylovora might be chromosomal rather than extrachromosomal.     

Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin

These studies represent the first systematic survey of the incidence of conjugative antibiotic resistance in Clostridium perfringens. Ninety-two antibiotic-resistant porcine strains were examined to see if they could donate their antibiotic-resistance determinants to sensitive recipient strains. Fifteen of the 89 tetracycline-resistant strains transferred their tetracycline resistance in mixed-plate mating experiments but no transfer of macrolide-lincosamide resistance was detected. The efficiencies of transfer of tetracycline resistance varied from 1.3 X 10(-3) to 1.9 X 10(-6) transconjugants per donor cell. Significantly higher transfer efficiencies were observed when both the donor and recipient strains were derivatives of strain CW 362. These values ranged from 3.7 X 10(-1) to 4.6 X 10(-2) transconjugants per donor cell. This high frequency transfer system should prove invaluable for further genetic studies on this microorganism.     

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.     

Interference of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains in the In Vitro Conjugative Transfer of R-Plasmids

Probiotic compounds, which are often constituted of lactobacilli, exert a number of health benefits through maintenance of the intestinal ecosystem balance. Among the important interactions that occur in the gut microbiota, plasmid transfer by mating is an increasing cause of concern, particularly when antibiotic-resistant genes are involved. Because lactobacilli seem to be able to influence this mechanism, the aim of the present work was to investigate the in vitro capability of two Lactobacillus plantarum strains (one bacteriocin producer and one nonproducer) to interfere with the conjugation processes. For this purpose different matings were performed adding to the donor and recipient cells L. plantarum 35d bac+ and L. plantarum 396/1 bac– as agents of interference. Conjugations added with a Staphylococcus aureus strain or without any agent of interference were used as controls. The results of our experiments demonstrated that both lactobacillus strains were able to decrease mating frequency. Statistically significant differences in the viable transconjugants were obtained in the presence and in the absence of the lactobacilli. The effect was almost the same with the two L. plantarum independent of bacteriocin production. In the trial performed with S. aureus, no decrease in mating frequency was observed, confirming that the capability to interfere with R-plasmid transfer ability could be a property of the tested L. plantarum strains.     

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4-6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.     

Biotic and abiotic factors affecting plasmid transfer in Escherichia coli strains.

The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.     

Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation.

Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3, between strains of B. thuringiensis subsp. israelensis were examined. Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h. Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006. However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B. thuringiensis subsp. israelensis. A specific protease-sensitive coaggregation between strains of B. thuringiensis subsp. israelensis was found to be unambiguously correlated with plasmid transfer. Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies. Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution. Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr- that is cells involved in aggregation formation is presented. Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr- cell. The aggregation phenotype was found to be transferred with high frequency (approximately 100%) in broth matings, and the appearance of Agr- isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid. No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.     

Biotic and abiotic factors affecting plasmid transfer in Escherichia coli strains.

The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.     

Induced variations in microorganisms

Genetic transfer of markers has been found to occur through a sexual cycle between intact cells in a mixed culture of two different genotypes. When rS (radiosensitive and streptomycin resistant) and Rs (radioresistant and streptomycin sensitive) cells ofRhizobium trifolii were brought into varying periods of cellular contact, the cell mixture yielded RS recombinants. The recombination frequency was higher in young mating cultures than in aged ones. Except for the acquisition of the R and S markers, the recombinants did not differ appreciably from the parental strains in other characters.     

中国香菇交配型和基因型的分析

自1987—1990,在长江以南九省(区)采集52个香菇品系,43株制作了孢子印用以检测交配型,又以检测A.B因子位点的等位基因方法测定了担孢子的基因型,保藏了以A_xB_x(或A_xB_y……)标记的标准单核菌丝体,这是建立中国香菇种质库的第一步,将为香菇杂交育种提供有效亲本。结果表明:每一菌株的四个交配型呈随机分配。具有四个交配型的菌株百分率是79%,有二个交配型的香菇菌株百分率是21%。有9个菌株的单核菌丝的基因型从A_1B_1……A_(18)B_(18)不同,A.B因子等位基因差异明显,这些菌株显示了A.B因子的很低的地区性重复频率。本项研究中建立了区分同宗A同宗B以及四个交配型的试验方法,遵循着核移动的规律,这个方法能够重复。     

Transfer of plasmids by conjugation in Streptococcus pneumonias

Transfer of resistance plasmids occurred by conjugation in Streptococcus pneumoniae (pneumococcus) similarly to the process in other streptococcal groups. The 20-megadalton plasmid pIP501 mediated its own DNase-resistant transfer by filter mating and mobilized the 3.6-megadalton non-self-transmissible pMV158. Pneumococcal strains acted as donors or as recipients for intraspecies transfers and for interspecific transfers with Streptococcus faecalis. Transconjugants contained the plasmids expected from their phenotypes and acted as donors for further transfers. Deficiency in an endonuclease essential for entry of transforming DNA did not affect the frequency of transfer. Transfer-deficient mutants of pIP501 have been found.