Conjugative transfer of plasmid pTO1 from Escherichia coli to Rhodococcus spp.

Summary We demonstrated the conjugative transfer of plasmid pTO1 from Escherichia coli S17-1 to different Rhodococcus spp. The plasmid contains the oriT fragment from RK2 and a fragment of Streptomyces C31 actinophage with the attachment site and the integration genes. Experiments on hybridization showed that plasmid pTO1 is chromosomally integrated into the Rhodococcus cells.     

Conjugative transfer of the Lactococcus lactis sex factor and pRS01 plasmid to Enterococcus faecalis

The low G+C gram-positive bacterium Lactococcus lactis harbours two highly similar conjugative elements: an integrative and conjugative element called sex factor and the pRS01 plasmid. Originally, it was believed that the host range of the sex factor was limited to L. lactis subspecies. Here, it is reported that pTRK28 cointegrates of a spectinomycin-marked L. lactis sex factor and of the pRS01 conjugative plasmid can be transferred from L. lactis to Enterococcus faecalis. These results demonstrate the conjugative transfer of these elements to other bacterial species. Furthermore, it is reported that Ll.LtrB, a mobile group II intron carried by both elements, can invade its recognition site upon pRS01 conjugative transfer to E. faecalis.     

Conjugative transfer of a shuttle plasmid for Escherichia coli to Mycobacterium smegmatis

Plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 10(4) transformants/microgram DNA. Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.     

Conjugative transfer of a plasmid from Escherichia coli to various strains of the order Actinomycetales

The conjugal transfer of autonomous and integrative plasmids from the donor strain Escherichia coli S17-1 to strains of genera Actinomadura, Arthrobacter, Kitasatoa, Micromonospora, Nocardia, Rhodococcus, Saccharopolyspora, and to 16 strains of the genus Streptomyces was demonstrated. The status of plasmids in recipient strains and the stability of their inheritance were analyzed. Plasmids constructed for strains of the genus Streptomyces were shown to function in a large number of strains belonging to the order Actinomycetales. The well-developed system of Streptomyces vector molecules and cloned genes of antibiotic biosynthesis allows their transfer to those microorganisms for which conventional techniques of plasmid transfer by regenerated protoplast transformation or electroporation have not been developed or are inefficient.     

Sex pheromones and plasmid transfer in Enterococcus faecalis

Plasmid-free Enterococcus faecalis excrete peptides (sex pheromones) which specifically induce a mating response in strains harboring certain conjugative plasmids. The response is characterized by the synthesis of a "fuzzy" surface material, visible by electron microscopy, which is believed to facilitate the aggregation of donors and recipients. Transconjugants which receive a specific plasmid shut down the production of endogenous pheromone; however, they continue to produce pheromones specific for donors harboring different classes of plasmids. In this review, we summarize what is known about the biochemistry and genetics of this phenomenon. Some emphasis is given to the hemolysin plasmid pAD1 and the regulation of its conjugal transfer.     

Enterococcus faecalis sufCDSUB complements Escherichia coli sufABCDSE

A total of 985 bacterial strains with different colony characteristics were isolated from the root of tree peony plants (variety 'Fengdan' and 'Lan Furong'); 69 operational taxonomic units were identified by amplified ribosomal DNA restriction analysis. Representatives of each group were selected for partial 16S rRNA gene sequencing and phylogenetic analysis. The major groups in the bulk soil, rhizosphere, and rhizoplane of Fengdan were Firmicutes (63.2%), Actinobacteria (36.3%), and Betaproteobacteria (53.0%), respectively. The major bacteria groups in the bulk soil, rhizosphere, and rhizoplane of Lan Furong were Actinobacteria (34.8%), Gammaproteobacteria (45.2%), and Betaproteobacteria (49.1%), respectively. In total, the bacterial isolates comprised 26 genera--14 in the bulk soil, 14 in the rhizosphere, and 11 in the rhizoplane. The most common genus in the bulk soil of Fengdan and Lan Furong was Bacillus (49.6% and 32.6%, respectively), in the rhizosphere Microbacterium (21.1%) and Pseudomonas (42.0%), and in the rhizoplane Variovorax (53.0% and 49.1%, respectively). The results show that there are obvious differences in the bacterial communities in the three root domains of the two varieties, and the plants exerted selective pressures on their associated bacterial populations. The host genotypes also influenced the distribution pattern of the bacterial community.     

Conjugative transfer of glycopeptide and macrolide resistant genes among Enterococci and from Enterococcus faecalis to Staphylococcus aureus

The resistance determinants were transferred from clinical strains of enterococci to Staphylococcus aureus strains. As recipients methicillin-resistant and methicillin-susceptible strains were used and the filter-mating procedure was performed. The transconjugants resistant to erythromycin were obtained in the case of all recipients, in one case the vanA determinant conferring the resistance to vancomycin was transferred together with erythromycin resistance. However the resistance was very unstable and the level was not as high as in the case of Enterococcus faecalis donor strain. The vanA determinant was easily transferred between enterococcal strains by the conjugation and a transfer occurred of vanA alone or together with erythromycin resistance.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.     

Conjugative transfer of Tn916 in Enterococcus faecalis: trans activation of homologous transposons.

Tn916 [carries tet(M)] is a 16.4-kb conjugative transposon that can establish itself in multiple copies in Enterococcus faecalis. To study the interaction of coresident homologous transposons during conjugation, an E. faecalis mutant defective in homologous recombination was utilized for construction of strains harboring Tn916 delta E (a derivative in which erm is substituted for tet) on the chromosome and Tn916 on a nonconjugative plasmid. When these strains were used as donors, the two transposons were able to transfer independently; however, they were found to transfer and become coestablished in the recipient up to 50% of the time. In contrast, cotransfer of a plasmid marker located outside the transposon occurred at a frequency of no greater than 0.5%. Separate experiments showed that mobilization of the nonconjugative plasmids pAM401 and pVA749 by chromosome-borne copies of Tn916 occurred only at low frequencies (generally less than 2% cotransfer). The data imply that the initiation of transposition of Tn916 results in a trans activation that is specific for homologous transposons present in the same cell.     

Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Sensitivity to detergents and plasmid curing in Enterococcus faecalis

This research reports the sensitivity of a clinical isolate of Enterococcus faecalis to sodium N-lauroylsarcosinate (sarkosyl) and sodium dodecyl sulfate (SDS), as well as the efficiency of these detergents in curing the strain. Compared to Escherichia coli, Enterococcus faecalis was very sensitive to both detergents, with minimum inhibitory concentrations (MIC) for the latter being 100 times lower than for Escherichia coli. The clinical isolate of Enterococcus faecalis used in this study exhibited plasmid-borne resistance to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50 μg/ml); 3% curing was observed after growth in the presence of sarkosyl but no curing was observed after growth in the presence of either SDS or acridine orange. In contrast, 35% curing of plasmid-bearing Escherichia coli was observed after growth in the presence of either SDS or acridine orange, but none was observed after growth in the presence of sarkosyl.     

High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria.

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.     

Pheromone-Responsive Conjugative Vancomycin Resistance Plasmids in Enterococcus faecalis Isolates from Humans and Chicken Feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10⁻³ per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3′), aph(6′), and aac(6′)/aph(2′), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.     

Rate of plasmid transfer among Escherichia coli strains isolated from natural populations.

The conjugative plasmid R1 was introduced into ten strains of Escherichia coli isolated from natural populations. Spontaneous nalidixic-acid-resistant mutants of the ten strains served as recipients. The ten donor and recipient strains were mated in all combinations and the rate at which R1 transferred between the strains was determined. The rate of transfer ranged from 5.2 x 10(-11)-1.1 x 10(-18) ml per cell h-1, and averaged 1.3 x 10(-15) ml per cell h-1. The results of these experiments suggest that the rates of conjugative transfer are far too low for plasmids to be maintained as parasites in their host populations. Infectious transfer is insufficient; plasmids must confer a selective advantage to their host to be maintained.     

Conjugal transfer of plasmid pAM beta 1 in Lactobacillus reuteri and between lactobacilli and Enterococcus faecalis.

The broad-host-range plasmid pAM beta 1 (erythromycin resistance) was transferred conjugally from Streptococcus lactis to Lactobacillus reuteri, L. murinus, and L. fermentum. Transfer of pAM beta 1 between two L. reuteri strains occurred, and lactobacillus transconjugants could act as donors of pAM beta 1 in crosses with Enterococcus faecalis JH2-2.     

Conjugative transfer of plasmid pTd33 in agrobacteria

Supramembrane structures that connect conjugating agrobacterial cells were visualized for the first time by transmission electron microscopy. The primary contact of cells during conjugation was shown to occur through the formation of long pili containing no VirB1 protein. Pretreatment of agrobacterial cells with acetosyringone resulted in a six-to tenfold increase in the transfer frequency of plasmid pTd33 at 19–25°C and had almost no effect at 30°C. The transfer of plasmid pTd33 fromA. tumefaciens strain GV3101 to plasmid-freeA. tumefaciens strain UBAPF-2 was 16 times decreased after the centrifugation of cells. The transfer efficiency of plasmid pTd33 fromA. tumefaciens strain LBA2525 (virB2::lacZ) to plasmid-freeA. tumefaciens strain UBAPF-2 was one order of magnitude lower than the transfer from the wild-typeA. tumefaciens strain GV3101. Treatment of donor cells with 0.01% SDS before mating decreased the transfer efficiency by a factor of 26. The role of pili in the establishment of contact between conjugating cells of agrobacteria is discussed.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

In vivo transfer of pAM beta 1 from Lactobacillus reuteri to Enterococcus faecalis

Trials were conducted to determine the in vivo transferability of plasmid-mediated antibiotic resistance between two strains of enteric Gram-positive bacteria. Germ-free mice were associated with the donor Lactobacillus reuteri DSM 20016 strain, carrying the broad host range pAM beta 1 plasmid, and with the Enterococcus faecalis JH2SS recipient strain. Analysis of faecal content of associated mice demonstrated that the in vivo transfer of this plasmid did occur and that frequencies of conjugation were affected by the presence of subtherapeutic levels of antibiotic in the diet.