Mobilization of the pACYC184 plasmid by hybrid pAS8-121 delta plasmids in Escherichia coli cells

The plasmid pACYC184 is shown to be mobilized for conjugal transfer in Escherichia coli cells by the deleted (Tn7-TcR) derivatives of the hybrid conjugative plasmid pAS8-121 (RP4-Co1E1). Both the mobilized and mobilizing plasmids are autonomously inherited by the recipient cells when the mobilizing plasmid carries single copy of IS8 (the plasmid pAS8-121 delta 16). Cointegrates pAS8-121 delta 16D:: ::pACYC184 are found in the recipient cells with pACYC184 being inserted between two repeats of IS8 if the derivate plasmid pAS8-121 delta 16D having the duplication of IS8 is used to mobilize pACYC184 for conjugal transfer. The insertion of pACYC184 between IS8 repeats in the plasmid pAS8-121 delta 16D eliminates the plasmid ability to be inserted with high frequency into the chromosome of the phototrophic bacterium R. sphaeroides 2R. The cointegrate pAS8-121 delta 16D:: pACYC184 is stable but can be resolved during the transformation deriving the plasmid pACYC184:: IS8. The latter may be used as a probe for isolation and analysis of IS8 DNA sequences and for constructing the vectors on the basis of pACYC184.     

Recombinant plasmid associated cell aggregation and high-frequency conjugation of Streptococcus lactis ML3

Lactose-positive (Lac+) transconjugants resulting from matings between Streptococcus lactic ML3 and S. lactis LM2301 possess a single plasmid of approximately 60 megadaltons (Mdal) which is nearly twice the size of the lactose plasmid of the donor. The majority of these Lac+ transconjugants aggregated in broth and were able to transfer lactose-fermenting ability at a frequency higher than 10(-1) per donor on milk agar plates or in broth. Lac+ transconjugants which did not clump conjugated at a much lower frequency. Lactose-negative derivatives of Lac+ clumping transconjugants did not aggregate in broth and were missing the 60-Mdal plasmid. The ability to aggregates in broth was very unstable. Strains could lose the ability to clump but retain lactose-fermenting ability. The majority of these Lac+ nonclumping derivatives of clumping transconjugants contained a plasmid of approximately 33 Mdal, the size of the lactose plasmid of the original donor ML3. These strains transferred lactose-fermenting ability at a frequency of approximately 10(-6) per donor, resulting in both Lac+ clumping transconjugants which contained a 60-Mdal plasmid and Lac+ nonclumping transconjugants which possessed a 33-Mdal plasmid. Our results suggest that the genes responsible for cell aggregation and high-frequency conjugation are on the segment of deoxyribonucleic acid which recombined with the 33-Mdal lactose plasmid in S. lactis ML3.     

Transfer and expression of the tetracyclin resistance transposon Tn925 in Acetobacterium woodii

The Enterococcus faecalis conjugative plasmid pCF10 was used to introduce Tn925 into Acetobacterium woodii by filter mating. Tetracycline resistance was transferred at frequencies of about 10(-6) per donor, but no plasmid DNA was found in the transconjugants. DNA hybridization analyses of HindIII-digested chromosomal DNA demonstrated the insertion of Tn925 at a variety of locations, whereas wild type DNA showed no hybridization at all. The transconjugants were used as donor in mating experiments with tetracycline-sensitive Bacillus subtilis. Transfer of tetracycline resistance was observed at frequencies of 10(-8) per recipient.     

Genetic mapping of the obligate methylotroph Methylobacillus flagellatum: characteristics of prime plasmids and mapping of the chromosome in time-of-entry units.

A pULB113 (RP4::mini-Mu cts) plasmid was used to generate a library of prime plasmids carrying fragments of the Methylobacillus flagellatum genome. The genes carried by these prime plasmids were identified by complementation after transfer to suitably marked Escherichia coli and Pseudomonas aeruginosa strains. The hybrid plasmids were used for complementation mapping with a range of E. coli, M. flagellatum, and P. aeruginosa mutants. A preliminary map of the M. flagellatum genome section with seven groups of linked markers was obtained. Three of seven groups contain an overlapping sequence of cloned genes and can be considered as one large group of linked genes. A high-frequency-of-recombination donor of M. flagellatum (strain MFK64) mobilized the chromosome in a polarized manner from a single transfer origin. The donor was used to construct a time-of-entry map of the M. flagellatum chromosome. This was achieved by determining the time of entry of six randomly dispersed markers, four of which are included in known groups of linked markers. The linear map of M. flagellatum reported here consists of 44 markers.     

Transfer in Marine Sediments of the Naturally Occurring Plasmid pRAS1 Encoding Multiple Antibiotic Resistance

The results of microcosm experiments performed with the fish-pathogenic bacterium Aeromonas salmonicida acting as a donor showed that promiscuous plasmid pRAS1, which encodes tetracycline resistance, is transferred at a high frequency in marine sediments even in the absence of a selective factor. The presence of oxytetracycline resulted in an increase in the transfer frequency compared with that of a microcosm to which no selective factor was added. Transfer frequencies of 3.4 × 10^-1 transconjugant per recipient and 3.6 transconjugants per donor cell were obtained in a microcosm to which oxytetracycline had been added. Hybridization with a DNA probe specific for plasmid pRAS1 revealed that 45.8% of the oxytetracycline-resistant isolates obtained from a microcosm with no selective pressure carried the plasmid, while 86.8% of the isolates obtained from a microcosm to which oxytetracycline had been added carried the plasmid. Phenotypic characterization of the transconjugants revealed that the plasmid had been transferred to a variety of different biotypes in both microcosms. The diversity among the transconjugants isolated from the microcosm to which oxytetracycline had been added was substantially lower than the diversity among the transconjugants isolated from the microcosm to which no selective agent had been added.     

Streptococcal plasmid pIP501 has a functional oriT site.

DNA sequence analysis suggested the presence of a plasmid transfer origin-like site (oriT) in the gram-positive conjugative plasmid pIP501. To test the hypothesis that the putative oriT site in pIP501 played a role in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompassed the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobilized by pVA1702, a derivative of pIP501, at a frequency of 10(-4) to 10(-5) transconjugants per donor cell, while pDL277 was mobilized at a frequency of 10(-8) transconjugants per donor cell. These results indicated that the oriT-like site was needed for conjugal mobilization. To demonstrate precise nicking at the oriT site, alkaline gel and DNA-sequencing analyses were performed. Alkaline gel electrophoresis results indicated a single-stranded DNA break in the predicted oriT site. The oriT site was found upstream of six open reading frames (orf1 to orf6), each of which plays a role in conjugal transfer. Taken together, our conjugal mobilization data and the in vivo oriT nicking seen in Escherichia coli argue compellingly for the role of specific, single-stranded cleavage in plasmid mobilization. Thus, plasmid mobilization promoted by pVA1702 (pIP501) works in a fashion similar to that known to occur widely in gram-negative bacteria.     

Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501.

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among gram-positive and gram-negative bacteria indigenous to the barley rhizosphere

The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 x 10(-2) +/- 3.84 x 10(-2). This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.     

Molecular cloning and transposition of a kanamycin resistance determinant from Campylobacter jejuni between replicons in Escherichia coli

This paper reports a restriction map of a fragment of DNA encoding kanamycin resistance cloned from plasmid DNA of Campylobacter jejuni ABA94 in the recombinant plasmid pRS9421-1. In transposition experiments, kanamycin-resistant R751::km9421 transconjugants appeared at frequencies of 10^-7 per donor cell. These transconjugants harboured a plasmid 4 kb larger than the parental 49 kb plasmid R751. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that the kanamycin resistant determinant had transposed from recombinant plasmid pRS9421-1 to plasmid R751.The authors are with the Department of Genetics and Cellular Biology, Faculty of Science, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Variants of the plasmid pAS8 delta with increased frequency of integration into Rhodopseudomonas sphaeroides chromosome--the result of IS8-element duplication

The possible participation of IS8 and IS elements of Rhodopseudomonas sphaeroides in cointegrate formation by chromosome of the purple bacterium and plasmid pAS8-121 delta has been studied. The plasmid derivatives having deleted Tn7 have been studied. Plasmid integration into the chromosome of the purple bacterium is shown to be mediated by IS8 element of the plasmid. Plasmid derivatives having the integration potential increased for two orders were isolated by a series of intergeneric conjugational crosses during which plasmid pAS8-121 delta was transferred from Rhodopseudomonas sphaeroides (cointegrate of plasmid and chromosome) to Escherichia coli (plasmid in an autonomous state) and back to Rhodopseudomonas sphaeroides. The restriction analysis of plasmid DNA digested by Hpal and Smal restriction endonucleases has revealed the tandem duplications of IS8 in plasmids capable of integration into the chromosome of the purple bacterium with a high frequency.     

Plasmid associated with diplococcin production in Streptococcus.

The ability to produce diplococcin (Dip+) was transferred by conjugation from Streptococcus cremoris 346 to two plasmid-free S. cremoris recipients at a high frequency (10(-1) per donor). Dip+ transconjugants from each mating gained a 54-megadalton plasmid. Spontaneous loss of this plasmid restored the Dip- phenotype.     

Plasmid associated with diplococcin production in Streptococcus

The ability to produce diplococcin (Dip+) was transferred by conjugation from Streptococcus cremoris 346 to two plasmid-free S. cremoris recipients at a high frequency (10(-1) per donor). Dip+ transconjugants from each mating gained a 54-megadalton plasmid. Spontaneous loss of this plasmid restored the Dip- phenotype.     

Transfer and expression of degradative and antibiotic resistance plasmids in acidophilic bacteria.

The genetic accessibility of selected acidophilic bacteria was investigated to evaluate their applicability to degrading pollutants in acidic environments. The IncP1 antibiotic resistance plasmids RP4 and pVK101 and the phenol degradation-encoding plasmid pPGH11 were transferred from neutrophilic bacteria into the extreme acidophilic eubacterium Acidiphilium cryptum at frequencies of 1.8 x 10(-2) to 9.8 x 10(-4) transconjugants per recipient cell. The IncQ antibiotic resistance plasmid pSUP106 was mobilizable to A. cryptum by triparental matings at a frequency of 10(-5) transconjugants per recipient cell. In the transconjugants, antibiotic resistances and the ability to degrade phenol were expressed. A. cryptum AC6 (pPGH11) grew with 2.5 mM phenol at a doubling time of 12 h and a yield of 0.52 g (dry cell weight) per g of phenol. A. cryptum harbored five native plasmids of 255 to 6.3 kb in size. Plasmids RP4 and pVK101 were transferred from Escherichia coli into Acidobacterium capsulatum at frequencies of 10(-3) and 2.3 x 10(-4) and to the facultative autotroph Thiobacillus acidophilus at frequencies of 1.1 x 10(-5) and 2.9 x 10(-6) transconjugants per recipient cell, respectively. Plasmid pPGH11 could not be transferred into the latter strains. T. acidophilus wild type contained six so far cryptic plasmids of 220 to 5 kb.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.     

Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7

Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27. The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc-. Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C. These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible. Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible. Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour). Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids. The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa). A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site. Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites. Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27. Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27.     

Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4-ColE1)

Summary We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by selection of Tra^- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants.Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: trakan-ColE1-amp-tet...Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA (TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8–17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1–9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.     

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.     

Evidence for a dissimilatory plasmid in Azotobacter chroococcum

Dissimilation of 2,4-dichlorophenoxyacetic acid in Azotobacter chroococcum is plasmid mediated. This dissimilatory plasmid designated pMSB1, was effectively cured by mitomycin C. The plasmid was transferred to another strain of Az. chroococcum at a frequency of 2.4 x 10(-3) transconjugants/donor cell. The cured cells did not utilize 2,4-D and its intermediates and lacked the plasmid DNA.     

Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.