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.     

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.     

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp.

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.     

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 multiple antibiotic resistance markers in Streptococcus pneumoniae.

Two antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-encapsulataed) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc [penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae recipients at a low frequency (10(-5) to 10(-6)). The resistance markers transferred were Tc (tetracycline); Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin), and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by chemical agents. All attempts to detect extra-chromosomal deoxyribonucleic acid from pneumococcal or streptococcal transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the wild-type group B Streptococcus host (20 x 10(6)).     

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.     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep, thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep. thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.     

Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25.

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.     

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.     

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.     

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.     

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.     

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.     

Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani

We report that the streptococcal resistance transposon, Tn916, is conjugally transferred to Clostridium tetani (Utrecht) in intergenic matings. Streptococcus faecalis CG180, harboring a 41-kb plasmid (pAM180) containing Tn916 (15 kb), transferred the transposon-associated tetracycline resistance (Tcr) to C. tetani in filter matings at a frequency of about 10(-4)/donor. An erythromycin resistance marker carried by pAM180 was not transferred, indicating lack of plasmid conjugation or stable inheritance of plasmid sequences. DNA extracted from C. tetani transconjugants was probed with radiolabeled Tn916 using Southern blot analysis and these results indicated that the transposon integrated at multiple host genomic sites. Tn916-carrying C. tetani strains were able to transfer Tcr to suitable recipient strains of C. tetani as well as to S. faecalis recipients. These results indicate that this transposon is able to be disseminated and expressed in obligately anaerobic gram-positive bacteria. Moreover, this system opens avenues for the implementation of transposon mutagenesis in this important pathogenic species.     

DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in Pneumococcus

Genes for chloramphenicol resistance (Cm^r) and tetracycline resistance (Tc^r), which are present as heterologous insertions in the chromosomes of some clinical isolates of Streptococcus pneumoniae (pneumococcus) and derivative strains, were transferred at a low frequency to other pneumococci by a DNase-resistant filter mating process that resembles conjugation. Cotransfer of Cm^r and Tc^r was the most common event. Tetracycline resistance was transferred alone from one Tc^r strain or rarely from Cm^rTc^r donors, whereas Cm^r was never transferred alone. Neither the donor strains nor the transconjugants contained detectable plasmids. Transconjugants acted as donors for transformation and for filter mating and had properties similar to those of the parent strain. The presence of the conjugative plasmid pIP501 in the donor did not appear to influence the transfer properties of the Cm^r or Tc^r determinants. No transfer of Cm^r or Tc^r toStreptococcus faecalis JH2-2 was observed.     

Conjugational plasmid transfer from A, B and H streptococci to N streptococci

Plasmid-mediated resistance to erythromycin and chloramphenicol was successfully transferred from group A, B and H streptococci to group N streptococci by a process akin to conjugation. The results showed that plasmids from streptococcal groups other than N were able to replicate in lactic streptococci as well. The transfer experiments were carried out by using a membrane filter mating technique. Four of the five plasmids used (pSM15346, pSM10419, pIP501, and pEL1) were transferred at frequencies ranging from 10(-1) to 10(-8) transconjugants per donor colony-forming unit. The highest transfer frequencies were obtained when S. pyogenes strain 15346 (pSM15346) served as the donor strain. The identy of transconjugants was verified by testing for the presence of unselected markers of the recipient strains, and both transduction and transformation were ruled out as the mechanisms of transfer.     

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus

Abstract Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus . Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis . No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.     

High-frequency chromosome transfer in Rhodopseudomonas sphaeroides promoted by broad-host-range plasmid RP1 carrying mercury transposon Tn501.

Insertion of the mercury resistance transposon Tn501 into broad-host-range plasmid RP1 greatly enhanced the ability of this plasmid to promote chromosome transfer in the photosynthetic bacterium Rhodopseudomonas sphaeroides. Compared with the wild-type RP1, which produced less than 10(-8) recombinants per donor cell, RP1::Tn501 produced between 10(-3) and 10(-7) recombinants per donor cell depending upon the marker selected. Plasmid RP1::Tn501 promoted polarized transfer of the chromosome from one or perhaps two origins on the chromosome, giving rise to two linkage groups. All of the biosynthetic and antibiotic resistance genes that have been mapped, including those involved in photosynthesis, occur on one or another of these linkage groups.