Translocation of sequences encoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez

Summary Salmonella ordonez strain BM2000 carries kanamycin (Km), ampicillin (Ap), spectinomycin (Sp), chloramphenicol (Cm), tetracyline (Tc), and sulfonamide (Su) resistance and production of colicin Ib (Cib). The Km and Cib characters were carried by a 97kb IncI1 plasmid (pIP565). In addition to the Km and Cib traits, all or part of the other antibiotic resistance (R) determinants could be transferred by conjugation from S. ordonez to Escherichia coli where all the acquired characters are borne by an IncI1 plasmid, designated complete or partial composite plasmid respectively. DNA from pIP565 and composite plasmids and total DNA from strain BM2000 were studied by agarose and polyacrylamide gel electrophoresis following digestion with restriction endonucleases, and by Southern hybridization. These comparative analyses enabled us a) to show that acquisition by pIP565 of resistance to all or some of the antibiotics was due to the insertion of a single DNA fragment into the receptor plasmid; b) to detect two types of composite plasmids with regard to the specificity of insertion into pIP565 and the mapping of the inserts; c) to demonstrate that the ApCmSpSuTc resistance determinants were integrated into S. ordonez BM2000 chromosomal DNA; d) to map the restriction fragments of the translocatable sequence integrated into strain BM2000 chromosome or into pIP565.The results obtained suggest that two distinct mechanisms for the translocation of the R determinants coexist in S. ordonez BM2000. Recombination between two of the four directly repeated copies of the IS-like sequence (IS1522) present in S. ordonez chromosome leads to the circularisation of all or part of the AmCmSpSuTc R determinants and is followed by either 1) a second recombination with the copy of IS1522 in pIP565 (Type I composite plasmids), or 2) transposition of precise groups of characters in various sites of pIP565 (Type II composite plasmids).     

Analysis of TwoStaphylococcus epidermidisPlasmids Coding for Resistance to Streptogramin A

The twoStaphylococcus epidermidisplasmids pIP1629 (7.5 kb) and pIP1630 (14.4 kb) contain thevgagene conferring resistance to streptogramin A. All the sequences of pIP1629, except two of the four 22-nt iterons preceding the replication gene, were found in pIP1630. The additional 6.9-kb fragment of pIP1630 is similar to the mobilizableS. epidermidisplasmid pSK639, carrying thedfrA-thyE-orf140operon and thought to replicate by an iteron controlled theta-type replication mechanism. The replication-mobilization elements of pIP1629 and pSK639 are very similar despite having been isolated in France and in Australia, respectively, showing that they are geographically widely dispersed inS. epidermidis.The genethyEencoding thymidylate synthetase carried by pSK639 is not present in pIP1630. pIP1630 probably arose by the recombination of two homologous plasmids carrying distinct resistance determinants.     

Nucleotide sequence of class D tetracycline resistance genes from Salmonella ordonez

Summary Plasmid pIP173, isolated from Salmonella ordonez strain BM2000, confers resistance to tetracycline and a number of other antibiotics. We determined the nucleotide sequence of the pIP173 tetR repressor and tetA resistance genes. The pIP173 tetR gene is essentially identical to the class D tetR gene from plasmid RA1. The pIP173 tet genes are flanked by directly repeated copies of the insertion sequence IS26. Interestingly, the 3 end of the tetR gene, encoding the C-terminal 16 amino acids of the TetR protein, extends into the flanking IS26 sequence. The relationships between the class A, B, C, and D TetA sequences parallel the relationships between the corresponding TetR sequences; class D is more closely related to class B than to either class A or C. Overall, the four TetA sequences show 38% identity and 57% similarity.     

Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant

Summary Streptococcal plasmid pGB301 is an in vivo rearranged plasmid with interesting properties and potential for the molecular cloning of genes in streptococci. Transformation of S. sanguis (Challis) with the group B streptococcal plasmid pIP501 (29.7 kb) gave rise to the deletion derivative pGB301 (9.8 kb, copy number 10) which retained the multiple resistance phenotype of its ancestor (inducible MLS-resistance, chloramphenicol resistance). Among the eight restriction endonucleases used to physically map pGB301 were four that cleaved the plasmid at single sites yielding either sticky (HpaII, KpnI) or bluntends (HpaI, HaeIII/BspRI). Passenger DNA derived from larger streptococcal plasmids (pSF351C61, 69.5 kb; pIP800, 71 kb) was successfully inserted into the HpaII site and, by blunt-end cloning, into the HaeIII/BspRI site. The gentamicin/kanamycin resistance gene of pIP800 was expressed by recombinant plasmids carrying the insert in either orientation. Insertion of passenger DNA into the HaeIII/BspRI site (but not the HpaII site) caused instability of adjacent pGB301 sequences which were frequently deleted, thereby removing the chloramphenicol resistance phenotype. The vector pGB301 has a remarkable capacity for passenger DNA (inserts up to 7 kb) and the property of instability and loss of a resistance phenotype following insertion of passenger DNA into the HaeIII/BspRI site should facilitate the identification of cloned segments of DNA when using this plasmid in molecular cloning experiments.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors

Summary Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66.pIJ101 was found to be self-transmissible by conjugation, to elicit lethal zygosis and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed.Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

Slow growth phenotype - A possible approach to improved plasmid maintenance in Saccharomyces cerevisiae

Summary Transformation-induced slow growth phenotype (SGP) in yeast is repressed in the presence of 2m plasmids. A full 2m-sequence-based recombinant plasmid (pJB502) was found to be more stable in a 2m-free- [cir] strain of Saccharomyces cerevisiae than in a cir⁺ strain. This could not be attributed to differences in growth rate calculated from kinetic analysis of plasmid loss, but transformed [cir] isolates, which had lost the recombinant plasmid, exhibited varying degrees of SGP in batch culture. One of these isolates was outcompeted in chemostat culture by the recombinant-plasmid-containing strain, suggesting that improved plasmid maintenance can result from SGP in cir hosts.     

Narrow host range of some streptococcal R plasmids

Nine R plasmids originally harbored by Streptococcus faecalis (pIP614, pIP655, pIP685, pIP686, pIP 1075, pIP1017),S. faecium (pIP716, pIP991), and group B Streptococcus (pMV120) wild-type hosts were transferred by conjugation into various recipients in order to study the extent of their intraspecies, interspecies, and intergeneric host range. Recipients were streptococci of groups A, B, C, D (S. faecalis, S. faecium, S. durans, S. bovis), and G, S. sanguis, two S. pneumoniae strains (encapsulated and nonencapsulated), and two strains of different genera, Staphylococcus aureus and Listeria inocua. The plasmids carried different antibiotic resistance markers: tetracycline, high levels of gentamicin and kanamycin or of streptomycin and kanamycin, and chloramphenicol. These R plasmids displayed narrow host ranges. They transferred into S. faecalis recipients and plasmid DNA could be detected in these transconjugants. Occasionally, the R plasmids also transferred into one or more other recipients, but no detectable plasmid DNA could be demonstrated in the new hosts.     

Plasmid-mediated resistance to fosfomycin in Staphylococcus epidermidis

Staphylococcus epidermidis strain BM2641, isolated from a patient, was resistant to penicillin G, methicillin, aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin B-type (MLS) antibiotics, and to high levels of fosmycin. Resistance to forsfomycin and/or to MLS was lost at low frequencies either spontaneously or after curing with novobiocin. The plasmid DNA from BM2641 and its cured derivatives was purified, analyzed by agarose gel electrophoresis and transferred to a nitrocellulose sheet. Comparative analysis of the resistance phenotypes with the plasmid content of the strains indicated that fosfomycin and MLS resistance were encoded by plasmids pIP1842 (2.5 kb) and pIP1843 (2.6 kb), respectively. Southern hybridization with a probe specific for gene fosA of Serratia marcescens showed that the fosfomycin resistance determinant in Staphylococcus is not homologous to that of Gram-negative bacteria.     

Evaluation of Plasmid Content and Tetracycline Resistance Conjugative Transfer in <Emphasis Type="Italic">Enterococcus italicus</Emphasis> Strains of Dairy Origin

Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 harbouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal species was able to acquire antibiotic resistance by conjugation, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid.     

A small cadmium resistance plasmid isolated from Staphylococcus aureus

We have isolated from Staphylococcus aureus a plasmid named pIP983, which measures 3.2 kb and specifies resistance to cadmium. The cad gene it carries is of the B type, as indicated by the level of resistance it confers on S. aureus and the sequence homology with the known cadB gene. Sequences homologous to pIP983 were found on several large S. aureus plasmids. They were localized close to the mcr region of pI/258 and pII147, and, at least in the case of the latter plasmid, were not contiguous, but interrupted by nonhomologous DNA.     

A nonconjugative mobilizable broad host range plasmid of Acinetobacter sp. that determines HgCl2 resistance

Summary HgCl₂-resistant strains of Acinetobacter sp. obtained from the soil at the Khaidarkan mercury mine (Kirghiz SSR) were found to contain, apart from large plasmids (60 kb), a small plasmid (7.5 kb) designated pKL1. It was established by conjugative crosses and transformation that pKL1 is a broad host range mobilizable plasmid and that it carries the Hg^r determinant. The restriction map of pKL1 was constructed; the site of the Hg^r determinant and the regions essential to replication were localized. A comparison of these results with earlier data suggests that microorganisms belonging to one microbiocenosis may carry Hg^r determinants on plasmids with highly different structures and properties.Deceased on July 16, 1985     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

Survey of plasmid profiles of <Emphasis Type="Italic">Shigella</Emphasis> species isolated in Malaysia during 1994–2000

Summary Plasmid profiling was used to characterize 219 strains of Shigellaspecies isolated from sporadic cases of shigellosis in Malaysia during the period 1994–2000. Heterogeneous plasmid patterns were observed in all Shigella spp. There was a correlation between plasmid patterns and serotypes of S. flexneri, S. dysenteriaeand S. sonnei. Five common small plasmids (>20.0 kb) were observed in S. flexneri1b and 2a, whereas six common small plasmids were found in serotype 3a. Some of these plasmids appeared to maintain their existence stably in each individual serotype. Plasmids of size 11.40 and 4.20 kb were present only in S. flexneri2a isolates, whereas the 4.40 kb plasmid was unique for serotype 3a. Large (>150 kb) or mid-range plasmid (20.0–150 kb) was not observed from any S. flexneri1b isolates. Eighty-nine percent of S. flexneriof various serotypes harboured the plasmid of 3.20 kb. All S. dysenteriaetype 2 isolates harboured the 9.00 kb plasmid, while four common small plasmids were found in S. sonneiisolates. The 2.10 kb plasmid was only seen in S. sonnei. Streptomycin resistance in S. dysenteriaetype 2 and multi-drug resistance in S. sonneimay be associated with the 9.00 and 14.8 kb plasmids, respectively. Plasmid profiling provided a further discrimination beyond serotyping and a useful alternative genotypic marker for differentiation of Shigellaspecies. To the best of our knowledge, this is the first report on the plasmid prevalence of the Malaysian Shigellaspecies.     

Restriction enzyme digestion patterns ofFrankia plasmids

Summary After the initial screening of more than 200Frankia strains, the plasmid DNA observed in eight Frankiae was analyzed.In situ lysis was performed to obtain an estimate of their copy number and molecular weight. Four plasmid classes were distinguished, 7–9, 18–20, 30–35 and 50–55 kb. Twelve plasmids were thus analysed with restriction enzymes to determine their plasmid restriction patterns.While someFrankia plasmids with comparable molecular weights were found to be heterologous in their restriction enzyme pattern, an 8 kb plasmid found in bothFrankia sp. ArI3, isolated fromAlnus rubra andFrankia sp. CpI1 isolated fromComptonia peregrina showed undistinguishable fingerprints. Furthermore, an 18 kb plasmid found in the same two strains, also showed homologous restriction enzyme patterns. However, the copy numbers of the two ArI3 plasmids were higher than those of the CpI1 plasmids.Similarly, strains ACN1^AG, , isolated fromAlnus crispa all contained a 50 kb plasmid, and the three plasmids were found upon restriction analysis to be undistinguishable.In one strain, ARgX17c isolated fromAlnus rugosa, it was found through restriction enzyme analysis that two plasmids of a similar molecular weight were in fact heterologous.The possible origin of the homologous plasmids and their potential as specificFrankia markers to be used in ecological studies are discussed.     

Comparative analysis of staphylococcal plasmids carrying three streptogramin-resistance genes: vat-vgb-vga.

Several staphylococcal plasmids (26-45 kb) carry all three streptogramin-resistance (Sg(R)) genes, vat, vgb, and vga. Seven such plasmids harbored by independent strains belonging to three taxa (Staphylococcus aureus, S. simulans, and S. cohnii subsp. urealyticum) were compared and the deleted derivative of one of them, pIP680 (11.3 kb), carrying the three streptogramin-resistance genes was sequenced. The seven native plasmids had in common a 12.1-kb part cocarrying the three Sg(R) genes. Sequence analysis of pIP680 revealed that the simultaneous presence of these three genes has probably resulted from cointegration of two plasmids: (i) a pAMbeta1-like plasmid harboring vat-vgb and whose replication gene has been inactivated by an IS257 insertion and (ii) a functional vga plasmid whose replication is similar to that of two staphylococcal plasmids, pSX267 and pSK41.     

Occurrence of plasmid DNA in the sepiolid squid symbiontVibrio fischeri

Because of the importance of plasmids in many bacterial associations with plants and animals, we determined the occurrence and distribution of plasmid DNA in symbioticVibrio fischeri from the light organ of the sepiolid squidEuprymna scolopes. Analyses of 225 isolates of symbioticV. fischeri from 25 individual squids revealed an overall plasmid-carriage rate of 56%. A large plasmid (39 kb) was detected in 96% of those isolates carrying plasmids, and multiple small plasmids were found to co-occur with one of the large plasmids in 81% of plasmid-carrying strains. In addition, these plasmids appear to be restricted toV. fischeri strains isolated fromE. scolopes and from seawater at sites of squid populations. We were unable to assign a role or function to these plasmids, but they do not carry genes required for the establishment of the light organ symbiosis. We conclude that the essential bacterial symbiotic determinants must be encoded on the chromosome and that the plasmids may carry genes that are important for the survival of theseV. fischeri strains outside of the symbiotic association.     

Conjugative Mobilization of the Rolling-Circle Plasmid pIP823 from Listeria monocytogenes BM4293 among Gram-Positive and Gram-Negative Bacteria

We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.     

Analysis of R-primes demonstrates that genes for broad host range nodulation of Rhizobium strain NGR234 are dispersed on the Sym plasmid

Summary R-prime plasmids carrying regions of the symbiotic (Sym) plasmid of the broad host range Rhizobium strain NGR234 were isolated in intergeneric matings with Escherichia coli K12. Three R-primes carrying approximately 180 kb (pMN23), 220 kb (pMN31) and 330 kb (pMN49) of Sym DNA were characterized in more detail. Restriction enzyme analysis and hybridization studies showed that these R-primes carried large overlapping regions of the Sym plasmid, and had the symbiotic genes (two copies of nifH, D and K; nodA, B, C and D; region II; host specific nodulation (hsn) genes) located over half of the 470 kb Sym plasmid. Only the largest of these R-primes (pMN49) contained the complete nodulation host range of the original parent strain NGR234. This broad host range was shown to be present on plasmid pMN49 by being expressed in Agrobacterium tumefaciens strain A136. Furthermore the R-prime plasmids were shown to contain different regions of distinctive host specific nodulation (hsn) for tropical legume infection and for the nodulation of the non-legume Parasponia. Nodulation of soybeans, however, required an additional region that was not essential for the nodulation of other tropical legumes. Strain NGR234 was also found to nodulate the stem and roots of the tropical legume Sesbania rostrata at a very low efficiency. However, the R-prime mini Sym plasmid constructions enabled a greater efficiency of nodulation of Sesbania rostrata to occur.     

Identification of new sex pheromone plasmids in Enterococcus faecalis.

Summary We describe the identification of the following new sex pheromone plasmids inEnterococcus faecalis: a haemolysin-bacteriocin plasmid, pIP964; three R plasmids, pIP1017, pIP1438 and pIP1440; and two cryptic conjugative plasmids, pIP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with DNA probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids.