Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.     

Plasmid-mediated horizontal gene transfer is a coevolutionary process

Conjugative plasmids are key agents of horizontal gene transfer (HGT) that accelerate bacterial adaptation by vectoring ecologically important traits between strains and species. However, although many conjugative plasmids carry beneficial traits, all plasmids exert physiological costs-of-carriage on bacteria. The existence of conjugative plasmids, therefore, presents a paradox because non-beneficial plasmids should be lost to purifying selection, whereas beneficial genes carried on plasmids should be integrated into the bacterial chromosome. Several ecological solutions to the paradox have been proposed, but none account for co-adaptation of bacteria and conjugative plasmids. Drawing upon evidence from experimental evolution, we argue that HGT via conjugation can only be fully understood in a coevolutionary framework.     

Chromosomally integrated conjugative plasmids are common in antibiotic-resistant Haemophilus influenzae.

Twenty-three highly antibiotic-resistant strains of Haemophilus influenzae and two of Haemophilus parainfluenzae without detectable large plasmids were examined for conjugative transfer of their resistance to H. influenzae strain Rd or to other strains. Very inefficient transfer was observed for 18 H. influenzae strains and 1 H. parainfluenzae strain. All H. influenzae transcipients carried a large plasmid, and they were in turn efficient donors of their resistances in standard conjugation crosses with isogenic recipients. This was not seen for the H. parainfluenzae transcipients. It is concluded that most of the original antibiotic-resistant cultures carried an integrated conjugative R plasmid which had been excised in a few cells in each population. It was these cells which transferred resistance in the primary crosses.     

Survey of modifying enzymes and plasmids in amikacin-resistant Serratia marcescens

Forty amikacin-resistant strains of Serratia marcescens isolated from four different hospitals (A, B, C, and D) were examined for modifying enzymes and plasmids. Twenty-one of the isolates produced acetyltransferase that modified amikacin. Eighteen of the 21 acetyltransferase-bearing isolates were from different inpatients in hospital A and the other three were from hospital C. Amikacin resistance was mediated by conjugative plasmid of 24 megadaltons in 15 of the 18 acetyltransferase-bearing isolates of hospital A and by nonconjugative plasmids, derivatives of the 24-megadalton plasmids, in the remaining three isolates of the same hospital. The 24-megadalton plasmid determined aminoglycoside acetyltransferase (6') IV. This plasmid-borne enzyme conferred amikacin resistance on S. marcescens but not on Escherichia coli K12. The frequency of transfer of the 24-megadalton plasmid from the S. marcescens isolate to E. coli K12 by conjugation was approximately 10(-7) (transconjugants/donors) and was 0.1% of that between E. coli strains. In acetyltransferase-bearing isolates from hospital C, the enzyme was mediated by a nonconjugative plasmid in one case and could not be associated with a plasmid in the remaining two cases. Neither enzymes nor plasmids could be associated with amikacin resistance of the isolates of the other two hospitals.     

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.     

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Effect of Genomic Location on Horizontal Transfer of a Recombinant Gene Cassette Between Pseudomonas Strains in the Rhizosphere and Spermosphere of Barley Seedlings

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 × 10⁻³ transconjugants/(donors × recipients)^1/2 in the rhizosphere and 4.57 × 10⁻² transconjugants/(donors × recipients)^1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 × 10⁻³ transconjugants/(donors × recipients)^1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Received: 21 July 2000 / Accepted: 21 August 2000     

Conjugative chromosomal gene transfer in Bacillus subtilis

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet(r)) and lincomycin (lin(r)) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative pl9cat plasmid producing a hybrid plasmid. Insertion frequency was 10(-4)-10(-5). Then genes tet(r) and lin(r) were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F'- and R' plasmids during conjugation in Escherichia coli and other gram negative bacteria.     

Direct stimulation of the transfer of antibiotic resistance by sex pheromones in Streptococcus faecalis

Clinical isolates of Streptococcus faecalis were examined for plasmid content and plasmid-related traits in order to determine whether such strains carried conjugative antibiotic resistance plasmids whose transfer was stimulated by sex pheromones. A 35-Mdalton tetracycline-resistance plasmid called pCF-10 was identified. pCF-10 carries genes that enable its host cell to clump and to act as a highly effective donor when exposed to sex pheromone preparations from recipients. The genetic background of the host cell markedly affects the expression of the pCF-10 fertility functions. The properties of this plasmid make it a good candidate for a detailed genetic analysis of streptococcal conjugation.     

Plasmid-mediated conjugative transfer of Klebsiella sp. rcs genesable to induce colanic acid capsular polysaccharide biosynthesis in Escherichia coli

Regulation of capsular biosynthesis (rcs) genes, encoding the ability to induce the production of a colanic acid polysaccharide capsule, were transferred to Escherichia coli by conjugation with Klebsiella pneumoniae (aerogenes) of capsular serotype K36. Transfer was mediated by a 58.4-MDa conjugative plasmid of incompatibility group IncM, which carried a copy of Tn7 (specifying resistance to trimethoprim and streptomycin) together with determinants for several further resistances. This plasmid did not carry the rcs genes itself, but mediated the conjugative recA-dependent transfer of part of the Klebsiella chromosome to E. coli. Once resident in E. coli, the rcs gene(s) could not be mobilised to other strains of E. coli, and the mobilising plasmid could be cured from capsulate transconjugants without loss of the ability to produce colanic acid. All such cured transconjugants contained an insertion of Tn7 in the chromosome, suggesting that the transposon might be involved in mobilisation of the rcs genes from Klebsiella sp. to E. coli. These findings explain previous observations that the ability to manufacture capsular polysaccharide could be transferred by plasmids between Klebsiella sp. and E. coli.     

Toxin Plasmids of Clostridium perfringens

SUMMARY

In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch''s postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract.     

R plasmids among Gram-negative bacteria with multiple drug resistance isolated in a general hospital.

The incidence of conjugative R plasmids in multiple drug-resistant strains of gram-negative bacteria isolated in 1973 from patients in a 700-bed general hospital in Tokyo and some properties of the R plasmids isolated are described. Conjugative R plasmids were found in 52 of the 96 strains (54%), from which 74 R plasmids were demonstrated. It is remarkable that the isolation frequency of R plasmids mediating quadruple- or five-drug resistance was rather low, and the complete pattern of multiple resistance of the original isolates was only rarely transferred by conjugation. These results revealed the existing state of the distribution of R plasmids among hospital strains with multiple drug-resistance.     

Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron

The Ll.LtrB group II intron from the low-G+C gram-positive bacterium Lactococcus lactis was the first bacterial group II intron shown to splice and mobilize in vivo. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Construction of conjugative gene transfer system between E. coli and moderately thermophilic, extremely acidophilic Acidithiobacillus caldus MTH-04

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-host-range IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-host-range IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The Km(r) and Sm(r) selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.