Distinct groups of plasmids correlated with bacteriocin production in Staphylococcus aureus

The genetic basis of bacteriocin (Bac) production by six strains of Staphylococcus aureus was examined. Gene transfer experiments (in which the plasmids were tagged with the erythromycin resistance transposon Tn551) and plasmid-elimination experiments by growth at 43 degrees C associated bacteriocin production with a particular plasmid in each strain. The Bac plasmids could be separated into two distinct groups: the first comprised plasmids larger than 40 kb, which did not specify immunity to bacteriocins; the second comprised small plasmids (8.0-10.4 kb) which also specified immunity to bacteriocins. The sequence relations among the small plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) were investigated by comparing restriction enzyme digest patterns and by hybridization. Plasmids pRJ10 and pRJ11 were indistinguishable and very closely related to plasmid pRJ9. Plasmid pRJ6, although different from the others, shared regions of sequence homology with them. No homology was found between plasmids pRJ6 or pRJ9 and the large Bac plasmids.     

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators. pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS 257 was not involved in the recombination events which gave rise to pRJ29.     

Interspecific Transfer of Streptomyces Giant Linear Plasmids in Sterile Amended Soil Microcosms

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30°C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 × 10⁻⁴ and 3.6 × 10⁻⁵ CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.     

Mobilization functions of the bacteriocinogenic plasmid pRJ6 of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT _pRJ6 was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT _pRJ6, is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT _pRJ6 of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra _pC223 site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.     

Interspecific transfer of Streptomyces giant linear plasmids in sterile amended soil microcosms

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30 degrees C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 x 10(-4) and 3.6 x 10(-5) CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.     

Incompatibility among α-hemolytic plasmids studied after inactivation of the α-hemolysin gene by transposition of Tn802

Five α-hemolytic plasmids were studied with respect to their molecular and genetic properties. Their molecular weights ranged from 48 to 93 Mdal. Digestion with HindIII restriction endonuclease indicated that they were all clearly different plasmids although similarities in their band patterns were observed. Plasmids pSU1, pSU105, and pSU316 produce F-like pili. Incompatibility studies between Hly plasmids were prevented by lack of markers other than α-hemolysin production. In order to overcome this problem, the inactivating properties of the transposable element Tn802 were used. Several Hly plasmids that have lost the ability to produce α-hemolysin were isolated after insertion of the ampicillin transposon Tn802. Incompatibility between the parental plasmids and their Tn802 derivatives suggests that α-hemolytic plasmids have spread over, at least, four incompatibility groups. Plasmids pSU1 and pSU105 were found to be incompatible with Hly-P212, the only representative, so far, of IncFVI. Plasmid pSU316 was incompatible both with ColB-K98 and R124, which suggests the existance of a FIII-FIV incompatibility complex. In addition, pSU5 and pSU233 were compatible with each other and with pSU316, pSU1, pSU105, and Hly-P212. They also produce a different type of pili from this test plasmids.     

Molecular relationships between trimethoprim R plasmids obtained from human and animal sources

A total of 65 trimethoprim R plasmids (35 obtained from human strains and 30 from animal strains of Escherichia coli ) were examined by the technique of restriction endonuclease fingerprinting. Plasmids belonging to incompatibility groups B, F_II and 1Δ obtained from human and animal sources showed close similarities within each group. Plasmids belonging to incompatibility groups 1α and P were also obtained from both human and animal sources, but there was no conclusive evidence of close relationships within the groups. Restriction endonuclease fingerprinting was found to be useful for obtaining information about the epidemiology of R plasmids. Its main limitation in this study related to broad host range plasmids of the P incompatibility group, some of which contained very few sites susceptible to cleavage by the restriction endonucleases tested.     

Short communication: Construction of a cloning vector for Paenibacillus polymyxa and P. azotofixans

A cloning vector that could replicate in Paenibacillus polymyxa, P. azotofixans and Bacillus subtilis was constructed using two Staphylococcus aureus plasmids. The recombinant plasmid confers chloramphenicol and erythromycin resistance and contains unique restriction sites for PvuII and BclI. The stability of pRJ45 was analysed.     

Control of the production of orthophosphate repressible extracellular enzymes in Neurospora crassa

Summary N-1, a plasmid isolated from a strain ofShigella flexneri in Japan more than 10 years ago, mediates the phage inhibition phenotype which has recently been found to be characteristic of plasmids of the H₂ incompatibility group. Using the criteria of phage inhibition, surface exclusion and incompatibility, the N-1 plasmid is shown to be closely related to H₂ plasmids isolated from non-typhoid salmonella and distantly related to H₁ plasmids isolated fromSalmonella typhi. Plasmids of other incompatibility groups did not show the H₂ type of phage inhibition.     

Nah plasmids of the IncP-9 group in natural Pseudomonas strains

Polymerase chain reaction studies showed that naphthalene-utilizing bacteria isolated from various localities of Belarus most often contained Nah plasmids of the P-9 incompatibility group and plasmids of indefinite systematics. The conventional incompatibility test and restriction enzyme analysis revealed three new IncP-9 subgroups: ζ, η, and IncP-9-like. In addition to the known nucleotide sequences of nahG and nahAc, two novel nahG variants were revealed by a restriction enzyme analysis of amplification products. An amplified rDNA restriction enzyme analysis (ARDRA) demonstrated that the native hosts of IncP-9 Nah plasmids were fluorescent bacteria of the genus Pseudomonas (P. fluorescens, P. putida, P. aeruginosa, and Pseudomonas sp.) and nonfluorescent bacteria of indefinite systematics.     

Molecular relationships between trimethoprim R plasmids obtained from human and animal sources

A total of 65 trimethoprim R plasmids (35 obtained from human strains and 30 from animal strains of Escherichia coli) were examined by the technique of restriction endonuclease fingerprinting. Plasmids belonging to incompatibility groups B, FII and I delta obtained from human and animal sources showed close similarities within each group. Plasmids belonging to incompatibility groups I alpha and P were also obtained from both human and animal sources, but there was no conclusive evidence of close relationships within the groups. Restriction endonuclease fingerprinting was found to be useful for obtaining information about the epidemiology of R plasmids. Its main limitation in this study related to broad host range plasmids of the P incompatibility group, some of which contained very few sites susceptible to cleavage by the restriction endonucleases tested.     

Plasmid-controlled resistance to copper in Escherichia coli.

The copper resistance of a strain of Escherichia coli isolated from the effluent of a piggery where pigs were fed a diet supplemented with copper sulfate was controlled by a conjugative 78-megadalton plasmid designated pRJ1004. Plasmid pRJ1004 exhibited surface exclusion and incompatibility with standard plasmids belonging to incompatibility groups I1 and K. Sensitive strains of E. coli K-12 were unable to form colonies on nutrient agar containing more than 4 mM copper, whereas transconjugants which harbored pRJ1004 were able to form colonies on medium containing up to 20 mM copper.     

Plasmid transfer to Bordetella pertussis: conjugation and transformation

Plasmids of the P and W incompatibility groups were introduced into Bordetella pertussis by conjugation. Plasmid DNA isolated from B. pertussis could be reintroduced by transformation. DNA isolated from Escherichia coli could not be introduced into B. pertussis by transformation if this DNA contained HindIII restriction sites. We have demonstrated that HindIII sites are modified by B. pertussis. Plasmids of the FI and FII incompatibility groups could not be introduced into B. pertussis by conjugation, and nonconjugative plasmids of the ColE1 and Q incompatibility groups could not be introduced by transformation. Our ability to introduce plasmids in the laboratory suggests that the apparent lack of plasmids in natural isolates of B. pertussis is not due to an inability to act as a plasmid recipient.     

In vivo formation of a plasmid cointegrate expressing two incompatibility phenotypes

Plasmids of the N- and M-incompatibility groups were identified in a clinical isolate of Shigella sonnei. The IncN plasmid pDT200 encoded resistance to trimethoprim, streptomycin, and sulfamethoxazole, and specified N-type pili which were synthesized constitutively. The IncM plasmid mediated resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. It was fi⁺ and was repressed for pilus synthesis. Pili specified appeared to be of the FII-type. A recombinant plasmid was formed on transfer of these plasmids from Sh. sonnei to Escherichia coli. The cointegrate plasmid expressed two incompatibility phenotypes and encoded two types of pilus. The incompatibility behavior of the cointegrate plasmid formed in vivo resembled that of composite plasmids constructed by in vitro linkage experiments. The finding that plasmids from clinical isolates may express two incompatibility phenotypes and two types of pilus, and that the pilus type and incompatibility group may not necessarily correspond, has important implications for plasmid classification schemes.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Incompatibility among alpha-hemolytic plasmids studied after inactivation of the alpha-hemolysin gene by transposition of Tn802.

Five α-hemolytic plasmids were studied with respect to their molecular and genetic properties. Their molecular weights ranged from 48 to 93 Mdal. Digestion with HindIII restriction endonuclease indicated that they were all clearly different plasmids although similarities in their band patterns were observed. Plasmids pSU1, pSU105, and pSU316 produce F-like pili. Incompatibility studies between Hly plasmids were prevented by lack of markers other than α-hemolysin production. In order to overcome this problem, the inactivating properties of the transposable element Tn802 were used. Several Hly plasmids that have lost the ability to produce α-hemolysin were isolated after insertion of the ampicillin transposon Tn802. Incompatibility between the parental plasmids and their Tn802 derivatives suggests that α-hemolytic plasmids have spread over, at least, four incompatibility groups. Plasmids pSU1 and pSU105 were found to be incompatible with Hly-P212, the only representative, so far, of IncFVI. Plasmid pSU316 was incompatible both with ColB-K98 and R124, which suggests the existance of a FIII-FIV incompatibility complex. In addition, pSU5 and pSU233 were compatible with each other and with pSU316, pSU1, pSU105, and Hly-P212. They also produce a different type of pili from this test plasmids.     

Inability of Some <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> Strains to Act as Recipients of Plasmid pRAS1 in Conjugal Transfer Experiments

Plasmids belonging to the IncU incompatibility group are mobile genetic elements isolated frequently from Aeromonas spp. These plasmids share structural and functional characteristics and often carry Class-1 integrons bearing antibiotic resistance genes. In this work the ability of two IncU plasmids, pAr-32 and pRAS1 to establish in different A. hydrophila strains after conjugal transfer was studied. In vitro transfer frequencies on solid surface ranged from 10⁻¹ to 10⁻⁶ for pAr-32 and from 10⁻³ to 10⁻⁵ for pRAS1. While carrying out these experiments we detected four strains unable to acquire plasmid pRAS1, indicating that the genetic background of recipients affects the establishment of the plasmid. We explored the possible reasons why these strains failed to yield transconjugants after mating experiments using A. salmonicida 718 as a donor. Factors included donor cell recognition, incompatibility, surface exclusion and restriction of incoming DNA. We found that none of these factors could explain the refractivity of non-receptive A. hydrophila strains to yield transconjugants. Although we do not know the reasons of this refractivity, we may speculate that these isolates lack a product necessary to replicate or stabilize plasmid pRAS1. Alternatively, these strains could contain a product that impedes plasmid establishment.     

Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli

The alpha-hemolytic Escherichia coli strain PM152 harbors three transmissible plasmids, which have molecular weights of 65 X 10(6) (pA152), 41 X 10(6) pHly152), and 32 X 10(6) (pC152). Plasmids pHly152 and pC152 belong to incompatibility groups J2 and N, respectively. By transforming E. coli K-12 with isolated plasmids, we showed that the genetic determinant required for hemolysis was located entirely on plasmid pHly152, and a physical map of this plasmid was constructed. By transposon mutagenesis, a deoxyribonucleic acid segment of about 3.5 X 10(6) daltons was identified as being essential for hemolysis. Most of the EcoRI and HindIII fragments of the hemolytic plasmid pHly152 were cloned by using pACYC184 and RSF2124 as vectors. Two classes of Tn3-induced hemolysis-negative mutants could be complemented by recombinant plasmids carrying fragments from the hemolysis region of pHly152, whereas a third class could be restored to hemolytic activity only by recombination between the mutant plasmids and a suitable recombinant deoxyribonucleic acid. These data suggest that there are at least three clustered cistrons which are required for hemolysis. Other EcoRI and HindIII fragments of pHly152 were identified as being essential for replication, incompatibility, transfer, and restriction.     

Distribution of sequences homologous to the impCAB operon of TP110 among bacterial plasmids of different incompatibility groups

Summary Mutagenic DNA repair is a function of many naturally occurring plasmids belonging to several different incompatibility groups. A DNA probe corresponding to the impCAB operon of the IncIl plasmid TP110, which encodes such functions, was used to investigate the distribution of homologous sequences in both related and unrelated plasmids. Southern blotting was used to demonstrate considerable sequence conservation amongst a number of plasmid types, with imp-related sequences being found on plasmids belonging to the I1, I1/B, B and FIV incompatibility groups. However, no homology was detected amongst plasmids of the N and L/M incompatibility groups, many of which carry functionally similar gene clusters. It appears that sequences determining mutagenic repair functions have been largely conserved within any one incompatibility group, but that significant divergent evolution has occurred between groups.