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.     

The molecular relatedness among alpha-hemolytic plasmids from various incompatibility groups

Deoxyribonucleic acid (DNA) reassociation studies among α-hemolytic (Hly) plasmids from FVI and FIII–IV incompatibility groups showed a close similarity between the nucleotide sequences of plasmids from the same group. With respect to R plasmids from the F overgroup, they have 20–26 Mdal in common, an amount of DNA close to the amount involved in the traF operon. No more extensive sequence homology was found between pSU316 (IncFIII–IV) and the incompatible plasmids ColB-K98 (IncFIII) or R124 (IncFIV). The IncIα I2 plasmid pSU5 has only the α-hemolytic region (5 Mdal) in common with plasmid pSU316 but it is much more closely related to IncFVI plasmids where the DNA in common amounts to 22 Mdal. Finally, the genetically unrelated plasmid pSU233 shares 66% of its nucleotide sequences (40 Mdal) with the IncFVI plasmids and has 16–23 Mdal in common with various F-like plasmids.     

Incompatibility properties of the IncFIII/FIV haemolytic plasmid pSU316 when integrated in the Escherichia coli chromosome

The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC::Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA::Tn802) was rapidly lost from strain SU51 both at 30 degrees C and 42 degrees C; (2) the IncFIII plasmid pSU397 (ColB-K98::Tn802) was lost from strain SU51 and at 42 degrees C but not at 30 degrees C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU51. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316.     

Genetics of the replication and maintenance functions of the hemolytic plasmid pSU316. Cloning of an IncFIII determinant

Two miniplasmids have been constructed from pSU306, a Tn802 insertion derivative of the IncFIII-IncFIV hemolytic plasmid pSU316. One of these, pSU3027, is a low copy number plasmid expressing both IncFIII and IncFIV incompatibilities, but is rather unstable, and probably lacks a putative par gene. The other, pSU3025, is maintained in about 340 copies per genome equivalent and expresses only IncFIII incompatibility. Most of the PstI-generated fragments from pSU3027 have been cloned in pBR322. One of the resulting plasmids, pSU3135, contains an insertion of 0.5 kb in the vector molecule, and expresses IncFIII, but not the IncFIV incompatibility. These results allowed us to identify and locate several genes involved in the control of pSU316 replication and stable plasmid maintenance.     

Isolation and evolutionary analysis of a RepFVIB replicon of the plasmid pSU212

We have isolated at least two different replication regions from pSU401, a Tn802 insertion derivative of the IncFVI plasmid pSU212. One of the replication regions (RepFVIB) is highly homologous to the RepFIIA replicon of IncFII plasmids, and thus belongs to the RepFIIA family. We have also cloned the incompatibility determinant (incFVI) and the copy number control gene (cop) from RepFVIB and determined their nucleotide sequences. The analysis of the sequences supports the idea of a modular evolution of RepFIIA plasmids.     

Purification of alpha-hemolysin from an overproducing E. coli strain

Summary The genetic determinant of the -hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions.-hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified -hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the -hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly^- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.     

Physical and functional map of the hemolytic plasmid pSU316

A restriction endonuclease analysis of the hemolytic plasmid pSU316 has allowed location of the cleavage sites for the endonucleases BamHI, XbaI, KpnI, BglII, SalGI, EcoRI, and HindIII. Hybridization experiments between pSU316 and pED100 have shown that the tra region of pSU316 lies in a segment comprising part of SalGI fragments S-1 and S-3 and the entire fragment S-4. The positions of other plasmid coded functions, namely the replication functions and α-hemolysin production, have been determined in the physical map.     

Relationship of the replication and incompatibility genes of an IncFVI haemolysin plasmid with other F-like haemolysin plasmids

The replication and incompatibility region of the IncFVI plasmid pSU502 has been isolated by in vitro DNA manipulation as part of a 12.6 kb plasmid, denominated pSU503. Plasmid pSU503 was strongly incompatible with its parental plasmid, pSU1, but was fully compatible with the haemolytic plasmids pSU316 (IncFIII/IV), pHly152 (IncI2) and pSU233 (Inc-pSU233). Furthermore, the 6.9 kb EcoRI fragment of pSU503 which carries the replication and incompatibility determinants of pSU1 did not show any detectable homology (less than 70%) with any of the haemolysin-determining plasmids with which it is compatible. Thus, homologous haemolysin determinants have become linked to apparently unrelated replicons.     

Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups

By using cloned deoxyribonucleic acid fragments from the hemolysis determinant of the hemolytic plasmid pHly152 as hybridization probes, a deoxyribonucleic acid segment of about 3.8 megadaltons was identified as a common sequence in several hemolytic (Hly) plasmids of Escherichia coli belonging in four different incompatibility groups. This segment contained the genetic information for the synthesis and secretion of the extracellular toxin alpha-hemolysin of E. coli. With the exception of pSU5, representing a composite plasmid, one part of which seems to be very similar to pHly152, the overall sequence homology of these Hly plasmids with pHly152 seems to be rather restricted. However, the Hly plasmid pSU316 showed sequence homology with pHly152 that did not extend beyond the hemolysis determinant. The two other plasmids, pSU233 and pSU105, also shared homology with pHly152 in the hemolysis determinant as well as in various other parts of this plasmid which did not seem to be directly linked to the hemolysis determinant. This suggests that the hemolysis determinant has spread to presumably unrelated plasmids of E. coli.     

Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex

Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac. A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage F0lac h. Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.     

Comparative study of non-conjugative R-plasmids from enterobacteria and Pseudomonas aeruginosa

Nonconjugative R-plasmids pBS76 and pBS94 (Sm Su), pBS95 and pBS96 (Sm Su Ap) isolated from clinical strains of Pseudomonas aeruginosa and plasmids pKMR281-pKMN284 (Sm Su), pKMR285-pKMR286 (Sm Su Tc) isolated from clinical strains of enterobacteria have been studied. Restriction maps of these plasmids are presented in the paper with some of plasmid genes for antibiotic resistance localized on them. The resistance determinants of plasmids pBS95 and pBS96 are shown to be included in transposon Tn3612 analogous to Tn3. Plasmids pBS76, pBS94-96 are of the wide host range and belong to incompatibility group P4 (IncQ). Plasmids pKMR281-pKMR286 are mutually incompatible and share the conspicuous DNA homology. They are inherited only by enterobacteria and are compatible with IncQ plasmids but in contrast to them are mobilized by RP4 plasmid with lower frequency.     

Analysis of IncF plasmids evolution: nucleotide sequence of an IncFIII replication region

We determined the nucleotide sequence of RepFIII, the IncFIII replication region of the plasmid pSU316. Our data confirmed that RepFIII belongs to the RepFIIA family. The comparison of the nucleotide sequences from several RepFIIA-family plasmids revealed that pSU316 and R1 replicons are almost identical (96% similarity). Most of the differences between them are clustered in the incompatibility determinant. Analysis of the rest of the sequence suggested that the divergence between R1 and pSU316 replicons is very recent.     

pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids

A new series of vectors, pSU2716, pSU2717, pSU2718, and pSU2719, has been constructed. The plasmids contain (i) the P15A replicon, (ii) the chloramphenicol acetyl transferase (CAT)-coding gene from Tn9, and (iii) the HaeII fragment which carries the multiple cloning site and the lacZ alpha reporter gene of pUC8, pUC9, pUC18 and pUC19, respectively. These vectors allow rapid and simple transfer of inserts from pUC plasmids, have an intermediate copy number (which allows regulated expression from the lac promoter), and are compatible with ColE1-derived vectors (and, therefore, can be used in studies requiring the joint expression of two genes, for example, in genetic complementation analysis). Furthermore, the accumulation of CAT instead of beta-lactamase, allows an easy visualization in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of proteins of 28-35 kDa, which can otherwise be obscured by the beta-lactamase.     

Incompatibility and surface exclusion properties of H1 and H2 plasmids.

Plasmids of the H incompatibility group showed two types of surface exclusion and incompatibility interactions. Strong incompatibility and surface exclusion were evident between plasmids within the same subgroup, and recombination frequently occurred between these plasmids after antibiotic selection for the presence of two plasmids in the same cell. Weaker interactions were seen between plasmids of the different subgroups, H1 and H2, and recombination was not detected. Incompatibility between H1 and H2 plasmids led preferentially to the loss of the H1 plasmid, irrespective of the order of entry of the plasmids. These data are consistent with the hypothesis that incompatibility is negatively controlled.     

Incompatibility between plasmids with independent copy control

Summary Incompatibility between autonomous plasmids has been attributed, for the most part, to interaction between plasmids' negative control systems and/or partitioning systems. In this report it is shown that indirectly regulated plasmids with non-interactive negative control systems are incompatible on the basis of their shared initiator protein. This principle was demonstrated for a family of Staphylococcus aureus plasmids whose copy number is regulated by inhibitory RNAs that control the production of a rate-limiting, trans-active, initiator protein. We have constructed a pair of plasmids that have the same regulation systems and different intiator proteins and another pair with different regulation systems and the same initiators. Both of these pairs of plasmids were shown to be incompatible.     

Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12

Representative plasmids for most incompatibility groups in Escherichia coli K-12 were transferred to a "bald" strain to compare transfer frequencies for liquid and solid media. Standard broth matings were used for a liquid environment, but for solid surface mating, conjugation was allowed to take place on nutrient plates before washing off the cells for transconjugant selection on plates containing appropriate drugs. Plasmids that determine rigid pili transferred at least 2,000x better on plates than in broth. Some plasmids that determine thick flexible pili transferred 45 to 470x better, whereas others transferred equally well in both environments, as did plasmids of the I complex, which determine thin flexible pili. These results clearly distinguished a number of surface mating systems where most plasmids were derepressed for transfer and determined conjugative pili constitutively. The temperature-independent IncH2 plasmid R831b transferred best on plates, but other IncH plasmids transferred equally well in broth. This inconsistency led to the reclassification of R831b as IncM.     

pHH502, a plasmid with IncP and IncI alpha characters, loses the latter by a specific recA-independent deletion event

Plasmid pHH502, of molecular weight 70 X 10(6), determined resistance to tetracycline, chloramphenicol, trimethoprim, sulphonamides and mercuric chloride and was incompatible with members of IncP and IncI alpha. It resembled other plasmids of IncI alpha in the following properties: it determined pili that were morphologically and serologically I alpha pili, whose production was repressed in established plasmid-carrying (R+) cultures; its transfer was equally efficient in liquid or on solid medium; it exerted surface exclusion against other IncI alpha plasmids; it was non-transferable to Proteus. In a reproducible, recA-independent event, pHH502 gave rise to pHH502-1, a plasmid of molecular weight 40 X 10(6), lacking determinants for resistance to tetracycline and chloramphenicol and all detectable IncI alpha characteristics. pHH502-1 was incompatible only with IncP plasmids and resembled other IncP plasmids in determining constitutive production of rigid pili, in its surface exclusion, in transferring at greater frequency on solid than in liquid medium and in being transmissible to Proteus mirabilis. It differed from other IncP plasmids in the morphology and serological type of its pili and in failing to transfer to Pseudomonas aeruginosa or Acinetobacter calcoaceticus. Small numbers of pHH502-1 rigid pili were present on bacteria carrying pHH502. Possible mechanisms for the generation of pHH502 and pHH502-1 are discussed.     

A method of plasmid classification by integrative incompatibility

A method of plasmid classification by integrative incompatibility has been developed. The characteristics of this system are as follows: (i) The conventional plasmids usually used as standards for incompatibility grouping were integrated into the host chromosome to increase stability and to minimize recombination with the superinfecting plasmid. Strains were constructed by integrative suppression which was in some cases facilitated by the introduction of Tn5 into the plasmid. (ii) The resulting Hfr strains were made deficient in the rec A function to eliminate homologous recombination between the resident and the superinfecting plasmids. A test plasmid is introduced into these rec A Hfr test strains in the stationary phase of growth. In an incompatible cross, the number of transconjugant colonies was usually less than 10^?2 of that in a compatible cross. Occasionally, an inhibitory mechanism, other than incompatibility was coded by the resident plasmid [e.g., restriction in R124 (inc FIV)]. This complicated the interpretation, but did not invalidate the experiment. The colonies arising in incompatible crosses were shown to carry drug resistance determinants coded by both the resident and superinfecting plasmids. These were presumably the result of rec-independent integration of all or part of the superinfecting plasmid into the host chromosome. Thus the reduced frequency of superinfectant formation in an incompatible cross is usually the consequence of incompatibility between the resident and the superinfecting plasmids. This integrative incompatibility system should be useful for epidemiological studies of R plasmids.     

Inhibition of Pore Formation by Blocking the Assembly of Staphylococcus aureus α-Hemolysin Through a Novel Peptide Inhibitor: an In Silco Approach

Staphylococcus aureus self-assembling α-hemolysin heptamer is an acute virulence factor that determines the severity of S. aureus infections. Hence, inhibiting the heptamer formation is of considerable interest. However, both natural and chemical inhibitors reported so far has difficulties related to toxicity, bioavailability, and solubility, which necessitate in identifying some alternatives. Hence, in this study, potential peptides for α-hemolysin inhibition was developed using in silico based approach. Haddock server was used to understand the residues involved in complex formation. Based on the key residues involved in the interaction, 20 peptides were designed and docked with the α-hemolysin monomer (Chain A). Further, the best scored Chain A-peptide complex was chosen and docked with Chain B to identify the ability of dimer formation in the presence of designed peptide. The stability of the Chain A–B dimer, Chain A-peptide and Chain A-peptide-Chain B complex was studied by performing molecular dynamic simulation over 3,000 ps. The peptide IYGSKANRQTDK was found to be binding efficiently with Chain A of α-hemolysin with highest binding energy and also revealed that the designed peptide disturbed the dimer formation, which provided useful information in developing promising lead for inhibiting α-hemolysin assembly in the future.     

Incompatibility between E colicin plasmids

We have tested the ability of pairs of colicin E plasmids to replicate stably in the same cell line. Although many of the pairs of E colicin plasmids were compatible, plasmids ColE3-CA38, ColE7-K317 and ColE8-J were mutually incompatible, as were ColE5-099, ColE6-CT14 and ColE9-J. Incompatibility between ColE6-CT14 and ColE5-099 or ColE9-J was asymmetrical, whereas incompatibility between the other plasmid pairs was symmetrical.