New incompatibility groups for Staphylococcus aureus plasmids

Incompatibility relationships between naturally occurring staphylococcal plasmids conferring erythromycin or kanamycin resistance have been studied making use of recombinants between these plasmids and pSA0301, a temperature-sensitive mutant plasmid determining tetracycline resistance. The four plasmids encoding kanamycin resistance fall in two incompatibility groups; similarly, the three plasmids responsible for erythromycin resistance belong to two other incompatibility groups. This brings the number of distinct incompatibility groups reported for Staphylococcus aureus plasmids to 13.     

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.     

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.     

Incompatibility groups of epsilon-caprolactam biodegradation plasmids from bacteria of Pseudomonas genus

Incompatibility of epsilon-caprolactam biodegradation plasmids pBS262, pBS263, pBS264, pBS265, pBS266, pBS267, pBS268, pBS270, pBS276, pBS269 with the tester plasmids of P-1, P-2, P-7, P-9 incompatibility groups in the system of strains of P. putida line BSA, as well as the character of plasmid interaction with the number of P. aeruginosa and P. putida bacteriophages have been studied. The majority of the studied plasmids belongs to IncP-7, IncP-9 or simultaneously to IncP-7 and IncP-9 incompatibility groups. The ability to restrict the growth of some bacteriophages of P. aeruginosa and P. putida has been demonstrated for some plasmids.     

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.     

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.     

RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids.

Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.     

Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker

Incompatibility relationships between staphylococcal plasmids carrying the same, single resistance marker were studied by means of appropriate recombinant plasmids. Naturally occurring plasmids encoding streptomycin, tetracycline, or chloramphenicol resistance, respectively, were used in this study, four of each phenotype. The plasmids responsible for tetracycline resistance proved to belong to a single incompatibility set. Similarly, the four streptomycin resistance plasmids fall in the same incompatibility set. On the other hand, plasmids encoding chloramphenicol resistance were divided in four distinct incompatibility sets, three of them being newly defined. Study of the molecular relationships between these plasmids by DNA-DNA hybridization and restriction endonuclease cleavage supported the conclusions from genetic tests that the four Tc^r and the four Sm^r plasmids are essentially identical, whereas the four Cm^r plasmids are diverse.     

Plasmid partition and incompatibility--the focus shifts

The mitotic apparatus that a plasmid uses to ensure its stable inheritance responds to the appearance of an additional copy of the plasmid's centromere by segregating it from the pre-existing copies: if the new copy arises by replication of the plasmid the result is partition, if it arrives on a different plasmid the result is incompatibility. Incompatibility thus serves as a probe of the partition mechanism. Coupling of distinct plasmids via their shared centromeres to form mixed pairs has been the favoured explanation for centromere-based incompatibility, because it supports a long-standing assumption that pairing of plasmid replicas is a prerequisite for their partition into daughter cells. Recent results from molecular genetic and fluorescence microscopy studies challenge this mixed pairing model. Partition incompatibility is seen to result from various processes, including titration, randomized positioning and a form of mixed pairing that is based on co-activation of the same partition event rather than direct contact between partition complexes. The perspectives thus opened onto the partition mechanism confirm the continuing utility of incompatibility as an approach to understanding bacterial mitosis. The results considered are compatible with the view that direct pairing of plasmids is not essential to plasmid partition.     

Evidence that IncG (IncP-6) and IncU plasmids form a single incompatibility group

The Escherichia coli IncG and IncU plasmid incompatibility groups were assigned in 1980 and 1981, respectively. Complete plasmid sequences have recently been published for both these groups, and revealed that their replication proteins are related. We show that when cloned at high-copy-number, putative iterons from the previously identified IncG replicon cause strong incompatibility with IncU plasmids. Incompatibility, albeit weaker, was also demonstrated between the two replicons at their normal low-copy-number. This suggests that a single incompatibility group exists. The only known IncG plasmid, Rms149, can replicate in Pseudomonas species where it is designated IncP-6. We recommend that the combined group be known as IncU (IncP-6 in Pseudomonas spp.).     

Host-specific factors determine the persistence of IncP-1 plasmids

Conjugative plasmids play a very important role in bacterial adaptation through the dissemination of useful traits. Incompatibility group P-1 (IncP-1) plasmids exhibit an extreme broad-host-range among Gram-negative bacteria and known to be one of the major agents to disseminate various phenotypic traits such as antibiotic resistance and xenobiotic degradation. Although the plasmids are believed to be very stable in most Gram-negative bacteria, little is known about the factors that affect their stability in various hosts, allowing their persistence in bacterial population. Here we show that the stability of the cryptic IncP-1β plasmid pBP136 differed greatly in four different Escherichia coli K12 host backgrounds (MG1655, DH5α, EC100, and JM109), whereas the closely related plasmid pB10 was stable in all four strains. The supply of the kleF gene, which is involved in the stability of IncP-1 plasmids but absent in pBP136, did not improve the stability of the plasmid. Our findings suggest that persistence of IncP-1 plasmids in the absence of selection is affected by strain-specific factors.     

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.     

Specification of characteristics for the classification of plasmids in incompatibility group U

Incompatibility group U is a new group of plasmids which has not been fully characterized. We describe the incompatibility relationships and phenotypic properties of four probable members including the proposed prototype, pAr-32.     

The plasmid heterogeneity of Shigella sonnei phase-I and -II strains

The plasmid composition of S. sonnei standard strains has been studied by the method of electron microscopy of the preparations of plasmid DNA. In S. sonnei cells I-941-HP, phase I, plasmids of 2,500; 5,000; 5,600; 6,100 and 6,800 base pairs, as well as plasmids of 85,000-117,000 and 170,000-235,000 base pairs have been detected. In S. sonnei cells, phase II, plasmids of 2,500; 4,900 and 6,100 base pairs, as well as plasmids of 85,000-109,000 base pairs, have been found. Thus, virulent S. sonnei in phase I contain additional plasmids of 5,600; 6,800; 110,000-117,000 and 170,000-237,000 base pairs. The range of plasmid lengths between 85,000-117,000 and 170,000-237,000 base pairs exceeds the usual background of electron-microscopic studies, which makes it possible to come to the conclusion on the intrastrain heterogeneity of these classes of plasmids. The suggestion has been made that the transition of S. sonnei from phase I to phase II is linked with the loss of fragments of the genetic material, limited by inverted DNA repetitions.     

Molecular relationships among plasmids of Bacillus thuringiensis: conserved sequences through 11 crystalliferous strains

Summary Screening for the plasmid content of 11 strains belonging to nine different serotypes ofB. thuringiensis was carried out by electron microscope examination and electrophoresis in agarose gels. All the strains contained at least two covalently closed, circular (CCC) DNA species. In one strain (berliner 1715), 17 extrachromosomal elements could be distinguished with regard to their size, ranging from 3.9 to 180 Mdal. Southern hybridisation experiments showed that most of these plasmids fell into two categories (inferior to 15 Mdal and superior to 15 Mdal) which have no homology between them. Within these two size groups there is partial conservation of DNA sequences through various serotypes. Further relationships among the plasmids were investigated by a two dimensional version of the Southern's blotting technique.Possible homology between plasmids and the chromosomal DNA was studied. It was shown that the smaller plasmids from the berliner 1715 and kurstaki HD₁ strains contained no sequence related to chromosomal DNA, whereas among the larger plasmids a few showed homologous sequences.Abbreviations cry^- tacrystalliferous mutant - GCC covalently closed circular DNA - OC open circular DNA - Mdal megadalton - kb 1,000 base pairs     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group.

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Genetic relationship between pUB110 and antibiotic-resistant plasmids obtained from thermophilic bacilli

The molecular relationship between pUB110 (Kmr, 4.4 kilobases (kb] and antibiotic-resistant plasmids from thermophilic bacilli, pTHT15 (Tcr, 4.5 kb) and pTHN1 (Kmr, 4.8 kb), were studied by blot hybridization. Extensive homology was observed between pUB110 and pTHT15 at the region which includes the replication origin. Incompatibility studies revealed that pTHT15 and pUB110 were slightly incompatible in Bacillus subtilis but that they were apparently compatible in B. stearothermophilus. This difference in incompatibility between pTHT15 and pUB110 in the two host cells might be due to a difference in the copy number of pTHT15 in the two organisms. From the results of blot hybridization, mode of kanamycin inactivation, and DNA sequencing, it was determined that pTHN1 encoded the identical gene for kanamycin nucleotidyl transferase as that of pUB110. All three plasmids pTHT15, pTHN1, and pUB110 shared a common DNA homology at the in vitro membrane-binding region.     

Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids.

A second autonomous replicon of P307, RepFIB, has been isolated that has significant homology with other replicons in IncFI group plasmids. Eleven homologous repeats of 21 base pairs are present on the sequence and flank an open reading frame capable of coding for a protein of about Mr = 40,000. This protein was identified by maxicell analysis of cloned RepFIB. A series of deletion mutations of RepFIB were inserted into a DNA polymerase I-dependent vector and examined for their replication proficiency in a polA1 strain. These experiments defined a minimal replication region of 1.6 kilobases which includes the three repeats immediately upstream and downstream of the open reading frame. Deletion of a second set of repeats further downstream doubled the copy number of a chimeric plasmid replicating under RepFIB control. It was concluded that these repeats control the copy number of the replicon. Incompatibility tests showed that all three sets of repeats could express incompatibility with a resident RepFIB plasmid.     

Retrotransfer of IncP1-like plasmids from aquatic bacteria

The first observation of plasmid retrotransfer by plasmids isolated from environmental sources is reported. A high incidence of retrotransferring ability amongst plasmids isolated from epilithic bacteria was found; some of these plasmids retrotransferred an IncQ plasmid at very high frequencies. Despite the broad host-range of the majority of the plasmids, only five out of 12 could be assigned to an incompatibility group by DNA hybridization. All five were designated IncP1; this revealed a limitation of probes derived from clinical sources for use with environmental isolates. Incompatibility testing by plate mating suggested that four additional plasmids displayed varying, albeit lower, degrees of incompatibility to the IncP1 plasmid RP1.