Molecular homology and incompatibility in the IncFI plasmid Group

The usual grounds for the inclusion of a plasmid in a particular incompatibility group are its mutual incompatibility with a type plasmid of that group, and, in some cases, the demonstration of shared regions of specific homology, presumed to be related to DNA replication. We have found that some plasmids classified as IncFI on genetical grounds share no homology with the previously described incompatibility regions of F on the basis of hybridization of specific radioactive probes to restriction enzyme digests of DNA from these plasmids. Others show homology with some or all of the regions of the F plasmid that can express incompatibility. The incompatibility behaviour of these plasmids has been examined to determine the relationship between the possession of regions of homology and the expression of incompatibility. Three plasmids, ColV3-K30, pHH507 and Entp307, show homology only with the secondary replicon of F and appear to use sequences homologous with the secondary F replicon in their replication. The results are consistent with the propositions that some contemporary IncFI plasmids arose by the integration of several replicons, and, in general, the replicon not being used for replicon expresses its incompatibility, as does the replicon being used for replication. We conclude that incompatibility of two plasmids with F does not necessarily demonstrate relatedness of the plasmids to each other, and that inclusion within the IncFI group can result from the possession of any of several combinations of inc sequences.     

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.     

Significance of filter mating in integrative incompatibility test for plasmid classification

For classification of plasmids in epidemiological studies, an integrative incompatibility test using liquid mating was developed by Sasakawa et al (Plasmid 3: 116-127, 1980). This test was designed to compare the relative mating frequency of a donor carrying a test plasmid with that of recA recipients carrying various integrated plasmids. To improve the accuracy of this method by increasing transfer frequency of a test plasmid, filter mating was introduced. A transfer frequency 10 to 30,000 times higher than that achieved by liquid mating was attained by filter mating. The degree of increase varied among the incompatibility groups and the majority of members belonging to the same incompatibility group exhibited a similar degree of increase. Standard plasmids were classified correctly with the integrative incompatibility test using filter mating. Out of 26 naturally occurring plasmids of poor transferability in liquid mating, all of domestic animal origin, 25 were correctly classified as IncH with the integrative incompatibility test using filter mating. Moreover, the method is capable of subdividing IncH plasmids directly into IncH1 and IncH2 , because IncH2 , but not IncH1 , plasmids showed incompatibility with the integrated plasmid, R478 , of the IncH2 group.     

Incompatibility Reactions of R Plasmids Isolated from Escherichia coli of Animal Origin

The incompatibility reactions of a group of 90 R plasmids, isolated from the fecal Escherichia coli of calves, pigs, and chickens, have been determined against reference plasmids of the incompatibility groups F(11), I, N, P, and W. Twenty plasmids belonged to incompatibility groups F(11), I, N, or P. Forty-nine plasmids were compatible with all of the five reference plasmids. Eight plasmids exhibited marked incompatibility with representatives of two or more of the above groups, whereas two strains showed incompatibility reactions consistent with the coexistence of two compatible plasmids. In addition, 19 plasmids remain untyped because they showed intermediate reactions with one or more reference plasmids.     

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.     

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.     

Comparative genetic organization of incompatibility group P degradative plasmids.

Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.     

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.     

Conjugative plasmids of enteric bacteria from many different incompatibility groups have similar genes for single-stranded DNA-binding proteins.

Among 30 conjugative plasmids of enteric bacteria from 23 incompatibility (Inc) groups, we found 19 (from 12 Inc groups) which can complement defects caused by a defective single-stranded DNA-binding protein of Escherichia coli K-12. The genes which are responsible for the complementation from three of these plasmids (Inc groups I1, Y, and 9) were cloned. These genes showed extensive homology with each other and with the E. coli F factor ssb gene (formerly denoted ssf), which codes for a single-stranded DNA binding protein. The proteins coded for by the cloned genes bound tightly to single-stranded DNA. Six other ssb- -complementing plasmids were tested for homology to the F factor ssb gene, and all of these showed homology, as did one of the ssb- -noncomplementing plasmids. Plasmids from a total of 13 different Inc groups of enteric bacteria were found to be likely to have genes with some homology to the ssb gene of the F factor. For plasmids from several different Inc groups, we found no evidence for strong homology with ssb of the F factor.     

Demonstration of a third incompatibility function on plasmids already incompatible with group P and group I plasmids

The addition of extra antibiotic resistance determinants to plasmids of a series previously shown to be incompatible in an atypical fashion with group I and group P plasmids, has enabled the existence of a third and major incompatibility function determined by these plasmids to be demonstrated. The in vitro construction of a plasmid consisting of the replication region of one of the plasmids, linked to the genes of the galactose operon, facilitated the identification of this third incompatibility function as similar to IncB 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.     

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.     

Molecular Comparison of Plasmids Encoding Heat-Labile Enterotoxin Isolated from Escherichia coli Strains of Human Origin

The molecular properties of enterotoxin (Ent) plasmids from 12 Escherichia coli strains of human origin were examined. Ten strains belonged to the O78 serogroup, and the remainder were of serogroup O7 or O159. Eleven plasmids coded for heat-labile enterotoxin (LT), and one coded for heat-stable enterotoxin (ST) and LT. The results of restriction enzyme digests and deoxyribonucleic acid reassociation experiments showed that all of the Ent plasmids were related, and supported the subdivision of the LT plasmids into three groups based on their genetic properties (M. M. McConnell et al., J. Bacteriol. 143: 158–167, 1980). Within group 1, two plasmids from South African strains were indistinguishable but differed in EcoRI and HindIII digests from the LT plasmid that originated from an Ethiopian strain. The three plasmids had >70% homology. The two non-autotransferring group 2 plasmids identified in O78.H11 strains from Bangladesh were indistinguishable. The group 3 plasmids were from strains belonging to serogroups O7 and O78 isolated in Bangladesh, India, and Thailand. They shared >95% homology but showed slight differences in fragment patterns when treated with EcoRI and HindIII. There was 60 to 70% homology between the plasmids of groups 1 and 3, and the group 2 plasmid had 40 to 50% homology with members of these two groups. The autotransferring Ent plasmids had up to 40% homology with R factors of incompatibility groups FI, FII, and FIV.     

A mathematical model of the incompatibility of plasmids using a mechanism of master replication

A mathematical model of incompatibility for plasmids that use master-replication was worked out. Formulas for calculating the segregation velocity of two incompatible plasmids and for determining the number of cell generations (l gamma), necessary to achieve a gamma-share of polyplasmid cells are given. It is shown that l gamma strives to -8 lg gamma with the increase of the copy number N, i.e. it does not depend on N. This is the main distinction of model presented from those by Novick at al. and Ishii et al., who consider the mechanisms of regular and random replication. For these mechanisms l gamma increases proportional to N. So, at N greater than 30, the difference between l gamma, calculated for the master-replication and the random replication exceeds 10. This allows to determine the mechanism of replication used by the plasmid by measuring the velosity of incompatible plasmids segregation.     

An unexpected incompatibility interaction between two plasmids belonging to the I compatibility complex

The plasmids R144-3 and R144, which belong to the Iα incompatibility group, were found to be incompatible with R621a, which is the exemplar of the Iγ incompatibility group. Extending the studies of Hedges and Datta (1), R621a and its derivative R621a.1 were shown to be compatible with other Iα plasmids. Hence R144 and R144-3 are not typical Iα plasmids.     

Effect of plasmids from various incompatibility groups on the development of bacteriophages specific to Pseudomonas aeruginosa and Pseudomonas putida

The aim of this work was to study the effect of plasmids belonging to different incompatibility groups on the growth of bacteriophages in Pseudomonas aeruginosa and Pseudomonas putida strains. The growth of bacteriophages was shown to be limited most often due to the presence in cells of plasmids belonging to the P-2 incompatibility group. Plasmids of the Inc P-2 group differed from one another in the spectrum of bacteriophages whose growth they limited. Phages whose growth was suppressed in strains containing plasmids of the P-5, P-9 or P-10 incompatibility groups were found. Some plasmids showed no specific interaction with bacteriophages. The plasmids investigated differed in the studied trait in P. aeruginosa and P. putida cells. In contrast to P. aeruginosa PAO, P. putida PpGI plasmid containing cells did not maintain the growth of donor-specific bacteriophages and, to a lesser degree, limited the growth of phages specific for P. putida PpGI.     

Characterization of conjugative plasmids belonging to a new incompatibility group (IncZ)

More than 30 conjugative R plasmids between 60 and 70 Md in size were identified in wild type strains of Enterobacteriaceae isolated in German Democratic Republic, Bulgaria, Mongolia, Poland, Ethiopia, Iraque, and Soviet Union. They have been characterized by means of several genetic and molecular techniques as members of a new incompatibility group, termed IncZ. The properties of these plasmids including digestion pattern after EcoRI treatment demonstrate a phylogenetic relatedness.     

Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups.

Two hundred ninety-seven bacteria carrying plasmids that range in size from 5 to 250 kb were identified from more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments. While some isolates contained numerous (three to five) small (5- to 10-kb) plasmids, the majority of the natural isolates typically contained one large (40- to 100-kb) plasmid. By the method of plasmid isolation used in this study, the frequency of plasmid incidence ranged from 24 to 28% depending on the samples examined. Diversity of the plasmids occurring in the marine sediment bacterial populations was examined at the molecular level by hybridization with 14 different DNA probes specific for the incompatibility and replication (inc/rep) regions of a number of well-characterized plasmid incompatibility groups (repB/O, FIA, FII, FIB, HI1, HI2, I1, L/M, X, N, P, Q, W, and U). Interestingly, we found no DNA homology between the plasmids isolated from the culturable bacterial population of marine sediments and the replicon probes specific for numerous incompatibility groups developed by Couturier et al. (M. F. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Our findings suggest that plasmids in marine sediment microbial communities contain novel, as-yet-uncharacterized, incompatibility and replication regions and that the present replicon typing system, based primarily on plasmids derived from clinical isolates, may not be representative of the plasmid diversity occurring in some marine environments. Since the vast majority of marine bacteria are not culturable under laboratory conditions, we also screened microbial community DNA for the presence of broad- and narrow-host-range plasmid replication sequences. Although the replication origin of the conjugally promiscuous broad-host-range plasmid RK2 (incP) was not detectable in any of the plasmid-containing culturable marine isolates, DNA extracted from the microbial community and amplified by PCR yielded a positive signal for RK2 oriV replication sequences. The strength of the signal suggests the presence of a low level of the incP replicon within the marine microbial community. In contrast, replication sequences specific for the narrow-host-range plasmid F were not detectable in DNA extracted from marine sediment microbial communities. With the possible exception of mercuric chloride, phenotypic analysis of the 297 plasmid-bearing isolates did not demonstrate a correlation between plasmid content and antibiotic or heavy metal resistance traits.     

Distribution of plasmid maintenance regions among IncFIV plasmids

The distribution of the IncFI basic replicons among IncFIV plasmids was assessed by DNA hybridization. In addition these and 20 other plasmids from 16 incompatibility groups were screened for the presence of IncIV, an incompatibility determinant recently found on the IncFIV plasmid R124. The IncIV determinant was found commonly but not universally among the IncFIV plasmids. It was also detected on the IncFI reference plasmid R386 and plasmids from IncB, IncI alpha and IncI gamma. The frequency and distribution of IncFI replicons among the IncFIV plasmids is similar to that observed in other F groups. The similarity of the IncFIV plasmids to plasmids of the other IncF groups and the failure to find replicons unique to IncFIV plasmids indicates that their division into a separate incompatibility group is not justified.