Cloning and characterization of a replicon region of the IncHII plasmid pHH1457

A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned. A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized. High incompatibility with IncHII plasmids was corroborated. Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette. pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups). Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes. Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids. This is the first report of the characterization of an IncHII plasmid maintenance region.     

Cloning and characterization of the Inc A/C plasmid RA1 replicon.

The Inc A/C plasmids, like Inc P and Inc Q plasmids, have a broad host range. However, their maintenance functions remain to be studied. An autoreplicative region of 2.79 kb named RepA/C, able to replicate both in the family Enterobacteriaceae and in Pseudomonas spp., was isolated and sequenced. The stability, copy number, and incompatibility expression of this replicon were determined. RepA/C and a nonautoreplicative fragment of 16 kb of this replicon were used as probes and showed specific hybridizations with the Inc P3-A/C plasmids from Pseudomonas spp. and members of the Enterobacteriaceae. These probes could be used as tools for identification of the plasmids of this epidemiologically important Inc group.     

Genetic analysis of transfer and incompatibility functions within the IncHI plasmid R27

This study was undertaken to establish a transfer complementation system for IncH plasmids and to locate regions of incompatibility within the HI1 plasmid, R27. Two regions of R27 were found to contribute to incompatibility as determined by incompatibility testing with fragments of R27 cloned in cosmid vectors. One of these regions hybridized with the IncHI1 rep probe (Couturier et al., Microbiol. Rev. 52, 375-395, 1988). Complementation analysis was carried out using transfer-deficient mutants of R27 in combination with pHH1508a. Cosmid vectors, which contained cloned restriction fragments of R27, were able to complement selected R27 Tra- mutants, enabling the transfer-deficient plasmid to transfer at near-normal frequencies. Complementation of R27 Tra- plasmids by pHH1508a at both 26 and 37 degrees C was shown to occur, but was host-dependent in its degree. These results suggest that the transfer mechanisms of IncHI and IncHII plasmids are related.     

Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties

Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.     

Molecular homology and incompatibility relationships between F and IncH1 plasmids

IncH1 plasmids and the F plasmid of Escherichia coli display one-way compatibility. An entering IncH1 plasmid is incompatible with a resident F plasmid, but is compatible when it is the resident plasmid. There is little molecular homology between IncH1 plasmids and the F plasmid. A single 5 MDal EcoRI restriction enzyme fragment from digests of several IncH1 plasmids hybridizes with probes constructed from the primary replication region of F. Homology can be demonstrated only with the gene for the essential replication protein of F (gene E), but the expression of incompatibility behaviour appears to be associated with the presence of the secondary replicon of the F plasmid. Thus R27 and F are compatible under growth conditions allowing replication and maintenance of F by the secondary replicon. However, a mutant F plasmid which lacks the secondary replicon of F is incompatible with R27 in both directions, irrespective of the growth conditions used.     

Isolation of the replication and partitioning regions of the Salmonella typhimurium virulence plasmid and stabilization of heterologous replicons.

Although the virulence plasmid of Salmonella typhimurium has a copy number of one to two per chromosome, plasmid-free segregants are produced at a rate less than 10(-7) per cell per generation. Three regions appear to be involved in the maintenance of this virulence plasmid. The first two, repB and repC, are functional replicons hybridizing with IncFII and IncFI plasmids, respectively, neither exhibiting the segregational stability of the parent virulence plasmid. The third region, par, cloned as a 3.9-kilobase Sau3A fragment, is not a functional replicon but exhibits incompatibility with the virulence plasmid. Subsequent tests revealed the ability of this 3.9-kilobase par insert to increase the stability of pACYC184 in S. typhimurium from less than 34% to 99% plasmid-containing cells after 50 generations. In addition, the par region increased the stability of oriC, R388, and repC replicons in both S. typhimurium and Escherichia coli hosts. The par region encodes 44,000- and 40,000-molecular-weight proteins essential for the Par+ phenotype but not for the Inc+ phenotype. Although actual sequestering of plasmids within the cell was not demonstrated, all results indicate that the par region described is an actual partitioning locus, similar in organization to those described for plasmids F, P1, and NR1.     

Analysis of a region in plasmid R386 containing two functional replicons

A miniplasmid has been obtained from R386 by ligating EcoRI fragments with a fragment carrying a kanamycin-resistance gene. It contains a 6.8-kb Eco fragment of R386 which hybridizes strongly with several IncFI plasmid DNAs but not with the primary or secondary replicons of the F plasmid. This mini-R386 is incompatible with certain IncFI plasmids, and it appears to be one example of a previously unidentified replicon widely distributed in the IncFI group. A region of R386 not closely linked to the 6.8-kb fragment is involved in copy number control of the mini-R386, and a sequence in the same region interacts with mini-F partition functions to cause incompatibility. The 6.8-kb fragment also restricts growth of T7 bacteriophage, and an adjacent fragment restricts phage T4 growth. A further R386 sequence, sharing homology with the F secondary replicon, is capable of autonomous replication. Hence R386, like F, contains at least two functional replicons.     

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86

Summary Many plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.     

Distribution of basic replicons having homology with RepFIA, RepFIB, and RepFIC among IncF group plasmids

Plasmids encoding F-like pili have been divided into groups on the basis of their incompatibility behavior. Three basic replicons have been recognized previously in the IncFI plasmid group and we have now examined their distribution in representative plasmids from 22 of the currently recognized incompatibility groups. The occurrence of these basic replicons was found to be rare outside of the IncF group, and significant hybridization was shown only for RepFIA to IncH1 and I group plasmids. Homology to the RepFIC basic replicon was found in all but one of the IncF group plasmids examined but RepFIA and RepFIB have a more restricted distribution. It appears likely that some plasmids carry vestiges of replicons which still express incompatibility but are incapable of replication. We suggest that evolutionary divergence among the plasmids of the IncF group has resulted from various genetic rearrangements among these basic replicons.     

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.     

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.     

Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035.

The large conjugative plasmid pIP501 was originally isolated from Streptococcus agalactiae. To study the molecular basis of pIP501 replication we determined the nucleotide sequence of a 2.2 kb DNA segment which is essential and sufficient for autonomous replication of pIP501 derived plasmids, in Bacillus subtilis cells. This region can be divided into two functionally discrete segments: a 496 bp region (oriR) that acts as an origin of replication, and a 1488 bp segment coding for an essential replication protein (RepR). The RepR protein, which has a molecular mass of 57.4 kDa, could complement in trans a thermosensitive replicon bearing the pIP501 origin. Chimeric Rep proteins and replicons were obtained by domain swapping between rep genes of closely related streptococcal plasmids belonging to the inc18 group (pIP501, pAM beta 1 and pSM19035). The chimeras were functional in B. subtilis.     

Nature of the compatible inheritance of hybrid plasmid pAS8 and its deletion mutants with replicon ColE1]

Hybrid plasmid pAS8, that consists of RP4 and ColE1 replicons, is incompatible with RP4 but co-exists with ColE1 replicon. The deletion mutants of pAS8, that replicates under the control of ColE1 replicon only, are incompatible with ColE1 derivatives, although the copy number of pAS8 and its deletion mutants in the cell is the same (1-3 per the chromosome). Incompatibility effect of plasmids is expressed in a sharp decrease in transformant's yield during selection of incoming and resident plasmids markers. In this case incompatibility is a very fast process, that leads to the elimination of resident or incoming plasmid just before plating on the selective medium. On the base of negative control theory, the repressors yield, synthesized in the presence of 1-3 copies of the deletion mutant is enough for the expression of ColE1-specific incompatibility. This ColE1 incompatibility is probably connected with the functional activity of ColE1 replicon. When ColE1 factor replicates under the control of RP4 replicon the expression of ColE1-specific incompatibility does not occur. Possibly, the incompatibility effect takes place when pAS8 deletion mutants cause the synthesis of ColE1-specific repressor. Also, the replicons of ColE1 may competein the membrane attachment site.     

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.     

Characterization of the basic replicons of the chimeric R/Ent plasmid pCG86 and the related Ent plasmid P307

Restriction-enzyme fragments that can replicate autonomously after circularization were isolated from the chimeric R/Ent plasmid pCG86 and the Ent plasmid P307. Two such regions containing a basic replicon were located in each plasmid. One of the basic replicons of P307, RepFIB, is almost identical with one of the basic replicons of pCG86. The other basic replicon in P307, RepFIC, is partly homologous with the second basic replicon in pCG86, RepFIIA/RepFIC. The latter is a hybrid basic replicon and is in addition partly homologous with RepFIIA, a basic replicon present in IncFII R plasmids. By restriction-enzyme mapping and nucleotide-sequence analysis we have determined a site in the hybrid replicon where it ceases to be homologous with the RepFIIA basic replicon contained in the IncFII miniplasmid pSM1. The 2410-bp region of homology with pSM1 corresponds with a segment containing the origin of replication and all the genes responsible for replication control of pSM1.