Study of the incompatibility and replication of the 70-kb virulence plasmid of Yersinia

The 70-kb virulence plasmid, vir, from four Yersinia enterocolitica and one Y. pseudotuberculosis strains are incompatible with IncFI plasmids FLac and R386 while they are compatible with plasmids representing nine other incompatibility groups. Hybridization experiments carried out on one of these virulence plasmids showed that it contains the F incompatibility determinant D, incD. This determinant was cloned onto pACYC184 and the recombinant clone expressed incompatibility with FLac. We conclude that the incompatibility observed between F or R386 and the 70-kb virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis is mediated by incD. Replication genes (rep) from the same plasmid were cloned independently in Escherichia coli. Rep and incD map on two different BamHI fragments. Surprisingly, the replicon isolated is not sensitive to inc D incompatibility. Apart from incD, vir and F share extremely little homology. In particular, there is no evidence for the presence of an F-like transfer operon on vir.     

F plasmid incompatibility and copy number genes: Their map locations and interactions

The genes coding for vegetative F plasmid replication, replication control, and incompatibility are known to map between the kilobase coordinates 40.3 and 49.3 (abbreviated 40.3–49.3F). We have subdivided this region of the F genome by a combination of in vivo and in vitro genetic techniques and have constructed F:pSC101 hybrid plasmids which contain the F DNA sequences having the approximate coordinates 41–43, 43–46, and 46–49F. We find that hybrids with regions 43–46 and 46–49F are incompatible with an F′lac⁺ plasmid while the hybrid with the region 41–43F is compatible. We have also constructed similar F:pSC101 hybrid plasmids with the regions 43–46 and 46–49F derived from mini-F plasmid copy number mutants. We find that hybrids made from three independent F copy number mutants show a loss of the incompatibility function associated with the 43–46F region and retention of the incompatibility function associated with 46–49F region. Moreover, spontaneous revertants, selected for regain of the 43–46F incompatibility function, have also regained normal control over their copy numbers. We also find that copy number mutations map in the 43–46F region. From our results we conclude (i) that F contains at least two inc⁺ loci, designated incA⁺ (46–49F) and incB⁺ (43–46F), and (ii) that gene(s) regulating F copy number may be related to the incB⁺ gene(s).     

Incompatibility between Flac, R386, and F:pSC101 recombinant plasmids: the specificity of F incompatibility genes.

The specificity of F incompatibility genes (inc⁺) has been studied with the Flac and R386 plasmids, members of the IncFI incompatibility group. Recently, two inc⁺ regions, incA (46.4–49.3F) and incB (43.1–46.4F) were identified by cloning these F sequences onto pSC101 and subsequently demonstrating incompatibility of the recombinants with Flac. It is shown here that the FincA⁺ recombinant is incompatible with both Flac and R386 while the FincB⁺ recombinant is incompatible only with Flac. Also, a plasmid mutant is described that has reduced incompatibility against Flac and R386. The mutation is located on the BamHI restriction fragment that contains the FincA region. These genetic findings are consistent with the deduction of Palchaudhuri and Maas, based on heteroduplex analysis of IncFI plasmids, that placed the IncFI determinant in the 46.4–48.6F region. The findings also indicate that the FincB⁺ gene product, which has been implicated in negative control of F copy number, is specific for the F replicon.     

Regions on the F plasmid which affect plasmid maintenance and the ability to segregate into Escherichia coli minicells

Certain derivative mini-F plasmids were found to segregate into Escherichia coli minicells, in contrast to the intact mini-F plasmid which does not. Segregation was not related to the presence or absence of the normal origin of vegetative replication, but appeared to be affected by regions of F which encode replication, incompatibility, copy number control, and partitioning functions. Segregation of mini-F plasmids into minicells was not random; the plasmid concentration in minicells did not correlate with the plasmid concentration in cells. Genes, or gene products, of F from the region spanning the sequences 44.1–49.3F appeared to affect the ability of mini-F plasmids to segregate into minicells. Segregation of mini-F plasmids into minicells was not directly related to stable plasmid inheritance. These results argue for the sequestration of mini-F plasmids in host cells.     

Plasmid mini-F encoded proteins

Summary Proteins specified by the mini-F plasmid (EcoRI restriction fragment f5) were labeled in Escherichia coli minicells and analyzed by SDS-PAGE. Four mini-F encoded proteins could be identified, having molecular weights of 44,000 (A), 36,000 (B), 34,000 (C), and 25,300 (D) daltons. The absence of certain proteins in deleted derivatives of mini-F, generated by treatment with various restriction endonucleases, allowed mapping of the proteins. The A protein maps between F-coordinates 45.7 and 47.9 kb. The gene locus for the B protein is located between 47.2 and 49.3 kb. The C protein maps on a BamHI fragment bordered by F-coordinates 41.5 and 42.8 kb, and finally the D protein maps between 42.8 and 43.8 kb. In addition our data confirm that there are two incompatibility loci on the mini-F genome, located between 45.7 and 47.2 kb (incA) and 44.0 and 45.7 kb (incB).We suggest that (i) the C and D proteins are positive control elements, interacting with origin I and origin II, respectively, (ii) that the incB locus is involved in plasmid partitioning, and (iii) that the A protein encoded by the incA locus is a negative control element.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. I. Localisation and mutual interactions of four Clo DF13 incompatibility regions

Summary The incompatibility properties of the bacteriocinogenic plasmid Clo DF13 have been examined. By using Clo DF13, Clo DF13 deletion, and transposon insertion mutants as well as compatible R plasmids into which Clo DF13 fragments have been cloned, we could identify and localise four different incompatibility regions on the Clo DF13 genome. These regions, designated incA, incB, incC, and incD are located in the following positions: incA about incD between 1.8% and 9% of the Clo DF13 genome. We studied the contribution of each of the four inc regions, separately and/or in combination with each other, to the incompatibility between two plasmid replicons. Two types of incompatibility can be distinguished: Type I evoked by incD, that overlaps the replication control area of Clo DF13 and type II, caused by incA, B and C. From our observations we present a model for plasmid incompatibility based on a combination of the existing repressor dilution and membrane attachment models.     

Partitioning of the F plasmid: Overproduction of an essential protein for partition inhibits plasmid maintenance

Summary Multicopy plasmids carrying the sopB gene of the F plasmid inhibit stable inheritance of a coexisting mini-F plasmid. This incompatibility, termed IncG, is found to be caused by excess amounts of the SopB protein, which is essential for accuratepartitioning of plasmid DNA molecules into daughter cells. A sopB-carrying multicopy plasmid that shows the IncG⁺ phenotype was mutagenized in vitro and IncG negative mutant plasmids were isolated. Among these amber and missense mutants of sopB, mutants with a low plasmid copy number and a mutant in the Shine-Dalgarno sequence for translation of the SopB protein were obtained. These results demonstrate that the IncG phenotype is caused by the SopB protein, and that the incompatibility is expressed only when the protein is overproduced. This suggests that the protein must be kept at appropriate concentrations to ensure stable maintenance of the plasmid.     

An essential gene for replication of the mini-F plasmid from origin I

Summary We constructed a series of defective mini-F plasmids, which have deletion(s) in the replication origin I and/or origin II, and their derivatives, which do not produce F3 protein, by insertion of the XhoI fragment of Tn5 into the XhoI site at 41.0 F (kilobases on the coordinate map of F-plasmid). Using these mutant mini-F plasmids, we found that F3 protein is essential for the replication of mini-F from origin I, but not from origin II.     

Copy number control and incompatibility of plasmid R1: Identification of a protein that seems to be involved in both processes

Summary Investigations into the genetic determinants for incompatibility of miniplasmids and hybrid replicons constructed from wild type and mutant R1 revealed the presence of an incompatibility function at the junction of two small PstI fragments. These two fragments were not distinguished in earlier experiments since they have the same mobility on agarose gels. This incompatibility function is distinct from other inc-determinants of R1 (Kollek and Goebel 1979; Molin and Nordström, 1980) and independent of R1-type replication. By means of specific deletions and subcloning of DNA fragments, the location of this new inc-determinant could be determined further. After deletion of this inc-determinant from miniplasmids, a 5-fold increase in copy number was observed which could then be reduced to a copy number of about 1 plasmid per cell by complementation with hybrid plasmids having this function. Incompatibility of miniplasmids deleted in this determinant is not reduced, whereas analogous deletions introduced into recombinant plasmids nearly abolish their incompatibility. This determinant seems to exert strong incompatibility only when cloned on pBR322. Therefore, its main function in plasmid R1 is probably restricted to copy control. The appearance of low copy numbers of miniplasmids carrying this determinant and of trans-acting copy control and strong incompatibility exerted by hybrid plasmids is consistently correlated with the presence of a protein of 11,000 molecular weight, synthesized in relatively large amounts in Escherichia coli minicells.     

Complementation of replication-deficient deletion derivatives of plasmid mini-F.

Deleted mini-F plasmids with defects in replication were constructed and tested to see whether they could be rescued through complementation by a helper plasmid. This allowed us to identify two genetic loci determining trans-acting functions required for stable maintenance of plasmid mini-F, one encoded by the PstI fragment from 45.7 to 47.3 F-coordinates (F) and the other most probably located in the region from 43.1 to 43.8 F. The smallest mini-F plasmid that could be established through complementation consists of the PstI fragment 44.0 to 45.7 F, encoding origin II and the incB locus.     

Probing plasmid partition with centromere-based incompatibility

Low-copy number plasmids of bacteria rely on specific centromeres for regular partition into daughter cells. When also present on a second plasmid, the centromere can render the two plasmids incompatible, disrupting partition and causing plasmid loss. We have investigated the basis of incompatibility exerted by the F plasmid centromere, sopC, to probe the mechanism of partition. Measurements of the effects of sopC at various gene dosages on destabilization of mini-F, on repression of the sopAB operon and on occupancy of mini-F DNA by the centromere-binding protein, SopB, revealed that among mechanisms previously proposed, no single one fully explained incompatibility. sopC on multicopy plasmids depleted SopB by titration and by contributing to repression. The resulting SopB deficit is proposed to delay partition complex formation and facilitate pairing between mini-F and the centromere vector, thereby increasing randomization of segregation. Unexpectedly, sopC on mini-P1 exerted strong incompatibility if the P1 parABS locus was absent. A mutation preventing the P1 replication initiation protein from pairing (handcuffing) reduced this strong incompatibility to the level expected for random segregation. The results indicate the importance of kinetic considerations and suggest that mini-F handcuffing promotes pairing of SopB-sopC complexes that can subsequently segregate as intact aggregates.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage φX174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5′ region has been identified and is functional in Bacillus subtilis.     

Maintenance of plasmids in HU and 1HF mutants of Escherichia coli

Summary Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the hupA-hupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the hupA-hupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.     

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2

Summary A DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc ₂ incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc ₂ incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.     

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.     

The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility

The incompatibitity that pSC101-derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmid's replication origin. We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superheliclty, can function as a cis-acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy. A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101-specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower-copy-number plasmid. We propose a model by which the par locus can enchance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.     

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.     

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.     

Identification of a partition region carried by the plasmid QpH1 of Coxiella burnetii

Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever in humans and other animals. Most of the isolates found carry plasmids which share considerable homology. Unfortunately all of these plasmids remain cryptic. Initial attempts to look for secreted or membrane proteins encoded by these plasmids using TnphoA mutagenesis revealed an open reading frame on the EcoRI-fragment C of the plasmid QpH1. Upstream DNA sequencing of the TnphoA insertions revealed a deduced peptide sequence with homology to the SopA protein which is encoded by the F plasmid in Escherichia coli. Maxi-cell analysis showed that fragment C encoded two proteins: one was 43.5 kDa in size and designated QsopA, and a second was 38 kDa in size. These proteins are similar in molecular weight to the SopA and SopB proteins, which are essential components of the partition mechanism of the F plasmid. The region appears to be conserved in plasmids QpRS, QpDV, and QpDG, but is absent in a plasmidless isolate in which plasmid sequences have integrated into the chromosomal DNA. Complementation studies demonstrated that fragment C has a plasmid partitioning function and can restore maintenance stability of the partition-defective mini-F plasmid. These data suggest that fragment C carries the plasmid partition region of the plasmid QpH1.