Conserved regions at the DNA primase locus of IncP alpha and IncP beta plasmids

Genes specifying DNA primases (pri) are common in all IncP plasmids examined so far. These plasmids suppress the thermosensitive character of the Escherichia coli dnaG3 mutation. The mechanism of suppression appears to be identical to that known for RP4 and IncI alpha plasmids. The DNA primases of both these plasmid types can substitute for the dnaG protein in chromosomal DNA replication. The pri genes of the alpha and beta subgroup of IncP plasmids are related to each other as judged from Southern hybridization and immunological data. Extensive DNA and protein sequence homology has been detected although the gene products of the alpha and beta subgroups exhibit substantial differences in size. The arrangement of overlapping genes at the pri locus of IncP alpha plasmids also appears to be present in the IncP beta group.     

Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids.

A physical map was constructed for the 250-kilobase plasmid pRiA4b, which confers the virulence properties of a strain of Agrobacterium rhizogenes for hairy root disease in plants. The complete HindIII and KpnI restriction map was determined from a collection of overlapping HindIII partial digest clones. Homologous regions with two well-characterized plasmids that confer virulence for crown gall disease, plasmids pTiA6 and pTiT37, were mapped on pRiA4b. As much as 160 kilobases of pRiA4b had detectable homology to one or both of these crown-gall-tumor-inducing plasmids. About 33 kilobases of pRiA4b hybridized to the vir region of pTiA6, a segment of DNA required for virulence of Agrobacterium tumefaciens. Portions of pTiA6 and pTiT37 transferred into plant cells in crown gall disease (T-DNA), shared limited homology with scattered regions of pRiA4b. The tumor morphology loci tms-1 and tms-2 from the T-DNA of pTiA6 hybridized to pRiA4b. A T-DNA fragment containing the tml and tmr tumor morphology loci also hybridized to pRiA4b, but the homology has not been defined to a locus and is probably not specific to tmr. A segment of pRiA4b T-DNA which was transferred into plant cells in hairy root disease lacked detectable homology to pTiA6 and had limited homology at one end to the T-DNA of pTiT37.     

Detection of tellurite-resistance determinants in IncP plasmids

Six IncP plasmids were tested for their ability to generate tellurite-resistant variants by plating bacterial strains harbouring them on medium containing potassium tellurite. Four plasmids, three of subgroup IncP alpha and one not allocated, formed variants that could transfer tellurite-resistance at the same frequency as plasmid-determined drug resistance. This property was not shared by two examples of subgroup IncP beta.     

Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.     

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.     

Interaction of the IciA protein with AT-rich regions in plasmid replication origins.

A set of AT-rich repeats is a common motif in prokaryotic replication origins. We have screened for proteins binding to the AT-rich repeat region in plasmids F, R1 and pSC101 using an electrophoretic mobility shift assay with PCR-amplified DNA fragments from the origins. The IciA protein, which is known to bind to the AT-rich repeat region in the Escherichia coli origin of chromosome replication, oriC, was found to bind to the corresponding region from plasmids F (oriS) and R1, but not to pSC101. DNase I footprint analysis showed that IciA interacted with the AT-rich region in both F and R1. When the IciA gene was deleted, the copy number of plasmid F increased somewhat, whereas there was no major effect on the replication of pSC101 and R1, or on the E. coli chromosome.     

Transfer of the gonococcal penicillinase plasmid: mobilization in Escherichia coli by IncP plasmids and isolation as a DNA-protein relaxation complex

A 4.4-megadalton penicillinase plasmid, pWD2, from Neisseria gonorrhoeae was transformed into Escherichia coli. pWD2 was efficiently mobilized by IncP plasmids in E. coli but not by Flac, R1drd-19, or R64drd-11. pWD2 could be isolated as a DNA-protein relaxation complex with properties similar to the well characterized ColE1 complex. The host range of pWD2 was shown to include gonococci, Enterobacteriaceae, and Hemophilus influenzae, but not Acinetobacter calcoaceticus or Pseudomonas aeruginosa. These findings suggest that P-group plasmids could have played a role in the dissemination of the TEM beta-lactamase to pathogenic gram-negative bacteria.     

Tumor-inducing (Ti) plasmids of Agrobacterium share extensive regions of DNA homology.

Labeled Ti plasmid DNAs from diverse Agrobacterium strains were hybridized to Southern blots of pTi-B6-806 plasmid DNA digest fragments of known map order. The map location of DNA sequences common to all Ti plasmids was found to be extensive, consistent with the view that Ti plasmids have evolved from a common ancestral plasmid.     

Behaviour of bacteriophage Mu-based IncP suicide vector plasmids in Rhizobium spp.

Abstract The ability of P-type plasmids, containing bacteriophage Mu, to survive in Rhizobia was examined under conditions in which the plasmids were not directly selected. The plasmid pRP4::Mu::Tn7 survived in all tested strains of Rhizobia and Agrobacteria . The surviving plasmids were analysed for expression of plasmid coded properties and for presence of both bacteriophage Mu and Tn7 DNA. A large variation in plasmid size and coding capacity was observed. In the parent plasmid Tn7 is inserted into the Mu DNA. The majority of surviving plasmids had lost Mu DNA but the Tn7 DNA was retained. Analysis of surviving plasmids which contained Mu DNA indicated that a small deletion of Mu is sufficient to allow plasmid survival. Deleted derivatives of pJB4J1 were also isolated in Rhizobium under similar conditions. The potential of these suicide vectors as potential vectors for transposon mutagenesis in Rhizobium is discussed.     

Cyanobacterial plasmids: Their widespread occurrence, and the existence of regions of homology between plasmids in the same and different species

Summary The results of screening of 29 diverse cyanobacterial (blue-green algal) strains for plasmid (CCC DNA) content are reported. Approximately one-half of the strains were shown to contain one or more CCC DNAs. CCC DNAs from four unicellular marine cyanobacteria were characterized in more detail. These strains contained multiple plasmids. Two kinds of Southern hybridization experiments allowed us to show that different plasmids within the same strain, and different plasmids within different strains, can (but do not always) contain restricted regions of sequence homology. We suggest that these regions of homology may be analogous to the transposable genetic elements of bacterial plasmids. This, together with indirect but compelling evidence for interspecific (or intergeneric) plasmid transfer, indicates that CCC DNAs (although as yet genetically cryptic) may play a role in the ecology and evolution of obligately autotrophic prokaryotes, as they do in the ecology and evolution of the better-known heterotrophic bacteria.     

Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids.

Minireplicons from the I-complex plasmids R387 (IncK) and pIE545 (IncZ) were constructed, and the nucleotide sequences of their replication regions were compared with that of the B plasmid, pMU720. The coding sequence of the putative replication protein, RepA, of each plasmid was located. RepA of K and B plasmids were homologous, whereas RepA of Z resembled RepA1 of FII plasmid. Sequences upstream of RepA were conserved in the three I-complex plasmids. Group B and Z plasmids were incompatible.     

A louse involved in the regulation of replication in plasmid pSC 101

The origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds iR1 and the RepA monomers that bind the RS sequences.     

Comparison of the nucleotide sequences of the vegetative replication origins of broad host range IncP plasmids R751 and RK2 reveals conserved features of probable functional importance.

An 864 bp EcoRI fragment carrying oriVR751, the vegetative replication origin of broad host range IncP plasmid R751, was cloned and sequenced. Only the trfA gene of the IncP plasmid RK2 was required in trans for the function of oriVR751. The sequence of oriVR751 showed 65% overall homology to that of oriVRK2 determined previously. Highly conserved regions of probable functional importance were apparent, including two sets of direct repeats postulated to be interaction sites for the trfA protein(s), a putative dnaA protein binding site and a downstream inverted repeat of unknown function.     

Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.     

Isolation of transmissible cointegrates of Yersinia pestis plasmids pYV and pYT with the plasmid RP4::Mu cts 62, IncP1

The transmissible cointegrates of the Yersinia pestis plasmids pYV and pYT with the broad host range plasmid RP4::Mu cts62 of the incompatibility group IncP have been constructed by the in vivo recombination. The cointegrative plasmid pKR14 (pYV76 omega RP4::Mu cts62) conferred on the transconjugants the properties of Ca2(+)-dependence at 37 degrees C, V-antigen synthesis, RP4 plasmid markers (ApR, KmR, TcR), immunity to the lysis by the bacteriophage Mu cts62 and incompatibility with the homologous replicon pYV76. Cointegrates pKR103 and pKR106 (pYT omega RP4::Mu cts62) conferred on the transconjugant clones the ability to synthesize the "mouse" toxin and fraction I. The capability of Escherichia coli cells to synthesize the latter products has been demonstrated together with the deficiency of these cells to transport the synthesized fraction I to the cell surface.     

Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication.

Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop. Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II). Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II. From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed.