Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis

Summary The nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acids; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA1 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA1 either encodes a gene product or structure information that is responsible for incompatibility.     

Characterization of the gene products produced in minicells by pSM1, a derivative of R100

Summary At least ten polypeptides larger than 6 kilodaltons (K) are produced in minicells from the miniplasmid pSM1 in vivo. pSM1 (5804 bp) is a small derivative of the drug resistance plasmid R100 (ca. 90 kb) and carries the R100 essential replication region as well as some non-essential functions. Cloned restriction fragments of pSM1 and plasmids with deletions within pSM1 sequences were used to assign eight of the ten oberserved polypeptides to specific coding regions of pSM1. Two of these polypeptides were identified as RepA1 and RepA2, proteins encoded by the essential replication region of pSM1/R100. The nucleotide sequence consisting of 885 bp outside the essential replication region is presented here. This sequence contains an open reading frame,orf4, for a protein 22.9 K in size, and one of the pSM1-encoded polypeptides was identified as theorf4 gene product. Five additional polypeptides were shown to be the products of other open reading frames mapping outside the essential replication region. Specific functions have been assigned to four of these polypeptides and tentatively to the fifth.     

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (amp^r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp^r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp^r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.     

Plasmid Co1IB contains an ssi signal close to the replication origin

Taking advantage of the plaque morphology method, we identified a single-strand initiation (ssi) signal in plasmid pSM32, a mini-Co1Ib plasmid. This ssi signal was situated in the 350-nt HaeIII segment of the 1.8-kb S7 fragment, and located nearly 400 nt downstream of the origin of DNA replication. Introduction of the ssi signal into a mutant of filamentous phage M13 lacking oric resulted in restoration of phage growth and RFI DNA synthesis. Interestingly, DNA homology studies showed that the nucleotide sequence of the ssi signal was extremely homologous with that of the "G4-type" ssi signal in plasmid R100.     

Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1

The 1.52-kb minimal replication origin of the 3.9-kb Streptomyces plasmid pSL1 was determined using a bifunctional derivative, pMCP44, of pSL1. Plasmids with linker insertions into the pSL1 part of pMCP44 were isolated from Escherichia coli. The sites of insertion were determined by restriction enzyme analysis and the ability of the mutant plasmids to replicate in S. lividans 66 was determined. All except one of the inserts in the 1.52-kb essential region inactivated replication. A 104-bp segment from this region could function as a replication origin in the presence of a helper plasmid containing a nonoverlapping pSL1 fragment. The sequence of this 104-bp fragment shows similarities to those of known plasmid replication origins.     

Characterization of the IncW cryptic plasmid pXV2 from Xanthomonas campestris pv. vesicatoria

The gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria strain Xv2 harbors an indigenous, cryptic plasmid pXV2 of 14.6 kb. This plasmid can only be maintained in Xanthomonas and is incapable of self-transmission. However, incompatibility testing classified it in IncW, a group containing the smallest number of naturally occurring, broad-host-range, conjugative plasmids. A pXV2 derivative containing only a 5.5-kb PstI fragment is stably maintained. Deletion of a 3.0-kb region from the PstI fragment causes a loss of plasmid stability. Nucleotide sequencing of the 2. 1-kb region essential for autonomous replication revealed a repA gene and a downstream noncoding region containing four iterons, two 17- and two 19-nt direct repeats, and an AT-rich region lying between the two sets of iterons. The sequence of the deduced RepA and the iterons shows homology to the RepA (39% identity) and the iterons, respectively, of the IncW plasmid pSa. Maxicell expression of the repA gene produced a protein of 35 kDa, a size similar to that deduced from the nucleotide sequence. Trans-complementation test confirmed that the repA gene and the iterons are indeed the essential elements for pXV2 replication.     

Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.     

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.     

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.     

Isolation and characterization of pHW15, a small cryptic plasmid from Rahnella genomospecies 2

A small cryptic plasmid designated pHW15 was isolated from Rahnella genomospecies 2 WMR15 and its complete nucleotide sequence was determined. The plasmid contained 3002 bp with a G+C content of 47.4%. The origin of replication was identified by deletion analysis as a region of about 600 bp. This region had an identity of 70% to the replication origin of the ColE1 plasmid at the nucleotide level. Sequence analysis revealed the typical elements: RNA I, RNA II and their corresponding promoters, a sequence allowing hybridisation of RNA II to the DNA and favouring processing by RNaseH, a single-strand initiation determinant (ssi) that allows initiation of lagging-strand synthesis, and a terH sequence required for termination of lagging-strand synthesis. The plasmid contained three expressed open reading frames, one of which showed homology to a ColE1 plasmid-encoded protein. Furthermore, a multimer resolution site was identified by sequence analysis. Its deletion resulted in formation of plasmid multimers during growth leading to an increased plasmid loss rate.     

Cloning the origin of transfer region of the resistance plasmid R1

The insertion of a 7.7-kb EcoRI fragment of the resistance plasmid R1 into pBR325 yielded a plasmid which is mobilizable by pDB12, a multicopy derivative of R1drd-19 lacking most of the resistance determinants. The vector alone was not mobilizable in this system. From this observation we conclude that we have cloned the origin of transfer (oriT) of R1. After inserting a 5.3-kb PvuII-EcoRI fragment of the 7.7-kb region into pUC9 the DNA was cleaved randomly with DNaseI and BamHI linkers were attached to the ends. A subsequent BamHI digestion and electrophoretic separation of the resulting DNA molecules by their size allowed us to generate an ordered series of stepwise shortened plasmids. Plasmids with a deletion of approximately 3400 bp could no longer be mobilized. Since the next larger plasmid with 284 additional base pairs could be mobilized, we are able to confine the oriT location within this extra nucleotide stretch. The DNA sequence of this region was determined. Dominant features within the DNA region are a high AT content and five inverted repeats, which might function as recognition or substrate sites for proteins of the conjugational transfer system.     

Molecular characterization of a DNA fragment carrying the basic replicon of pTUC100, the natural plasmid encoding the peptide antibiotic microcin J25 system

The Escherichia coli plasmid pTUC100 encodes production of, and immunity to, the peptide antibiotic microcin J25. In the present study, an approximately 8-kb fragment immediately adjacent to the previously sequenced microcin region was isolated and its DNA sequence was determined. The main features of the newly characterized region are: (i) a basic replicon which is almost identical to that of the RepFIIA plasmid R100; (ii) two ORFs with 96% identity to two ORFs of unknown function on pO157, a large plasmid harbored by enterohemorragic E. coli, and a large ORF which does not show significant homology to any other reported nucleotide or protein sequence; and (iii) two intact insertion sequences, IS1294 and IS1. Sequence analysis, as well as that of the G+C content of both the 8-kb fragment and the previously sequenced microcin locus, lead us to propose that plasmid pTUC100 is a composite structure assembled from DNA elements from various sources.     

A comparison of the origin of replication of pSa with R6K

Summary The plasmid pOri3 is a derivative of the origin of replication of pSa. Replication is defective as a result of a truncated repA gene, the product of which is required for plasmid replication. The defective replication is complemented by the presence of the intact repA gene of pSa, or by the presence of the plasmid R6K. The basis of this complementation has been examined by comparing the nucleotide sequence of the origin of pSa with that of R6K. A 13 base pair sequence present twice in the origin of pSa has homology with a 13 base pair sequence that is present fourteen times in the origin of R6K. These sequences may be the binding sites for the initiator proteins of these two plasmids. The location of these binding sites relative to the genes for the initiator proteins suggests that an autoregulatory control mechanism for the synthesis of the initiator proteins may also play a role in the control of plasmid copy number.     

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.     

In vitro deletion mapping of the viral strand replication origin of Pseudomonas bacteriophage Pf3.

The origin of viral strand replication of the filamentous bacteriophage Pf3 has been characterized in Escherichia coli by in vitro deletion mapping techniques. The origin region was functionally identified by its ability to convey replicative properties to a recombinant plasmid in a polA host in which the replication origin of the vector plasmid is not functional. The origin of Pf3 viral strand replication is contained within a DNA sequence of 139 bp. This sequence covers almost completely one of the intergenic regions of the Pf3 genome, and it specifies both replication initiation and termination functions. Although no nucleotide sequence homology is present between the Pf3 origin of viral strand replication and that of the E. coli filamentous phages Ff (M13, f1, and fd) and IKe, their map positions and functional properties are very similar.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Nucleotide sequence of the Thiobacillus ferrooxidans chromosomal gene encoding mercuric reductase

The nucleotide sequence of the Thiobacillus ferrooxidans chromosomal mercuric-reductase-encoding gene (merA) has been determined. The merA gene contains 1635 bp, and shares 78.2% and 76.6% sequence homology with the transposon, Tn501, and plasmid R100 merA genes, respectively. From the sequence, a 545-amino acid (aa) polypeptide was deduced, and comparison with those of Tn501 and R100 revealed 80.6% and 80.0% homology, respectively, at the aa sequence level. Divergence among the three merA aa sequences was clustered within a specific region (aa positions 41-87). By analysis of codon usage frequency, it is speculated that the T. ferrooxidans merA gene originated from Tn501, R100, or a common ancestral gene, but not from T. ferrooxidans itself.     

Sequence analysis of the plasmid pColG from the Escherichia coli strain CA46

The complete 4715 nucleotide sequence of the plasmid pColG from the Escherichia coli strain CA46, which was originally assumed to code for colicin G activity, has been determined. Based on the nucleotide sequence homology of the 1828bp replication region, with an average G+C content of 48%, pColG was classified as a ColE1-like plasmid. Computer assisted analysis of the remaining 2887bp nucleotide sequence with an average G+C content of 34% revealed three putative OFRs. To find out whether one or all of the three ORFs code for a possible bacteriocin, a DNA fragment encompassing these ORFs has been cloned and the recombinant colonies tested for bacteriocin production. None of the colonies had an inhibitory activity against E. coli strains DH5, HB101 and MC4100. The assumption that the plasmid pColG from the E. coli strain CA46 codes for a bacteriocin thus could not be confirmed.