Nucleotide sequence of the replication region of plasmid R401 and its incompatibility function

The plasmids R401 and Rtsl belong to the same incompatibility group, IncT. The nucleotide sequence of the basic replicon of R401 consisting of 1,857 base pairs was determined and compared with that of mini-Rtsl previously reported. The mini-R401 was found to be composed of two clusters of direct repeated sequences flanking a large open reading frame that could encode a 33,000 Mr protein (RepA protein) consisting of 288 amino acids. This structure of mini-R401 is quite similar to that of mini-Rtsl. Furthermore, the nucleotide sequence of mini-R401 is identical to that of mini-Rtsl except for eleven nucleotides; three are located near the carboxyl terminus portion of the RepA coding region (repA) and four are in the repeated sequences (incI) located downstream from repA. Incompatibility study showed that mini-R401 plasmid coexisted stably with the cloned incI repeats of mini-Rtsl, suggesting that mini-R401 RepA protein binds to incI repeats of mini-Rtsl less efficiently than does mini-Rtsl RepA protein.     

Unidirectional replication of plasmid R100

We isolated a 284 base-pair BamHI fragment of plasmid R100 that supports initiation of replication of a plasmid regardless of the orientation of the fragment. Analysis of the specific radioactivity of restriction fragments from 32P-labeled replication intermediates synthesized in vitro shows that replication of the plasmid carrying the 284 base-pair fragment is unidirectional. The direction of replication depends on the orientation of the fragment present in the plasmid. The 5' ends of the leading-strand DNA formed in the early stage of replication were mapped to a region downstream from the 284 base-pair fragment in the direction of replication. The lagging-strand DNA products were also identified and their 3' ends mapped to unique sites within the 284 base-pair fragment causing unidirectional replication of R100.     

The nucleotide sequence of the replication origin beta of the plasmid R6K

We h ave identified by molecular cloning a region of 283 base pairs of the HindIII 2 fragment of R6K which corresponds to the region of the replication origin beta. This 283 base-pair DNA fragment, when present contiguously with the structural gene for the replication initiation protein of R6K, encoded in the HindIII 9-15 and part of HindIII 2 restriction fragments, will support the replication of a plasmid chimera containing the pBR322 replicon in a pol Ats host at the nonpermissive temperature. The nucleotide sequence of the region of replication origin beta has been determined. The nucleotide sequence has some homology with the ori gamma region of R6K; it has a 15-base-pair homology with the replication origin of Escherichia coli.     

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.     

Direction of deoxyribonucleic acid transfer and replication in a derivative of plasmid R100-1.

The site of integration and the molecular orientation of a prophage Mu integrated within the resistance transfer factor component of plasmid R100-1 have been determined on the physical map of the plasmid. This allowed us (i) to determine the direction of deoxyribonucleic acid transfer from oriT during conjugation and (ii) to demonstrate the unidirectionality of replication in conditions of exponential growth (by determining the strand preference of Mu-specific Okazaki fragments).     

Genetic analysis of the inter-relationship between plasmid replication and incompatibility

Summary The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050. Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties. Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety. Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1.The inability of the composite plasmid to replicate at 42° in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component. Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell. Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nevertheless retain pSC101 incompatibility. In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability.     

The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19

Summary The region of plasmid R1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wildtype R1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.     

Nucleotide sequence analysis of the complement resistance gene from plasmid R100

The multiple antibiotic resistance plasmid R100 renders Escherichia coli resistant to the bactericidal action of serum complement. We constructed a plasmid (pOW3) consisting of a 1,900-base-pair-long restriction fragment from R100 joined to a 2,900-base-pair-long fragment of pBR322 carrying ampicillin resistance. E. coli strains carrying pOW3 or R100 were up to 10,000-fold less sensitive to killing by serum complement than were plasmid-free bacteria or bacteria carrying pBR322. Nucleotide sequencing revealed that 875 of the 1,900 bases from R100 correspond exactly to part of the bacterial insertion sequence IS2. The remaining 1,075 bases contained only one sizeable open reading frame; it covered 729 base pairs (243 amino acids) and was preceded by nucleotide sequences characteristic of bacterial promoters and ribosome binding sites. The first 20 amino acids of the predicted protein showed features characteristic of a signal sequence. The remainder of the predicted protein showed an amino acid composition almost identical with that determined for the traT protein from the E. coli F factor. Southern blot analysis showed that the resistance gene from R100 does not hybridize to the serum resistance gene from ColV,I-K94 isolated by Binns et al.; we concluded that these genes are distinct.     

The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region.

The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21. pRR12 produced an altered incompatibility product and also had an altered incompatibility target site. The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication. The incompatibility properties of pRR21 were indistinguishable from those of NR1. Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene. In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region. Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1. The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production. For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived. This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.     

Complete nucleotide sequence of an exogenously isolated plasmid, pLB1, involved in gamma-hexachlorocyclohexane degradation

The alpha-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, gamma-hexachlorocyclohexane (gamma-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of gamma-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS6100 composite transposon containing two copies of linB, and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other alpha-proteobacterial strains but not to any of the beta- or gamma-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for gamma-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in gamma-HCH degradation.     

Cloning of replication, incompatibility, and stability functions of R plasmid NR1.

The region of R plasmid NR1 that is capable of mediating autonomous replication was cloned by using EcoRI, SalI, and PstI restriction endonucleases. The only EcoRI fragment capable of mediating autonomous replication in either a pol+ or a polA host was fragment B. SalI fragment E joined in native orientation with the part of SalI fragment C that overlapped with EcoRI fragment B, and also two contiguous PstI fragments of sizes 1.6 and 1.1 kilobases from EcoRI fragment B-mediated autonomous replication. When these individual SalI fragments were cloned onto plasmid pBR313 or the individual PstI fragments were cloned onto plasmid pBR322, none of these single fragments could rescue the replication of the ColE1-like vectors in a polA host, even in the presence of a compatible "helper" plasmid derived from a copy mutant of NR1. In contrast to the results reported for closely related R plasmid R6, EcoRI fragment A of NR1 could not rescue the replication of ColE1 derivative RSF2124 in a polA(Am) mutant or in a polA(Ts) mutant at the restrictive temperature. Although capable of autonomous replication, EcoRI fragment B of NR1 (or smaller replicator fragments cloned from it by using other restriction enzymes) was not stably inherited in the absence of selection for the recombinant plasmid. When EcoRI fragment B was ligated to EcoRI fragment A of NR1, the recombinant plasmid was stable. Thus, EcoRI fragment A contained a stability (stb) function. The stb function did not act in trans since EcoRI fragment B was not stably inherited when a ColE1 derivative (RSF2124) ligated to EcoRI fragment A was present in the same cell. A cointegrate plasmid consisting of EcoRI fragment B of NR1 ligated to RSF2124 was also not stably inherited, whereas only EcoRI fragment B was unstable when both RSF2124 and EcoRI fragment B coexisted as autonomous plasmids in the same cell. The incompatibility gene of NR1 was shown to be located within the region of overlap between SalI fragment E and the PstI 1.1-kilobase fragment. A copy mutant of NR1 (called pRR12) was found to have greatly reduced incompatibility with NR1; this Inc- phenotype is cis dominant.     

The nucleotide sequence of the replication origin of plasmid NTP1.

The sequence of the DNA of the origin region of NTP1 has been obtained. Analysis of the sequence indicates that: (1) there is great sequence homology in the DNA upstream from the origin in NTP1, ColE1, CLODF13, PBR345 AND PBR322; (2) only seven base pairs of NTP1 are identical with the sequence downstream from the origin in ColE1, although some homology exists for 140 bases downstream; (3) two ten base pair direct repeats are present in NTP1 which are also conserved in all four plasmids named above; (4) probably no polypeptide greater than fifteen amino acids in length is encoded by the NTP1 origin region, since no single open reading frame is conserved in all five plasmids.     

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Genetic loci responsible for incompatibility on a co-integrate plasmid, R100-1.

An R plasmid, R100-1, was mapped previously (Yoshikawa, 1974) by transduction from an integratively suppressed Hfr strain to a recipient with a mutation in gene dnaA. By this method various types of transductants of plasmid R100-1 that exist autonomously or in the integrated state were obtained. Seventy-one such transductants were used in the present study to map gene inc, which is responsible for incompatibility. The results obtained can be explained by either of the following: (i) R100-1 has only a single gene or gene cluster (inc) despite previous work suggesting that this plasmid is a co-integrate of two replicons; (ii) R100-1 possesses more than one inc locus located between the repA and tra loci.