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.     

Repressor gene finO in plasmids R100 and F: constitutive transfer of plasmid F is caused by insertion of IS3 into F finO.

Fertility factor F confers bacterial conjugation, a process which involves at least 20 tra genes. Resistance plasmids such as R100, R6-5, and R1 have homology with F in the tra region. Conjugal transfer of these plasmids is, however, repressed, while transfer of F is constitutive. Repression of R transfer is due to the existence of the two genes, called finO and finP; constitutive transfer of F is believed to be due to a lack of finO in F. In this paper, we report the identification and DNA sequence of the finO gene of R100, encoding a protein of 21,265 daltons. We show that F does actually encode finO, but the gene has been inactivated by insertion of IS3. Lederberg and Tatum (Nature [London] 158:558, 1946), who discovered sexuality in bacteria, may have had an Escherichia coli K-12 strain harboring such an finO F factor, which facilitated the generation of recombinant progeny useful for genetic analysis of bacteria and established the foundation for molecular genetics.     

Integration host factor and conjugative transfer of the antibiotic resistance plasmid R100.

Transfer of plasmid R100-1 was reduced 100-fold in the absence of integration host factor.     

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.     

Derepression of conjugal transfer of the antibiotic resistance plasmid R100 by antisense RNA.

Conjugal transfer of the normally repressed antibiotic resistance plasmid R100 was derepressed by fragments of R100 that carried the traJ promoter and the traJ leader but lacked the finP promoter.     

Transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid R6K.

Transformation experiments showed that spontaneous deletions which result in loss of streptomycin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10(-4) in plasmid molecules of R6K. Similar deletions were thus readily selected by conjugal transfer of R6K, and their appearance was dependent upon recA+ activity in either donor or recipient host. The deoxyribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA+ strains. In four plasmids examined after transfer to a recA host, an inverted repeat of the preexisting TnA element was shown to have been inserted at a similar location and was in two instances associated with deletions which extended from the same direction as those described above. The deletions are ascribed to the result of recA+-dependent recombination between direct repeats of TnA.     

Replication of antibiotic resistance plasmid R6K DNA in vitro.

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.     

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 F plasmid carries an IS3 insertion within finO

DNA-DNA hybridization studies support the conclusion that coding sequences of the fertility inhibition gene, finO, are present on the F plasmid and map downstream of the transfer region, entirely within 99.19-2.0 F. In addition, the results indicate that the IS3a element at 100/0-1.26 F is inserted within the F finO coding region. This insertion may have inactivated the finO gene on the F plasmid.