PSM2, PSM1, and PSM15 are small plasmids derived from R100 by spontaneous deletions at either end of the insertion sequence IS1. These plasmids were used to identify regions neighboring IS1 as well as the IS1 DNA itself, by cleavage with EcoR1, HindIII, Hae III, Hpa II, Hha I, Hinf, and AIu I. The nucleotide sequencing results demonstrate that IS1 contains 768 bases. About 30 bases at the ends of IS1 were found to be repeated in an inverted order. The deletions occurring at the ends of IS1 were found to be due to illegitimate recombination. The hypothesis that RNA polymerase could play an important role in such recombination phenomena is discussed based on the nucleotide sequences surrounding the recombinational hot spots.

https://www.pnas.org/content/pnas/75/2/615.full.pdf

Nucleotide sequence of an insertion element, IS1

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, derived from an outbreak in Sakai city, Japan in 1996, possesses two kinds of plasmids: a 93-kb plasmid termed pO157, found in clinical EHEC isolates world-wide and a 3.3-kb plasmid termed pOSAK1, prevalent in EHEC strains isolated in Japan. Complete nucleotide sequences of both plasmids have been determined, and the putative functions of the encoded proteins and the cis-acting DNA sequences have been analyzed. pO157 shares strikingly similar genes and DNA sequences with F-factor and the transmissible drug-resistant plasmid R100 for DNA replication, copy number control, plasmid segregation, conjugative functions and stable maintenance in the host, although it is defective in DNA transfer by conjugation due to the truncation and deletion of the required genes and DNA sequences. In addition, it encodes several proteins implicated in EHEC pathogenicity such as an EHEC hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP) and type II secretion system. pOSAK1 possesses a ColE1-like replication system, and the DNA sequence is extremely similar to that of a drug-resistant plasmid, NTP16, derived from Salmonella typhimurium except that it lacks drug resistance transposons.

Complete Nucleotide Sequences of 93-kb and 3.3-kb Plasmids of an Enterohemorrhagic Escherichia coli O157:H7 Derived from Sakai Outbreak

DnaA protein directs the binding of DnaB protein in initiation of DNA replication in Escherichia coli.

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.

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

Identification of the DNA sequence from the E. coli terminus region that halts replication forks

Unidirectional replication in Escherichia coli of three small plasmids derived from R factor R12.

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Unidirectional replication in Escherichia coli of three small plasmids derived from R factor R12.