Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome.

Between 59 and 60 min on the Escherichia coli genetic map, there is a highly conserved sequence of 29 base pairs, containing an inverted repeat of seven base pairs that appears 14 times, 32 or 33 base pairs apart, downstream of the iap gene coding region. About 24 kilobase pairs downstream of the 14 repeats, a similar 29-base-pair sequence with a spacing of 32 base pairs appears seven times. Nucleotide sequences hybridizing with the 29-base-pair fragment were also detected in Shigella dysenteriae and Salmonella typhimurium but not in Klebsiella pneumoniae or Pseudomonas aeruginosa.     

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.     

Mapping of insertion element IS5 in the Escherichia coli K-12 chromosome. Chromosomal rearrangements mediated by IS5

We identified phage clones containing insertion element IS5 in a set of 476 lambda phage clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K-12 W3110. Precise locations and orientations of IS5 were then determined by cleavage analysis of phage DNAs containing them. We mapped 23 copies of IS5 (named is5A to is5W) on the W3110 chromosome. Among them, ten were identified as the common elements present at the same locations in both chromosomes of W3110 and another E. coli K-12 strain, JE5519. While most of the mapped IS5 elements were scattered over the W3110 chromosome, four copies of IS5 (designated is5L, is5M, is5N and is5O) were in a region representing tandem duplication of a DNA segment flanked by two copies of IS5. Interestingly, one unit of this DNA segment as well as a portion of it was seen also in a tandem array in a different region where two copies of IS5 (designated is5P and is5Q) were present. In particular two pairs of the mapped IS5 elements may have been involved in inversion of the chromosomal segments in two of the E. coli K-12 derivatives.     

DNA duplications detect the hidden transposition of the Tn5 element in the Escherichia coli K12 genome

The frequency of Tn5 transposition localized in an arm of a tandem duplication was estimated as 1.3 X 10(-2) per cell per generation, two orders of magnitude higher than usual one. Approximately thirty per cent of all transpositions usually registered occur from the spontaneous duplications. The effect revealing latent transpositions is in good accordance with a conservative transposition model permitting some interesting predictions: 1. Composite transposons can be a reason for the double stranded cuts in DNA. 2. The transposition frequency in cis for composite elements seems to be many times higher than in trans. 3. Partially transpositions in cis can be recA dependent. 4. The estimation of Tn5 transposition in cis presented in the paper is a minimal one.     

Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9

HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated. The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants. The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold. The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain. This mutation does not stimulate excision of Tn5 and Tn10. LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold. The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed.     

Specificity of introducing the transposon that determines chloramphenicol resistance (Tn9) into the chromosome of Escherichia coli K-12]

Translocation of Tn9 coding for chloramphenicol-resistance from lambdaatt80 genome into bacterial chromosome was studied. Three preferential sites of Tn9 integration, attTn9A, ATTTn9B and attTn9C, were found on the chromosome of Escherichia coli K-12; attTn9A was mapped between purD and rpoB loci, attTn9B was cotransducible with argG, attTn9C was located between 61 and 66 minutes of the standard E. coli K-12 genetic map. The integration of Tn9 in these att sites occurs with almost equal probability. Tn9 integrated in attTn9A shown significantly higher frequency of excision and P1-transduction frequency than Tn9 integrated in either of two other attTn9 sites.     

Escherichia coli K-12 mutation that inactivates biodegradative threonine dehydratase by transposon Tn5 insertion.

From a collection of kanamycin-resistant mutants of Escherichia coli K-12 isolated by transposon Tn5 mutagenesis, we have identified a mutant that lacks functional biodegradative threonine dehydratase (EC 4.2.1.16) by direct enzyme assay and by the loss of cross-reacting material with affinity-purified antibodies against the purified enzyme. Aerobic and anaerobic growth of this strain on various carbon sources failed to reveal a phenotype. Evidence for the insertional inactivation of threonine dehydratase by Tn5 was obtained by cloning the DNA segments flanking the Tn5 insertion site into pBR322 and hybridizing the cloned DNA to a synthetic oligodeoxynucleotide probe complementary to the DNA segment coding for a unique hexapeptide at the amino terminus end of the enzyme; the region of homology to the synthetic cDNA sequence appears to be located within about 500 nucleotides from one end of Tn5. Genetic analysis with the transposon element that caused insertional inactivation located the tdc gene at min 67 on the E. coli chromosome.     

Identification of a novel genetic element in Escherichia coli K-12.

Induction of the SOS repair processes of Escherichia coli K-12 caused a 14.4-kilobase species of circular deoxyribonucleic acid, called element e14, to be excised from the chromosome. To aid further characterization of this species, an 11.6-kilobase segment of e14 was inserted into the HindIII site of plasmid pBR313. To map e14 on the E. coli K-12 chromosome, the recombinant plasmid, pAG2, was used to transform a polA recipient, an event which required integration of pAG2 into the recipient chromosome. This recombinational event was dependent upon the region of homology between the incoming plasmid and the chromosome, as no transformants were scored when either a strain cured of the element was the recipient or pBR313 was the transforming deoxyribonucleic acid. Using these transformants, we have shown that e14 maps between the purB and pyrC loci near min 25. Several strains of E. coli K-12 were found to contain e14; however, one strain, Ymel trpA36, did not. In addition, e14 was found to be absent in both E. coli B/5 and E. coli C. The approach to mapping developed for this work could be used to map other fragments of E. coli deoxyribonucleic acid which have no known phenotype.     

Characteristics of Escherichia coli K-12 mutants with altered effectiveness of excision of Tn5 and Tn9 transposons

The properties of Escherichia coli K-12 mutans HFETn5, HFETn9 and LFETn9 have been studied. The majority of mutations were shown to have pleiotropic effect. Some of them increase cell sensitivity to UV light and mitomycin C and affect efficiency of homologous recombination in transduction and conjugation. The level of spontaneous mutagenesis is increased in a number of mutants. None of the mutations isolated affect frequency of transposition of Tn5 from bacteriophage lambda::Tn5 into the chromosome. Based on analysis of properties of hfeTn5-09 and hfeTn9 mutations and on the date of preliminary mapping of hfeTn5-09 mutation, these mutations were considered to be novel. It is shown that the processes of precise excision of Tn5 and Tn9 transposons may be accomplished by at least two pathways, one of them being dependent on recA gene functions.     

Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tn1 and Tn3.

Escherichia coli K-12 minicells were employed to examine polypeptides encoded by plasmids carrying wild-type and mutant Tn1 or Tn3 transposition elements. Tn1- and Tn3-containing minicells express high levels of four transposon-specified polypeptides. Three, of molecular weights 30,000, 28,000, and 25,000, are related immunologically to beta-lactamase, the enzyme responsible for ampicillin hydrolysis. A fourth polypeptide of molecular weight 19,000 is encoded by the Tn1 or Tn3 region which spans the BamHI cleavage site. Mutant transposons which no longer produce this polypeptide transpose at higher than wild-type frequencies to give aberrant transposition products (Gill et al., J. Bacteriol. 136: 742--756, 1978; Heffron et al., Proc. Natl. Acad. Sci U.S.A. 72:3632--3627, 1975). No expression could be detected from a region of the transposons extending from the inverted repeat sequence distal to the beta-lactamase gene to more than half the distance into the Tn1 or Tn3 sequence.     

Transposon (Tn5)-mediated suppressive integration of ColE1 derivatives into the chromosome of Escherichia coli K12 (dnaA)

While integration of ColE1 had not been observed previously by ordinary suppressive integration, a $\text{dnaA}$ (Ts) $\text{E. coli}$ strain with Tn$\text{5}$ at various sites of the chromosome and ColE1 or its mini-derivative, pAO3, but not pSC101, inserted by the same transposon produced integratively suppressed strains depending on the RecA function. In contrast to Hfr strains made with a stringently controlled plasmid, they contained the plasmid not only in an integrated but in an autonomous state at an amount comparable to the strain containing the plasmid only autonomously. Introduction of a RecA-deficient mutation to the strain with an integrated ColE1 derivative through conjugation failed. This is likely to be due to lethality of such a strain without RecA-dependent excision of the integrated high copy number plasmid or to quantitative deficiency of DNA polymerase I in addition to the $\text{recA}$ mutation.     

Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12.

In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.