Further mapping of IS2 and IS3 in the lac-purE region of the Escherichia coli K-12 genome: structure of the F-prime ORF203.

The sequence organization of the F-prime ORF203 was determined by heteroduplex analysis. This large, type II F-prime (Scaife, 1967) contains lac, proC, and purE genes derived from the W1485 subline of Escherichia coli K-12. The IS3 and IS2 elements previously found in the lac-proC-purE region derived from the 58-161 subline (Hu et al., 1975) are also present in the same locations in the bacterial deoxyribonucleic acid (DNA) from the W1485 subline. Recombination between the IS2 region of F and an IS2 element located between lac and proC on the bacterial DNA apparently led to the formation of the perental Hfr, OR21. IS2 is thus directly repeated, with one copy of each element appearing at each of the two junctions between F and the bacterial sequences on ORF203. The F plasmid is found together with ORF203 in the plasmid DNA, and this probably forms from ORF203 by recombination between the directly repeated IS2 elements. ORF203 appears to have been excised from the Hfr chromosome by recombination between the IS3 sequence alpha3beta3 located counterclockwise of lac and the directly repeated IS3 sequence alpha4beta4 located clockwise of purE.     

Specificity in the formation of delta tra F-prime plasmids.

Twenty-three independent delta tra F-prime plasmids from three different Escherichia coli K-12 sublines were isolated from Hfr strains whose points of origin coincided with the IS3 element alpha 3 beta 3 or alpha 4 beta 4 in the lac-purE region of the E. coli chromosome. Electrophoretic analysis of plasmid deoxyribonucleic acid digested with EcoRI and hybridization analysis of plasmid deoxyribonucleic acid digested with BglII revealed that at least 14 of these plasmids were formed by processes involving specific bacterial and F loci. Two of the specific bacterial loci involved in delta tra F-prime formation were located at approximately 3.3 and 11.7 min on the E. coli chromosomal map. Two of the delta tra F-prime plasmids contained bacterial deoxyribonucleic acid with circularization endpoints that mapped very near the termini of the IS2 element that is normally located between lac and proC.     

Specificity in formation of type II F'' plasmids.

Eight new F' plasmids derived from Hfr strains in which F is integrated at the chromosomal element alpha 3 beta 3 have been isolated and subjected to restriction enzyme, hybridization, and electron microscope heteroduplex analysis. Plasmids carrying extensive amounts of bacterial deoxyribonucleic acid were produced even though they were obtained by selection for transfer of lac, which is closely linked to F in the parental Hfr strains. Seven plasmids were type II Flac+ proC+ purE+ plasmids, and one was a type I Flac+ proC+ plasmid. Five of the Flac+ proC+ purE+ plasmids contain approximately 284 kilobases of bacterial deoxyribonucleic acid, which is identical for all five within the resolution of the restriction enzyme analysis. Theses results indicate that type II F' plasmids are the predominant tra+ F' type from this region of the Escherichia coli K-12 chromosome and that the recombination events leading to formation of these plasmids exhibit site specificity.     

Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. IV. Recombination characteristics of Gamr mutants

In the radiation-resistant Gamr444 mutant the inheritance frequency of long F' episomes ORF1 (purE+ tsx+ procC+ lac+) and F'14 (ilv+--argE+) is lower, and the frequencies of chromosome mobilization and integrative suppression of temperature-sensitive dnaA46 mutation by the sex factor F are much higher than those in the wild-type strain AB1157 and another radiation-resistant mutant Gamr445. In this respect, the mutant Gamr444 is very similar to the recRC sbcB mutant (RecF-pathway of recombination).     

Ultraviolet Sensitivity Gene of Escherichia coli B

The ultraviolet sensitivity gene of Escherichia coli B was introduced into a K-12 recipient by transduction with phage P1. The uvs gene of E. coli B is cotransducible with the proC locus of K-12, is closely linked to tsx, is not linked to lacZ, and only rarely to purE. The transductants are mucoid, filamentous on irradiation, and show plating-medium response. The order of markers is lacZ proC tsx uvs purE.     

Mapping by Transposons of the Inversion Termini in Escherichia Coli K-12 Strain 1485in

Strain 1485IN carries a chromosomal inversion which corresponds to 35% of the chromosome and includes proC, trp and his genes. The termini of the inversion lie between the lac and proC loci and between his and cdd of the normal strain. Using Tn10 and Tn5 in transduction crosses between the normal and inversion strains, the termini were mapped to sites located approximately 0.25 min and 1.6 min away from proC and his, respectively within a region of roughly 4 kb long. The crosses where the normal strains carrying Tn10 near the terminus are donors and the inversion strain is a recipient, yielded unusual Tetr His- recombinants, which arose from illegitimate recombination leading to the replacement of a chromosomal his+ region with a transducing fragment carrying proC. Another rearrangement was detected between the normal and inversion strains in a region outside the inverted segment near the cdd locus.     

Formation of delta tra F' plasmids: specific recombination at oriT

Delta tra F' plasmids can be isolated from matings between Hfr donors and recA- recipients, with selection for transfer of proximal chromosomal genes. Previous experiments indicate that F DNA from the neighborhood of the transfer origin up to the proximal junction with the chromosomal DNA is present on these plasmids, together with chromosomal segments, some of which belong to distinct size classes. We have sequenced across the novel joints contained in five delta tra FproA+ plasmids and in five delta tra FpurE+ plasmids, and we have compared these with the F sequence near oriT and with a chromosomal site near purE. The previously reported specificity in formation of some of these classes is confirmed at the nucleotide sequence level. The F DNA in nine of these novel joints extended beyond the nicking sites identified by others in lambda oriT+ bacteriophages up to a position between two sequenced oriT- mutations. Small plasmids containing these novel joints are mobilized in trans by pOX38 at frequencies less than 5 X 10(-7) times the mobilization frequencies for similar plasmids that contain oriT. The relations of these findings to the location of the nicking site at oriT are discussed.     

Further genetic mapping of the phoA-phoR region for alkaline phosphatase synthesis in Escherichia coli K 12

Summary Genetic mapping of the E. coli chromosomal region carrying the structural gene (phoA) and the regulatory gene (phoR) for alkaline phosphatase synthesis was carried out by conjugation. Recombinant colonies were selected and the segregation frequency of outside markers was determined. The genetic order lac phoA proC phoR tsx lon is proposed.     

Genetic Analysis of an ESCHERICHIA COLI Mutant with a Lesion in Stable RNA Turnover

A mutant that rapidly degrades more than 80% of its rRNA and tRNA under defined conditions was genetically analyzed. Two genes, srnA and srnB, are separately located, and the mutated alleles of both are required for degradation of stable RNA in cultures treated with rifampicin at 42 degrees . srnA is closely linked to tsx by matings and transduction tests; by P1 transduction, the gene order is lac (9 min) proC (9.55 min) tsx (9.8 min) srnA (about 10 min) purE (12 min) rnsA (14.4 min). srnB is not yet completely mapped, but is outside the lac-rnsA region, probably in the region between 75 and 90 min.-The product of the rnsA gene, RNase I, is a potent endonuclease of E. coli, and the only one known that can attack ribosomes and tRNA. However, not only are the srn lesions genetically separate from rnsA, but also, derivatives of an srn strain were prepared lacking RNase I, and they retain the Srn(-) phenotype. Thus, no correlation of rapid RNA turnover and RNase I activity has been found.     

Molecular cloning and characterization of the purE operon of Escherichia coli

The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene.     

Mapping of chromosomal IS5 elements that mediate type II F-prime plasmid excision in Escherichia coli K-12.

Three IS5 elements were mapped in overlapping chromosomal segments on a series of F-prime plasmids by restriction analysis and hybridization. IS5A was located clockwise of proA near 6 min, IS5B was located clockwise of purE near 12 min, and IS5C was tentatively located near 14 min on the Escherichia coli K-12 map. The physical structures of nine type II F-prime plasmids that contain chromosomal DNA from this region indicated that these plasmids were excised from the chromosome by recombination between pairs of IS5 elements.     

Inversions and deletions generated by a mini-gamma delta (Tn1000) transposon.

Intramolecular transposition by an engineered derivative of the transposon gamma delta (Tn1000) is described. This 1-kb element contains inverted repeats of the 40 bp of the delta end of gamma delta, bracketing a kan gene, but it contains no resolution site. Transposition was analyzed in two plasmids; one contained two contraselectable (conditional lethal) genes (thyA and sacB) adjacent to the mini-gamma delta element in a 13.0-kb pBR322/pUC-based two-component plasmid (a heterodimer), and the other contained a different contraselectable gene (strA [rpsL]) in a 13.2-kb three-component plasmid (a heterotrimer). Selection for loss of function of a single contraselectable gene yielded inversions and deletions. Each inversion plasmid was 1 kb larger than the parent plasmid: it had a second copy of mini-gamma delta inserted in the contraselected gene, with that copy plus the intervening segment inverted, and the 5-bp target site duplicated. Each deletion plasmid was smaller than the parent plasmid and had a deletion that extended from one transposon end into or through the contraselected gene for distances of up to 9.4 kb. The frequencies of deletions versus inversions ending in a single target gene were similar, although overall, deletions outnumbered inversions because deletions, but not inversions, into sites beyond the contraselected gene inactivate it. This work also demonstrates that thyA (which encodes thymidylate synthetase) is a useful contraselectable marker.     

Deletion Map of the Escherichia coli K-12 Sex Factor F: the Order of Eleven Transfer Cistrons

A series of Hfr deletion mutants was isolated. These mutants contain deletions which extend from a lambda prophage into an Flac which is integrated into the gal operon. Transfer-deficient deletion mutants were found to fall into four different phenotypic groups when tested for male- and female-specific phage resistance. Conjugational and transductional complementation tests with Flac point mutants deficient in transfer (tra(-)) were performed, and the order of 11 tra cistrons was determined. The tra genes are all located between an F gene for the inhibition of female-specific phages and the transposed lac operon originally carried by the Flac. The order of genes in the Hfr studied was established to be: proC... phi(II) (R)... traJ traA traE traK traB traC traF traH traG traD traI...lac...attlambda...bio.     

Inversion in the lactose region of Escherichia coli K-12.

A spontaneous mutant of Escherichia coli K-12, strain SY99, with an inversion in the lactose region was isolated and partially characterized. The inversion was detected due to inverse chromosomal conjugational transfer after introduction of an F42 (F'lac) episome. The termini of the inversion are between proAB and lac on one side and lac and proC on the other. The inverse conjugational transfer in SY99 did not appear to be absolute but was always accompanied by a residual "normal" counterclockwise mobilization. This residual transfer was further shown to be caused by the intrinsic instability of this region (at least in the line W3110). The possible involvement of IS3 elements flanking the lactose operon is discussed.     

Deletions within E. coli plasmids carrying yeast rDNA.

Deletions occur in recombinant DNA plasmids that contain yeast ribosomal DNA (rDNA) inserted into the E. coli plasmids pSC101 and pMB9. Deletions within a pMB9 plasmid containing an insert longer than one tandem rDNA repeat apparently are due to homologous recombination because (1) all of the independently derived deletion products of this plasmid lost one complete rDNA repeat (8.6 kb) and retained only a single copy of the segment repeated at the ends of the original insert and (2) deletions were detected only when the insert had terminal redundancy. Deletions also occur within a pSC101 plasmid containing a tandem duplication of a segment (4.7 kb) including both pSC101 DNA and rDNA. Once again these deletions appear to be due to the presence of a duplicated region because all deletion products have lost one complete repeat. Deletions within both of these plasmids took place in both rec+ and recA- host cells, but occurred more frequently in rec+ cells. Oligomerization of the deletion products also occurred in both hosts and was more frequent in rec+ cells.     

A physical map of the bioAB region in the lambda bio transducing phage

The center of the pBopA promoter-operator region for the bioABFCD operon is located 1.71 kb clockwise from the att lambda site on the Escherichia coli genome, as determined by the position of the p131 (IS1) insertion. The order of several bio endpoints to the right of p131 is lambda bio267, 122, 169, 74, 1, and 69. The endpoints of the two bio deletions, delta 61 in bioA and delta 3h in bioB, were also determined.     

Modified Map Positions for lac and the pro Markers in Escherichia coli K-12

Interrupted mating experiments were performed with Hfr strains H and C and three leu lac purE recipient strains derived from a common parent and carrying, respectively, the proA(-), proB(-), and proC(-) mutations. It was concluded that if leu is placed at 1.5 min and purE at 12 min from thr, the origin on the Taylor-Trotter map, lac is at about 7.5 min and the pro genes are at about 6.0, 6.6, and 8.4 min, respectively. Both conjugational and transductional data suggest that the strain carrying the proB(-) mutation also carries a second mutation close to the proA site which independently confers a Pro(-) phenotype. The times before the onset of transfer of chromosomal deoxyribonucleic acid by both Hfr strains B4 and B8 were approximately 3 min.     

Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes.

To study expression of uncG, the gene coding for the gamma subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the alpha subunit, and uncG. Two deletions which fused uncA and uncG coded for alpha-gamma fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region. Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E. coli. The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked protons. The deleterious effect was eliminated by making a nonpolar deletion in the upstream F0 gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the F0 genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability. A plasmid which carried F0 genes and the gene for the delta subunit caused deleterious proton leakiness in unc+ cells but not in cells from which the unc operon was deleted. The proton leakiness caused by these different plasmids was therefore due to the production of a leaky F0 proton channel and required the presence of F1 genes. The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the F0 proton channel.     

Origin of Escherichia coli K-12 Hfr B7.

Several F' plasmids encoding resistance to tetracycline have been derived from a trg::Tn10 Hfr B7 strain of Escherichia coli K-12. One of these plasmids, JGF312, was analyzed by restriction endonuclease digestion and Southern blot hybridization to cloned chromosomal fragments. This analysis revealed that JGF312 was formed by Tn10-promoted deletion from the Tn10 insertion (31.4 min) to within the prophage rac at 30.1 min. Hfr B7 was shown to result from recombination between IS2 of F delta (33-43) and a chromosomal IS2 located within the rac-man region at 30.9 min on the genetic map.