DNA rearrangement of the shufflon determines recipient specificity in liquid mating of IncI1 plasmid R64.

The shufflon is a unique DNA rearrangement found in plasmid R64. R64 shufflon consists of four DNA segments, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any inverted repeats results in a complex DNA rearrangement where four DNA segments invert independently or in groups. The shufflon is a biological switch to select one of the seven C-terminal segments of the pilV gene. To examine the biological significance of the shufflon, R64 derivatives, where the pilV gene was fixed in seven C-terminal segments, were constructed and used as donor cells for liquid mating. The transfer frequencies depended markedly on the combinations of recipient bacterial strains and C-terminal segments of the pilV gene in donor cells, indicating that the shufflon determines the recipient specificity in liquid mating of R64. The products of the pilV genes were found to be a component of thin pili produced by R64.

Effect of diltiazem on silent ischemic episodes, plasma bradykinin and prostaglandin metabolism.

Plasma bradykinin and prostaglandin metabolism are related to the anginal pain modulating system in patients with ischemic heart disease. We carried out a placebo controlled single blind test of diltiazem (30 mg three times a day) in 15 patients with chronic stable angina. The effect of diltiazem was evaluated by exercise treadmill testing and 48-h ambulatory electrocardiographic monitoring. Plasma bradykinin, thromboxane B2, and 6-keto-prostaglandin F1 alpha levels were determined by radioimmunoassay prior to and during diltiazem therapy. Diltiazem significantly increased the exercise time and reduced episodes of angina. Diltiazem, however, did not appreciably improve either the frequency of silent myocardial ischemic episodes or the total duration of the silent myocardial ischemic episodes. Diltiazem also tended to decrease plasma bradykinin, thromboxane B2, and 6-keto-prostaglandin F1 alpha levels. When ischemic episodes on ambulatory electrocardiographic monitoring are categorized according to heart rate change at the onset of episode (type A, preceded by heart rate increase > or = 5 beats/min; type B, no preceding heart rate increase), diltiazem was only effective on type A ischemic episodes as well as on symptomatic ischemia. Further, bradykinin was significantly decreased by diltiazem only in patients with exercise-induced silent ischemia or no exercise-induced ischemia, while the thromboxane B2/6-keto-prostaglandin F1 alpha ratio was unaffected by the administration of diltiazem. Thus, silent and symptomatic ischemia may be associated with different bradykinin and prostaglandin responses.

Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64.

Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.

Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.

Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64.

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.

Distribution of shufflon among IncI plasmids.

A shufflon or clustered inversion is a novel type of DNA rearrangement originally discovered in the IncI1 plasmid R64 (T. Komano, A. Kubo, and T. Nisioka, Nucleic Acids Res. 15:1165-1172, 1987). In a 1.95-kilobase region of R64 DNA, four DNA segments inverted independently or in groups, resulting in a complex DNA rearrangement. We found similar types of shufflon in other IncI1 plasmids, including delta, pIP111, pIP565, pIP112, pIP186, R144, R163, R483, and R621a. A variant type of shufflon occurs in the IncI1 plasmid ColIb.

Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames.

The IncI alpha plasmid R64 was found to bear a highly mobile DNA segment which was designated as a clustered inversion region (J. Bacteriol. 165, 94-100, 1986). The clustered inversion region consists of four DNA segments designated respectively as A, B, C and D which differ in molecular size and restriction sites. The four DNA segments invert independently or in groups resulting in a complex DNA rearrangement. We now show the nucleotide sequence of the clustered inversion region of R64. The present results suggest that the clustered inversion region is a biological switch to select one of seven open reading frames whose primary structures at the region proximal to N-termini are constant while those at the C-terminal region are variable. A name, "Shufflon" was proposed to call this kind of the clustered inversion region.

Highly mobile DNA segment of IncI alpha plasmid R64: a clustered inversion region.

When R64 DNA was digested with EcoRI, two DNA fragments not equimolar to the plasmid DNA were produced. A DNA region including these fragments was cloned (pKK009), and the pKK009 DNA sample was found to be a mixture of six or more DNA species with EcoRI, PstI, and AvaI cleavage sites at different positions, suggesting a complex rearrangement of DNA. When a part of the pKK009 DNA was removed by HindIII digestion, 33 different types of plasmids (pKK010-series plasmids) were obtained out of 58 clones tested, but no DNA rearrangement could be observed. On the basis of a comparison of the detailed restriction maps of these pKK010-series plasmids, we propose a model in which four DNA segments invert independently or in groups within the 1.95-kilobase region of R64, so that the arrangements of these four segments change randomly. The fixed pKK010-series plasmid DNA was again rearranged in the presence of R64, indicating that trans-acting gene function may be present to mediate the DNA rearrangement. The gene (tentatively designated as rci) was located on a 4.5-kilobase E9' fragment of R64.

Physical and genetic analyses of the Inc-I alpha plasmid R64.

A 126-kilobase (kb) physical and genetic map of the Inc-I alpha plasmid R64 was constructed by using the restriction enzymes, BamHI, SalI, XhoI, HindIII, and EcoRI. The replication (Rep) and incompatability (Inc) functions of this plasmid were located in a 1.75-kb segment of an EcoRI fragment, E10 (3.3 kb). In addition, the genes determining growth inhibition of phage BF23 (Ibf), suppression of dnaG ( Sog ), resistance to tetracycline (Tetr), and resistance to streptomycin ( Strr ) were located on the 5.5-kb HindIII-XhoI fragment, the 8.1-kb EcoRI fragment (E5), the 4.6-kb HindIII fragment (H8), and the 4.1-kb HindIII fragment (H10), respectively. The map of R64 was compared with that of ColIb, which belongs to the Inc-I alpha group.

Molecular recombination between R-factor deoxyribonucleic acid molecules in Escherichia coli host cells.

THREE PREVIOUSLY STUDIED R FACTORS WERE USED: 222/R4, controlling transmissible resistance to sulfonamide, streptomycin, chloromycetin, and tetracycline (SU(r) SM(r) CM(r) TC(r)); 222/R3, a derivative of 222/R4 (now termed 222/R3W) having lost TC(r); and R15, controlling infectious resistance to SU and SM only. Two types of derivative R factors were isolated from 222/R4 by serial subculture in Salmonella species. One derivative, termed 222/R1, lost resistance to SU, SM, and CM, and the other, termed 222/R3N, lost only TC(r). Each factor was transferred to a standard Escherichia coli K-12 host. Recombinant factors of 222/R4 phenotype were isolated by selection after mixed culture of E. coli (222/R1)(+) and (222/R3N)(+) strains. Density-gradient equilibrium centrifugation of lysates of E. coli R(+) hosts in the presence of ethidium bromide separated R-factor deoxyribonucleic acid (DNA) as a heavy satellite peak which was subjected to electron microscopy or analytical density gradient centrifugation. Each DNA comprised a unimolecular species of circular DNA. The contour of R15 measured 22.3 mum [equivalent to 46 x 10(6) atomic mass units (AMU)], and that of 222/R4 measured 33.6 mum (70 x 10(6) AMU). 222/R3W appeared to be a point mutant or small deletion of 222/R4 with an almost identical size, whereas 222/R3N had lost a DNA segment of about 3 mum, and measured 30.3 mum or 63 x 10(6) AMU. The 222/R1 factors also appeared to have arisen by loss of DNA from 222/R4, 222/R1A being 22.3 mum or 46 x 10(6) AMU, whereas all other 222/R1 factors appeared to be duplicates, measuring 25.6 mum or 53 x 10(6) AMU. The DNA from six recombinant factors of R4 phenotype was indistinguishable in size and configuration from the parental 222/R4. In most cases, the number of R-factor copies (present as covalently closed circular molecules) per copy of the E. coli chromosome was less than 2, ranging from 1.2 to 3.3.

Composite circular forms of R factor deoxyribonucleic acid molecules.

Two R factors, one (R15) conferring resistance to streptomycin and sulfonamide (SM(r)SU(r)) and the other (222/R3) to streptomycin, sulfonamide, and chloramphenicol (SM(r)SU(r)CM(r)), were transferred to a Proteus mirabilis strain, and deoxyribonucleic acid (DNA) extracted from these strains was subjected to density-gradient centrifugation. R15-DNA formed a single satellite band at a density of 1.709 g cm(-3). Electron microscopy of samples from this band showed circular molecules of one type, with a contour length of 18 mum (35 x 10(6) daltons). 222/R3-DNA formed a satellite band with three peaks at densities 1.708, 1.711 and 1.717 g cm(-3). Electron micrographs revealed circular structures from each band with contour lengths, respectively, of 29 (54 x 10(6) daltons), 36 (68 x 10(6) daltons), and 6 mum (12 x 10(6) daltons). "Supertwisted" forms of several molecular species were found. It is suggested that 222/R3 DNA comprises either a single 36-mum molecule or two individual molecules, 29 and 6 mum in length, and that this may reflect the evolutionary development of R factors.

Early abortive lysis by phage BF23 in Escherichia coli K-12 carrying the colicin Ib factor.

Growth of phage BF23 was restricted in Escherichia coli K-12 strains carrying a colicin I factor (ColIb); most infected cells lysed early without producing progeny phages. Either addition of chloramphenicol before phage infection or ultraviolet irradiation of phage prevented early abortive lysis, an indication that certain phage functions are required for this phenomenon. Very little or no phage-induced lysozyme was synthesized in the infected ColI(+) cells. This result suggests that early abortive lysis was not due to the lysozyme action. A small fraction (0.05) of BF23-infected ColI(+) cells showed normal phage growth. This "escaped growth" may reflect the physiological state of the host bacteria rather than the heterogeneity of the infecting phage. Host-controlled modification was not observed. A phage mutant, BF23hI, able to grow on ColI(+) cells, was isolated and was characterized to be recessive to the wild-type BF23 in its ability to undergo early abortive lysis. Among the T series phages, T5 induced early abortive lysis, and growth of T5 was restricted upon infection to ColI(+) cells. These results and the other observations, including the occurrence of phenotypic mixing between BF23 and T5, suggest that these two phages are related to each other even though the receptor sites for BF23 and T5 are apparently different.