Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. IV. The F sequences in F14

The structure of F14, in particular the arrangement of the F sequences on this plasmid, has been studied by the electron microscope heteroduplex method. F14 has a molecular size of 311 ± 10 kilobase pairs (M = (206 ± 8) × 10⁶daltons). It contains all of F (94.5 kilobases). A sequence of length 5.7 kilobases, which occurs once in F (with co-ordinates 2.8 to 8.5F), is directly repeated in F14. It occurs at the two junctions of F DNA with chromosomal DNA. Thus, F14 contains about 211 ± 10 kilobases of chromosomal DNA. A previously unidentified direct repeat has also been discovered on F itself; the sequence with co-ordinates 93.2 to 94.5F is directly repeated at 13.7 to 15.0F. Physical observations indicate that the population of closed circular plasmid molecules extracted from F14-containing strains is heterogeneous. In addition to F14 itself, molecules the size of F and 2.3 times the size of F were found. The latter molecules contain all the chromosomal sequences of F14 and one copy of the 2.8 to 8.5F segment. Such heterogeneity was observed in both recA^? and recA⁺ backgrounds. It is proposed that this heterogeneity is due to intramolecular recombination events occurring within F14 between the duplicated 2.8 to 8.5F sequences. Such recombination can account for the previously observed genetic instability of F14. Another F prime plasmid, F186, independently isolated from the Hfr parent of AB313, was found to be identical to F14.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. VII. Mapping the ribosomal RNA genes of plasmid F14

Escherichia coli episome F14 contains two 16 S + 23 S ribosomal RNA gene sets. Their positions and structure have been studied by electron microscope heteroduplex methods. The A gene set maps from 81.4 to 86.7 kilobases from the counterclockwise junction of F DNA with bacterial chromosomal DNA of F14; the B gene set maps very close to the clockwise junction, that is, at map position 204.4 to 209.7 kilobases from the counterclockwise junction. The structure of the rRNA-rDNA hybrids indicates that each gene set consists of one 16 S gene (r16) of length 1.28 ± 0.12 kilobases, a spacer (rsp) of length 0.57 ± 0.12 kilobase and a 23 S gene (r23) of length 2.60 ± 0.20 kilobases. We propose the genetic notation rrn for the entire gene set. The structure of the DNA/DNA duplexes between the rrnA set and the rrnB set shows that there is a region of non-homology of length 0.29 ± 0.06 kilobase within the otherwise homologous rrnA and rrnB sequences. This non-homology region is probably part, but not all, of the spacer between the 16 S and 23 S genes.     

Genetic studies of F plasmid maintenance genes

We have used the mutagenic potential of the ampicillin resistance transposon Tn3 and in vitro deletion techniques to study essential regions for maintenance of mini-F plasmids. Our parental mini-F plasmid contains the 40.3 to 40.8F and 43.1 to 49.3F sequences, a total of 6.7 kilobases (kb). From a spectrum of insertion and deletion mutants, we find only two insertion regions; they map near the 45.8- and 46.4-kb coordinates. In each region the orientation of Tn3 insertion is unique and different from that of the other region. Spontaneous deletions extend from either region in a common direction which is toward the 49.3-kb coordinate. One deletion plasmid, pBK138-2, which arose from a combination of in vitro and spontaneous deletion events, contains just the 44- to 45.8-kb sequences and the ampicillin resistance gene of Tn3. As shown by J. Wechsler and B. C. Kline (1980, Plasmid 4, 276–280), the 45.8- to 46.3-kb sequences specify F sensitivity to the plasmid curing agent, acridine orange. Since sensitivity to acridine orange is a property of normal F maintenance, the 45.8- to 46.35-kb sequences also likely are required for normal plasmid maintenance.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: VIII. The structure of bacteriophage φ80d3ilv+su+7, including the mapping of the ribosomal RNA genes

The sequences present on the DNA of the transducing phage, φ80d₃ilv⁺su⁺7 have been mapped by electron microscope heteroduplex methods. In addition to some φ80 sequences, the phage DNA contains sequences from the extreme counterclockwise region and from the extreme clockwise region of the bacterial chromosomal part of F14. The former includes ilv, the latter a 16 S and a 23 S ribosomal RNA gene. These two regions are joined on the transducing phage DNA by the 2.8 to 8.5F sequence.By direct observation of the structure of the rRNA/DNA hybrids, the 16 S and 23 S genes have lengths of 1.38 ± 0.14 and 2.66 ± 0.17 kilobases. They are separated by a spacer of length 0.57 ± 0.13 kilobases.The rRNA genes (rrn) of φ80d₃ilv⁺su⁺7 are derived from and are identical with the rrnB gene set of F14. In heteroduplexes between the rrnB gene set of φ80d₃ilv⁺su⁺7, and the rrnA gene set of F14 we observe that there is a region of non-homology of length 0.25 ± 0.06 kilobases within the spacer sequence. This confirms observations in the preceding paper on the structure of out-of-register duplexes of the two rRNA gene sets of F14.A model for the integration and excision events involved in the formation of φ80d₃ilv⁺su⁺ 7 from φ80dmet(K) is proposed.     

Deletion mutants of F, F deletion (8.5--17.6) and the mechanism of their formation.

Heteroduplex analysis shows that a cryptic plasmid extracted from the male phage-sensitive strain, RP42, is a deletion mutant of F and lacks the segment with F-coordinates 8.5F to 17.6F. Consideration of the sequence deleted in F shows that the molecular recombination event between γδ and ?ζ DNA sequences actually occurs only between specific short segments at the ends.     

Regulation of the F-factor pif operon: pifO, a site required in cis for autoregulation, titrates the pifC product in trans.

F factor pifC, pifA, and pifB gene expression is subject to negative regulation by the product of the pifC locus (J.F. Miller and M. H. Malamy, J. Bacteriol. 156:338-347, 1983). In this paper, we describe the properties of a new regulatory site in the pif region, pifO, which is required in cis for autoregulation of pif gene expression. Spontaneous pifO mutations were isolated that allow expression of a pifC-lacZ protein fusion in the presence of pifC product in trans. Recombination of these pifO mutations onto F'lac results in increased pifA and pifB activity. Thus, a single regulatory element, pifO, regulates pifC, pifA, and pifB expression in cis. The presence of multiple copies of a fragment from the pif region carrying wild-type pifO sequences (F coordinates 42.9 to 42.43 kilobases) in trans to F'lac results in an increase in pifA and pifB activity as measured by inhibition of T7 plating. When the pifO mutations are recombined onto a plasmid carrying pifO, the resulting recombinants are greatly decreased in the ability to increase F'lac pif expression. These results suggest that increased F'lac pifA and pifB expression caused by pifO sequences in trans is a consequence of titration of pifC product and derepression of the pif operon.     

Physical mapping of genes on the F plasmid of Escherichia coli responsible for inhibition of growth of female-specific bacteriophages.

By correlating the resistance or sensitivity to female-specific phages of strains carrying F plasmids with deletions for part of the region 32.6 to 42.9 F, cloned F fragments, and other plasmids, it was shown that the pif loci are located near and clockwise to a point on F with coordinate 38.3 F.     

Incompatibility between Flac, R386, and F:pSC101 recombinant plasmids: the specificity of F incompatibility genes.

The specificity of F incompatibility genes (inc⁺) has been studied with the Flac and R386 plasmids, members of the IncFI incompatibility group. Recently, two inc⁺ regions, incA (46.4–49.3F) and incB (43.1–46.4F) were identified by cloning these F sequences onto pSC101 and subsequently demonstrating incompatibility of the recombinants with Flac. It is shown here that the FincA⁺ recombinant is incompatible with both Flac and R386 while the FincB⁺ recombinant is incompatible only with Flac. Also, a plasmid mutant is described that has reduced incompatibility against Flac and R386. The mutation is located on the BamHI restriction fragment that contains the FincA region. These genetic findings are consistent with the deduction of Palchaudhuri and Maas, based on heteroduplex analysis of IncFI plasmids, that placed the IncFI determinant in the 46.4–48.6F region. The findings also indicate that the FincB⁺ gene product, which has been implicated in negative control of F copy number, is specific for the F replicon.     

Cloning of the pif region of the F sex factor and identification of a pif protein product

This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages. We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F. All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases. A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103. A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103. Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein.     

An oligopurine sequence bias occurs in eukaryotic viruses.

Twenty four DNA and RNA viral nucleotide sequences, comprising over 346 kilobases, have been analyzed for the occurrence of strings of contiguous purine or pyrimidine residues. On average strings greater than or equal to 10 contiguous purines or pyrimidines are found three and a half times more frequently than would be expected for a random distribution of bases. Detailed analysis of the 172 kilobase Epstein-Barr viral sequence shows that the bias in favor of contiguous purine residues increases with the length of the purine string. These findings are similar to those seen for genomic DNA from higher eukaryotes. In contrast no overrepresentation of oligopurine or oligopyrimidine strings is observed in 52 kilobases from eight bacteriophage and E. coli DNA sequences.     

Novel incompatibility and partition loci for the REPI replication region of plasmid ColV-K30.

The minimum pColV-K30 REPI region necessary for replication was located within a ca. 1.3-kilobase DNA segment. Adjacent to the essential replication sequences, there are two DNA regions that express incompatibility with plasmids containing the F secondary replicon of the F EcoRI fragment f7. One of these regions corresponds to incE, already described in that F plasmid fragment which expresses incompatibility with f7-containing plasmids. The other is a novel sequence that we designated incF, which confers incompatibility with REPI, P307, and f7 derivatives, cis-acting pColV-K30 sequences conferring stability to REPI-containing plasmids were also identified and localized noncontiguous to REPI, ca. 20 kilobases downstream from the aerobactin iron transport genes, which were thus flanked by REPI and its partition (par) sequences.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. V. ilv+ Deletion mutants of F14

The structure of a number of F′ilv episomes derived from F14 by bacteriophage P1-mediated transduction have been determined by the electron microscope heteroduplex method. F16, F25, F310 and F312 are all simple deletion mutants of F14. F316 is essentially the same but contains a small insertion (0.8 kilobase) of DNA of unknown origin within the F sequences at 78.6 F. The length of these plasmids are all about the same as that of phage P1 DNA itself. The sequences of F and the sequences of bacterial DNA that are present on the episomes are contiguous on the parental F14. Thus, their structures are consistent with the usual model for the mechanism of P1 transduction. The physical order of ilv genes is also consistent with previous genetic mapping. From this order one can determine the polarity of the Escherichia coli K12 chromosomal sequences on F14 and its F′ilv derivatives relative to the F sequences. This order is consistent with the known counterclockwise transfer order of the parental Hfr AB313. F′ilv episomes carry only one copy of the 2.8 to 8.5 F sequence, which is present as a direct duplication on F14. The F′ilv episomes are genetically stable, whereas F14 is unstable because of reciprocal recombination between the two duplicate sequences. The strain F316/AB2070 is different in several respects. All of the bacteria carry P1 phage DNA. As noted above, F316 itself carries a small insertion. Two transfer-defective deletion mutants, F316Δ(65.4-78.6) and F316Δ-(78.6-0.6) are also present in the population of F316/AB2070 cells. In each case, the deletion borders on one of the junctions of inserted DNA and F14 DNA in F316. Thus, these junctions appear to be hot spots for deletion formation.     

Genetic mapping of the F plasmid gene that promotes degradation of stable ribonucleic acid in Escherichia coli.

F+ Escherichi coli cells that contain an srnA mutant allele degrade their stable ribonucleic acid (RNA) extensively after RNA synthesis is blocked at 42 degrees C. The relevant gene promoting degradation of stable RNA, srnB+, or its promoter was mapped between 1.7 and 2.8 kilobases on the F plasmid by using deleted F' plasmids and chimeric plasmids composed of pSC101 and fragments of F plasmid.     

alphabeta sequence of F is IS31.

Previous studies have shown that there is a deoxyribonucleic acid (DNA) segment, of length 1.3 kb and denoted as the alphabeta sequence, which occurs twice on the F plasmid at corrdinates 93.2 to 94.5/OF kb and 13.7 to 15.0F kb. In the present investigation, heteroduplexes were prepared between a phage DNA carrying the insertion sequence IS3 and suitable F-prime DNAs. The hybrids formed show that IS3 is the same as alphabeta. This result plus previous studies support the view that: (i) the insertion sequence IS2 and IS3 occur on F and, in multiple copies, on the main bacterial chromosome of Escherichia coli K-12; and (ii)these IS sequences on the main bacterial chromosomes are hot spots for Hfr formation by reciprocal recombination with the corresponding sequences of F.     

Deletion mutants of F, FΔ(8.5–17.6) and the mechanism of their formation

Heteroduplex analysis shows that a cryptic plasmid extracted from the male phage-sensitive strain, RP42, is a deletion mutant of F and lacks the segment with F-coordinates 8.5F to 17.6F. Consideration of the sequence deleted in F shows that the molecular recombination event between γδ and εζ DNA sequences actually occurs only between specific short segments at the ends.     

Identification of F0 subunits in the rat liver mitochondrial F0F1-ATP synthase.

In order to identify the subunits constituting the rat liver F0F1-ATP synthase, the complex prepared by selective extraction from the mitochondrial membranes with a detergent followed by purification on a sucrose gradient has been compared to that obtained by immunoprecipitation with an anti-F1 serum. The subunits present in both preparations that are assumed to be authentic components of the complex have been identified. The results show that the total rat liver F0F1-ATP synthase contains at least 13 different proteins, seven of which can be attributed to F0. The following F0 subunits have been identified: the subunit b (migrating as a 24 kDa band in SDS-PAGE), the oligomycin-sensitivity-conferring protein (20 kDa), and F6 (9 kDa) that have N-terminal sequences homologous to the beef-heart ones; the mtDNA encoded subunits 6 (20 kDa) and 8 (less than 7 kDa) that can be synthesized in isolated mitochondria; an additional 20 kDa protein that could be equivalent to the beef heart subunit d.     

Identification of the pifC gene and its role in negative control of F factor pif gene expression

The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection. We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene. A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis. This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map. Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB.