Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F100, F152, and F8 and mapping of the Escherichia coli chromosomal region fep-supE-gal-attlambda-uvrB.

The genetic and physical structures of commonly used F-prime factors carrying the galactose region of the Escherichia coli chromosome were analyzed. Deletions in the chromosomal DNA sequences in the F-prime factors were found to be frequent events. A genetic method was developed to reconstruct the original F-prime factors from deletion variants. Heteroduplex analysis of the reconstructed F-prime factors confirmed the derivation of the F-prime factors F100 and F152, from the same Hfr, and finally determined the normal E. coli chromosomal sequence in the region between fep and uvrB, containing about 5 min in genetic units and about 246.5 in kilobase units (kb). This sequence could be connected with the DNA sequences of the lac-purE region, which had been physically determined previously. Together they constituted a total of 528.6 kb. From these combined sequences, the distance from lacPO to galK was calculated to be 412.9 kb, which corresponds to 8.8 min in genetic units.     

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.     

Expression of a mutation affecting F incompatibility in the integrated but not the autonomous state of F.

Previously we have described a mutant Hfr strain in which incompatibility between the integrated F factor and an autonomous F-prime (F') factor was abolished. The mutation (inc) was located in the integrated F factor. F-prime factors isolated from the mutant Hfr strain have the same incompatibility behavior as those isolated from normal Hfr strains. Reintegration of these F' factors into the chromosome restores the Inc- phenotype characteristic of the mutant Hfr. The inc mutation thus affects incompatibility between integrated F and autonomous F(Fi-Fa incompatibility) but not incompatibility between two autonomous F factors (Fa-Fa incompatibility). The implications of this finding for the mechanism of plasmid incompatibility are discussed.     

Electron Microscope Analysis of Partial Denaturation of F Factor Deoxyribonucleic Acid

Partial denaturation pattern of sex factor deoxyribonucleic acid of Escherichia coli was studied by electron microscopy. Clustering of the adenine-plusthymine-rich regions in one part of the molecule was revealed. The positions of these regions were located on the physical map of F by analyzing the partial denaturation pattern of heteroduplexes between F and F-prime factors with various parts of F sequences deleted.     

Gene dosage effects of the structural gene for a lipoprotein of the Escherichia coli outer membrane.

The gene dosage effects of the structural gene (lpp) for the lipoprotein of the Escherichia coli outer membrane were examined. A novel F-prime factor containing the lpp gene was constructed. The amount of the free-form lipoprotein in the merodiploid strain carrying the F-prime factor was found to be about two times as great as that in the corresponding haploid strain. On the other hand, the amount of the bound-form lipoprotein, which is vovalently linked to the peptidoglycan, was not significantly different in the merodiploid strain as compared with the corresponding haploid strain. The present results suggest that the lpp gene is expressed constitutively in contrast to another major protein of the E. coli outer membrane, tolG protein (protein II, D. B. Datta et al., J. Bacteriol. 128:834-841, 1976). The F-prime factor isolated may include a portion of the E. coli chromosome (located between 33 and 36 min on the genetic map) that is not covered by any other F-prime factor.     

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.     

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.     

Evidence for two distinct pyruvate kinase genes in Escherichia coli K-12

A strain of Escherichia coli K-12 defective in pyruvate kinase F has been produced. The existence of this mutant, in conjunction with earlier results, strongly suggests that the two pyruvate kinases in this bacterium are distinct forms and not interconvertible. Either form of pyruvate kinase appeared to be equally effective in the glycolytic conversion of phosphoenolpyruvate to pyruvate. Genes specifying pyruvate kinase A and pyruvate kinase F were present on the small F-prime F506 and the locus for pyruvate kinase F was found to be at minute 36.5 on the E. coli genetic map.     

Evidence for involvement of pyrH+ of an Escherichia coli K-12 F-prime factor in inhibiting construction of hybrid merodiploids with Salmonella typhimurium

Transconjugants were not recovered in matings between Salmonella typhimurium and Escherichia coli K-12 strains carrying the chromosomal region between leu and argF on an F-prime factor, even when a restriction-deficient recipient was used. A mutant F-prime factor compatible for transfer to S. typhimurium was constructed by transposon mutagenesis and characterized as being deficient in directing the synthesis of UMP kinase (encoded by pyrH). Other compatible F-prime factors were readily constructed by employing a procedure designed to select for strains carrying F-prime factors harboring pyrH mutations.     

Identification of a membrane protein associated with expression of the surface exclusion region of the F transfer operon.

Membrane preparations from radioactively labeled male and female strains of Escherichia coli K-12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. An intensely labeled band corresponding to a protein of molecular weight of 24,000 was readily apparent in preparations from Hfr and F-prime strains but not in those from female strains. When preparations from a series of Hfr strains containing transfer operon deletions were examined, presence of the band was found to be associated with retention of the region of the F transfer operon between ilzA and traD. Thus, the band ("protein S") appears to be the product of an F tra operon activity corresponding to traS (the gene for surface or entry exclusion), or an unknown gene in its vicinity. As predicted, protein S was subject to Fin+ control; only a faint band was detectable if the repressed plasmid R100 was also present in the F lac strain. A 24,000-dalton protein was also found in membrane preparations from strains carrying the derepressed plasmids R100-1 and R1-19 but not in those from strains carrying the repressed plasmids R100 or R1. Thus, the appearance of protein S in the membrane may be a general phenomenon resulting from transfer operon expression of F-like plasmids.     

Effect of tra mutations on F factor-specified immunity to lethal zygosis

Summary Hfr, F⁺, and F-prime cells are, unlike F^– cells, insensitive to an excess of Hfr donor cells, indicating that there is an F factor mediated immunity to lethal zygosis (Ilz). Results with Flac episomes carrying traJ, traS or various polar mutations in the tra region indicate that this immunity is independent of surface exclusion, of traJ control, and of all known genes within the tra operon. However, analysis of a series of strains with deletions in the F factor, extending from the right into the tra region, suggests that a gene for immunity to lethal zygosis is located within the tra region. We therefore conclude that Ilz is genetically complex, and present a hypothesis to account for these results.     

Insertion element IS121 is near proA in the chromosomes of Escherichia coli K-12 strains.

Insertion element IS121 was mapped between proA and a previously mapped IS5A element in two F-prime plasmids. Results of hybridizations of IS121 to chromosomal DNA from four other strains suggest that IS121 is normally present at this position in the chromosomes of Escherichia coli K-12 strains.     

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.     

Targeted mutagenesis of the b subunit of F1F0 ATP synthase in Escherichia coli: Glu-77 through Gln-85.

Subunit b of Escherichia coli F1F0 ATP synthase contains a large hydrophilic region thought to be involved in the interaction between F1 and F0. Oligonucleotide-directed mutagenesis was used to evaluate the functional importance of a segment of this region from Glu-77 through Gln-85. The mutagenesis procedure employed a phagemid DNA template and a doped oligonucleotide primer designed to generate a predetermined collection of missense mutations in the target segment. Sixty-one mutant phagemids were identified and shown to contain nucleotide substitutions encoding 37 novel missense mutations. Mutations were isolated singly or in combinations of up to four mutations per recombinant phagemid. F1F0 ATP synthase function was studied by mutant phagemid complementation of a novel E. coli strain in which the uncF (b) gene was deleted. Complementation was assessed by observing growth on solid succinate minimal medium. Many phagemid-encoded uncF (b) gene mutations in the targeted segment resulted in growth phenotypes indistinguishable from those of strains expressing the native b subunit, suggesting abundant F1F0 ATP synthase activity. In contrast, several specific mutations were associated with a loss of enzyme function. Phagemids specifying the Ala-79----Pro, Arg-82----Pro, Arg-83----Pro, or Gln-85----Pro mutation failed to complement uncF (b) gene-deficient E. coli. F1F0 ATP synthase displayed the greatest sensitivity to mutations altering a single site in the target segment, Ala-79. The evidence suggests that Ala-79 occupies a restricted position in the enzyme complex.     

Interactions of group H resistance factors with the F factor

The four R factors described in this paper form a single compatibility group which has been previously designated group H. Recombination was demonstrated between any pair of the plasmids TP117, 123, and 124. In contrast, TP116 did not appear to recombine with any other members of the group. TP117, 123, and 124 usually displaced the F factor from Escherichia coli K-12F(+), while TP116 and F coexisted stably in that strain. Deoxyribonucleic acid reassociation experiments showed minimal homology between F and the four group H plasmids. The results indicate that there are limitations to using incompatibility alone for classification of bacterial plasmids.     

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.     

The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector.

A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.     

Deletion analysis of the F plasmid oriT locus.

Functional domains of the Escherichia coli F plasmid oriT locus were identified by deletion analysis. DNA sequences required for nicking or transfer were revealed by cloning deleted segments of oriT into otherwise nonmobilizable pUC8 vectors and testing for their ability to promote transfer or to be nicked when tra operon functions were provided in trans. Removal of DNA sequences to the right of the central A + T-rich region (i.e., from the direction of traM) did not affect the susceptibility of oriT to nicking functions; however, transfer efficiency for oriT segments deleted from the right was progressively reduced over an 80- to 100-bp interval. Deletions extending toward the oriT nick site from the left did not affect the frequency of transfer if deletion endpoints lay at least 22 bp away from the nick site. Deletions or insertions in the central, A + T-rich region caused periodic variation in transfer efficiency, indicating that phase relationships between nicking and transfer domains of oriT must be preserved for full oriT function. These data show that the F oriT locus is extensive, with domains that individually contribute to transfer, nicking, and overall structure.