Heteroduplex analysis of tra delta f'' plasmids and the mechanism of their formation.

Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient.     

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.     

Genetic control of the formation of plasmid F'. I. Effect of recA- and seg-2 mutations in an Hfr donor strain on the character of plasmid F' formation]

Assuming the similarity of the processes of illegitimate recombination, such as deletion formation, with the process of F' plasmid formation, we have undertaken the study of the influence of recA- and seg- alleles of Hfr donor on the F' plasmid formation. The data obtained demonstrate the strong influence of donor genotype on the frequency of F' plasmid formation and on the nature of F' plasmids formed, thus demonstrating that the most of F' plasmids have been formed via recombination in Hfr donor cells. The recA- mutation decreased the total yield of F' plasmids selected using both proximal and distal Hfr markers and affected drastically the distribution of the F' plasmids inheriting different proximal unselected markers. The existence of recA-dependent and recA-independent modes of F' plasmid formation was demonstrated. The Escherichia coli chromosome contains regions which involve preferentially in recA-dependent (between proA and gal, and clockwise from gal) or recA-independent (between leu and proA, and the region counterclockwise from argE) recombination. The seg-2 mutation causes only partial block of both recA-dependent and recA-independent recombination pathways, however it causes dramatic decrease of genetic exchanges leading to the formation of the type II F' plasmids. Both seg- and recA- mutations decrease the frequency of the formation of Tra+ F' transconjugants. The percent of Tra- transconjugants, which remain sensitive to MS2 and Q beta donor specific phages, also drops significantly under the influence of the recA- and seg- alleles. Thus, the recombination involving the F structure in wild type strains and seg- mutants occures preferentially in the points of F outside the regions essential for transfer and sensitivity to male specific phages, while in recA- and recA-ges- strains the points inside these regions (tra operon) frequently involved in F' plasmid looping out. There exist more strict correlation between the fertility and sensitivity to phage Q beta than to phage MS2.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis.

Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr). When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses. However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA. These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli. A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype.     

Description of an incompatibility mutant of Escherichia coli

A mutant Hfr strain of Escherichia coli which has an impaired incompatibility function but is normal for other F factor functions has been isolated. This Inc(-) Hfr permits the maintenance and transfer of both the integrated F factor and an F' factor. F' factors have been isolated from the integrated F factor of the Inc(-) Hfr strain. When these episomes were tested in matings with Hfr or F' strains, they did not differ in any observed way from wild-type F' factors.     

Convenient construction of recA deletion derivatives of Escherichia coli.

Two Escherichia coli K-12 Hfr strains have been constructed which transfer a recA deletion, which is highly linked to a Tn10 insertion conferring tetracycline resistance, early during conjugation. These strains transfer the recA deletion in opposite directions with different origins of transfer, allowing for preservation of desirable recipient strain markers either clockwise or counterclockwise of recA.     

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.     

Derivation of an F-Merogenote and a φ80 High-Frequency Transducing Phage Carrying the Histidine Operon of Salmonella

An F' factor, FS400, carrying the his operon, the gnd gene, and the rfb gene cluster of Salmonella typhimurium was isolated. FS400 was introduced into an Escherichia coli strain having a lengthy deletion of the his gene region. From this strain, Hfr derivatives were isolated which had the F' factor integrated in the tonB locus near the attachment site of phi80. One of the Hfr strains was lysogenized with a heat-inducible, h mutant of phi80, and from this strain a high-frequency transducing phage carrying the his genes and the gnd gene of Salmonella was isolated.     

Isolation of F' plasmids carrying a portion of the Salmonella typhimurium histidine transport operon.

The transposable drug resistance element Tn10 was employed as a region of homology to direct the insertion of Tn10-containing derivatives of F'ts114 lac into the chromosome of a Salmonella typhimurium strain that carries a Tn10 insertion in the histidine transport operon. Based on the direction of transfer of the resulting Hfr strains, the chromosomal Tn10 insertion was determined to be in orientation "A." New F' plasmids were selectively generated from one of the Hfr strains. The F' factors carry an intact dhuA hisJ portion of the histidine transport operon. A Southern hybridization revealed that one of the F' plasmids was formed by a type II excision event.     

Replication of Fpoh+ plasmid in mafA mutants of Escherichia coli defective in plasmid maintenance.

A class of F' plasmids, designated Fpoh+, was previously shown to be able to replicate extra-chromosomally on Hfr strains by virtue of carrying the specific site or region poh+ (permissive on Hfr) of the E. coli chromosome (Hiraga, 1975, 1976a). These plasmids were now found to replicate on E. coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids. The derivatives of Fpoh+ that have lost the poh+ site, on the other hand, failed to replicate on mafA mutants. These mutants harboring Fpoh+ (but not Poh- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh+ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons. It is tentatively concluded that the poh+ site is required for F' plasmids to replicate on mafA mutants as well as on Hfr strains. In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh+ region contains the replication origin of the E. coli chromosome.     

Isolation of a hybrid F' factor-carrying Escherichia coli lactose region and Salmonella typhimurium histidine region, F42-400 (F' ts114 lac+, his+): its partial characterization and behavior in Salmonella typhimurium.

Episome F' ts114 lac+ (F42-114) was transferred into Salmonella typhimurium carrying an F'his+ (FS400) episome, and fused episome F' ts114 lac+, his+ (F42-400) was obtained. Episome F42-400 could be transferred to S. typhimurium, Escherichia coli and Klebsiella pneumoniae. Identification of the episome was based on: (i) temperature sensitivity of the Lac+ and His+ phenotypes; (ii) the fact that F- segregants, obtained after temperature curing or acridine orange curing, were simultaneously Lac- and His-; and (iii) linkage of lac+ with his+ in episomal transfers to E. coli and S. typhimurium. The frequency of episome transfer was influenced by the genotype of the donor. Plasmid LT2, prevalent in S. typhimurium LT2 strains, was suggested to be responsible for the low fertility of S. typhimurium donors. Episome F42-400 was capable of chromosome mobilization, and the extent of chromosome mobilization was not influenced by the presence or absence of the histidine region on the donor chromosome. Growth in a defined medium with acridine orange was able to cure F42-400. The frequency of curing was increased (the frequency of His+ cells was 0.0001%) if the cells were grown at 40 C in the presence of acridine orange. Selection for temperature-resistant Lac+, His+ derivatives in a strain without histidine deletion yielded Hfr strains. However, similar and stronger selections in strains without the chromosomal histidine region failed to yield Hfr strains. Our inability to obtain Hfr's in strains without the chromosomal histidine region was explained by assuming that the episome F42-400 has lost the F sites involved in integration into the S. typhimurium chromosome.     

Formation of stable Hfr strains in R factor RP1 integration with the chromosome of E. coli K12 recA and their use for R-plasmid selection

Analysis of thermoindependent derivatives of E. coli K12 JC1553 recA (p VD1) carrying a replication thermostable mutant pVD1 of R factor RP1 IncP Ap Km Tc showed that formation of about 5 per cent of them was associated with stable integration of the plasmid with the bacterial chromosome. The respective bacteria had the following features: (1) preserved all the markers of plasmid pVD1, (2) according to the data of the electrophoretic analysis had no extrachromosomal DNA on prolonged cultivation under nonselective conditions, (3) were effective donors of the chromosomal genes, (4) had a low rate of the plasmid marker transfer on crossing with R- recipient. The latter feature was suggested to be used as a test for identification of stable Hfr strains. Investigation of the properties of the transconjugants obtained on crossing of stable Hfr strains with R-recipients rec+ showed that same of them had plasmid DNA with a higher molecular mass as compared to that of plasmid pVD1 DNA. The presence of this DNA was connected with formation of R' plasmid as a result of an irregular exclusion of plasmid pVD1 from the chromosome of stable Hfr bacteria. On the basis of the results a simple method was proposed for selection of R' plasmids having a number of advantages over the classical ones. The perspectives of using thermostable derivatives of RP1 for cloning the chromosome genes are discussed.     

Construction of an Hfr strain useful for transferring recA mutations between Escherichia coli strains.

Strain JC10240 (Hfr PO45 srlC300::Tn10 recA56 thr-300 ilv-318 rpsE300) was constructed. On account of the close linkage of Tn10 to recA56, the latter can be moved to other Escherichia coli (and closely related) strains in transductional or conjugational crosses selecting resistance to tetracycline.     

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin.

The Escherichia coli F plasmid gene required for amino-terminal acetylation of F-pilin subunits was identified. Using Western blots (immunoblots), we assayed the reaction of monoclonal antibodies with F-pilin polypeptides in inner membrane preparations from various F mutant strains. It was known that JEL92 recognizes an internal pilin epitope and JEL93 recognizes the acetylated amino-terminal sequence (L.S. Frost, J.S. Lee, D.G. Scraba, and W. Paranchych, J. Bacteriol. 168:192-198, 1986). As expected, neither antibody reacted with inner membranes from F- cells or Flac derivatives that do not synthesize pilin. Mutations that affected the individual activities of F tra genes traA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -P, -R, -U, -V and -W or trb genes trbA, -B, -C, -D, -E, -G, -H, and -I did not prevent JEL92 or JEL93 recognition of membrane pilin. However, Hfr deletion mutants that lacked the most-distal transfer region genes did not express pilin that reacted with JEL93. Nevertheless, all strains that retained traA and traQ did express JEL92-reactive pilin polypeptides. Analysis of strains expressing cloned tra segments showed that traA and traQ suffice for synthesis of JEL92-reactive pilin, but synthesis of JEL93-reactive pilin is additionally dependent on traX. We concluded that the traX product is required for acetylation of F pilin. Interestingly, our data also showed that TraA+ TraQ+ cells synthesize two forms of pilin which migrate at approximately 7 and 8 kDa. In TraX+ cells, both become acetylated and react with JEL93. Preparations of wild-type F-pilus filaments contain both types of subunits.     

Temperature-sensitive recA mutant of Escherichia coli K-12: deoxyribonucleic acid metabolism after ultraviolet irradiation.

A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles. It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.     

Mutants of Escherichia coli that do not contain 1,4-diaminobutane (putrescine) or spermidine.

Strains of Escherichia coli K12 have been constructed which do not contain any of the polyamines normally present in a wild type strain, namely, 1,4-diaminobutane (putrescine) and spermidine. This phenotype arises as a consequence of the assembly into these strains of deletion mutations in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). The polyamine-deficient strains grow indefinitely in the absence of polyamines but with a growth rate one-third of that found in the presence of polyamines. These strains can act as hosts for bacteriophages T4, T7, and f2, although the latter phage is poorly adsorbed; they can also maintain F' factors, ColE1 and P1 plasmids, and lysogeny by bacteriophage lambda. In contrast, the production of bacteriophage lambda in the absence of polyamines is strikingly decreased (greater than 99%) either after infection of a nonlysogen or after induction of a lysogen. A polyamine-deficient Hfr strain can transfer its chromosome to a recipient at a normal rate, but the number of recombinants observed in a cross is decreased approximately 300-fold. No such effect is observed when only the F- recipient strain in a cross is polyamine deficient.     

Mutations in the purine nucleoside phosphorylase (pup) gene of Escherichia coli K-12 characterized by leaky damage to enzymatic activity and a pleiotropic effect]

The phenotype of earlier obtained mutants AIR38 and AIR6 is caused by leaky mutations of the structural gene for purine nucleoside phosphorylase (pup). These mutants are unable to grow on the medium with inosine as the only carbon source in the presence of thymidine. In contrast to ordinary leaky mutations, AIR38 and AIR6 are dominant in heterozygotes. When the strain F' with mutations AIR38 or AIR6 on episomes was used for conjugational matings with F- recA (pup+), recombinants with unexpected phenotype were observed: about 20% of recombinants F' became pup+ and thus the convertion of AIR38 and AIR6 alleles to pup+ took place. AIR38 mutation, unlike AIR6, is mapped in the proximal region of the pup gene and is characterized by pleyotropic effect on deo-genes: the inosine-induction of purine nucleoside phosphorylase in AIR38 mutant is absent and the induction of thymidine phosphorylase by exogenous thymidine is decreased. A speculation was made that the mutation AIR38 altered the structure of purine nucleoside phosphorylase in the site responsible for its interaction with membrane.     

Linkage distortion following conjugational transfer of sbcC+ to recBC sbcBC strains of Escherichia coli.

Conjugational recombination in Escherichia coli depends normally on RecBCD enzyme, a multifunctional nuclease and DNA helicase produced by the recB, recC, and recD genes. However, recombination can proceed efficiently without RecBCD in recB or recC strains carrying additional mutations in both the sbcB and sbcC genes. Recombination in these strains, sometimes referred to as the RecF pathway, requires gene products that are not essential in the RecBCD-dependent process predominating in the wild type. It has also been reported to produce a different spectrum of recombinant genotypes in crosses with Hfr donors. However, the sbcC+ gene was unknowingly transferred to the recipient strain in some of these crosses, and this may have affected the outcome. This possibility was examined by conducting parallel crosses with Hfr donors that were either wild type or mutant for sbcC. Transfer of sbcC+ from an Hfr donor is shown to alter the frequency of recombinant genotypes recovered. There is a severe reduction in progeny that inherit donor markers linked to the sbcC+ allele and an increase in the incidence of multiple exchanges. Colonies of mixed genotype for one or more of the unselected proximal markers are also much more prevalent. Since the yield of recombinants is lower than normal, these changes are attributed to the reduced viability of recombinants that inherit sbcC+ from the Hfr donor. When the Hfr donor used is also mutant for sbcC, the yield of recombinants is greater and the frequencies of the different genotypes recovered are similar to those obtained in crosses with a rec+ sbc+ recipient, in which transfer of sbcC+ has no apparent effect. Earlier studies are re-examined in light of these findings. It is concluded that, while recombination in recBC sbcBC strains involves different enzymes, the underlying molecular mechanism is essentially the same as that in the wild type.     

带有IS1的mini—F质粒所发动的染色体复制对于recA基因的依赖性

我们曾报道整合的F′质粒所发动的大肠杆菌染色体复制依赖于recA基因,而整合的F质粒则不。构建带有IS1的mini-F质粒,它们的复制起点分别来自F或F′质粒。这些质粒的整合抑制菌株中都有约20％是recA依赖的,不管这一mini-F质粒的复制起点来自F或F′质粒,也不管这一质粒在游离状态中的复制方向是单向或双向。实验结果说明,质粒的整合位置是决定由整合质粒所发动的染色体复制对recA基因的依赖性的主要因素。