Pasteurella Bacteriophage Sex Specific in Escherichia coli

Phage H, thought to be specific for Pasteurella pestis, was shown to plate efficiently on F⁻ strains of Escherichia coli but not on F⁺, F′, or Hfr strains. The phage was adsorbed rapidly to F⁻ strains but was not adsorbed to strains carrying F. Comparison with seven other reported female-specific phages showed that, although phage H was similar to the other phages in some characteristics, the exceptionally low efficiency of plating (<10⁻⁹) on F-containing cells makes phage H a particularly useful female-specific phage.     

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

Summary A class of F plasmids, designated Fpoh ⁺, was previously shown to be able to replicate extrachromosomally 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.     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

Specific Action of Sodium Dodecyl Sulfate on the Sex Factor of Escherichia coli K-12 Hfr Strains

A specific action of sodium dodecyl sulfate (SDS) on the sex (F) factor in the integrated state of Escherichia coli K-12 Hfr H strain is reported. Growth of Hfr cells in Penassay Broth containing SDS results in the elimination of part or all of the F factor, yielding low and nonfertile variants of defective Hfr type and F⁺ cells and also F⁻ derivatives. Appearance of such variants was generally observed after the culture reached stationary phase. The frequencies of F⁻ cells then increased. F⁻ cells were usually isolated as the major population among survivors. Some defective variants of Hfr cells with an intermediate fertility between standard Hfr and F⁺ cells had lost sensitivity toward the male-specific ribonucleic acid phage M12. Other defective Hfr variants with as much or less fertility than standard F⁺ cells had also all lost sensitivity to phage M12. On single-colony isolation, they segregated nonfertile female H cells which, when infected with F, could restore high fertility with oriented transfer of the chromosome the same as that of the original Hfr H. Also, sensitivity to phage M12 was regained. Female H cells were characterized as those lacking fertility but still retaining a small segment of F or sfa locus at the original part of the chromosome, where newly infected F could attach. Similar results were obtained with two other Hfr strains. A possible mechanism of the specific action of SDS is discussed.     

Drug Resistance of Enteric Bacteria VIII. Chromosomal Location of Nontransferable R Factor in Escherichia coli

The R₂₁(TC) factor, obtained by transduction of the R₁₀(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R₂₁(TC)⁺ transductants are infected with the F₁₃ factor, the nontransferable R₂₁(TC) factor acquires transmissibility by conjugation. R₂₁(TC)⁺ conjugants of Escherichia coli K-12, to which only the R₂₁(TC) factor was transmitted by cell-to-cell contact from an F′ R⁺ donor, were still unable to transfer their R₂₁(TC) factor by conjugation. In crosses between Hfr and F⁻E. coli K-12 strains containing R₂₁(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.     

Structure of the core regions in lipopolysaccharides from Escherichia coli K12 W2252-11U−, the Ter-15 mutant,and Ter-15 (F′-lac) and Ter-15 (F+) cells

From Escherichia coli K12 W2252-11U^? cells, the Ter-15 mutant, the Ter-15 (F′-lac) and the Ter-15 (F⁺) cells, lipopolysaccharides were isolated and the primary structure of its core oligosaccharides was elucidated. When the F′-lac episome is transferred to the Ter-15 mutant by conjugation, the structure of the glucose III(1 → 3)glucose II(1 → 3)glucose I residue and the galactose I(1 → 2)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 mutant changes into the structure of the glucose IV(1 → 6)glucose III(1 → 2)glucose II(1 → 3)glucose I residue and the galactose I (1 → 6)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 (F′-lac) cells, but the core oligosaccharide in the Ter-15 (F⁺) cells is the same structure with that of the core oligosaccharide from the Ter-15 mutant when F⁺ episome is transferred to the Ter-15 mutant. Also, the core oligosaccharide from the Ter-15 (F′-lac) cells shows the same structure with that of the core oligosaccharide from E. coli K12 W2252-11U^? cells (the parent cells). As the result, the ability to produce the structure of the core oligosaccharide in E. coli K12 W2252-11U^? cells is recovered in the Ter-15 (F′-lac) cells by the dominant expression of lac gene or its containing DNA segment in F′-lac episome.     

Plasmid replication and Hfr formation in strains of Escherichia coli carrying seg mutations.

Summary Several conditional-lethal mutations that do not permit the replication of F-factors ofEscherichia coli K-12 are located at a site calledseg. This gene is located on theE. coli chromosome betweenserB andthr. It is unrelated to other known genes involved in DNA replication. Strains carryingseg mutations were unable to replicate F-lac⁺, several F-gal⁺s, F-his⁺ and bacteriophage at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C.When the kinetics of the loss of F-primes is studied inseg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F⁺ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F⁺ state did not show F-genote formation.F-genote formation fromseg Hfr strains is dependent of a functionalrecA gene, as F-genote formation was not seen with aseg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F-gal⁺, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.     

Detection of transcribable recombination products following conjugation in rec+, reCB- and recC-strains of Escherichia coli K12

By crossing Hfr and F^? strains of Escherichia coli which carry non-identical (but non-complementing) lacZ^? mutations, the detection of β-galactosidase produced from LacZ⁺ recombination products is possible, beginning 60 minutes after transfer of the Hfr lac^? allele. This system was used to show that when the F^? cells carry recB^?, almost normal amounts of LacZ⁺ enzyme are formed even though the number of viable recombinants is less than 1% of the Rec⁺ level. A similar result is found when the F^? cells carry recC^?. In contrast, LacZ⁺ enzyme activity is not detected either when RecA^? F^? cells are used or in a stable RecA^? merodiploid carrying the two lacZ^? alleles.     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

Behavior of Some Episomal Elements in a Recombination-Deficient Mutant of Escherichia coli

Conjugal transfer and autonomous replication of some episomes occurred normally in a recombination-deficient (Rec^–) mutant of Escherichia coli K-12. Transduction with phage Plbt of an R factor also occurred normally in this Rec^– mutant, but complete or abortive transduction with Plbt of chromosomal genes did not occur. In contrast, transduction of galactose genes by phage λ_dg occurred in the Rec^– bacteria as frequently as in the Rec⁺ strain. It was shown that phage Plbt does not grow at all on the Rec–bacteria. Recombination between two different R factors, two mutants of phage λ and two mutants of phage T4 occurred normally in the Rec^– bacteria, but did not give a Rec⁺ phenotype to the host bacteria. Colicinogenic factor I made the Rec^– host bacteria more resistant to ultraviolet light but the colicinogenic strain was still infertile in the crosses with the Hfr srains of E. coli K-12.     

Alteration of a Host Character by Infection with RNA Phage Qβ

An F + strain of E. coli K 12, W-2241 has a genetic constitution lac^– (i⁺ z⁺ y^–), str-r. The inability of this strain to utilize lactose is due to a deletion at the y locus controlling formation of an enzyme permease and therefore, a true lac⁺ revertant can not be expected. The strain, however, produces many colonies on minimal lactose agar medium, when it is plated after infection with RNA phage Qβ, while much fewer colonies are observed on the same medium seeded with uninfected cells. In analyzing this phenomenon, we discovered that these LAC⁺ colonies were not the result of a mechanism similar to transduction but produced by a mechanism related to phage liberation, and that the colonies originated from Qβ⁺, const cells. The data suggest the following working hypothesis for the mechanism by which LAC⁺ colonies are produced in a population of W-2241 cells infected with phage Qβ. Constitutive cells carrying Qβ produce intracellular β-galactosidase and at the stage of phage liberation, the enzyme is liberated into the medium, hydrolyzing lactose to form glucose. Since glucose could be utilized by all of the cells on the medium, the Qβ⁺, const cell acts as a colony forming center. The phenomenon described in this paper shows a new type of virus-host relationship which may have some bearing on the influence of animal or plant viruses on host tissues.     

Isolation of High-Frequency Recombining Strains from Escherichia coli Containing the V Colicinogenic Factor

The isolation and characterization of high-frequency recombining strains from different Escherichia coli host cells containing either the F factor or the Col V factor are described. The strains (with one exception) formed from three of the V⁺ parents showed the same origin and polarity of transfer (xyl-arg-pro-trp-his-mal). The Hfr strains formed from the one remaining V⁺ and the F⁺ host cells showed a greater variety in their points of origin. In addition, several Hfr strains isolated from V⁺ parents lost the ability to produce colicin V. F_v⁺ segregants of these were isolated, and the F_v factors appeared to retain their preferential site for Hfr formation, but they lacked other propertes controlled by the Col V factor. Chromosomal integration of episomes and its relation to the fertility of F⁺ and V⁺ strains are discussed. Production of colicin V appeared to be uninfluenced by the state of the Col V factor within the cell.     

Genetic Factors Affecting Recovery of Nonpoint Mutations in the Region of a Gene Coding for Ornithine Transcarbamylase: Involvement of Both the F Factor in Its Chromosomal State and the recA Gene

Mutants of E. coli K12 that overproduce ornithine transcarbamylase can be identified in Car^- strains because they permit utilization of citrulline as a carbamyl phosphate source, due to reversal of the normal OTCase reaction; they are called Cut mutants (citrulline utilizers). Hfr strains that carry the F factor adjacent to argF (one of two duplicate genes that code for ornithine transcarbamylase in E. coli K12) yield more Cut mutants than do F⁺ or F^- strains, or Hfr strains in which the F factor is not adjacent to argF. When Hfr strains in which the F factor is integrated adjacent to argF are made recA, they yield few Cut mutants. Many of the Cut mutants recovered from one of the Hfr strains used in the investigation (Hfr P4X) are unstable; the properties of these unstable mutations suggest that they carry aberrations in the region of the argF gene. Thus, the increased yields of Cut mutants probably result from aberrations that occur when the F factor is integrated adjacent to argF. The nature of these aberrations is not yet known. The unstable Cut mutants are to a large extent stabilized by recA; such stabilization is one of the properties of duplications. Other data indicate that the aberrations may be more complex than simple gene duplications; in particular properties of segregants and some recombinants derived from unstable Cut mutants are most easily interpreted by assuming that segregation from, and possibly formation of, the unstable mutants occurs in several stages.     

A new activity in the Ftra operon which is required for F-pilin synthesis

Summary Membrane preparations from a series of Hfr mutant strains of Escherichia coli K12 deleted in the promoter distal end of the F transfer operon were analyzed. Deletions which extended into traG, as expected, had no discernible effect on synthesis of membrane F-pilin. A more extensive deletion in strain KI777 which eliminated traH activity similarly had no effect on F-pilin synthesis. Membranes from three other TraF⁺ TraH^- deletion strains, as well as membranes from all strains carrying deletions extending into traF or further, lacked F-pilin, however. Since traH amber mutations do not affect synthesis of membrane pilin (Moore et al. 1981 b) we conclude that a gene required for F-pilin biosynthesis is located between traF and traH. We have named this gene traQ.Further evidence for traQ and an assay for its activity was obtained by examining the products of a TraM⁺ TraJ⁺ TraA⁺ lambda transducing phage, KI13, in UV irradiated cells. Infection of F^- cells with KI13 does not result in F-pilin synthesis. Membrane pilin is synthesized as a product of the transducing phage if an Flac or Hfr irradiated host is used, however. Mutant analysis demonstrated that this synthesis is independent of host expression of traA, traL, traE, traK, traB, traV, traW, traC, traU, traF, or traH, but dependent on expression of the traF-traH region. We interpret our data to indicate that an activity encoded by traQ is required for the conversion of traA product to F-pilin.     

Ter mutation and susceptibility to phi X174 phage in E. coli K12.

The Ter-15 mutant derived from E. coli K12 W2252-11U^? RC^str (wild type I) is found to be sensitive to φx174 phage infection. Lipopolysaccharide extracted from this mutant inactivates the phage, and has core oligosaccharides identical in amounts to those in the lipopolysaccharide from wild type cells.In contrast, the Ter-21 mutant derived from E. coli K12 W2252-11U^? RC^rel (wild type II) is not sensitive to this phage infection, and its lipopolysaccharide does not inactivate the phage. Its lipopolysaccharide sugars are found to be D-glucose and D-ribose, thus differing from the lipopolysaccharide sugars of the wild type cells.     

Growth of coliphage BF23 on rough strains of Salmonella typhimurium: the bfe locus

Summary Coliphage BF23 develops in Salmonella typhimurium rough strains. The phage is neither restricted nor modified by S. typhimurium. The growth patterns of the phage were slightly different in S. typhimurium than in Escherichia coli, although phage propagated on S. typhimurium is identical to the phage propagated in E. coli by several criteria used. Mutants of S. typhimurium resistant to BF23 were isolated and found to map (by P22-and Plmediated transduction) in the same position as bfe mutants of E. coli. The order of genes was: metB-argC-bfe-rif-purD-metA.Phage BF23 does not form plaques on smooth S. typhimurium strains, since the phage fails to adsorb irreversibly to smooth cells. Nevertheless, on solid agar, the phage prevents growth of many (but not all) smooth strains. Moreover, UV-and alkali-inactivated phage BF23, although unable to form plaques on sensitive hosts, retains the ability to prevent growth of the host on solid medium. This ability is sensitive to protease and resistant to DNAse and RNase. Heat treatment of the phage causes rapid loss of the cell-growth-preventing-ability whereas the ability to form plaques is lost much more slowly. These results lead to a proposal that phage BF23 virions carry a colicin-like factor that kills sensitive cells.     

Mapping of a gene causing resistance to chlorate inSalmonella typhimurium

Chlorate-resistant mutants ofS. typhimurium LT2 and LT7 and ofS. abony have been isolated, which are deficient in the biosynthesis of nicotinic acid and thiamin and in the fermentation of inositol. These mutants could be divided into 5 groups. The most likely gene order isnicB-chlG-thiB-inlB. This segment is transferred early in conjugation experiments with Hfr H2 and Hfr B2 as donors. In time-of-entry experiments with Hfr B2 as donor the segment entered about 3 minutes afterpur C. Consequently this segment maps in the 79- to 82-minutes region of the genetic map. From recombinant analysis of nic⁺ recombinants obtained in a four-point cross between Hfr B2 and ahis carBpur C del (nic chl G) acceptor the incorporation frequency of the transferred donor fragment was calculated to be about 0.41. The number of crossing-over events per minute length of the chromosome was about the same as in similar crosses betweenE. coli Hfr and F^–. However, between thenic and thepur C markers it was much higher; it may therefore be inferred that there is a higher probability for a crossing-over event in the regions adjacent to the region that is deleted in the recipient.In crosses betweenS. abony Hfr H2 del (nic thi inl chl) and F^– strains no recombinants were observed which have obtained the deletion from the donor. Nearly all auxotrophic or nic⁺ recombinants obtained in a cross between Hfr B2 and a F^– del (nicBthiBchlGinlB) strain have inherited all markers of the donor, which are present in the deletion of the recipient.     

Early transfer of genes determining transfer functions by some Hfr strains in Escherichia coli K12

Summary Sixty-eight Hfr strains were examined for their ability to transfer early in conjugation the transfer genes carried by the integrated sex factor. This was measured by mating these strains with F^- phenocopied recipient cultures of strains carrying transfer-deficient Flac ⁺ factors, and then measuring the ability of the recipient strains to transfer lac ⁺ to a further recipient strain. Most Hfr strains did not complement the missing transfer functions, though in some strains complementation was observed. It is concluded that on the sex factors of different Hfr strains either the site at which integration occurs or the origin of transfer must vary.     

Transfer ofF′ fromEscherichia coli K 12 toEscherichia coli B and to strains ofParacolobacter andKlebsiella

The transfer of theF episome fromEscherichia coli K 12 toE. coli B,Paracolobacter andKlebsiella was studied. The frequency of transfer of the episomal markers toE. coli B was very low. The large majority ofE. coli B cells which had received the episomal markerslac ⁺ orgal ⁺ were F^–, which indicates that the episomal markers were stably integrated on the chromosome. Recombinants from K 12 F⁺ × B F^– crosses were mostly F^–. These results suggest that the multiplication of theF-factor ofE. coli K 12 is restricted inE. coli B. The transfer of theF-lac ⁺ Ad ⁺ episome fromE. coli K 12 toParacolobacter andKlebsiella strains was in most cases only possible when donor and acceptor strain were plated together on selective media. Stable incorporation of episomal markers was also found withParacolobacter coliforme. Paracolobacter aerogenoides andKlebsiella aerogenes strains could be infected withF-lac ⁺ Ad ⁺. The episomal markers were not incorporated and the episomes were easily lost, which indicates that these strains contained theF factor in the autonomous state.     

Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).