Chromosome transfer and Hfr formation by F in rec+ and recA strains of Escherichia coli K12.

We attempted to assess the role of Hfr clones in chromosome transfer by F⁺ populations. We thought that any Hfr-independent component of fertility might be affected to a different extent by the recA mutation than was the Hfr component. However, the rate of Hfr formation and the efficiency of chromosome transfer were reduced to an equal extent (× 100-fold) by the recA mutation. Such experiments therefore provide no evidence for an Hfr-independent component. It appeared that Type II strains, which were thought to suffer a defect in Hfr formation, actually produced fertile clones but had a secondary defect which affected the persistence of these clones. Thus, evidence from Type II strains is also not useful for examining the quantitative contribution of Hfr cells to F⁺ transfer.     

Inversion of Transfer Modes and Sex Factor-Chromosome Interactions in Conjugation in Escherichia coli

A study was made of the mating properties of an unusual system of interconvertible donor strains of Escherichia coli K-12: Ra-1, Ra-2, and RaF⁺. The Ra-1 and Ra-2 strains are Hfr strains whose origins are widely separated on the chromosome and whose transfer modes proceed in the opposite direction from one another. When Ra-1 cells were mated with females, a small fraction of the donors transferred markers via the Ra-2 mode. This effect was enhanced by preconjugal ultraviolet (UV) treatment of the Ra-1 cells. Among the survivors of UV-treated Ra-1 cells, a few stable Ra-2 cells were found. When Ra-2 cells were used as the donors, some of them were found to mate via the Ra-1 mode, in analogy with the Ra-1 to Ra-2 alteration with inversion of F mentioned above. Related experiments suggested that the inversion occurs by detachment of the F factor from one Hfr origin locus, followed by reassociation of the F factor with the other Hfr origin locus. Both the Ra-1 and Ra-2 strains reverted spontaneously to an F⁺ strain, called RaF⁺. Cultures of RaF⁺ cells were found to mate primarily according to the Ra-1 and Ra-2 transfer modes, with smaller contributions also coming from transfer modes with origins elsewhere on the chromosome in a way which is similar to the transfer of markers from a normal F⁺ strain. The RaF⁺ sex factor was found to be wild type, whereas the chromosome was found to carry irregularities (sex factor affinity loci) at the locations of the Ra-1 and Ra-2 origins. Only about 10% of the donor capacity of the RaF⁺ strain was due to stable spontaneous Hfr cells in cultures of RaF⁺ cells.     

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.     

Evolution of a Site of Specific Genetic Homology on the Chromosome of Escherichia coli

Integration of the factors F(v) and F into the chromosome of a substrain of Escherichia coli K-12 has been studied. The F(v) factor is a fertility factor derived from Col V, lacking the ability to govern the production of colicin V. The derivatives of an Hfr(v) (Hfr isolated from a V colicinogenic parent) strain, PK2 (initially isolated from C600 V(+)), were shown to retain a unique bidirectional sex factor affinity locus between recA and pheA. This site shows no affinity for the E. coli K-12 F factor as shown by inability to isolate Hfr strains with origins in this region from a parental strain containing a cytoplasmic F factor. However this area exhibits two regions of homology to the V colicinogenic factor. One gives rise to Hfr(v) strains identical to the original Hfr(v) strain, PK2, with an origin and polarity of transfer designated pheA-CC injecting markers in the order pheA-his-trp-pro. The second gives rise to strains apparently originating at the same site but with reverse polarity designated recA-C, transferring markers in the order recA-thyA-str-xyl. For strains possessing the F(v) factor only the second homology is apparent. A model for the evolution of these strains is presented.     

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.     

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.     

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.     

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.     

Unique Activity Spectrum of Colicin FY: All 110 Characterized Yersinia enterocolitica Isolates Were Colicin FY Susceptible

Colicin F_Y is a plasmid encoded toxin that recognizes a yersinia-specific outer membrane protein (YiuR) as a receptor molecule. We have previously shown that the activity spectrum of colicin F_Y comprises strains of the genus Yersinia. In this study, we analyzed the activity of colicin F_Y against 110 Yersinia enterocolitica isolates differing in geographical origin and source. All isolates were characterized through analysis of 16S rRNA genes, serotyping, biotyping, restriction profiling of genomic DNA, detection of virulence markers and susceptibility to antibiotics. This confirmed the broad variability of the collection, in which all 110 Y. enterocolitica isolates, representing 77 various strains, were inhibited by colicin F_Y. Although isolates showed variable levels of susceptibility to colicin F_Y, it was not associated with any strain characteristic. The universal susceptibility of Y. enterocolitica strains to colicin F_Y together with the absence of activity towards strains outside the Yersinia genus suggests potential therapeutic applications for colicin F_Y.     

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.     

Isolation and characterisation of Hfr strains from a recombination-deficient strain of Escherichia coli

Summary A T6^s RecA^- strain carrying a lac proB deletion and F_ts lac ⁺ was challenged with phage T6 and survivors which were both T6^r and Lac⁺ at 42° were tested for fertility. Among these were a number of Hfr strains which had their points of origin at or near the tsx locus and which still carried the recA allele. These arose in comparable frequencies in the RecA^- strain and in a Rec⁺ analogue. We conclude that such integration does not require the RecA function. The rate of chromosome transfer was similar in one such RecA^- Hfr and its Rec⁺ derivative; the yield of recombinants from the RecA^- strain was slightly lower than from its Rec⁺ derivative.     

Sex-determined chemotaxis inSalmonella typhimurium LT2

Chemotactic interaction between F^? and Hfr strains ofSalmonella typhimurium is described. Under the experimental conditions used, the motile Hfr cells are attracted by the F^? cells.     

Participation of RNase I in MS2 Phage Growth

Participation of RNase I in the growth of phage on infection with bacteriophage MS2 was studied.

Some strains of uracil-requiring E. coli were isolated, grown in MS broth, and transferred to a minimal medium to exhaust the pool of nucleotides. The phage was then added to the cells grown in uracil-deficient medium. The growth of phage was observed to occur at the burst size of two hundreds in strains of E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, which possessed RNase I, but not in strains, A19 (Hfr) U^? and Q13 (Hfr) U^?, which lacked RNase I.

A marked increase in acid-soluble fraction was observed with E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, whereas the increase was little with E. coli A10 (Hfr) U^? and Q13 (Hfr) U^? Conditions for the growth of phage in uracil-deficient medium were investigated and the effect of antibiotics were also investigated.     

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.     

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.     

A Comparative Study of Two F-like Colicin Factors, ColV2 and ColV3, in Escherichia coli K-12

Of three colicin factors, each determining the synthesis of a colicin V in three wild-type Escherichia coli strains studied, two were shown to have sex-factor activities. In E. coli K-12, these activities resembled those of the F sex factor (including rapid and efficient self-transmission in exponentially growing cultures, adsorption of "male-specific" ribonucleic acid phage, production of "female phenocopies," elimination by acridine orange, and chromosomal transfer dependent upon recombination with host bacterium) and differed in this way from those of the colicin I sex factor (ColI). The two V factors, ColV2 and ColV3, differed in their efficiency of plating male-specific phage and in the pattern of transfer of chromosomal markers. Furthermore, although neither factor could stably coexist with F within the same cell, they showed markedly different exclusion effects. In general, ColV2 excluded F and ColV3 was excluded by F, irrespective of which sex factor was preestablished in the cell. An exception to this was the ability of ColV2 to stabilize in any one of a series of Hfr strains, giving rise to strains which in the majority of cases showed normal Hfr and colicinogenic properties.     

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.     

Chlorophyll fluorescence and gas exchange measurements in field research: an ecological case study

We tested whether cheap and quick chlorophyll (Chl) fluorescence can be used in ecophysiological field studies as proxies for gas-exchange measurements. We measured net photosynthetic rate at saturating irradiance and ambient atmospheric CO₂ concentrations (P_Nsat), maximum carboxylation rate (V_cmax), maximum quantum yield of PSII (F_v/F_m), the performance index (PIabs), leaf nitrogen (Narea), and carbon isotope discrimination (Δ¹³C) within four herbaceous species along two elevational gradients. We analysed the relationship between Chl fluorescence and gas-exchange parameters and their link to indirect assessment of plant performance via ecophysiological traits. F_v/F_m showed no relationship to P_Nsat and only weak relationships to V_cmax. PI_abs was positively related to P_Nsat and V_cmax. PI_abs, P_Nsat, and V_cmax were positively associated with N_area and negatively to Δ¹³C, whereas F_v/F_m showed no relationship to Narea and a positive to Δ¹³C. Thus, PIabs might be suitable to characterize the photosynthetic activity when aiming on large numbers of samples.     

Characterization of V-Prime Factors in Escherichia coli K-12

An episome derived from an Hfr_v (Hfr isolated from a V colicinogenic parent) strain of Escherichia coli K-12 was isolated and characterized. The direction of gene transfer was inverted from that in the original parental strain.     

Induction of Colicin Production by High Temperature or Inhibition of Protein Synthesis

Escherichia coli K-12 colicinogenic for ColE1 yielded mutants that appeared to produce colicin at 43 C but not at 30 or 37. These mutants proved to have the mutation recA⁻ Further study revealed that both recA⁻ and recA⁺ bacteria, when carrying ColE1 or ColE2, produce more colicin during growth at higher temperatures or after brief exposure to temperatures beyond the growth range. Counts of lacunae demonstrated that the increase of colicin production is due to an increase in the number of cells that yield colicin. Heat treatment causes lacunae to increase by the same factor in recA⁺ and recA⁻ cells, although recA⁻ bacteria produce 500 times fewer lacunae than recA⁺. Inhibition of protein synthesis, notably by chloramphenicol, also induces colicin production in as much as 90% of the cells after removal of inhibition (to permit colicin synthesis). Induction of colicin production by chloramphenicol requires that ribonucleic acid synthesis continue during the period of inhibition. These results are discussed in relation to the regulation of colicin production.