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.     

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.     

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.     

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.     

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.     

Physiology of Escherichia coli K-12 During Conjugation: Altered Recipient Cell Functions Associated with Lethal Zygosis

The number of viable F⁻ cells decreases when Escherichia coli recipient cells are mixed with an excess of Hfr cells. Evidence is presented showing that lethal zygosis was accompanied by changes in the physiology of the recipient cells, including (i) inhibition of deoxyribonucleic acid synthesis, (ii) inhibition of β-galactosidase induction, (iii) altered transport and accumulation of galactosides, and (iv) leakage of β-galactosidase into the supernatant fluid. The results are discussed in terms of possible conjugation-associated changes that, at high Hfr to F⁻ ratios, lead to lethal zygosis.     

Delayed lysis with a mutant of salmonella bacteriophage p22

A mutant of bacteriophage P22 (Lys⁻) was isolated which shows a plaque morphology on mixed plates comparable to the r⁺ plaques of the T-even phages. When Lys⁻ and normal Lys⁺ plaques are juxtaposed on a petri dish, the Lys⁺ plaque exhibits a flat side adjacent to the Lys⁻ plaque. The mutant is identical to P22 under an electron microscope, is inactivated at the same rate by antiserum and heat, and has the same kinetics of attachment. It does not plate on Salmonella lysogenic for phage P22 nor on strain St/22. In liquid culture, the lysis of mutant infections in M9CAA medium is delayed between 20 and 40 min. Cells mixedly infected in M9CAA with Lys⁻ and Lys⁺ phage lyse later than Lys⁺-infected cells and even later than Lys⁻-infected cells. In unsupplemented M9 medium, however, mixedly infected cells again lyse later than Lys⁺-infected cells, but Lys⁻-infected cells require more than 3 hr to lyse. In supplemented and unsupplemented M9 media, intracellular phage development and endolysin synthesis proceed in Lys⁻ infections at least as rapidly as in Lys⁺-infected cells. In diluted infections, the latent and eclipse periods of Lys⁻ and Lys⁺ infections are indistinguishable. The possible mechanisms involved in the control and timing of lysis are discussed.     

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.     

Some Properties of Excision-defective Recombination-deficient Mutants of Escherichia coli K-12

Strains of Escherichia coli that carry the mutation uvrA6 show no measurable excision of pyrimidine dimers and are easily killed by ultraviolet (UV) light, whereas strains that carry recA13 are defective in genetic recombination and are also UV-sensitive. An Hfr strain carrying uvrA6 was crossed with an F⁻ strain carrying recA13. Among the recombinants identified, one carrying uvrA recA proved to be of exceptional sensitivity to UV light. It is estimated from the UV dose (0.2 erg/mm² at 253.7 nm) required to reduce the number of colony-forming cells by one natural logarithm that about 1.3 pyrimidine dimers were formed in a genome of 5 × 10⁶ base pairs for each lethal event. This double mutant is 40 times more UV-sensitive than the excision-defective strain carrying uvrA6. The replication of one pyrimidine dimer is generally a lethal event in strains carrying recA13. Spontaneous breakdown and UV-induced breakdown of the deoxyribonucleic acid (DNA) of cells of the various genotypes were estimated by growing the cells in medium containing ³H-thymidine and measuring both acid-precipitable and acid-soluble radioactivity. The UV-induced degradation in strains with recA13 did not require the uvr⁺ genes and hence appears to depend upon a mechanism other than dimer excision. The greater level of survival after irradiation in Rec⁺ as compared to Rec⁻ bacteria may be due to a recovery mechanism involving the reconstruction of the bacterial chromosome through genetic exchanges which occur between the newly replicated sister duplexes and which effectively circumvent the damaged bases remaining in the DNA.     

Chromosome Transfer and Recombinant Formation with Deoxyribonucleic Acid Temperature-Sensitive Strains of Escherichia coli

Haploid recombinant yield is reduced in matings conducted at 42.5 C between deoxyribonucleic acid (DNA) temperature-sensitive [dna⁻(TS)] recipients unable to synthesize DNA at 42.5 C and any of the three major donor types (Hfr, F⁺, F′) of Escherichia coli. No such reduction is observed in matings conducted at 42.5 C when the dna⁻(TS) mutation is in the donor parent. Evidence is presented which indicates that chromosome transfer from donors to recipients unable to replicate DNA at 42.5 C during vegetative growth occurs at normal frequencies when the mating is conducted at 42.5 C. It is concluded that some stage in haploid recombinant formation is adversely affected in dna⁻(TS) recipients mated at the temperature restrictive for DNA synthesis.     

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.     

Some Effects of Nalidixic Acid on Conjugation in Escherichia coli K-12

The role of deoxyribonucleic acid (DNA) synthesis in the Escherichia coli conjugation system has been studied using nalidixic acid (NAL) to specifically inhibit DNA synthesis in matings between reciprocal combinations of male (Hfr) and female (F⁻) mutants resistant and sensitive to NAL; the physiological action of NAL on the strains utilized was also studied. Matings between combinations of mutants resistant (Nal^r) and sensitive (Nal^s) to NAL allow various parental functions to be established: pair formation studies show that the female cells are responsible for the slight decrease in pair formation when NAL is present in Hfr(Nal^s) × F⁻ (Nal^s) matings. Preformed mating pairs are stable in the presence of NAL. In matings between Hfr(Nal^s) and F⁻(Nal^r), the transfer gradient constant increases linearly with low NAL concentration (0.1 to 0.6 μg of NAL per ml). Higher concentrations of NAL (5 μg/ml) act on Nal^s males to rapidly stop chromosome transfer; under these conditions, however, DNA degradation is unmeasurable as determined from single-strand nicking in the male cells. This result is consistent with a model for chromosome transfer which requires DNA synthesis in the male cell. Inhibition of DNA synthesis (by 85%) in the female has no effect on conjugal chromosome transfer. High concentrations of NAL (>20 μg/ml) produce slight inhibition in chromosome transfer for the Hfr(Nal^r) × F⁻(Nal^r) mating tested; this effect is probably caused by action of NAL on the male. The inhibition of chromosomal transfer by NAL appears to be irreversible in the normal sense. A pulse of NAL, applied during transfer, immediately stops the transfer which is in progress. On removal of the NAL block, the temporal appearance of recombinants is consistent with the idea that a new round of transfer has commenced from the sex factor location on the male chromosome.     

Stabilization of Lactose Metabolism in Streptococcus lactis C2

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac⁺) transductants of lactose- and proteinase-negative (Lac⁻ Prt⁻) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac⁺ transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac⁻ segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac⁺ transductants showed 12 to 88% conversion from Lac⁺ to Lac⁻ within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac⁻ variants in 168 h, while S. lactis C2 had 96% conversion from Lac⁺ to Lac⁻ in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac⁺ transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac⁺ transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.     

Mapping of a Mutation, polB100, Affecting Deoxyribonucleic Acid Polymerase II in Escherichia coli K-12

Direct assay for deoxyribonucleic acid polymerase II in extracts has been used to screen recombinants for the polB allele in Hfr × F⁻ crosses, F-ductants in episome transfer, and transductants in generalized transduction by phage P1. The polB gene is located at 2 min on the Escherichia coli linkage map; it is 39 to 64% co-transducible with leu. A mutant, E. coli polA1 polB100 polC (ts), deficient in deoxyribonucleic acid polymerases I and II and having a thermolabile deoxyribonucleic acid polymerase III, has been prepared by the P1-mediated cross: P1 (HMS85 polB100) × E. coli BT1026 polA1 polC (ts) leu⁻.     

Dose-Dependent Effects of Lactobacillus rhamnosus on Serum Interleukin-17 Production and Intestinal T-Cell Responses in Pigs Challenged with Escherichia coli

The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 10⁹ CFU/ml) or high (1 × 10¹¹ CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4⁺ ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3⁺ CD4⁺ CD8⁻ T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4⁺ ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3⁺ CD4⁺ CD8⁻ cells and Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells. The percentage of ileal intraepithelial CD3⁺ CD4⁻ CD8⁺ cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4⁺ ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor β1 (TGF-β1), and ileal IL-10 was upregulated in the low-dose piglets after F4⁺ ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3⁺ CD4⁺ CD8⁻ T cells, the expansion of Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells, and the attenuation of F4⁺ ETEC-induced increase in CD3⁺ CD4⁺ CD8⁺ T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4⁺ ETEC challenge, thus eliciting a strong proinflammatory response.     

Nature of the genetic determinant controlling exfoliative toxin production in Staphylococcus aureus

Phage group II Staphylococcus aureus has been identified as the etiological agent of the staphylococcal scaleded skin syndrome. The development of an animal model system permitted fulfillment of Koch's postulates and recognition of exfoliative toxin (ET) as being responsible for some of the clinical manifestations of this syndrome. Initial studies directed toward associating a lysogenic phage with the genetic control of ET synthesis failed to support this hypothesis. Growth of two Tox⁺ strains at 44 C was more effective than growth in ethidium bromide or sodium dodecyl sulfate in eliminating the ability to produce ET. The early and rapid accumulation of ET-negative (Tox⁻) variants during growth of strain UT 0007 at 44 C, the lack of any selective advantage of the Tox⁻ variants over Tox⁺ cells during growth at 44 C, and an enhanced elimination frequency at 44 C of 97.9% over the spontaneous frequency of loss strongly suggest that the gene for ET synthesis is extrachromosomal. Additional evidence suggests that this gene is located on a plasmid which is not associated with genes for penicillinase synthesis and cadmium resistance. Two Tox⁺ strains harbored lysogenic phage capable of transducing cadmium resistance, but not penicillin resistance, to specific Tox⁻ recipients.     

Specific Effects of a recB Mutation on the HfrH Strain of Escherichia coli

The recB268::Tn10 mutation was introduced into the HfrH strain of Escherichia coli. Compared with recB F⁻ and recB F⁺ cells, the viability of this mutant strain was much lower. Compared with wild-type HfrH, the recB derivative donated much shorter fragments of its chromosome to the recipient. It is suggested that the recB gene product (i.e., RecBCD enzyme) participates in Hfr transfer.     

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.     

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.     

A high‐fat diet exacerbates the Alzheimer's disease pathology in the hippocampus of the App NL−F/NL−F knock‐in mouse model

Insulin resistance and diabetes mellitus are major risk factors for Alzheimer''s disease (AD), and studies with transgenic mouse models of AD have provided supportive evidence with some controversies. To overcome potential artifacts derived from transgenes, we used a knock‐in mouse model, App^{NL−F/NL−F} , which accumulates Aβ plaques from 6 months of age and shows mild cognitive impairment at 18 months of age, without the overproduction of APP. In the present study, 6‐month‐old male App^{NL−F/NL−F} and wild‐type mice were fed a regular or high‐fat diet (HFD) for 12 months. HFD treatment caused obesity and impaired glucose tolerance (i.e., T2DM conditions) in both wild‐type and App^{NL−F/NL−F} mice, but only the latter animals exhibited an impaired cognitive function accompanied by marked increases in both Aβ deposition and microgliosis as well as insulin resistance in the hippocampus. Furthermore, HFD‐fed App^{NL−F/NL−F} mice exhibited a significant decrease in volume of the granule cell layer in the dentate gyrus and an increased accumulation of 8‐oxoguanine, an oxidized guanine base, in the nuclei of granule cells. Gene expression profiling by microarrays revealed that the populations of the cell types in hippocampus were not significantly different between the two mouse lines, regardless of the diet. In addition, HFD treatment decreased the expression of the Aβ binding protein transthyretin (TTR) in App^{NL−F/NL−F} mice, suggesting that the depletion of TTR underlies the increased Aβ deposition in the hippocampus of HFD‐fed App^{NL−F/NL−F} mice.