Isolation and characterization of Hfr strains of Erwinia amylovora

Hfr strains (Hfr 159 and its derivatives, Hfr 160 and Hfr 161) were constructed from Erwinia amylovora ICPB EA178 by introducing an Escherichia coli F'his+ plasmid and then selecting for integration of F'his+ after treatment with acridine orange. The Hfr strains were relatively stable upon repeated transfers on nonselective media. Interrupted mating experiments and analyses of inheritance of unselected markers showed that his+ is transferred by Hfr 159 as the proximal marker at a relatively high frequency (about 5 x 10(-4) recombinants per input donor cell), followed by ilv+, orn+, arg+, pro+, rbs+, met+, trp+, leu+, ser+, and thr+ (not necessarily in that precise order). The donor strains, previously constructed in E. amylovora by integration of F'lac+ from E. coli transfer cys+ as the proximal marker followed by ser+. Further analysis of one of those earlier donor strains, Hfr99, showed that ser+ is followed by arg+, orn+, met+, pro+, leu+, ilv+, rbs+, his+, trp+, and thr+ (not necessarily in that precise order). Thus, the Hfr strains constructed by integration of F'his+ are different, in terms of origin and direction of transfer, from those derived from integration of F'lac+. The applicability of these Hfr strains to mapping the genes on the E. amylovora chromosome is indicated.     

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.     

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.     

Recombinant F′ Factors from Escherichia coli K-12 Strains Carrying recB or recC

The frequency of genetic exchanges between F' factors and the bacterial chromosome was studied in recombination-deficient Escherichia coli mutants under conditions in which the recombinant F' factors were immediately transferred to new hosts. In a series of double matings, F101-1 thr(+)leu(-) episomes were first transferred into each of four intermediate F(-)thr(-)leu(+) strains carrying various rec alleles. After the original F' donors were killed with phage T6, the F101-1 episomes were then transferred from the intermediate cells to F(-)thr(-)leu(-)Str(R)recA(-) females. Recipients of nonrecombinant episomes formed Thr(+) (Str(R)) colonies, and recipients of recombinant episomes formed Leu(+)(Str(R)) colonies. A comparison of the numbers of Leu(+)(Str(R)) and Thr(+)(Str(R)) colonies shows that recB(-) males formed 18 to 21% and recC(-) formed 47 to 60% of the wild-type level of recombinant episomes that could be detected after transfer. No recombinant episomes were detected using a recA(-) intermediate strain. If the intermediate strains harboring the F101 episomes were purified, allowed to grow for 50 generations, and then mated with the recA(-) recipient, recombinant episomes were transferred at 8% of the wild-type level for recB(-) and 13% for recC(-). In contrast, only 0.4 and 0.6% of the normal number of recombinants were obtained from crosses between Hfr Cavalli donors and the same recB(-) and recC(-) strains. Recombinant episomes were detected with greater frequency among newly formed rec(+), recB(-), and recC(-) partial diploids than in those which were 50 generations old.     

Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus

Myxophage MX8 can initiate a lysogenic cycle in Myxococcus xanthus. The lysogenic phage was gentically stable in vegetative cells and persisted in the latent state through many cell generations in the absence of extracellular phage reinfection. The latent state also was stable during the host developmental cycle, since myxospores transmitted latent MX8 genetic information to future progeny cells. DNA hybridization experiments to probe the structure of the lysogenic phage provided physical evidence that MX8 formed a prophage. During lysogenization, MX8 DNA was cut at a specific site (attP) on phage DNA, and we have concluded that genetic recombination between attP and a bacterial DNA site (attB) leads to integration of MX8 DNA and formation of stable MX8 prophage. The genetic and physical properties of MX8 that we describe should make MX8 useful in the analysis of development of M. xanthus by genetic methods.     

Transfer of episome F'lac+ and chromosomal trp+ genes from Erwinia amylovora to Salmonella typhimurium.

The F'lac+ episome of Escherichia coli origin was transferred by conjugation with frequencies of 10(-7) to 10(-5) from Erwinia amylovora to 14 out of 15 Salmonella typhimurium trp female parents. The chromosomal trp+ genes were transferred with frequencies of 10(-7) to 10(-6) only to one trpB and 2 trpD female parents, which have a point mutation in the 2nd and fourth structural genes, respectively, of the tryptophan operon. The transferred male trp+ genes became integrated at the selected sites of the S. tryphimurium chromosome. The resulting Trp+ hybrids were phenotypically stable, lacked a cryptic trp allele selected against in the female parent, had high genetic homology values in the tryptophan region, and showed biochemical reactions and pathogenicity typical of S. typhimurium.     

The tripartite associations between bacteriophage, Wolbachia, and arthropods

By manipulating arthropod reproduction worldwide, the heritable endosymbiont Wolbachia has spread to pandemic levels. Little is known about the microbial basis of cytoplasmic incompatibility (CI) except that bacterial densities and percentages of infected sperm cysts associate with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage WO-B induces CI. However, current hypotheses have not considered the separate roles that lytic and lysogenic phage might have on bacterial fitness and phenotype. Here we describe a set of quantitative approaches to characterize phage densities and its associations with bacterial densities and CI. We enumerated genome copy number of phage WO-B and Wolbachia and CI penetrance in supergroup A- and B-infected males of the parasitoid wasp Nasonia vitripennis. We report several findings: (1) variability in CI strength for A-infected males is positively associated with bacterial densities, as expected under the bacterial density model of CI, (2) phage and bacterial densities have a significant inverse association, as expected for an active lytic infection, and (3) CI strength and phage densities are inversely related in A-infected males; similarly, males expressing incomplete CI have significantly higher phage densities than males expressing complete CI. Ultrastructural analyses indicate that approximately 12% of the A Wolbachia have phage particles, and aggregations of these particles can putatively occur outside the Wolbachia cell. Physical interactions were observed between approximately 16% of the Wolbachia cells and spermatid tails. The results support a low to moderate frequency of lytic development in Wolbachia and an overall negative density relationship between bacteriophage and Wolbachia. The findings motivate a novel phage density model of CI in which lytic phage repress Wolbachia densities and therefore reproductive parasitism. We conclude that phage, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis. Clarifying the roles of lytic and lysogenic phage development in Wolbachia biology will effectively structure inquiries into this research topic.     

Mechanism of conjugation and recombination in bacteria. XVIII. Polarity of donor DNA strands transferred to the recipient as determined by DNA-RNA hybridization.

Polarity of donor DNA strand transferred into recipient during conjugation in Escherichia coli K-12 was determined by DNA-3H-RNA hybridization. Lambda prophage was used as a marker. The defective lysogen Hfr H (lambdat11) as a donor and thermosensitive F- CR34 dnaB strain as recipient were used. Two sets of hybridization experiments, with 1-strand specific lambda mRNA and lambda mRNA specific for both phage strands but with large excess of r-strand specific mRNA, were carried out. Strand 1 of lambda DNA was detected preferentially in recipient cells mated at restrictive temperature, when Hfr transferred its genophore in the order gal-lambda-bio. Thus the genophore is transferred with 5'OH at its origin.     

Replication of Coliphage M-13 I. Effects on Host Cells After Synchronized Infection

Techniques have been described for synchronization of bacteriophage M-13 infection of host cells. The latent period in infected cells was 10 min, and no appreciable number of intracellular phage was observed. Phage production proceeded in three phases after release of the starvation block: an initial rapid exponential rate of progeny phage release without cell lysis, a period of rate transition accompanying the resumption of host cell division, and a second, slower exponential rate of phage production which paralleled the rate of host cell division. The size of infected cells was not affected by infection, but the generation time was increased by 25%. Starved infected cells exhibited a much longer lag in attaining an exponential rate of growth upon the addition of nutrients than did an uninfected control culture.     

Altered growth properties of normal human cells induced by phorbol 12,13-didecanoate

The tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cells with 10(-7) or 10(-8) M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures. Removal of PDD from the culture medium while the cells were at a high cell density resulted in a return to near-normal saturation density by the subsequent passage. Anchorage independent growth of normal human cells in methylcellulose was also promoted by PDD in a dose dependent manner, with prior subculturing in the presence of PDD being required for maximal colony formation. The structural analog 4 alpha-phorbol 12,13-didecanoate failed to elicit similar cellular responses.     

Cell growth and hemoglobin synthesis in murine erythroleukemic cells propagated in high density microcapsule culture

Summary A new microencapsulation technology, developed for the encapsulation of living cells, has been demonstrated to be useful for the study of growth and differential gene expression using Friend erythroleukemic cells cultured at high cell densities. Using this technology, cultures of FL Clone 745 cells were encapsulated within semipermeable membranes composed of cross-linked alginic acid and poly-l-lysine. Cell growth studies measuring total cell number demonstrated an average generation time of 8.5 h in 5% (vol/vol) microcapsule cultures vs. 8.0 h in suspension cultures. Similar microcapsule cultures were serially propagated for more than 90 cell generations (13 sequential passages) with no significant change in this growth rate. In addition, final culture densities of greater than 1.0×10⁸ cells/ml of intracapsular volume were attained using a 3% (vol/vol) microcapsule culture in conjunction with a standard refeeding schedule. Comparison of the level of dimethyl sulfoxide-induced hemoglobin production in suspension and microcapsule cultures demonstrated that the total amount of hemoglobin produced on a per cell basis was comparable in both systems. Due to the retention characteristics of the semipermeable membrane, the concentration of detergent-released hemoglobin, relative to other released protein, was approximately twofold higher in microcapsule cultures than in control suspension cultures.     

Loss of R Factors Promoted by Bacteriophage M13 and the M13 Carrier State

The infection of different Hfr strains of Escherichia coli bearing derepressed R factors of the fi(+) or fi(-) type can result in the loss of the R factor and the conversion of the infected cells to the R(-) state. This extends earlier observations on the elimination of F' factors by bacteriophage M13 infection. Variability in the efficiency of this conversion can arise because of genetic factors independent of the R factor being eliminated. A fraction of the infected but unconverted R(+) cells were M13 carrier strains. The carrier state had an intracellular basis, and single R(+) cells could maintain the carrier state.     

Interaction of RNA-containing bacteriophages with the host cells: MS2-induced E. coli mutants and formation of DNA-containing derivatives of MS2 bacteriophage

Sensitive cells of Escherichia coli AB 259 Hfr 3000 infected with RNA-containing phage MS2 produce phage particles and continue to divide showing segregation of sensitive cells maintaining new infection cycles. Phage multiplication in sensitive cells gives rise to phage resistant forms in their progeny. The described phenomenon has been shown to be due not to pre-existing phage-resistant cell selection but is a result of interaction of the phage and the cell. In contrast to the usual spontaneous or chemically induced Escherichia coli mutants MS2-induced phage-resistant cells are genetically unstable. During their reproduction they segregate new MS2-resistant types carrying more significant changes in the region coded by the sex factor. Cells belonging to two final MS2-induced mutants also produce a new type of phages; they are DNA-containing forms neutralized, however, by anti-MS2 serum. Production of such phage proves that genetic moiety of RNA-containing phage is able to be expressed as a part of the DNA structure.     

Increased Fragility of Escherichia coli After Infection with Bacteriophage M13

Male strains of Escherichia coli infected with filamentous phage M13 released the progeny phage particles from intact cells. At the same time, the cells continued to grow and multiply at a slightly lower rate than the uninfected cells. Concomitant with the phage release, lipopolysaccharide from the cell wall of the infected cells was also released. The buoyant density of E. coli HfrC in diaginol, 1.25 g/cc, did not change as a result of infection. Detergents like sodium dodecyl sulfate and Sarkosyl specifically lysed the infected cells. The infected cells showed enhanced fragility as indicated by inactivation by various stresses, namely heat, osmotic shock, and freezing and thawing. It is concluded that the infection with M13 causes certain alterations in the surface structure of E. coli, thus making the cells more fragile.     

Nature of the carrier state of bacteriophage SP-10 in Bacillus subtilis

Although the association of phage SP-10 with Bacillus subtilis W-23-S(r) persists in heat- and antiserum-resistant form through the spore stage, it is unstable in vegetative cells and frequently terminates in loss of the carried phage or in lysis. On low-tonicity media, the plating efficiency of carrier cells is low. However, high concentrations of succinate or sucrose or a slowed growth rate preserve viability: on 0.48 m succinate-agar, the viable count per optical density unit is the same as that of a noncarrier control culture. Carrier clones retain phage on 0.48 m succinate-agar. At higher succinate levels, many colonies emerge free of phage; at 1 m succinate, all are cured, probably because high succinate inhibits reinfection. Growth of carrier cells in liquid medium with antiphage serum results in rapid curing; events in such cultures with and without succinate were studied quantitatively by tracing the emergence of sensitive cells, the multiplication and induction of carrier cells, and the sensitivity of carrier cells to superinfection with virulent phage. During log phase, 40 to 70% of the carrier cells became sensitive to virulent phage, although the same cells were insensitive during lag and stationary phase. Apparently, fluctuations in repressor levels are responsible. Spontaneous induction of carrier cells followed a qualitatively similar pattern, perhaps in response to changes in level of the same repressor. Production of sensitive segregants by carrier followed a different course, presumably because the repressor does not affect segregation. Many sensitive cells were found two to three divisions after inoculation in antiserum medium. This suggests that each inoculum cell contained one or only a few phage replicons. The data are compatible with the idea that the carrier state in media without antisera is maintained entirely by reinfection and without replication of phage in the latent state. Alternative models which involve replication of latent phage are not ruled out, however.     

Cell culture density dependent toxicity and chromatin changes upon cadmium treatment in murine pre-B-cells

Murine pre-B-cells grown in the presence of lower (1 μM) or higher (5 μM) concentration of cadmium chloride were separated into 13 fractions by centrifugal elutriation. The rate of DNA synthesis after cadmium treatment determined in permeable cells was dependent on cell culture density during cadmium treatment. Cell cycle analysis revealed a shift in the profile of DNA synthesis from replicative to repair DNA synthesis upon cadmium treatment. The study of the relationship between cell culture density and cell diameter at lower and higher cell densities in the presence of 1 μM cadmium chloride concentration showed that a. at 5×10⁵ cell/ml or lower densities cells were shrinking indicating apoptotic changes, b. at higher cell culture densities the average cell size increased, c. the treatment of cells with low CdCl₂ concentration (1 μM) at higher cell culture density (>5×10⁵ cell/ml) did not change significantly the average cell diameter. At 5 μM cadmium concentration and higher cell culture densities (>5×10⁵ cell/ml) the average cell size decreased in each elutriated fraction. Most significant inhibition of cell growth took place in early S phase (2.0–2.5 C value). Apoptotic chromatin changes in chromatin structure after cadmium treatment were seen as large extensive disruptions, holes in the nuclear membrane and stickiness of incompletely folded chromosomes.     

Escherichia coli Hfr-DNA degradation in endonuclease I-deficient minicells.

[3H]Thymidine (dThd)-labelled Hfr DNA was transferred by conjugation into Escherichia coli F- minicells harvested from an endonuclease I-deficient (endI-) strain and its iosgenic wild type (endI+) parent. The susceptibility of this DNA to attack by DNAase was examined. The kinetics of in vivo conversion of [3H]dThd-labelled DNA into acid soluble radioactivity was examined. This activity, attributed to exonuclease action was the same for both strains. Contribution of endonuclease I was measured by an analysis of changes in weight-average (Mw) and number-average (Mn) molecular weight distribution of DNA molecules recovered from minicells. Reduction in Mw was greater in the endI-strain. The ratio Mn/Mw changed drastically during the incubation period of endI- minicells, but remained unchanged in the endI+ strain. These experiments suggest that the presence of the endI- mutation in minicell-producing strain chi1268 leads to a greater loss in M2 of Hfr DNA conjugally transferred into the minicells.     

Isolation of a mycoplasma-specific binding peptide from an unbiased phage-displayed peptide library

An important goal in medicine is the development of methods for cell-specific targeting of therapeutic molecules to pathogens or pathogen-infected cells. However, little progress has been made in cell-specific targeting of bacterially infected cells. Using a phage display approach, we have isolated a 20-mer peptide that binds to Mycoplasma arginini infected pancreatic beta-cells in tissue culture. This peptide binds to M. arginini infected beta-cells 200 times better than a control phage and is specific for the infected cells. Furthermore, transferring the M. arginini contamination to another cell line renders the newly infected cell line susceptible to peptide binding. Immunolocalization experiments suggest that the peptide is binding to M. arginini adhered to the cell surface. The free synthetic peptide retains its binding in the absence of the phage vehicle and tetramerization of the peptide increases its affinity for the infected cells. Efforts have been made to use this peptide to eliminate Mycoplasma from infected cell lines using ferromagnetic beads coated with the selected peptide. A ten-fold reduction of infection was accomplished with one fractionation via this approach. Our results suggest that this peptide, isolated from an unbiased selection, may be of utility for the detection and reduction of Mycoplasma infection in cultured cells. Furthermore, a general implication of our findings is that phage display methods may be useful for identifying peptides that target a broad array of other biological pathogens in a specific fashion.     

A new electro-optical approach to rapid assay of cell viability

A new electro-optical (EO) approach was developed and applied to rapidly assay cell viability by using phage M13K07. Since phage M13K07 can replicate only in living bacteria and cannot replicate in the presence of inhibitors, the difference between the EO signals obtained in the presence and absence of the phage can be used as an important factor for evaluating cell viability. Variation in the electrophysical parameters of Escherichia coli XL-1 during its interaction with phage M13K07 was studied under exposure of the cells to various inhibitors of cellular metabolism. Significant changes in the EO signal were found during incubation of living E. coli cells with phage M13K07. At the same time, no changes were recorded during cell incubation with the phage after pretreatment of E. coli XL-1 cells with sodium azide, carbonyl cyanide 3-chlorophenyl hydrazone, chloramphenicol, and kanamycin. This finding can be explained by the decrease in the number of living cells in the culture after preliminary incubation with the chemical agents, and it was confirmed by colony counts by conventional plating onto solid LB medium before and after treatment of the cells with the inhibitors. The EO approach can be used as a rapid method for evaluation of the inhibitory effects of various chemical agents and drugs, and it has the potential for the study of the molecular mechanisms underlying cell death.     

F deoxyribonucleic acid superinfected into phenocopies of donor strains.

When F+ donor cells of Escherichia coli are conjugated with F-, F+, or Hfr recipients under the conditions of phenocopy mating, the male recipients are found capable of accepting the F episome as effectively as the F- recipients. The F deoxyribonucleic acid (DNA) superinfected into the male recipients is converted to the covalently closed, circular duplex form, as in the F- recipients. It is also found that the synthesis of the strand complementary to the transferred single strand and its subsequent conversion to the covalently closed, circular duplex occur effectively in male recipients as well as in female recipients. Under these mating conditions, F-ilv+ episome superinfected to F+ and Hfr cells is diluted out during growth, whereas F-ilv+ transferred into F-cells is replicated and established in almost all progeny cells. These results suggest that the incompatibility of the F episome is not due to the reduction in the rate of the conversion of transferred single-straned F DNA to covalently closed, circular duplex, but, rather, to an inhibition of further replication of the covalently closed, circular F DNA.