Evidence for heterogeneity in populations of T5 bacteriophage. II. Some particles are unable to inject their second-step-transfer DNA

Hulen, Christian; Labedan, Bernard; Legault-Démare, J

doi:10.1128/jvi.36.3.633-638.1980

Cited by 7 publications

(9 citation statements)

References 19 publications

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“…Injection of T5 am DNA could also be tested by measuring the percentage of radioctive A2-DNA retained by superinfected bacteria after centrifugation and DNase treatment. We previously showed that, due to the heterogeneity of T5 phage stocks (20,34), only about 25% of the radioactivity initially adsorbed is actually injected. Table 6 shows that the percentage of radio- a The amounts of radioactivity present in the different pellets are expressed as percentages of the radioactivity initially contained in the P5 pellet, usually defined as the adsorbed radioactivity (29, 38).…”

Section: Concentrated Bacteria (5 X 109 Cells Per Ml)mentioning

confidence: 99%

Host cell metabolic energy is not required for injection of bacteriophage T5 DNA

Maltouf¹,

Labedan²

1983

J Bacteriol

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The addition of various metabolic inhibitors (uncouplers, cyanide, arsenate, ionophores) separately or together (for example, arsenate and an uncoupler) or even harsher methods of energy depletion did not prevent bacteriophage T5 from injecting its first-step-transfer DNA (a DNA segment 3 ,um long) into the cytoplasm of host cells. The same indifference to metabolic energy was observed if first-step-transfer DNA was decapsidated and uncoiled before injection, thus precluding any energetic help from the phage capsid or from some tension stored in DNA tightly packed in the head. Penetration of the second-step-transfer DNA across the cytoplasmic membrane was studied by determining injection of superinfecting T5 A2-amber phages into Sup-bacteria containing proteins Al and A2 previously encoded by the first-step-transfer DNA of a primary wild-type phage. The addition of various metabolic inhibitors after synthesis of proteins Al and A2 but before superinfection did not prevent this penetration of second-steptransfer DNA. Thus, we conclude that traversal of the cytoplasmic membrane by the entire T5 DNA (a molecule 34 ,m long) must occur by diffusion through protein channels.

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Section: Concentrated Bacteria (5 X 109 Cells Per Ml)mentioning

confidence: 99%

Host cell metabolic energy is not required for injection of bacteriophage T5 DNA

Maltouf¹,

Labedan²

1983

J Bacteriol

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“…The number of T5 receptors was determined from the plateau of saturation curves. In the stock of phages used here, lytic phages amounted only to 50%o of the total population, whereas the other half consisted of killer phages able to adsorb to the host cell but not to form plaques (19). To determine the total number of T5 receptors in a cell, the number of receptors for lytic phages was multiplied by 2. In experiments with whole cells, bacteria were first centrifuged and then washed with buffer A; incubation with T5 phages was performed for 30 min in the presence of 300 iLg of spectinomycin per ml to stop protein synthesis.…”

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confidence: 99%

Integration of the overproduced bacteriophage T5 receptor protein in the outer membrane of Escherichia coli

Menichi¹,

Buu²

1983

J Bacteriol

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The tonA gene codes for an outer membrane protein, a receptor of phage T5, the TonA protein. Strains harboring pLG513, a multicopy plasmid in which the tonA gene has been cloned, overproduced TonA protein, which appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell envelope proteins as a 78,000-molecular-weight protein. Identical results have been observed by Plastow et al. (FEBS Lett. 131:262-264, 1981) with plasmid pLC19-19, in which the tonA gene has also been cloned. The activity of the TonA protein, measured by its capacity to inactivate phage T5, increased by five- to sixfold in purified envelopes of cells harboring pLG513 compared with cells lacking the plasmid. Solubilization of the cytoplasmic membrane by Triton-Mg2+ treatment did not increase this activity. However, partial solubilization of outer membrane proteins by Triton-EDTA unmasked further T5 receptor activity, resulting in a final increase of around 50-fold, a value more consistent with the expected gene dosage effect. Treatment of whole cells by trypsin in conditions in which trypsin is allowed to enter the outer membrane revealed that part of the overproduced T5 receptors were embedded in the outer membrane and masked by a trypsin-sensitive protein. In addition, no T5 receptor activity was detected in either the periplasmic space or the cytoplasm. These results suggest that all of the overproduced TonA molecules were synthesized in an active form and integrated in the outer membrane, but only a small fraction could be reached or recognized by phage T5 in vivo.

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“…1B). Moreover, in a high (10 spermine or 1 mM spermidine, which in the presence of i0-5 mM) magnesium concentration, spermidine showed the M Mg2+ is known to condense DNA (7), showed a partially same effect. It has been verified that the presence of .…”

Section: Resultsmentioning

confidence: 99%

“…This last figure was obtained by incubating 1011 lytic phages with decreasing quantities of TonA suspension containing 1% Triton X-100 for 1 h at room temperature and titrating the unadsorbed phages (15). The number of T5 receptors was determined from the plateau of the saturation curve, taking into account that, in our phage stocks, lytic phages amounted to only 50% of the total population, whereas the other half 214 TOSI, LABEDAN, AND LEGAULT-DEMARE of the total population was able to adsorb but form plaques (10,13). The activity of such preparations remained constant for more than 1 year when the preparations were kept at 4°C in 1% Triton X-100.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the coliphage T5 DNA ejection process with free and liposome-associated TonA protein

Tosi¹,

Labedan²,

Legault-Démare³

1984

J Virol

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Outer membrane protein TonA, the receptor for coliphage T5, has been partially purified and incorporated into the phospholipid bilayer of liposomes. Adsorption of the phage to its receptor in either a free or liposome-associated form is fast and sufficient to trigger the ejection of encapsidated DNA. In both in vitro systems the exit of DNA from the phage capsid is a very slow process. Ejected DNA can partially accumulate inside the liposome aqueous compartment, but the transfer from the phage head to the liposome internal space is never complete, perhaps because the liposome volume is too small. The presence of polyamines or divalent cations (magnesium) or both in the incubation medium diminished the extent of DNA ejection, possibly by stabilizing DNA inside the head. DNA movement was slowed as the temperature was decreased from 37 to 18°C. Furthermore, incubation at 4°C totally prevented this DNA movement, even if a large part of the DNA had already exited the capsid.

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Evidence for heterogeneity in populations of T5 bacteriophage. II. Some particles are unable to inject their second-step-transfer DNA

Cited by 7 publications

References 19 publications

Host cell metabolic energy is not required for injection of bacteriophage T5 DNA

Host cell metabolic energy is not required for injection of bacteriophage T5 DNA

Integration of the overproduced bacteriophage T5 receptor protein in the outer membrane of Escherichia coli

Analysis of the coliphage T5 DNA ejection process with free and liposome-associated TonA protein

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