An improved filamentous helper phage for generating single-stranded plasmid DNA

The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites. Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence. We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding. Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype. Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis. The corresponding mutation, M to I at position 108 (M108I), is located in a putative ␣-helical structure, which in the homologous ␥␦ resolvase has been implicated in dimerization. The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction. The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion. The corresponding mutations are located in different domains of the protein. The properties of these mutants in connection with the role of Fis in inversion will be discussed.

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“…The M13 helper phage used to isolate the mixed lysate for hydroxylamine mutagenesis of pGP1188 was R408 (28).…”

Section: Methodsmentioning

confidence: 99%

Gin mutants that can be suppressed by a Fis-independent mutation

et al. 1995

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“…The lysates were harvested by adding 3 to 4 ml of broth to each plate, gently agitating the plates on a gyratory platform shaker for 60 min, centrifuging the recovered liquid to remove suspended cells, and heating the supernatant at 65°C for 10 min to kill residual viable cells. The fl-type transducing particles were prepared from plasmid-containing K38 with R408 helper phage (20). The Iketype transducing particles were prepared from pKUN1-containing K906 with Ike-9 helper phage.…”

Section: Methodsmentioning

confidence: 99%

Low-frequency infection of F- bacteria by transducing particles of filamentous bacteriophages

et al. 1988

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Filamentous particles containing single-stranded plasmid and bacteriophage DNA are able to infect FEscherichia coli at frequencies of approximately 10-6. This infection is dependent on an intact particle and requires the products of the tolQ, tolR, and toUl genes of the bacteria. The addition of CaCl2 can increase the frequency about 100-fold, presumably by increasing the concentration of particles at the bacterial surface.The filamentous bacteriophages fl, fd, and M13 form plaques only on Escherichia coli that have F pili, while the related filamentous phage Ike forms plaques only on E. coli that have N pili. These pili are extracellular structures that confer donor activity in bacterial conjugation (4, 10, 18). Pilin, the major structural protein of pili, and additional proteins required for its assembly are encoded by genes carried on the F or N conjugative plasmids. The filamentous phages require pili for entry of the genome into the bacterium but not for replication and assembly, as E. coli lacking the F factor produce phage efficiently when fl DNA is introduced into them by transformation (13).A number of studies have strongly suggested that infection by filamentous phage is a multistep process. Initially, the particle binds to the tip of the pilus via an interaction with the phage-encoded gene III protein located at one end of the phage particle (for reviews, see references 15 and 19). It is postulated that the pilus subsequently depolymerizes into the membrane, thereby bringing the tip of the phage particle to the surface of the bacterium. There the particle must interact with additional proteins in order for the viral DNA to translocate into the cytoplasm (24,25). This step in the entry pathway is defined by mutations in the tolQ, tolR, and tolA genes of E. coli which render F+ bacteria uninfectable by filamentous phages. These mutant bacteria are still fully competent to undergo conjugation and are able to bind filamentous phages to the tips of their pili (24). In addition, tol mutants are not killed by (they are tolerant to) the colicins El, E2, and E3 (16, 25), although they retain the ability to bind the colicins to a specific receptor defined by the bacterial btuB locus (5, 11).These observations suggest that pili serve as the primary receptors for the filamentous phages, providing both an attachment site and perhaps a mechanism for bringing the particle close to the bacterial membrane where the ToIQ, ToiR, and TolA proteins are located (24). This paper supports this idea by demonstrating that transducing particles of filamentous phages can bypass the pilus and infect F-E. coli at low efficiencies.MATERIALS AND METHODS Strains and plasmids. The F-strains of E. coli used in this study are shown in Table 1 and are all derivatives of the K-12 strain C600. The mutations in orf-J, tolQ, toIR, and tolA were introduced into K17 by P1 transduction. The * Corresponding author. rfaC2::TnJO allele from CS1700 (provided by C. Schnaitman) was introduced into A593 by P1 transduction. The plasmids pD8, pKUN1, pKA...

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“…The pKT0/NS1 expression vector carries the packaging signals for the filamentous bacteriophage fl (Dotto et al, 1981) enabling the rescue of ssDNA following superinfection of bacterial cells (bearing the plasmid) with phage R408 (Russel et al, 1986). Oligonucleotide-directed mutagenesis (Kunkel, 1985) was carried out as described by Brierley et al (1989).…”

Section: Methodsmentioning

confidence: 99%

A region of the coronavirus infectious bronchitis virus 1a polyprotein encoding the 3C-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate

Tibbles¹,

Brierley²,

Cavanagh³

et al. 1995

Journal of General Virology

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In order to investigate the mechanisms involved in the processing of infectious bronchitis virus polyproteins, several candidate regions of the genome have been cloned and expressed in vitro. During these studies it was observed that the translation product encoded by one of these clones (pKT205) was poorly expressed. Biochemical and genetic analyses revealed that the basis for the poor expression was a post-translational event involving ubiquitination of the protein and degradation by an ATP-dependent system operating in the reticulocyte lysate used for the in vitro expression. Two independently acting regions which conferred instability were identified, one of which mapped to the predicted 3C protease domain, contained within the 5' end of the clone, while the other, more C-terminal region, was effective in conferring instability upon a heterologous protein to which it had been transferred. These regions may influence the stability of the authentic viral protein(s) in vivo and hence allow for the control of their expression and/or function at the level of proteolysis by cellular protease(s).

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An improved filamentous helper phage for generating single-stranded plasmid DNA

Cited by 341 publications

References 7 publications

Gin mutants that can be suppressed by a Fis-independent mutation

Gin mutants that can be suppressed by a Fis-independent mutation

Low-frequency infection of F- bacteria by transducing particles of filamentous bacteriophages

A region of the coronavirus infectious bronchitis virus 1a polyprotein encoding the 3C-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate

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