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The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism.

Martinsohn JT, Radman M, Petit MA - PLoS Genet. (2008)

Bottom Line: However, the precise molecular processes underlying this mosaicism are unknown.To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into lambda.The recombination editing proteins, MutS and UvrD, showed only marginal effects on lambda recombination.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine R. Descartes, INSERM U571, Université Paris Descartes, Paris, France.

ABSTRACT
Genome mosaicism in temperate bacterial viruses (bacteriophages) is so great that it obscures their phylogeny at the genome level. However, the precise molecular processes underlying this mosaicism are unknown. Illegitimate recombination has been proposed, but homeologous recombination could also be at play. To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into lambda. High yields of recombinants between 22% diverged genes have been obtained when the virus Red Gam pathway was active, and 100 fold less when the host Escherichia coli RecABCD pathway was active. The recombination editing proteins, MutS and UvrD, showed only marginal effects on lambda recombination. Thus, escape from host editing contributes to the high proficiency of virus recombination. Moreover, our bioinformatics study suggests that homeologous recombination between similar lambdoid viruses has created part of their mosaicism. We therefore propose that the remarkable propensity of the lambda-encoded Red and Gam proteins to recombine diverged DNA is effectively contributing to mosaicism, and more generally, that a correlation may exist between virus genome mosaicism and the presence of Red/Gam-like systems.

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Related in: MedlinePlus

Both λ Nec3 and λ Nec6 produce rolling circle intermediates.DNA extracted during a kinetics of λ infection was loaded on a gel (30 µl), besides a λ ladder size marker (shown as L, 1 ng in the middle lane, 10 ng on the right side lane). UV photograph of the gel is shown in the left panel, and Southern blot in the right panel (the last lane was omitted for blotting). Mo, Di and RC: monomer, dimer and rolling-circle forms of λ respectively. C: E. coli C600 chromosomal DNA.
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pgen-1000065-g006: Both λ Nec3 and λ Nec6 produce rolling circle intermediates.DNA extracted during a kinetics of λ infection was loaded on a gel (30 µl), besides a λ ladder size marker (shown as L, 1 ng in the middle lane, 10 ng on the right side lane). UV photograph of the gel is shown in the left panel, and Southern blot in the right panel (the last lane was omitted for blotting). Mo, Di and RC: monomer, dimer and rolling-circle forms of λ respectively. C: E. coli C600 chromosomal DNA.

Mentions: λ replicates by two distinct modes, theta type and rolling-circle type, which may be different substrates for recombination. However, all derivatives analysed in this work contain a Chi site, so they should produce rolling circle intermediates, even in the absence of Gam, as is the case when pL is inverted. We verified by Southern analysis that both types of constructs, with pL inverted or not, produced rolling circle intermediates in our growth conditions. To do so, phages were adsorbed to 1 ml of C600 cells grown to an OD of 0.5 (in TB medium supplemented with 0.1% maltose and thiamin 1 µg/ml), at an MOI of 1, at 37°C without agitation. Samples were withdrawn 0, 30 and 60 minutes after adsorption, cells were pelleted and resuspended in 100 µl of SET buffer (20% sucrose, 50 mM Tris pH 7.5, 50 mM EDTA, 0.5 mg/ml lysozyme), and incubated 10 min at 37°C. Lysis was then completed by adding 100 µl of SET supplemented with 5% SDS and bromophenol blue. Crude extracts were vortexed 1 minute, and loaded (30 µl) on a 15 cm-long, 0.5% TBE agarose gel supplemented with 40 µg/ml ethidium bromide. To achieve best separation, electrophoresis proceeded in TBE buffer with 40 µg/ml ethidium bromide for 3 h at 150 volts. This high migration voltage heated considerably both buffer and gel, and this appeared necessary to achieve best resolution, as the same gels run in the cold room did not allow to separate λ from the bulk of chromosomal DNA as nicely. Under such conditions, the dimer and trimer of λ, prepared by partial ligation, migrated faster than the rolling-circle intermediates, which co-migrated with the upper limit of the bulk of chromosomal DNA. Transfer, and hybridisation, followed classical protocols (the whole λ genome was taken as a probe). Results on Figure 6, right panel, show that both λNec3 (Gam−) and λNec6 (Gam+) produce rolling circle intermediates as a function of time, with the Gam- phage producing less than the Gam+ phage, as expected. Monomeric molecules migrate ahead of rolling-circle products, and dimer molecules of λ are absent. A similar result was obtained when phages were adsorbed to the strains used for recombination scoring (P2 lysogen for Nec3, and recA mutant for Nec6).


The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism.

Martinsohn JT, Radman M, Petit MA - PLoS Genet. (2008)

Both λ Nec3 and λ Nec6 produce rolling circle intermediates.DNA extracted during a kinetics of λ infection was loaded on a gel (30 µl), besides a λ ladder size marker (shown as L, 1 ng in the middle lane, 10 ng on the right side lane). UV photograph of the gel is shown in the left panel, and Southern blot in the right panel (the last lane was omitted for blotting). Mo, Di and RC: monomer, dimer and rolling-circle forms of λ respectively. C: E. coli C600 chromosomal DNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2327257&req=5

pgen-1000065-g006: Both λ Nec3 and λ Nec6 produce rolling circle intermediates.DNA extracted during a kinetics of λ infection was loaded on a gel (30 µl), besides a λ ladder size marker (shown as L, 1 ng in the middle lane, 10 ng on the right side lane). UV photograph of the gel is shown in the left panel, and Southern blot in the right panel (the last lane was omitted for blotting). Mo, Di and RC: monomer, dimer and rolling-circle forms of λ respectively. C: E. coli C600 chromosomal DNA.
Mentions: λ replicates by two distinct modes, theta type and rolling-circle type, which may be different substrates for recombination. However, all derivatives analysed in this work contain a Chi site, so they should produce rolling circle intermediates, even in the absence of Gam, as is the case when pL is inverted. We verified by Southern analysis that both types of constructs, with pL inverted or not, produced rolling circle intermediates in our growth conditions. To do so, phages were adsorbed to 1 ml of C600 cells grown to an OD of 0.5 (in TB medium supplemented with 0.1% maltose and thiamin 1 µg/ml), at an MOI of 1, at 37°C without agitation. Samples were withdrawn 0, 30 and 60 minutes after adsorption, cells were pelleted and resuspended in 100 µl of SET buffer (20% sucrose, 50 mM Tris pH 7.5, 50 mM EDTA, 0.5 mg/ml lysozyme), and incubated 10 min at 37°C. Lysis was then completed by adding 100 µl of SET supplemented with 5% SDS and bromophenol blue. Crude extracts were vortexed 1 minute, and loaded (30 µl) on a 15 cm-long, 0.5% TBE agarose gel supplemented with 40 µg/ml ethidium bromide. To achieve best separation, electrophoresis proceeded in TBE buffer with 40 µg/ml ethidium bromide for 3 h at 150 volts. This high migration voltage heated considerably both buffer and gel, and this appeared necessary to achieve best resolution, as the same gels run in the cold room did not allow to separate λ from the bulk of chromosomal DNA as nicely. Under such conditions, the dimer and trimer of λ, prepared by partial ligation, migrated faster than the rolling-circle intermediates, which co-migrated with the upper limit of the bulk of chromosomal DNA. Transfer, and hybridisation, followed classical protocols (the whole λ genome was taken as a probe). Results on Figure 6, right panel, show that both λNec3 (Gam−) and λNec6 (Gam+) produce rolling circle intermediates as a function of time, with the Gam- phage producing less than the Gam+ phage, as expected. Monomeric molecules migrate ahead of rolling-circle products, and dimer molecules of λ are absent. A similar result was obtained when phages were adsorbed to the strains used for recombination scoring (P2 lysogen for Nec3, and recA mutant for Nec6).

Bottom Line: However, the precise molecular processes underlying this mosaicism are unknown.To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into lambda.The recombination editing proteins, MutS and UvrD, showed only marginal effects on lambda recombination.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine R. Descartes, INSERM U571, Université Paris Descartes, Paris, France.

ABSTRACT
Genome mosaicism in temperate bacterial viruses (bacteriophages) is so great that it obscures their phylogeny at the genome level. However, the precise molecular processes underlying this mosaicism are unknown. Illegitimate recombination has been proposed, but homeologous recombination could also be at play. To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into lambda. High yields of recombinants between 22% diverged genes have been obtained when the virus Red Gam pathway was active, and 100 fold less when the host Escherichia coli RecABCD pathway was active. The recombination editing proteins, MutS and UvrD, showed only marginal effects on lambda recombination. Thus, escape from host editing contributes to the high proficiency of virus recombination. Moreover, our bioinformatics study suggests that homeologous recombination between similar lambdoid viruses has created part of their mosaicism. We therefore propose that the remarkable propensity of the lambda-encoded Red and Gam proteins to recombine diverged DNA is effectively contributing to mosaicism, and more generally, that a correlation may exist between virus genome mosaicism and the presence of Red/Gam-like systems.

Show MeSH
Related in: MedlinePlus