Bacteriophage ϕ29 infection of Bacillus subtilis minicells

Garcı́a, Juan Antonio; Salas, Margarita

doi:10.1007/bf00268058

Cited by 7 publications

(3 citation statements)

References 23 publications

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“…The 029 mutant sus14(1242), that produces a delayed lysis phenotype, was as described (28). Minicells were prepared from B.subtilis CU403 thyA thyB metB div IV Bi (29) as described (30).…”

Section: Bacterial Strains Plasmids and Phagementioning

confidence: 99%

Overproduction and purification of protein P6 ofBacillus subtilisphage φ29: role in the initiation of DNA replication

et al. 1985

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A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda. A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction. This protein, representing about 4% of the total E. coli protein after 1 h of induction, was obtained in a highly purified form. The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination. Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer. The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein).

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“…The 029 mutant sus14(1242), that produces a delayed lysis phenotype, was as described (28). Minicells were prepared from B.subtilis CU403 thyA thyB metB div IV Bi (29) as described (30).…”

Section: Bacterial Strains Plasmids and Phagementioning

confidence: 99%

Overproduction and purification of protein P6 ofBacillus subtilisphage φ29: role in the initiation of DNA replication

et al. 1985

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“…To study whether the increased fluorescence observed over time is due to synthesis of new fluorescent proteins within minicells, we conducted an assay measuring translation activity in purified minicells. Previous evidence of gene expression within minicells have been reported in the forms of expression from heterologous plasmid DNA [31][32][33][34][35], producing transcription and translation activity from minicell lysate [41,44], or via susceptibility to phage infection and production of phage particles [45][46][47][48][49]. We sought to directly verify and quantify gene expression within minicells using a fluorescence-based assay.…”

Section: Heterologous Expression Measured In Bulk Minicell Preparationsmentioning

confidence: 99%

Minicells as a potential chassis for engineering lineage-agnostic organisms

Wei

Jackson-Smith

Endy

2020

Preprint

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BackgroundGenomes encode for organisms and thus genome synthesis implies the possibility of organismal synthesis, including the synthesis of organisms without constraint to lineage. Current genome-scale engineering projects are focused on minimization, refactoring, or recoding within the context of existing natural lineages. Minicells arise naturally as anucleate cells that are devoid of heritable genetic material but are capable of gene expression. Thus, minicells may serve as a useful starting point for developing lineage-agnostic organisms encoded by newly-designed synthetic genomes. However, the composition and expression capacity of minicells is fixed at the time of their formation. The possibility of reestablishing cellular growth and division starting from minicells and entirely heterologous synthetic genomes is unknown.ResultsWe observed expression and segregation of functional proteins among mixed populations of reproducing cells and so-derived anucleate minicells via fluorescence microscopy. By adapting and integrating established methods of preparation and purification we were able to isolate minicells from a growing population of progenitor cells with a purity of at least 500 minicells per progenitor. We then used heterologous expression of plasmid-encoded green fluorescent protein to estimate the absolute expression capacity of minicells. We found that minicells can support the formation of 4.9 ± 4.6 × 108 peptide bonds prior to exhausting their initial intrinsic expression capacity.ConclusionsMinicells can be produced in large numbers with high purity and can also harbor and express engineered plasmids. The observed variation in minicell gene expression capacity suggests that about 13% of gene-expressing minicells can support the formation of more than one-billion peptide bonds, an amount sufficient to replicate known prokaryotic proteomes. Stated differently, while most minicells would require a sophisticated genetic ‘boot’ program to first increase minicell-specific expression capacity sufficient to instantiate newly reproducing lineages, a subset of minicells may be able to directly support whole genome, lineage-agnostic organism engineering.

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“…Infection of anucleate minicells has become established as a powerful technique in determining the gene products encoded by a wide range of natural bacteriophages (4,5,9,10,13,18,19) and by in vivoand in vitro-constructed X transducing phages (15). Studies of the regulation of phage expression in infected minicells have, however, produced several anomalous observations.…”

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confidence: 99%