DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors

Michel, Bertrand; Palla, Emanuela; Niaudet, B.; Ehrlich, S. Dusko

doi:10.1016/0378-1119(80)90025-6

Cited by 32 publications

(11 citation statements)

References 21 publications

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“…Plasmid introduction in B. subtilis requires a multimeric donor molecule (2), although monomers have been shown to be active in S. pneumoniae (3,4). Nevertheless, cloning ofchromosomal genes by introduction of a recombinant plasmid has been achieved in B. subtilis (5,6). However, a more promising approach may be the transformation of an endogenous plasmid by recombinant DNA (7).…”

mentioning

confidence: 99%

Cloning of chromosomal genes in Streptococcus pneumoniae.

Stassi¹,

López²,

Espinosa³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

104

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A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the maiM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid, pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme.A 10-kilobase restriction fragment containing the sd-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment ofa recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtiis are considered and compared.Despite the fact that bacterial transformation was discovered in Streptococcus pneunwniae, this species has not until now been used to clone recombinant DNA. In the bacterium commonly used for cloning, Escherichia coli, the artificial method of transformation allows uptake of DNA without alteration, whereas the naturally evolved DNA uptake systems of S. pneumoniae and Bacillus subtilis process the incoming DNA (1). The cutting of DNA during its binding and entry and the necessity to reconstruct a plasmid from single-stranded fragments are impediments to cloning in these Gram-positive bacteria. Plasmid introduction in B. subtilis requires a multimeric donor molecule (2), although monomers have been shown to be active in S. pneumoniae (3, 4). Nevertheless, cloning ofchromosomal genes by introduction of a recombinant plasmid has been achieved in B. subtilis (5, 6). However, a more promising approach may be the transformation of an endogenous plasmid by recombinant DNA (7). The latter process can be called plasmid transformation, as opposed to the former process of plasmid transfer, in which a new plasmid replicon is established in the cell. The recent introduction of multicopy plasmids into S. pneumoniae (4, 8) has made cloning in this species practicable.Our approach to cloning in S. pneumoniae depended on previous analysis of the maltose-utilization system (9, 10). The mal region-a cluster of genes concerned with maltosaccharide utilization-includes the maiM gene, which specifies amylomaltase, and a regulatory gene. Restriction fragments, either unfractionated or enriched for the maiM gene, were ligated to the plasmid pMV158 (11) and used to transform recipient cells with and without endogenous plasmid.Successful cloning of the wild-type allele of the malM gene was achieved. A recom...

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mentioning

confidence: 99%

Cloning of chromosomal genes in Streptococcus pneumoniae.

Stassi¹,

López²,

Espinosa³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

104

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“…The factors behind this observation (in the study cited, the homologous sequences were on a resident plasmid) will be a source of bias against insertions mediated by small fragments. (ii) There may also be a bias against larger fragments similar to that encountered in cloning random fragments in B. subtilis (27). (iii) If auxotrophic operons are on average very long in B. subtilis, and the trp operon is estimated to be 6 kb in length (17), the use of longer fragments in the range which successfully transforms may not greatly decrease the probability that any fragment will contain an internal fragment of such an operon.…”

Section: Discussionmentioning

confidence: 99%

Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis

O’Kane¹,

Stephens²,

McConnell³

1986

J Bacteriol

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An integrable plasmid, pOK4, which replicated independently in Escherichia coli was constructed for generating transcriptional fusions in vivo in Bacillus DNA. It did not replicate independently in Bacillus subtilis, but it could be made to integrate into the chromosome of B. subtilis if sequences homologous to chromosomal sequences were inserted into it. It had a selectable marker for chloramphenicol resistance and carried unique sites for EcoRI and SmaI just to the 5' side of a promoterless alpha-amylase gene from Bacillus licheniformis. When B. subtilis DNA fragments were ligated into one of these sites and the ligation mixture was used to transform an alpha-amylase-negative B. subtilis strain, chloramphenicol-resistant transformants could be isolated conveniently. Many of these were alpha-amylase positive, owing to the fusion of the plasmid amylase gene to chromosomal operons. In principle, because integration need not be mutagenic, it is possible to obtain fusions to any chromosomal operon. The site of each integration can be mapped, and the flanking sequences can be cloned into E. coli. The alpha-amylase gene can be used to detect regulated genes. We used it as an indicator to detect operons which are DNA-damage-inducible (din), and we identified insertions in both SPI3 and PBSX prophages.

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“…wt. 1 x 10 6 ) of DNA inserts found in recombinant plasmids is only one-third of the mean size of the fragments in the EcoRI or HindIII digested donor DNA (Michel et al 1980). It is not yet clear if this phenomenon reflects a preferential transformation of recombinant plasmids with small inserts or is due to posttransformational deletions.…”

Section: Molecular Cloning With Plasmid Vectorsmentioning

confidence: 99%

Cloning Vectors Derived from Plasmids and Phage of Bacillus

Kreft¹

1982

Gene Cloning in Organisms Other Than E. Coli

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DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors

Cited by 32 publications

References 21 publications

Cloning of chromosomal genes in Streptococcus pneumoniae.

Cloning of chromosomal genes in Streptococcus pneumoniae.

Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis

Cloning Vectors Derived from Plasmids and Phage of Bacillus

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