Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities

Mulder, J A; Venema, Gerard

doi:10.1128/jb.152.1.166-174.1982

Cited by 23 publications

(3 citation statements)

References 27 publications

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“…Conceivably, subunit a might be involved in binding, because it is the polypeptide lacking in binding-deficient mutants (23), and the nuclease subunit b, which is present in these mutants, might be involved in DNA entry. The idea of a protein complex involved in both binding and entry of donor DNA in B. subtilis was strongly supported by more recent experiments (submitted for publication), which indicated that subunit b was in fact the nuclease that is absent in DNA entry-deficient mutants of B. subtilis (18).…”

Section: Discussionmentioning

confidence: 99%

“…In Diplococcus pneumoniae binding to competent cells seems to result in the introduction of single-stranded nicks in the donor DNA (11,12). In addition, in both D. pneumoniae and B. subtilis nuclease activity seems to be required for entry of bound DNA (13,18). It was of interest to study whether the purified DNA-binding protein complex exhibited nuclease activity.…”

Section: --mentioning

confidence: 99%

“…The mechanism of DNA entry in Bacillus subtilis, as studied so far, seems to involve steps analogous to those in S. pneumoniae (9,18). The presence of a nuclease activity that is loosely bound to the cellular surface of B. subtilis competent cells and that is missing in entry-deficient mutants (17) has been described (18). Steps involved in DNA binding in B. subtilis are less well characterized.…”

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Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein

Smith¹,

Wiersma²,

Bron³

et al. 1983

J Bacteriol

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In DNA binding-deficient mutants of Bacillus subtilis a competence-specific protein with a subunit molecular weight of 18,000 was absent. The native protein containing this subunit was purified from B. subtilis membranes by chromatography on hydroxyapatite, DEAE-cellulose, and Sephacryl S-200. This protein appeared to be complexed with a second protein of slightly lower molecular weight (17,000) and a different isoelectric point. The native protein complex (apparent molecular weight, 75,000) contained approximately equal amounts of the two polypeptides and showed a strong DNA-binding activity. Incubation of the complex with plasmid and bacteriophage DNA revealed nuclease activity, specifically directed toward double-stranded DNA. Predominantly single-stranded nicks and a limited number of double-stranded breaks were introduced in the presence of Mg2+ ions. In the presence of Mn2+ ions the complex produced low-molecular-weight breakdown products from the DNA.

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Section: Discussionmentioning

confidence: 99%

Section: --mentioning

confidence: 99%

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Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein

Smith¹,

Wiersma²,

Bron³

et al. 1983

J Bacteriol

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Intracellular effects of phage ϕW-14 DNA on transformation of Bacillus subtilis

et al. 1984

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Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage phi W-14 DNA (in which half the thymine residues are replaced by alpha-putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37 degrees C after DNA uptake at 30 degrees C in the presence of low concentrations of phi W-14 DNA (0.1 microgram/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of phi W-14 DNA was enhanced by an increase in its concentration (to 0.5-1 microgram/ml), or by increasing the temperature of uptake (to 37 degrees C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 microgram/ml) or untreated with phi W-14 DNA, and incubated for 20 min at 37 degrees C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with phi W-14 DNA. The influence of a high concentration of phi W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Heterospecific transformation in Bacillus subtilis: protein composition of a membrane-DNA complex containing unstable heterologous donor-recipient complex

Riele

Venema

1984

Mol Gen Genet

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Previously it was demonstrated that, in contrast to the homologous donor-recipient complex, the unstable heterologous donor-recipient complex remains bound to the cellular membrane. To examine whether proteins known to be involved in the processing of transforming DNA in Bacillus subtilis are associated with membrane fragments which carry chromosomal DNA, a crude membrane-DNA complex was subjected to electrophoresis through a sucrose gradient. This resulted in the separation of membrane fragments associated with DNA and free membrane fragments. By means of two-dimensional gel electrophoresis several proteins, either uniquely present or considerably enriched in the purified membrane-DNA complex, were detected. Among these proteins we identified the 45 kD recE gene product, required for recombination, the 18 kD binding protein involved in the binding of transforming DNA and a 17 kD nuclease involved in the entry of transforming DNA. These results suggest that the membrane sites at which donor DNA integrates into the recipient chromosome are in the vicinity of the sites of entry of donor DNA through the membrane.

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Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities

Cited by 23 publications

References 27 publications

Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein

Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein

Intracellular effects of phage ϕW-14 DNA on transformation of Bacillus subtilis

Heterospecific transformation in Bacillus subtilis: protein composition of a membrane-DNA complex containing unstable heterologous donor-recipient complex

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