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

Abstract

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.

Differential expression of two closely related deoxyribonuclease genes, nucA and nucB, in Bacillus subtilis

“…2). The ATC codon has been shown to function as an initiation codon in B. subtilis (Chen and Paulus, 1988) and has been implicated previously as a start codon for another competence-induced gene (Albano et al, 1989), The nucA gene would Ihus encode a protein with a molecular mass of 16,2kDa and an isoeiectric poini of 4.8, which is in agreement with the actual obseived characteristics of this protein (Smith et al, 1984). tn addition, the deduced NucA protein possesses an W-terminal hydrophobic region, the presence of which had been suggested previously in order to interpret the anchoring of the protein to fhe cytoplasmic membrane (Smitii et al.…”

“…B. subtilis mutants blocked early in stage II of sporuiation did not exhibit this DNase activity (Akrigg and Mandelstam, 1978). The substrate specificify and properties of the two DNases discussed above are quite similar: both etizymes are manganese-stimulated endonucleases degrading both double-stranded linear and covalently closed circular DNA and liave no detectable activity towards singlestranded DNA (Akrigg, 1978;Smith et aL,_ 1983b). In an attempt to identify gene(s) homologous to nucA, we cloned a DNase-encoding gene, nucB, which was shown to specify the previously identified sporulation-specific, extracelluiar DNase.…”

Transformation in Bacillus subtilis: further characterization of a 75,000-dalton protein complex involved in binding and entry of donor DNA

“…both polypeptides in the native 75,000-dalton complex was required. The nuclease activity was restricted to polypeptide b (21). Analysis of the nuclease subunit b on DNAcontaining polyacrylamide gels revealed nuclease activity at positions identical to those of the major competence-specific nuclease activities which were previously implicated in the entry of donor DNA during transformation (14,22).…”

“…The products in lanes 1 and 2 were visualized i brilliant blue; those in lanes 3 and 4 were visualized b Preparation of competent cultures and transf procedures used for the preparation of compi and transformation were as described previou Purification of the 75,000-dalton protein polypeptides a and b. The procedure used 75,000-dalton protein complex was as describ (21). Samples containing the purified 75,000-dz were separated into subunits on two-dimensi4 polypeptides a and b were extracted from described previously (22).…”

“…The purified 75,000-dalton protein complex showed strong DNA binding as well as nuclease activity. The nuclease activity was dependent on the presence of divalent cations and was directed specifically toward double-stranded DNA (21). Analysis of the separated polypeptides a and b revealed that for effective DNA binding, the interaction of * Corresponding author.…”