Megacinogenic plasmid from Bacillus megaterium 216

Abstract

Ability to produce megacin A, a bacteriocin of B. megaterium, was transferred from the strain B. megaterium 216 into auxotrophic derivatives of the strain B. megaterium KM via protoplast fusion and polyethylene-glycol-induced protoplast transformation by plasmid DNA, respectively. A 30.9 megadalton plasmid was detected in cells with MegA phenotype, and the loss of this phenotype was accompanied in each case with the elimination of that plasmid. The megacinogenic plasmid pBM309 has a single site for both BamHI and XhoI. It is cleaved by the endonucleases SalI, BglII, PstI, PvuII, and EcoRI into 3, 3, 4, 4, and 9 fragments, respectively. The physical map of this plasmid is presented.

Cloning and Characterization of the DNA Region Responsible for Megacin A-216 Production in Bacillus megaterium 216

“…The largest protein (termed ␥ chain) is the full-length product of ORF293A, whereas the shorter ␣ and ␤ chains appear to be products of proteolytic cleavage between Gln-185 and Val-186 of the ␥ chain. The calculated molecular masses of the ␥, ␣, and ␤ chains (32,855,21,018, and 11,855 Da, respectively) are substantially lower than the values deduced from electrophoretic mobility in denaturing gels. The likely reason of the anomalous migration, at least in the cases of the ␣ and ␥ chains, is the highly acidic composition (pI 3.92 and 4.51, respectively).…”

“…Previous studies described several aspects of megacinogeny in the B. megaterium 216 strain, including the microbiological phenomenon of megacin A-216 production and location of the megA genes on one of the large plasmids present in the host strain (14,17,19,28,30,32,(39)(40)(41). The main goal of the present work was the cloning and characterization of the genes involved in megacin A-216 production and immunity.…”

“…A study using protoplast fusion and protoplast transformation demonstrated that the genetic determinants of megacin A-216 production, immunity to megacin A-216, and inducibility of megacin A-216 production were transferable into nonmegacinogenic B. megaterium strains, and acquisition of the MegA phenotypes was associated with the transfer of an ϳ47-kb plasmid (pBM309), one of the 10 indigenous plasmids present in B. megaterium 216 (32). Fragments of pBM309 were cloned in B. megaterium, and the genes responsible for megacin production were localized to an approximately 10-kb region of pBM309; however, this DNA region was not analyzed beyond establishing a restriction map (30,41).…”

Sequencing and Characterization of pBM400 from Bacillus megaterium QM B1551

“…Three restriction enzyme libraries as follows were constructed from the pBM400 plasmid: (i) a partial Sau3A library in pGEM3-Z f(ϩ) (Promega, Madison, Wis.) was used for shotgun cloning and sequencing in E. coli DH10B, and (ii) EcoRI and (iii) PstI fragment libraries in pJM103 (39) were used for closing sequence gaps. B. megaterium was transformed by protoplast fusion based on the method of Von Tersch and Carlton (59) and regenerated in regeneration medium (43,59) as described by English and Vary (11).…”

Cloning Vectors Derived from Plasmids and Phage of Bacillus

“…Extrachromosomal DNA in Bacillus was first demonstrated in B. megaterium and since then many reports have been made of plasm ids in this species (Carlton and Smith 1974;Rostas et al 1980), B. subtilis (Lovett and Bramucci 1975;Tanaka and Koshikawa 1977;Bernhard et al 1978;Uozumi et al 1980), and B. pumilus (Lovett et al 1976). However, the majority of these plasmids, in most cases isolated from culture collection strains, lack readily identifiable markers and are thus not of immediate use as cloning vehicles.…”