Cloning and characterization of the DNA region responsible for Megacin A-216 production in Bacillus megaterium 216.

Upon induction, Bacillus megaterium 216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills B. megaterium and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in B. megaterium. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (megA) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the megA gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gln-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the megA gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the megA gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.

The inhibition of SOS-responses and MDR by phenothiazine-metal complexes.

The gene of multidrug resistance (mdr) is inducible by different environmental stresses (SOS gene). We tested the inhibitory action of some new metal complexes of phenothiazines on megacin encoding bacterial gene induced by mitomycin-C as an example of "SOS induction" and on efflux pump of mouse lymphoma cells. The interaction of compounds to DNA was measured by thermal stability of DNA. It was found that metal co-ordination complexes of trifluoperazine (TFP) and chlorpromazine (CPZ) added before mitomycin administration have an inhibitory action on megacine induction. The TFP-V(IV) complex was effective at a lower concentration than TFP alone. The inhibitory effect of some metal coordinating complexes (TFP-Cu(II) and TFP- V(IV)) exceeded the action of TFP alone on efflux pumps. We propose that these compounds can form a complex with the regulatory protein or DNA resulting in the inhibition of SOS response and inhibit the mdr function by inactivating the P-glycoprotein as well.

A new bacteriocinogenic activity: megacin BII encoded by plasmid pSE 203 in strains of Bacillus megaterium.

Mesophilic strains producing a new bacteriocin: Megacin BII, have been isolated from strains of Bacillus megaterium. Facultatively thermophilic strains producing Megacin BI were less sensitive to this new activity than non-producing mesophiles and strains producing Megacin BII were also more resistant to Megacin BI. Strains producing Megacin BII contained a large plasmid of 36.10(6):pSE 203. This plasmid was introduced into non-megacinogenic acceptor strains by protoplast transformation, they then became megacin producers and immune to Megacin BII. Plasmid pSE 203 has been mapped with endonucleases. No similarity to the Megacin A plasmids pBM 309 [Rostás et al. (1980) and pBM 113 (von Tersch and Carlton (1983 b)] was evident.

Molecular cloning of structural and immunity genes for megacins A-216 and A-19213 in Bacillus megaterium.

A host-vector system was developed for molecular cloning in Bacillus megaterium and used to clone the structural and immunity genes for megacins A-216 and A-19213. Recombinant clones that expressed immunity only or both immunity to and production of each megacin were obtained. Restriction mapping of native megacinogenic plasmids and recombinant clones was used to construct physical and genetic maps of megacinogenic plasmids pBM309 and pBM113. Limited sequence homology between pBM309 and pBM113 was detected by Southern blot hybridization and was mapped to, at most, a 6.4-kilobase-pair region of pBM309 and a 6.1-kilobase-pair region of pBM113.

Deletion mutants of megacinogenic plasmid pBM309 from Bacillus megaterium.

A 46.8-kb plasmid, pBM309, of Bacillus megaterium determines the production of a bacteriocin, megacin A, and confers immunity against this antibiotic on the host cells. The megacin A (megA) and megacin A-immunity (megAim) genes were mapped on the physical map of pBM309 by using its deletion derivatives. Both genes were isolated as a 10.6-kb PstI fragment and cloned in Bacillus subtilis vector plasmid pBD9 for expression in B. megaterium.

Bacteriocin from Bacillus megaterium ATCC 19213: comparative studies with megacin A-216.

A bacteriocin produced by Bacillus megaterium ATCC 19213 was identified, purified, and compared with megacin A from B. megaterium 216. The ATCC 19213 bacteriocin was inducible with mitomycin C and showed phospholipase A activity. Both megacin A-216 and megacin A-19213 contained two dissimilar polypeptide subunits. Megacin A-216 contains a 30,000-dalton alpha subunit and a 15,000-dalton beta subunit. Megacin A-19213 is composed of an alpha subunit 18,000 daltons in mass and a beta subunit about 7,500 daltons in mass. No sequence similarities between alpha and beta subunits of either megacin were detected. The two megacins were further distinguished by quantitative differences in activity spectra and by immunodiffusion analyses.

Megacinogenic plasmids of Bacillus megaterium.

Megacins A-216 and A-19213 in Bacillus megaterium are plasmid encoded, as shown by analysis of cured, non-megacinogenic (Meg-) derivatives of strains 216 and ATCC 19213 and by polyethylene glycol-mediated protoplast transformation of Meg- bacteria with plasmid DNA. The results of both techniques implicated a 31-megadalton plasmid, pBM309, in megacin A-216 production and a 29-megadalton plasmid, pBM113, in megacin A-19213 production.

Improved purification and some properties of megacin Cx, a bacteriocin produced by Bacillus megaterium.

Megacin Cx is a bacteriocidal protein, previously described by Durner and Mach (Durner, K. (1970) Z. Allg. Mikrobiol. 10, 93-102; Durner, K. (1970) Z. Allg. Mikrobiol. 10, 373-382; Durner, K., and Mach, F. (1966) Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig. 2, 120, 565-575), which is released into the medium during growth by Bacillus megaterium strain 337. We have optimized the culturing conditions to achieve reproducibly high yields of this protein; under these conditions, megacin Cx represents about one-half of the protein secreted into the medium. We have developed improved methods of purifying the protein to homogeneity. The active form of megacin Cx is a monomeric protein, a single polypeptide of Mr = 210,000, as determined by analytical ultracentrifugation and gel electrophoresis in sodium dodecyl sulfate. Like many other proteins secreted by Gram-positive organisms, megacin Cx contains remarkably little methionine and cysteine. The purified protein blocks protein synthesis in sensitive cells, but has little immediate effect upon nucleic acid synthesis. Our purification procedure separates megacin Cx from another activity in the culture medium which blocks the synthesis of RNA and DNA, as well as synthesis of protein, in sensitive cells.

Megacinogenic plasmid from Bacillus megaterium 216.

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.

Temperature range variants of Bacillus megaterium.

Facultatively and obligately thermophilic variants were isolated from 3 out of 12 tested mesophilic Bacillus megaterium strains. The variants occurred at a frequency of 10(-8)-10(-9). The ability to grow at elevated temperatures was cured by means of treatment with acridine orange. Stable revertants were isolated from facultatively and obligately thermophilic variants. An unknown type of megacin was produced by the facultative thermophiles. This megacin attacked mesophilic and obligately thermophilic strains. The thermophiles displayed a few divergent taxonomic characteristics but a close relationship between the strains was indicated by the megacin spectrum and sensitivity to phage. Arrhenius plots revealed that the strains could be considered as temperature range variants and that the temperature characteristic increased with growth at a higher temperature range. The case for a plasmid involvement in the phenomenon is discussed.

Production of megacins C and Cx: presumptive evidence extrachromosomal control.

Exposure of growing cultures of Bacillus megaterium C4A(-) to ethidium bromide or an elevated growth temperature was found to eliminate megacin C production. Ethidium bromide resulted in a cure rate of up to 13%. Growth at 43 degrees C gave a cure rate of up to 99%. Megacin C production was lost spontaneously at a rate of 4% or less. There was a greater rate of spontaneous loss of megacin Cx production by B. megaterium 337, up to 14%. Growth at 43 degrees C resulted in a cure rate of up to 24% in this organism. Reversion to the Meg(+) state by cured clones has never been demonstrated. These observations support the hypothesis that production of megacins C and Cx is plasmid mediated. Meg(-) clones adsorbed more megacin than either parent strain and were more susceptible to megacin action.

Studies on megacinogeny in Bacillus cereus. I. Multiplication of phage wx causing lysogenic conversion to megacin A (phospholipase A) production.

Phage wx capable of reconverting Bacillus cereus strain W derivatives, cured to lose megacin A (phospholipase A) production into megacin A-producing cultures, exhibits unusual kinetics of multiplication; its clear mutant, phage wxc, behaves similarly. The phages are not adsorbed by stationary phase indicator bacteria. As sonicated bacteria fail to inactivate the phages, the absence of adsorption cannot be attributed to an undersurface localization of the receptors. Multiplying bacteria exert a slow and slight degree of phage adsorption. Cells inhibited by chloramphenicol produce no receptors. It has been assumed that the receptor, produced from a precursor involved in bacterial cell synthesis, either absorbs the phage in the nascent state or is incorporated in the cell and loses its phage-adsorbing capacity.

Studies on megacinogeny in Bacillus cereus. II. Bacillus cereus isolates characterized by prophage-controlled production of megacin A (phospholipase A).

Five out of a number of Bacillus cereus strains isolated from soil produced high titre specific bacteriocin (megacin A) in mitomycin C-induced cultures. In the course of cultivation with ethidium bromide, the strains gave off segregants not producing bacteriocin (cin-). The lysate of two wild strains formed plaques on the corresponding cin- bacteria. The two phages (wx23 and wx26) were identical in antigenic structure with phage wx was present in the lysate of B. cereus strain W, and converted cin- derivatives into cultures producing megacin A (phospholipase A). The phages produced plaques at 26 degrees C but not at 37 degrees C. In the lysates of the remaining three strains phages were not detected with biological and morphological methods; these cultures have been assumed to carry defective prophage genome. As the corresponding prophages are responsible for the determination of inducible phospholipase A production, phages named wx seem to form a separate group of B. cereus phages.

Electron microscopy of phages liberated by megacin A producing lysogenic Bacillus megaterium strains.

Mitomycin C was added at fairly high concentration (5-10 mug/ml) to exponentially growing cultures of selected strains of Bacillus megaterium. Lysis of the bacteria followed, associated by liberation of phage and megacin A production. In contrast, a low concentration (0.5 mug/ml) of mitomycin induced only megacin A production. Electron microscopic examination of the lysates induced by 5-10 mug/ml of mitomycin in 19 strains of B. megaterium showed them all to contain phages; most of the strains proved polylysogenic. Their lysates contained distinct complete phages of different structures and dimensions. A few strains released defective phage particles. The significance of the electron microscopic findings is discussed in relation to megacinogeny.