Molecular Characterization of Plasmid pBM300 from Bacillus megaterium QM B1551

Abstract

Strain QM B1551 of Bacillus megaterium contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G؉C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea. oriT and ssoT-like regions were detected near two mob genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.

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).…”

“…The GC content of the sequenced region is 30.85%, lower than the average GC content of B. megaterium DNA (37%) (29) and lower than that of two B. megaterium plasmids, pBM300 (35.2%) and pBM400 (36.5%), whose sequences have become available recently (21,34).…”

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

“…There are also genes for fatty acid metabolism, cell wall hydrolysis, sigma factors, and cell division, as well as integrons, insertion sequence (IS) elements, and transposons. Because B. megaterium is frequently found with Pseudomonas species in contaminated environments, it has long been suspected of having the capability of metabolizing unusual substrates of possible bioremedial use (30,52,61). To that extent, B. megaterium plasmids carry genes for heavy metal resistance, including Cu and Cd export, and genes such as styrene monooxygenase are present.…”

“…Plasmids pBM100 and pBM200 of QM B1551 were previously sequenced and deposited in GenBank. Plasmids pBM300 and pBM400 have been analyzed (30,52,63) and have been resequenced to higher coverage and reannotated for this study (see Fig. S1 in the supplemental material).…”

Complete Genome Sequence of the Industrial Strain Bacillus megaterium WSH-002

“…Compared with the previously published genomes of vitamin B 12 producers B. megaterium DSM 319 and ATCC 12872/ QMB1551 (5,9,14), the genome of strain WSH-002 carries most of the core genes and pathways of B. megaterium (5,269 ORFs). A notable characteristic of strain WSH-002 is that about 9.26% of its genes are responsible for the synthesis and secretion of proteins (508 ORFs).…”