Sequencing and Characterization of pBM400 from Bacillus megaterium QM B1551

Bacillus megaterium is a spore-forming bacterium found in soil, seawater, sediments, rice paddies, dried food, honey, and milk (56). The economic importance of B. megaterium includes its production of vitamin B12 and penicillin amidase, its ability to express foreign proteins without degradation, and its use in AIDS diagnostics (56; C. Ginsburgh, D. Spaulding, G. Robey, M. A. Shivakumar, O. R. Vanags, L. Katz, and J. L. Fox, Abstr. Int. Conf. AIDS, abstr. 674, 1989). Recently, a strain of B. megaterium has been found that produces plasmid-borne oxetanocin, a potential antiviral agent (35). Plasmidless strains are used in industrial and research settings as hosts for efficient expression of foreign proteins. Most B. megaterium strains contain more than four plasmids (56), yet only a few of their plasmid genes are known. B. megaterium strain QM B1551 has an array of seven plasmids that have been shown to harbor germination, sporulation, megacin, and rRNA genes (23, 24, 51; D. M. Stevenson and P. S. Vary, Abstr. 11th Int. Spores Conf., abstr. 60, 1992). Many plasmids in Bacillus and other gram-positive bacteria are cryptic in nature. While the small rolling circle replicating plasmids of gram-positive bacteria have been extensively studied (20, 22), only a few of the plasmid genes or replicons of the large theta plasmids have been characterized (for reviews, see references 10 and 16). Plasmids usually provide the host cell with a selective advantage, such as antibiotic resistance (17), resistance to toxic heavy metals (49), or degradation of unusual compounds (14, 44). In addition, some plasmids produce bacteriocins (for review, see reference 19). Bacteriocin (megacin) genes have been reported to be carried by a 47-kb plasmid of B. megaterium 216 (43), a 45-kb plasmid of ATCC 19213 (59), and a plasmid in strain QM B1551 (23). Gram-positive bacteria may also harbor plasmids that carry virulence genes, i.e., the insecticidal toxins of B. thuringiensis and B. sphaericus (46) and the anthrax toxins produced by B. anthracis from plasmids pXO1 and pXO2 (36, 42). B. megaterium QM B1551 carries over 11% of its cellular DNA as stable plasmid DNA under laboratory conditions without known selection (23). The seven indigenous plasmids are 5.4, 9.1, 26.3, 54, 71, 108, and 165 kb (23) and are designated pBM100 to pBM700, respectively. A plasmidless derivative of QM B1551, strain PV361, has been isolated (58). The complexity of the array of plasmids in B. megaterium offers a unique opportunity to study plasmid interactions, replication, compatibility, plasmid-borne genes, and possible horizontal transfer. It also presents a challenge for the isolation and sequencing of plasmids with no known selectable markers and widely varying copy numbers within a multiplasmid array. When strain QM B1551 of B. megaterium is cured of its seven indigenous plasmids, there are few obvious phenotypic effects under laboratory conditions besides a defect in germination (52) and production of a megacin (23). QM B1551 is known to be very efficient at sporulation and germination (12). The plasmidless strain PV361 can grow normally on rich media, sporulate normally, and germinate on rich media but cannot germinate on single germination triggers because of a plasmid-borne germination gene (52). The plasmids are dispensable under laboratory conditions, yet they are fairly stable upon subculturing. To begin to understand plasmid interactions and the role of plasmid genes in B. megaterium plasmid DNA, we cloned and sequenced fragments containing six of seven compatible replication origins. The two smallest plasmids have been completely sequenced and identified as rolling circle replicating plasmids with replicon similarities to the pTX14-3 plasmid of B. thuringiensis and the pXO1-89 plasmid gene of B. anthracis (G. Baisa, Y. Zhou, and P. Vary, unpublished data). The remaining four sequenced replicons on clones I, II, III, and IV are iteron-type theta replicons from the larger plasmids and are very similar in both Rep proteins and iterons (all five large plasmid replicons cross-hybridize) (24, 51, 57; M. Kunnimalaiyaan and P. Vary, unpublished data). The 12.3-kb replicating clone II fragment and its adjacent rRNA operon (6.3 kb), located just upstream from this replication clone, have been sequenced and characterized from pBM400 (54 kb) (24). To date, this is the only rRNA operon found in nonessential DNA, and its presence has considerable evolutionary significance. Since B. megaterium produces many unusual enzymes and is often found in contaminated environments along with degradative-plasmid-bearing pseudomonads (56), we are greatly interested to see if any similar metabolic genes are plasmid-borne. The complete sequencing of pBM400 and the larger cryptic plasmids of B. megaterium will enable comparative plasmid genomics and the study of plasmid horizontal transfer while expanding our understanding of plasmid genetics in gram-positive bacteria. This paper presents the recombinational labeling, isolation, and sequencing of the remaining 35 kb of pBM400. It reports the initial description of several interesting plasmid-borne genes, including a possible integrase, an insertion sequence (IS) element, unusual metabolic genes, and several large inverted and direct repeats (DRs).