Abstract
Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs) of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb) in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp) was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp) at the mid-exponential growth stage (OD600 = 2.0) of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp) and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively). These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.
Highlights
Plasmids are extra-chromosomal DNA elements that can be characterized by their copy number in the host [1,2]
We designed three pairs of primers on rep (22308–22447), orf4 (41233– 40373), and orf19 (62239–62365) of pBMB67 to determine the plasmid copy numbers (PCNs). The results from this experiment showed that the PCNs of pBMB67 targeted at three different fragments were 21.3, 20.1, and 21.0, respectively, with a relative standard deviation of 3.0%
The PCN of pBMB2062 in strain YBT-1520 was almost 1.83-fold of that in strain HD-136. These results indicate that the PCN of a plasmid varies in different hosts, at least for the plasmid included in this study
Summary
Plasmids are extra-chromosomal DNA elements that can be characterized by their copy number in the host [1,2]. Plasmids are usually considered nonessential for the survival of bacteria, they metabolically burden their hosts [2,3,4]. To minimize this metabolic load and coexist stably with their hosts, plasmids must control their replication; the plasmid copy number (PCN) of a given plasmid is typically fixed within a given host and under defined cell growth conditions [3,5]. PCN is defined as the number of copies of a plasmid present per chromosome in a cell [6,7]. There are a few studies on PCN of multiple native plasmids in the same host, but with cruder tools [9]
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