Determination of Plasmid Copy Number Reveals the Total Plasmid DNA Amount Is Greater than the Chromosomal DNA Amount in Bacillus thuringiensis YBT-1520

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.

Evolution and dynamics of megaplasmids with genome sizes larger than 100 kb in the Bacillus cereus group

“…Strains of this group typically contain several plasmids, with some strains containing more than 10 [6,14,15]. Plasmids of this group are crucial for the phenotype and virotype of strains.…”

pXO16 from Bacillus thuringiensis serovar israelensis: Almost 350 kb of terra incognita

“…These insecticidal genes are often carried by large plasmids, as other virulence traits in the B. cereus group that are also frequently plasmid-encoded (Gonzalez and Carlton, 1980;Hoton et al, 2005;Kolstø et al, 2009). In this context, most B. thuringiensis strains possess particularly large sets of plasmids, harboring up to 17 different plasmids with size ranging from 2 to 600 kb (Lereclus et al, 1982;Reyes-Ramirez and Ibarra, 2008;Zhong et al, 2011). The conjugative transfer of these plasmids has been extensively studied in the light of the transfer of cry genes (Battisti et al, 1985;Gonzalez et al, 1982;Reddy et al, 1987).…”

Mining New Crystal Protein Genes from Bacillus thuringiensis on the Basis of Mixed Plasmid-Enriched Genome Sequencing and a Computational Pipeline

“…Previous reports showed that B. thuringiensis strain YBT-1520 contains a total plasmid genetic content of 988 kb (51). To acquire high-quality sequencing data, we mixed 21 plasmid-enriched genomic DNA sequences from B. thuringiensis strains for Illumina sequencing (Ͼ50ϫ coverage), and theoretically the average cost for the identification of a cry gene would be about 5% of that of a wholegenome shotgun sequencing strategy.…”