Evaluation Of Plasmid DNA Amount In Bacillus Thuringiensis Strain YBT-1520

Bacillus thuringiensis subsp. kurstaki strain YBT-1520 (H3abc), which is highly toxic to lepidopteran pests, was isolated by our lab and protected by patent. We have already finished sequencing the whole genome of the strain (unpublished), and found that this strain contains 11 endogenous plasmids with vary sizes (ranging from 2 kb to 416 kb) and modes of replication (rolling-circle replication [RCR] andθreplication) and its chromosome consists of 5,401,320 bp. However, why the strain YBT-1520 contains so many plasmids, what the functions of the 11 native plasmids and the impact on their host are, remain still unknown. In this thesis, we determined the plasmid copy numbers (PCNs) of 11 plasmids using real-time qPCR and evaluated the total plasmid DNA amount in strain YBT-1520. In addition, we studied the function of the genes harbored on plasmids through bioinformation analysis. These studies will help us further understand the biological actions of plasmid. The research results were summarized as following:1. Three pairs of primers on rep (22308-22447), orf4 (41233-40373), and orf19 (62239-62365) of pBMB67 were designed to determine the PCNs of pBMB67. The results showed that the PCNs of pBMB67 targeted at three different fragments were 21.7, 20.1, and 21.0, respectively, with a relative standard deviation of 3.0%. These findings showed the different targeted fragments on plasmid for real-time qPCR would not affect the results for PCN and demonstrated the reliability of our experimental results.2. The PCNs of the 11 plasmids in strain YBT-1520 were calculated at the mid-exponential growing stage (OD600=2.0) by the absolute quantification method. The PCNs of all 11 native plasmids were found to range from 1.38 to 172. Of note, the PCN reduced as the molecular weight increased, and thus the PCN negatively correlated to the molecular mass of plasmids. However, the PCNs of some plasmids (For example, pBMB7635 and pBMB8240) deviated from this trend. As far as we know, this study on PCN of multiple native plasmids in the same host with sensitive tools is the first instance.The plasmid DNA amount was calculated by multiplying the plasmid size by the PCN for each of the 11 plasmids from strain YBT-1520. The data showed that the total DNA amount of the 11 plasmids was equivalent to 8746 kb at the mid-exponential growing stage (OD6oo=2.0). To our surprise, the calculated total plasmid DNA amount was 1.62-fold greater than the chromosomal DNA amount (5401 kb), this finding provided insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.3. Three native plasmids (pBMB2062, pBMB67, and pBMB293) with different sizes (2 kb,67kb, and 293 kb, respectively) and different replication mechanisms (RCR andθreplication, respectively) were selected to assay the PCNs of these plasmids over the entire life cycle (from spore germination to the spore stage) of strain YBT-1520. The PCNs of all three plasmids were found dynamically shifting at different growth phases. Specifically, the PCNs ranged from 71 to 190 for pBMB2062, from 8 to 23 for pBMB67, and from 1 to 3 for pBMB293. The PCNs of all three plasmids were lowest during the germination phase (1～2 h), and then increased rapidly across the logarithmic phase. The PCN of pBMB2062 reached its maximum at the mid-exponential growth stage, while the values of pBMB67 and pBMB293 peaked at the early stationary stage. However, the PCNs of all plasmids decreased rapidly during the late stationary phase and remained stable and close to the minimum after presporulation (18 h).4. Based on the PCNs of pBMB2062, pBMB67, and pBMB293 in the spore stage, the PCNs of the other plasmids in the spore stage were deduced and the total plasmid DNA amount of YBT-1520 in the spore stage was calculated to be 5674 kb, which was slightly more than the chromosomal DNA amount (5401 kb).5. The pBMB2062, which is the smallest plasmid (2062 bp) and has the highest PCN of those tested, were selected to monitor the PCN of the same plasmid in different hosts. The data showed that The PCNs of pBMB2062 in strain YBT-1520, HD-1, and HD-136 were 172,115, and 94 at the mid-exponential growth stage (OD600=2.0), respectively. These results indicated that the PCN of a plasmid varies in different hosts.6. GO annotation functional gene analysis were carried out using the software BLAST2GO online.117 proteins among 399 annotated ORFs were found to be beneficial for the host survival.These findings described above help us understand the characteristic of plasmid and the relationship between plasmid and host.