Nucleotide Sequence And Replication Properties Of The Bacillus Sphaericus Cryptic Plasmid PLG

Bacillus sphaericus is an important pathogen against mosquitoes. The study of their cryptic plasmids is neglected because the toxin protein genes had been proved to be located in the chromosome and the small plasmid can provide only little genetic information. With the aim of exploring the background of its molecular genetics, we undertook the cloning, sequence analysis of the cryptic plasmid pLG isolated from B. sphaericus LP1-G strain. The purified plasmid DNA was prepared by the modified QIAGEN kit protocol. According to the enzyme digestion map, we cloned two HindIII fragments into the vector pUC18 and then have them sequenced, and the complete sequence of the plasmid is acquired through primer walking cross the gap and provided data to identify the direction.It is a circular molecule of 11,066 bp, with a C+G% content of 37.92%, displaying 23 ORFs that exhibited a significant coding probability. The overview of its basic information shows the gene coding density is 2.078 genes per kb, average length is 416 bases per gene, coding percentage is 86.6%. The Genbank accession number of the whole sequence of the plasmid pLG is AY325804.Using the blast software to compare and search the gene, we found 4 known ORFs in the pLG. Among those, rep gene is of the most important for plasmid replication ability. Homology analysis indicated that pLG rep gene was similar to those of B. thuringiensis plasmid pBMB9741 and pGI1. Based on the sequence similarities among minimal replicon modules, pLG belong to Group III family in the RCR plasmid. The ORF 16 is composed of 630 nucleotides, encoding a protein of 210 amino acids, which has deduced amino acids sequence similar to B. thuringiensis plasmid pGI3. A unique P-loop structure was found in this ATP/GTP binding motif.Furthermore, ORF7 and ORF8 possibly encoding exported protein and hydrolase-related protein, both having a peptide signal, were identified. To determine its replicon's character, we use enzyme to digest pLG DNA, then cloned different fragments to vector pAW001. After transforming the host Bt 4Q-7, we acquire the initial result that show pLG's replicon locate in a HindIII fragment, with transformation frequency of 3x10~3个/(g DNA in Bt 4Q-7.