Identification Of Extrachromosomal DNA Of Paenibacillus Mucilaginosus KNP414

Paenibacillus mucilaginosus, a potassium-and phosphate-dissolving and nitrogen-fixing bacterium in soil, has long been used in agriculture, bioleaching engineering and wastewater treatment. However, the lack of researches of molecular mechanism has greatly restricted the application of P. mucilaginosus. We have sequenced the whole chromosomal DNA of P. mucilaginosus KNP414and completed the relevant bioinformatics analysis. This study involves the identification of the extrachromosomal DNA of strain KNP414. In order to know about the molecular mechanisms of special functions of strain KNP414, we investigated the stability, molecular structure and predictive functions of the endogenous plasmid. Besides, we did an experiment to induce prophage. All these are the supplements to the whole genome genetic information of strain KNP414.Firstly, we studied the stability of plasmid of strain KNP414(pKNP414) under different incubation time and medium. The optimal medium to get more plasmid pKNP414included1%maltose,0.2%(NH4)2SO4,0.1%K2HPO4,0.05%MgSO4,0.001%CaCO3. And the optimal incubation time is16h. We obtained the plasmid with high quality using the improved kit method. Based on the circularity of the plasmid, the specialapproach of "purify-enzyme cleavage-purify" could be used to purify plasmid DNA. At last, we obtained unique plasmid DNA without genome and polysaccharide.Secondly, we constructed the restriction pattern of pKNP414preliminarily usingoR I, ApaL I, Himd III, Pst I, BamH I, EcoR V and NotI. Then digest the plasmid using double-enzyme cleavage of Pst I and BamU I and cloned the fragment. The full-length sequence of pKNP414was obtained by reverse-primers design, reverse PCR and gene walking. And the characters of the plasmid were analyzed by a series of bioinformatics software. The results showed that the full-length of pKNP414was7363bp containing a rRNA gene cluster of6000bp and the GC content was49.3%. The analysis of restriction enzyme sites accorded closely with our prediction by enzyme cleavage. Besides, the plasmid contained7ORFs. One (ORF1) of them encoded the replication origin protein of phage and its3'end overlapped with5s_rRNA gene. ORF3which was related to infection of phage coded lywallzyme. These results indicate that pKNP414is closely associated with phage. So we can deduce that pKNP414might came from the phage on the genome.Thirdly, we determined the copy number of pKNP414in strain KNP414by real-time PCR which is popular in recent years. Single copy gene repX on the pKNP414is chosen for target gene while single copy gene gyrB on the genome for reference gene. This result reveals pKNP414has six copies, belonging to low copy plasmid.Finally, we obtained six fragments of the prophage sequences by cloning based on the prophage sequences from the chromosomal DNA of strain KNP414. The result suggested that the prophage sequences are not correlated with the plasmid sequences. At the same time, the phage of strain KNP414could be induced by mitomycin C (MMC). The phage DNA bands were detected by2.5xSDS-EDTA gel electrophoresis, while no plaque was observed during the double board experiment. So, strain KNP414was preliminarily assigned to lysogenic bacteria which can be induced.In conclusion, the full-length of a plasmid pKNP414was obtained from Paenibacillus mucilaginosus KNP414, which is a good supplementary to the genome and will be a potential vector in the genetics of Paenibacillus mucilaginosus.