| Research backgroundLactobacillus plantarum was the most abundant and diverse strain of Lactobacillus,which was also the model strain to study Lactobacillus.These plasmids were likely to play a very important role in the growth,development,inheritance and evolution of Lactobacillus,so natural plasmids can provide an ideal research object for promoting the study of Lactobacillus.The strain of Lactobacillus plantarum PC518 strain from Sichuan pickle contained multiple plasmids.Our team had analyzed and published two plasmids,plasmid pLp18 and plasmid pLp60.In this study,two new plasmids were found.One plasmid belonged to the circle-replicating,and the other belonged to theta-replicating.A comprehensive analysis of the sequence and genetic information of the new plasmids,not only increased the number of natural plasmid databases,but also had important significance for the study of molecular biology of Lactobacillus plantarum.The whole genome sequencing could realize the comprehensive scanning of the variation information among the Lactobacillus genome,and could develop a large number of gene markers at one time.Lactobacillus futsaii was also a kind of Lactobacillus.In this paper,the whole genome sequence of Lactobacillus futsaii Y97 isolated from fu-tsai in Taiwan was analyzed which provided a theoretical basis for the next comparative study with Lactobacillus futsaii subsp.Chongqingii subsp.nov CQ16Z1.Objective1.The new plasmids of Lactobacillus plantarum PC518 strain was extracted and detected.2.The new plasmids performed sequence analysis and biological characteristic analysis.3.Sequence analysis of the whole genome of Lactobacillus futsaii Y97.Materials and methods1.The plasmids of Lactobacillus plantarum PC518 were extracted by the improved method.The new plasmids were detected by BLAST comparison at NCBI site,plasmid DNA library and inverse PCR.2.Use software such as Snap Gene Viewer,Mega X,DNA Man and Primer 5.Using the tools and websites in the NCBI databases,such as BLAST(htty://www.blast.ncbi.nlm.nih.gov/)and looking for open reading frame(ORF)website(https://www.ncbi.nlm.nih.gov/orffinder/).The characteristics of ORF,restriction endonucleases sites and replication mechanism were analyzed.3.The bacteria of Lactobacillus futsaii Y97 which cultivated and collected were sent to BGI company for the whole genome sequence sequencing.Result1.Two new plasmids were detected from Lactobacillus plantarum PC518 strain,one was plasmid pLP325 and the other was plasmid plp75 TA.2.The plasmid pLp325 belonged to the rolling-circle replication pC194 family,which encoded two ORFs,replication protein and mobile protein.There were reverse repeats in the upstream and downstream of nick in plasmid pLP325.The 43 plasmids of pC194 family were collected and analyzed by multiple sequence alignment and Rep protein clustering.It was showed that the bind site of dso from plasmid in the pC194 family was always characterized by a pair of reverse repeat sequences,which may be upstream or downstream of the nick site.43 pC194 family hosts belonged to 7 genera and 21 species.3.The plasmid plp75 TA was a theta-replicating plasmid and was a member of the RepA_N family,which encoded 10 ORFs.7 ORFs contained a rep protein,a mobile protein,two toxins,an antitoxin,a DNA binding helix turn helix transcription regulator,and a site-specific integrase.Beyond that,the functions of the other three ORFs encoded two hypothetical proteins and a relaxase which cannot be inferred from the GenBank database.A pair of direct repeat sequences were found at both ends of the toxin and antitoxin systems and site-specific integrase(TAI)of plasmid plp75 TA.61 plasmids containing TAI were collected,and their similarity and coverage were over 95%.Only56 plasmids could establish the rep phylogenetic tree.Rep proteins were clustered into 7clusters by phylogenetic tree.The similarity of plasmids between clusters were different.The highest similarity was 71.13% and the coverage was 91%.The lowest similarity was 0.The host range of 56 plasmids belonged to Lactobacillus and Pedicoccus.In the upstream of the TAI plasmid plp75 TA,there was also a toxin and DNA binding helixturn helix transcription regulator(TAITR).No direct repeat sequence was found at both ends of TAITR.22 plasmids containing TAITR with similarity and coverage greater than 95% were collected.Only 21 plasmids could establish phylogenetic tree through rep.In phylogenetic tree,rep proteins were clustered into four clusters.And the similarity between plasmid pL2022-1 in three clusters and plasmid pL11995-7 in four clusters was 66.67%,while the coverage rate was only 1%.The host range of 21 plasmids only belonged to Lactobacillus.4.The genome consisted of one chromosome of 2.56 Mb and three plasmids.The sizes of the three plasmids were 37,880 bp,27,087 bp and 25,584 bp,respectively.The genome contains 2 622 genes which make up 87.06 % of genome.The number of tandem repeat sequences is 71;the total length of tandem repeat sequences is 4 575 bp which makes up 0.1727 % of genome.43 minisatellite DNAs,3 microsatellite DNAs,56 tRNAs,12 rRNAs and 7 sRNAs were predicted.Conclusion1.Plasmid pLP325 should belong to pC194 rolling-circle family.The bind of origin of replication belonged to pC194 family replication was characterized by reverse repeat sequence,which could be located upstream or downstream of nick.It provided a new view for the position of bind.The host range of pC194 family was narrow.2.The sequence analysis indicates plasmid plp75 TA belonged to the theta-replicating plasmid.It showed that the TAI and TAITR could act as transposons through the analysis of TAI and TAITR of plp75 TA.The TAI and TAITR may transpose between plasmids..3.Through the analysis of the whole genome sequence of Lactobacillus futsaii Y97,it was found that the strain contained one chromosome and three plasmids.The genomic information provided can be compared with Lactobacillus futsaii subsp.Chongqingii subsp.nov CQ16Z1 studied by our research group which is helpful to further study Lactobacillus futsaii subsp.Chongqingii subsp.nov CQ16Z1. |
PDF Full Text Request