Genome Analysis Of Leptospira Borgpetersenii 56604 And Plasmids Identification In Leptospira

The genus Leptospira includes at least 21 species clustered into three groups — pathogens,non-pathogens,and intermediates — based on 16 S rRNA phylogeny.Leptospirosis,caused by pathogenic Leptospira,is a worldwide zoonotic infection.Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens.Recent identification of extrachromosomal elements besides the two chromosomes in L.interrogans has provided new insight into genome complexity of the genus Leptospira.The large size,low copy number,and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly.In this study,we sequenced the complete genome of L.borgpetersenii serogroup Ballum serovar Ballum str.56604 and first characterized two circular plasmids lbp1 and lbp2 co-existing in this strain.However,there were no extrachromosomal elements identified in the genus of L.borgpetersenii and the genome of it only contains two chromosomes.Then we provided further confirmation of the prensence and stability of plasmids through in silico analysis combined with pulsed-field gel electrophoresis(PFGE)and Southern blot.There are 15 Leptospira strains endemic in China,comprising L.borgpetersenii,L.weilii,and L.interrogans.We improved the experimental conditions and established an effective method to successfully separate and identify the plasmids by S1 nuclease digestion following PFGE analysis,independent of genome sequence data.Southern blot hybridization with extrachromosomal element-specific probes,designated as lcp1,lcp2 and lcp3-rep,further confirmed their occurrences as extrachromosomal elements.In total,24 plasmids were detected in 13 out of 15 tested strains,which can all hybridize with the three probes.It seems that the plasmids in Chinese epidemic strains can be divided into three types: lcp1,lcp2 and lcp3.Blastp search of the lcp1,lcp2 and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates.Based on the plasmids identified,shuttle vectors constructed with the rep genes can be successfully transformed into some pathogenic Leptospira,yet genetic incompatibility is supposed to hinder successful transformation.Therefore,S1-PFGE provides a method for rapid identification of plasmid in Leptospira and might contribute to guide for the genetic transformation of Leptospira.