Function Of The Regulatory Genes Of DNA Phosphorothioate Modification In Pseudomonas Fluorescens Pf0-1

Phosphorothioation is a unique epigenetic modification of DNA,in which the non-bridged oxygen atoms are replaced by sulfur in a stereospecific and sequence-selective manner.This is the first reported physiological modification on the DNA sugarphosphate backbone.In addition,sulfur is also the first element found in DNA since the ever have been regarded common chemical elements such as hydrogen,oxygen,phosphorus,and nitrogen.Phosphorothiolate modification was originally reported for the DNA degradation phenotype of Streptomyces lividans and has been detected in more than 200 bacterial and archaea species up to date.Even as a relative widespread modification,the frequency of modification is different in strains and exhibits varied modification sequences specificities.The physiological PT modifications are endowed by a large family of five-gene clusters termed as dndA-E.Recently,the application of single-molecule real-time sequencing has revealed that the phosphorothioation modification was evenly distributed over the entire genome and was a partial,low frequency and dynamic genomic modification,which intriguing the question on the mechanism to maintain this low abundance modification suitable for cellular activities.The negatively transcriptional regulator Dpt B in Salmonella enterica serovar Cerro 87 has been reported,however,the reported regulated sequence of Dpt B is absent in other strain such as Pseudomonas fluorescens Pf0-1and Streptomyces lividans.For a better understanding of the regulatory mechanism of PT modifications,this work mainly focus on the candidate regulator SpfB and characterized is potential regulator mechanism.Firstly,we constructed both of the interruption and complementation genetic strains,respectively,for indentify the regulatory role of spfB.The contrastive analysis of the iodine-induced cleavage of genome DNA suggested the similar negative regulatory function for the PT modification.Then,in order to reveal the regulatory mechanism,the operons within spf gene cluster were grouped by RT-PCR,which turned out to be a single shared operon.Subsequently,based on RT-PCR results,the transcription level of spf operon of ?spfB mutant increased13-fold compared with that of wild type.Besides,the complementation of spfB contributed to the drop of the transcription to the similar level with that of wild type strain.Consistently,all these data supported that SpfB directly down-regulated the transcription of the operon,and showed negative trgulatory role of SpfB.Secondly,the bioinformatics analysis of SpfB revealed the the same conserved DGQHR motif and showed high sequence identity with Dpt B.To determine whether spfB directly regulates the spf operon,the spfB was expressed and purified for the electrophoretic mobility shift assay(EMSA)experiment.Expectedly,the purified SpfB binding to the upstream of spfB and generated significantly shifted bands.DNase I footprinting assay with FAM-labeled primers uncovered two protected regions with the same conserved motif 5'-TGTTTGT-3',which could not be found in the binding promoter region of Dpt B.In summary,this work here revealed the negative regulatory protein SpfB encoded by spf cluster for the DNA PT modification in P.fluorescens Pf0-1.The identified binding sites of SpfB in the upstream region of the co-transcribed spfBCDE operon depicted the strict host-specific DNA-protein interaction.These findings pave the way for the study of other possible regulatory proteins in so many poorly characterized bacteria and will benefit the expanding of the knowledge of PT modifications.