The DNA Phosphorothioation In Human Microbiome

DNA phosphorothioation(PT)is a natural modification in which the nonbridging oxygen in the phosphate backbone of DNA is replaced by sulfur.It is widely distributed in bacteria and archaea.It can endow cells with a variety of physiological functions,including restricting gene transfer and antioxidation.Two gene clusters,dnd A-E and ssp A-D,have been found to be responsible for catalysing this modification in strains of Escherichia coli,Salmonella,Vbrio,Pseudomonas.Among them,dnd A-E can confer cells with 5?-G_psAAC-3?/5?-G_psTTC-3?or 5?-G_psGCC-3?on consensus sequences while ssp A-D can confer cells with 5?-C_psCA-3?on the single strand.Although the distribution,physiological function and catalytic mechanism of PT have been preliminarily studied,its distribution in human microbiome has not been reported yet.In addition,little is known about the mechanism of PT dependent restriction,and the possible existence of its regulatory mechanism remains to be elucidated.This study aims to explore the distribution of PT in human microbiome so as to supply direct support for the generality of PT and explore the possible regulation mode of restriction gene dnd FGH.Firstly,the distribution of two PT modifying gene clusters in human microbiome was investigated.In this study,dnd CDE and ssp BCD were used as query sequences.Through multiple sequence alignment,more than 2,000 strains containing PT modifying gene clusters were found in the database,and these strains were distributed in the human intestinal tract,mouth,vagina,skin and other parts.The PT modifying genes have a certain genera preference,and are widely distributed in Escherichia coli,Mycobacterium,Clostridium difficile and so on.In addition,unknown genes related to PT were found.Secondly,the abundance of PT in the human intestinal bacteria DNA was analyzed by mass spectrometry and gene sequencing.In this study,six novel PT-linked dinucleotides were found in the DNA of human intestinal bacteria,namely C_psG,C_psT,A_psG,T_psG,G_psC and A_psT.Meanwhile,DNA analysis of intestinal bacteria from obese volunteers showed that after dietary intervention,the abundance of PT modifying genes,the abundance of strains harboring PT modifying genes,and the types and total amount of PT-linked dinucleotides were significantly decreased,which proved that there was a positive correlation between PT modifying genes and PT products.A regulation mode of PT contents in the human intestinal bacteria was proposed.Finally,a regulatory protein DndBi of dnd FGH(PT restricting genes)was found from the unknown genes acquired in this study.The genetic and biochemical operations were carried out on Aeromonas TH0426 containing the protein,and DndBi was initially identified as the negative regulatory protein of dnd FGH.Then,the promoter binding site of DndBi was determined as ATCTG by DNase I footprints.In conclusion,this study investigated the distribution of PT in human microbiome for the first time,and preliminarily revealed the extensive distribution and species preference of PT modifying genes in human microbiome.Through the analysis of gene sequencing results and PT products,the study of PT was extended from single strain level to intestinal bacteria level for the first time,which provided a reference for further study about the effect of PT on human microbiome.Through the study of the functional analysis,DndBi was initially identified as the negative regulatory protein of dnd FGH,and the regulatory mechanism of PT restriction function was explored for the first time.This study provides strong data support for further study on the universality and diversity of PT,and also lays a foundation for the study about the function of PT dependent restriction.