Study On The Convergence Of SBDHNH And DNA Phosphorothioation-In Proteus Mirabilis 1166＿PMIR

DNA Phosphorothioation（PT）modification is a new type of epigenetic modification that occurs naturally and widely in bacteria and archaea.Its chemical nature is that a sulfur atom replaces a non-bridging oxygen atom on the DNA deoxyribose-phosphate backbone with sequence selectivity and R_Pconfiguration specificity.So far,the PT modification systems that have been discovered include two types,one is the modification that occurs on the double strand of DNA conferred by the expression product of the dnd ABCDE gene cluster,and the other is the ssp ABCD gene cluster,whose expression products conferred modification that occurs on the single strand of DNA.The PT modification conferred by the Dnd ABCDE protein is called orphan phosphorylation modification when it exists alone.When it is present with its homologous restriction enzyme Dnd FGH,it can form a restriction-modification（RM）system to protect cells from foreign invasion of non-PT-DNA to maintain the stability of its own genetic information;PT modifications conferred by Ssp ABCD protein can be combined with its homologous protein Ssp E to form a new set of PT-sensitive bacterial defense systems with wide anti-phage activity.DNA modification is usually recognized by a specific protein encoding a modified biological function,and only a specific protein that recognizes the modified substrate DNA and performs restriction cleavage is called modification dependent restriction endonucleases（MDREs）.The fusion-type MDREs consist of identifying specific modified domains and catalytic domains.SBD is a recently revealed domain that specifically recognizes and binds sulfur with R_Pcofiguration in PT modifications.In this project,Proteus mirabilis 1166＿PMIR was selected as the research object.Genomic sequence analysis of P.mirabilis 1166 found that it had both sbdhnh gene and PT modification gene cluster——dnd BCDE,and the two were close to each other.LC-MS/MS detection found that its PT modification type was d(G_PSA)/d(G_PST),presumably its modified core sequence was 5’-G_PSAAC-3’/5’-G_PSTTC-3’.In vivo experiments found that SBDHNH can recognize PT modifications of four common sequence patterns:5’-G_PSAAC-3’/5’-G_PSTTC-3’,5’-G_PSATC-3’/5’-G_PSATC-3’,5’-G_PSGCC-3’/5’-G_PSGCC-3’and 5’-C_PSCA-3’.When heterologously coexpressed SBDHNH and PT modifications containing these four sequence patterns respectively and once induced SBDHNH expression,it caused the growth of host bacteria to be inhibited or not to grow.At the same time,the reverse transcription-PCR results showed that in P.mirabilis 1166,sbdhnh was hardly expressed under normal culture conditions,which was consistent with the coexistence of dnd gene cluster and sbdhnh gene in the strain.The key site mutations and partial frame deletions of SBDHNH confirmed that SBD was the key domain for identifying PT modification.In addition,there is a complete PT restriction-modification system——Dnd BCDE-Dnd FGHI system in P.mirabilis 1166.Phage infection experiments showed that the system can act as a R-M system to resist the invasion of foreign non-PT-DNA.To explore the physiological significance of the coexistence of SBDHNH protein and PT modification,we heterologously coexpressed SBDHNH and PT system of P.mirabilis1166 to the same host bacteria,and conducted antiviral function studies on them.The results did not show that the coexistence of SBDHNH protein and PT proteins had obvious antiviral synergy.