A Novel DNA Restriction-Phosphorothioation System In Bacteria

A novel DNA sulfur modification, firstly discovered in the Gram-positive bacteria Streptomyces lividans, has been identified as one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides swapped with sulfur (RP configuration). As the first uncanonical physiological modification, the sulfur is unprecedently discovered on DNA backbone but not base. The DNA isolated from Streptomyces lividans is degraded during normal electrophoresis or pulsed-field gel electrophoresis (PFGE),which was designated as the phenotype of Dnd (DNA degradation), and determined by dnd gene cluster carrying five open reading frames (ORFs, dndA-E). DNAs from many bacteria are degraded during normal electrophoresis or pulsed-field gel electrophoresis in the presence of Tris. Homologous dnd gene cluster can be found in a large array of representative strains.A region of 6,665 bp DNA carrying purely five ORFs (dndA-E) was minimized as a sole determinant for the DNA modification to form phosphorothioate on the DNA backbone in Streptomyces lividans, leading to a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. An efficient mutation-integration-complementation system was developed to allow for the detailed functional analysis of these dnd genes. Abolished Dnd phenotype by the specific in-frame deletion of the dndA, C, D, and E genes or aggravation of the Dnd phenotype by the in-frame deletion of dndB gene could be restored by the expression vectors carrying independent and counterpart dnd gene(s). Interestingly, overdosage of DndC or DndD, but not other Dnd proteins in vivo was found to be detrimental to the cell viability.Simlar electrophoresis related DNA degradation is found in many Gram-negative bacteria Salmonella enterica strains. The Dnd phenotype of Salmonella enterica serovar Cerro 87 is observed by modified plasmid pUC18 and pSET152. The sed gene cluster (sedB-E) in Salmonella enterica serovar Cerro 87, which encoded four predicted homologous genes with dndB-E in Streptomyces lividans, was cloned with degenerate primers. sed gene cluster in plasmid pJTU1238 is expressed successfully in E. coli DH5α.A novel restriction- phosphorothioation system was found in S. enterica 87. In comparison with modified plasmid isolated from wt S. enterica 87 or engineered E. coli DH5α, plasmid isolated from S. enterica 87 mutant XTG102, dnd gene cluster deletion, or wt E. coli DH5αcould be generated only one percent of transformants in wt S. enterica 87. However, the restriction phenotype loses in mutant XTG102, there was no distinct difference among the four plasmid transformation. Interestingly, the restriction system of XTG102 was restored by complemention with dnd gene cluster in pJTU1238. All these support that dnd gene cluster is responsible for the restriction system besides controling DNA phosphorothioation. pJTU1238 itself could not restricts unmodified plasmid,so a lineage novel dnd gene cluster which encoded four predicted genes dndF-I was found. Indenpendent knock-out of dnd G, H, and I, instead of dndF, could result in abolishment of the restriction phenotype. The conserved restriction- phosphorothioation gene cluster in many bacterial genera and similar restriction- phosphorothioation system in E. coli B7A suggested that there was a universal restriction-modification system in bacterial genera.