Characteristic And Mechanistic Studies On DNA Phosphorothioation Specificity In Streptomyces Lividans 66

A phosphorothioation modification of genomic DNA was discovered in bacteria. The modification, involving the incorporation of sulfur into DNA, is determined by a five genes, constituting dnd gene cluster. Phosphorothioation DNA, such as that of Streptomyces lividans 66, degraded during electrophoresis in Tris containing buffer. This phenotype is called Dnd phenotype (DNA degradation).The Dnd phenotype can be analyzed by DNA electrophoresis in TAE buffer (Tris, acetic acid and EDTA), but the analyzing process is not stable and the phenotype is not always reproducible. Here, a fast, simple and efficient method of Dnd phenotype analyzing was presented. PAA (peracetic acid) was first produced by combination of acetic acid and H2O2. PAA was then used to react with TAE to produce the phosphorothioated DNA backbone cleaving reagent PAA-TAE. The PAA-TAE can cleave DNA phosphorothioation site accurately and efficiently, thus make Dnd phenotype analyzing a simple process.Bioinformatic analysis revealed that the dnd clusters are wide spread among bacteria. DNA degradation is also often observed during Pulse Field Gel Electrophoresis of bacteria typing. Dnd phenotype of the corresponding bacteria strains was analyzed and results showed that most of the strain contains DNA phosphorothioation. The observation suggests that DNA phosphorothioation is common phenomenum among bacteria.Physiologically happened DNA phosphorothioation is a site chosen process. One site of pHZ209 from S.lividans 66 is preferentially modified while other potential sites have a low modification frequency. After dndB gene was in-frame deleted, frequency at the preferred site on pHZ209 decreased while other sites increased. Although there are multiple potential modification sites on the plasmid, at most, only one of them was chosen to be modified if the modification happens.After cloning and sequencing, a totally 14 modification sites were identified on pHZ209. Comparison of DNA sequences traversing the modification sites revealed a consensus sequence: 5'-c—cGGCCgccg-3'. Systematic mutagenesis indicates that the central core sequence 5'-GGCC-3'is absolutely required by the modification activity while the flanking sequence and the'gccg'are required only in dndB mutant. Site directed mutation on them will diminish the modification frequency in dndB mutant, suggesting that dndB gene participate directly or indirectly this site chosen process.Complement of single dndB gene to its mutant only partially restore the modification frequency on the preferred site, while complement of dnd cluster or dnd cluster with only one of the gene in-frame deleted restored the frequency totally. Changing the flanking sequence traversing the preferred modification site diminished the corresponding modification frequency in S.lividans while in dndB mutant the preferred site was totally eliminated. These results suggest that there are multiple cis-acting elements and trans-acting factor participate the site chosen process.Transcription of dnd genes were analyzed by RT-PCR. The dnd promoter was divergently transcribed, with dndA in one direction and dndBCDE transcript in another direction, supporting the bioinformatics analysis result. Using rrnA gene of 16s rRNA in S.lividans as internal standard, transcription of dnd gene during different developmental stage was semi-quantified. dnd genes were transcribed actively in the log phase of development and the transcription decrease sharply at late log phase and stationary phase. RT-PCR results also implied that dndB might negatively regulate the expression of dndA. A short N-terminal affinity tag StrepTagII was introduced into pET-15b, generating pJTU2036. dndA, dndB and dndC were constructed into the vector and the corresponding protein expressed in E.coli soluble. pIB139, contain ermE promoter, was also modified to include a StrepTagII tag generated pJTU2032. dnd genes were constructed into pJTU2032 and then conjugated into S.lividans. The resultant strains will be used to analyze protein-protein interaction of Dnd protein.Physiological involvements of dnd genes were also explored. Growth rates comparison showed that the growth of HXY1 and HXY6 were delayed while HXY2, HXY1 and HXY6 have a longer stationary phase. L-Ala inhibits the growth of HXY1 and HXY6. Branched amino acids, such as L-Valine, L-Isoleucine and L-Threonine, to a less extent, also inhibited the growth of them. HXY1 and HXY6 showed some extent of thiostrepton resistance. When dndA was introduced into S.coelicolor M145, an earlier production of red pigment production was observed. Under the condition of oxidation stress, similar effects were observed, implying the pleiotropic effects of dndA. The above results provide clues for further investigation of physiological meaning of DNA phosphothioation.