| A novel DNA modification discovered in Streptomyces lividans is different from DNA methylation. This unusual modification causes wild type S. lividans DNA sensitive to site-specific oxidative double-strand cleavage (Dnd phenotype, DNA degradation). Preliminary study reveals that such DNA modification involves incorporation of sulphur.The chief aim of the present study is to characterize the deleted region of S. lividans mutant strain ZX1 which lacks Dnd phenotype and sulphur modification, to localize and analyze the entire dnd cluster and to study the biological function and regulation of dnd genes.S. lividans mutant strain ZX1 lacks this novel modification because of a large chromosomal deletion. With a series of hybridization and subcloning, the two deletion end-points of this deleted region and deletion junction were localized precisely and cloned. The two end-points were localized on a 4.3kb BamHl-EcoR1 fragment of a cosmid 16C3 and a 1.2kb Sail fragment of a cosmid 17G7 respectively. The deletion junction was localized on a 2.8kb BamHI fragment of the ZX1 chromosome. A 12kb region involved in Dnd phenotype was confirmed to be deleted completely from the chromosome of ZX1.Six subclones varying in size from the 12kb region involved in Dnd phenotype were cloned onto an integrative vector pSET152 and were introduced into ZX1. The entire functional dnd gene cluster was minimized to an 8.3kb insert in pHZ1904, because pHZ1904 could complement the Dnd phenotype of S. lividans mutant strain ZX1 as well as Streptomyces parvulus ATCC12434 and Streptomyces nanchangensis NS3226, two naturally Dnd -deficient Streptomyces strains.Using Erase-a-Base System, a series of ExoIII progressive deletion mutants were prepared for sequencing dnd cluster. Analysis of the sequence revealed that it contained three ORFs. The first ORF (named dndA, 1.1kb in size) resembled nifS of nitrogen-fixing bacteria; the second ORF (dndB, 1.0kb) and third ORF (dndC, 3.2kb) showed no obvious similarity to any known genes in the DNA and protein databases.Two 5. lividans mutant strains LA1 and LA2 generated by gene replacement in dndB and dndC were obtained respectively. Both of these two mutants lost Dnd phenotype because of disruption of a respective dnd gene. This fact suggested that dndB and dndC were directly involved in the novel DNA modification in wild type S. lividans. S. lividans mutant strains ZX1 (dnd cluster deleted) and ZX64 (dndA disrupted) had pleiotropic mutations including low mel expression and poor sporulation. It was speculated that dndA together with its downstream DNA (2.5kb) might be involved in these two phenotypes because dndA together with its downstream DNA could restore normal sporulation and melexpression to ZX64, while dndB and dndC had no such effect because LAI and LA2 showed no obvious difference in these two phenotypes from wild type S. lividans 1326.Dnd phenotype could be detected when the entire dnd cluster was integrated in the chromosome or carried by the low copy-number plasmid in ZX1. However, when dnd cluster was carried by a plasmid with a copy-number around or higher than c.10 copies in ZX1, Dnd phenotype could not be observed. The dnd cluster seemed to express when it was carried by the high copy-number plasmid because complementation of individual dndA, dndB or dndC mutation could be observed and such expression did not seem to influence the normal dnd expression in wild type S. lividans 1326. We can thus speculate that the simultaneous over-expression of all three or even two of the three dnd genes may have bad effect on the cell and is thus under the tight control, and dosage of the Dnd proteins in the cell can not exceed the acceptable limit. On the other hand, the acceptable and balanced dosage of the coordinately expressed Dnd proteins may have inhibitory activity to the further over-expression of the dnd genes. Self-regulation of the expression level might also be the sensoring activity of one of the three genes. Such a self-regulatory mechanism suggests that this novel sulp...
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