The Dnd (DNA degradation) phenotype, reflecting a novel DNA modification by sulfur in Streptomyces lividans 1326, was strongly aggravated when one (dndB) of the five genes (dndABCDE) controlling it was mutated. Electrophoretic banding patterns of a plasmid (pHZ209), reflecting DNA degradation, displayed a clear change from a preferential modification site in strain 1326 to more random modifications in the mutant. Fourteen randomly modifiable sites on pHZ209 were localized, and each seemed to be able to be modified only once. Residues in a region (5'-c-cGGCCgccg-3') including a highly conserved 4-bp central core (5'-GGCC-3') in a well-documented preferential modification site were assessed for their necessity by site-directed mutagenesis. While the central core (GGCC) was found to be stringently required in 1326 and in the mutant, 'gccg' flanking its right could either abolish or reduce the modification frequency only in the mutant, and two separate nucleotides to the left had no dramatic effect. The lack of essentiality of DndB for S-modification suggests that it might only be required for enhancing or stabilizing the activity of a protein complex at the required preferential modification site, or resolving secondary structures flanking the modifiable site(s), known to constitute an obstacle for efficient modification.