Localization And Rudiment Studies Of Biological Function Of DNA Degradation Gene Cluster Add From Streptomyces Avermitilis

The restriction map of pHZl318 carrying the entire add gene cluster from S. avermitilis NRRL8165 was made. Its two subclones pHZ2104 and pHZ2105 were introduced into S. lividans mutant ZX1. The entire functional add gene cluster was shortened to an 11.6kb insert in pHZ2104, because pHZ2104 could complement Dnd phenotype of mutant S. lividans ZX1 as well as S. parvulus ATCC12434 and S. nanchangensis NS3226, two naturally Dnd-deficient Streptomyces strains, whereas pHZ2105 can not confer ZX1 Dnd phenotype. The derivatives of pHZ2104 resulting from the disruption of the three putative ORFs respectively by aadA can not confer Dnd phenotype on ZX1.Using Erase-a-Base System, a series of ExoIII progressive deletion mutants were prepared for sequencing of add gene cluster. Analysis of the sequence revealed that addA resembled nifS of nitrogen-fixing bacteria and dndA. The entire add gene cluster showed 78% identity to dnd gene cluster from S. lividans.In order to facilitate the study of biological function of add gene cluster, E. coli-S. avermitilis intragenus conjugation system was established. In addition, pHZ2114 for the replacement of the entire add gene cluster and pHZ2130 for disruption of addA were constructed.