Objective: Streptomyces sp. X335, harboring a 4. 3-kb plasmid pDYM4.3k, was isolated from Tibet sample. Cloning, sequencing, analyzing and functional investigations of pDYM4. 3k.
Methods: Prime walking to obtain plasmid sequence, BLAST to predict gene function, Southern hybridization to determine replication intermediates, and mating to test conjugal function of plasmid.
Results: The complete nucleotide sequence of pDYM4. 3k consisted of 4346 bp, encoding 3 genes of which one resembled Streptomyces major conjugative gene tra and other two were with unknown functions. Experiments demonstrated that a new gene of pDYM4. 3k and its upstream c. 300 bp were required for plasmid replication. Single-stranded pDYM4. 3k as a replication intermediate was detected, indicating it rolling-cirle replication mode. pDYM4.3k could conjugal transfer among Streptomyces lividans.
Conclusion: Streptomyces small plasmid pDYM4. 3k was able to autonomous replication and conjugal transferring.