Functional analysis of a small cryptic plasmid pYS1 from Nocardia

Bacteria of the genus Nocardia cause opportunistic infections of lung, brain and central nervous system, and cutaneous tissue. They are also producers of antibiotics and industrially important enzymes. As studies describing plasmids in this genus are limited, we have characterized a 4326 bp cryptic plasmid pYS1 from Nocardia aobensis IFM 10795. Three open reading frames (ORFs) were predicted. Both sequence analyses and detection of single-stranded intermediates suggested a rolling-circle mechanism as the mode of replication of pYS1. Mutageneses and deletion analyses revealed both the predicted double- and single-stranded origins to be indispensable in replication, suggesting a lack of secondary signals for leading and lagging strand synthesis. The replicon of pYS1 is broad-host-range and compatible to that of pAL5000 of mycobacteria, making it potentially useful in genetic manipulation of various actinomycetes. Insertion analyses showed orf1, despite its sequence similarity to plasmid transfer genes, is involved in plasmid stability rather than conjugation and is lethal in the absence of a functional orf3. This situation is somewhat analogous to the kil/kor system of pIJ101 of Streptomyces, except that orf3 was unrelated to korA and was shown by promoter-probe assays to encode a novel transcriptional repressor negatively regulating orf1 expression.