Identification and functional analysis of the transfer region of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica.

Abstract

Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) that is present either as an integrated element or as an autonomously replicating plasmid. Conjugational transfer of pMEA300 results in pock formation, zones of growth inhibition that become apparent when plasmid-carrying donor cells develop in a confluent lawn of plasmid-lacking recipient cells. A 6.2-kb pMEA300 DNA region specifying the functions of conjugation and pock formation was sequenced, revealing 10 open reading frames. This is the first sequence of the transfer region of a plasmid from a nonstreptomycete actinomycete. No clear similarities were found between the deduced sequences of the 10 putative Tra proteins of pMEA300 and those of Streptomyces plasmids. All Tra proteins of pMEA300 thus may represent unfamiliar types. A detailed mutational analysis showed that at least four individual proteins, TraG (9,488 Da), TraH (12,586 Da), TraI (40,468 Da), and TraJ (81,109 Da), are required for efficient transfer of pMEA300. Their disruption resulted in a clear reduction in the conjugational transfer frequencies, ranging from (5.2 x 10(1))-fold (TraG) to (2.3 x 10(6))-fold (TraJ), and in reduced pock sizes. At least two putative proteins, TraA (10,698 Da) and TraB (31,442 Da), were shown to be responsible for pock formation specifically. Specific binding of the pMEA300-encoded KorA protein to the traA-korA intragenic region was observed.