Figure 3

Deletion derivatives of pB264 used to identify domains involved (A and B) replication and (C) conjugal transfer. The linear representation of the pB264 element indicates relative positions of the restriction sites for Xho I (X), Sac II (Sc), and Sph I (Sp), as well as the positions and orientations of ORFs 1 through 9. (A) Deletion derivatives of pB264 that retain the ability to replicate in Rhodococcus. The entire pB264 element is represented in the plasmid pAL298. For this plasmid and all derivatives of pAL298, only the portions derived from pB264 are shown (as black boxes), while deleted regions are represented as dotted lines. Note that in plasmid 2 (and all derivatives of plasmid 2), the region bounded by the two Sac II sites denoted by asterisks (*) was inverted during plasmid construction. Plasmids 1–8 could all be propagated by themselves in Rhodococcus sp. I24. Plasmid 9, however, could only be maintained in Rhodococcus sp. I24 when either pAL312 or pAL314 were present. Plasmids 10 and 11 could only be maintained in Rhodococcus sp. I24 when pAL314 was present. (B) Regions of pB264 supplied in trans to support replication of plasmids 9, 10, and 11. (C) Deletion derivatives of pB264 that retain the origin of conjugal transfer. Plasmid 16 (pJANET) contains the bulk of the pB264 element, lacking only a 136 bp fragment at the 5' end. While pAL281 can not be transferred from Rhodococcus sp. B264-1 to other rhodococci via conjugation, plasmids 15 through 20 are readily transferred.