CONSTRUCTION AND PROPERTIES OF CLONING VECTORS BASED ON A 7,2-KB RHIZOBIUM-MELILOTI CRYPTIC PLASMID

Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were construc ted by extension of the host range of a 7.2-kb Rhizobium meliloti cryp tic plasmid (pRm1132f) with the ColE1-based plasmids, pBR322, pACYC177 , pACYC 184, pSUP301, or pHC179; mobilization was facilitated by intro duction of the oriT region from pRK2, a broad-host-range plasmid. The vector plasmids transferred readily into a wide range of gram-negative bacteria and had relatively low copy number in R. meliloti; two const ructs, pFD1001 and pFD1212, were completely stable in R. meliloti isol ated from nodules of alfalfa (Medicago sativa). A representative of th e vector constructs (pFD1001) could be maintained in R. meliloti in th e presence of the broad-host-range shuttle plasmid pRK290. These two v ector plasmids could be introduced into X. meliloti, either simultaneo usly or singly when pRK290 was the resident plasmid; however, entry of pRK290 was blocked when pFD1001 was the resident plasmid. The cloning vectors constructed in this study should prove to be useful for the g enetic manipulation of Rhizobium. (C) 1995 Academic Press, Inc.