Construction and properties of cloning vectors based on a 7.2-kb Rhizobium meliloti cryptic plasmid.
Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRM1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction 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 constructs, pFD1001 and pFD1212, were completely stable in R. meliloti isolated from nodules of alfalfa (Medicago sativa). A representative of the vector constructs (pFD1001) could be maintained in R. meliloti in the presence of the broad-host-range shuttle plasmid pRK290. These two vector plasmids could be introduced into R. meliloti, either simultaneously 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 genetic manipulation of Rhizobium.
Froissard
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Plant Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario.
Bromfield
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Whitwill
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Barran
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LR
D016960:Agrobacterium tumefaciens / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D005822:Genetic Vectors; D007711:Klebsiella pneumoniae / Q000235:genetics; D000455:Medicago sativa / Q000382:microbiology; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics; D015183:Restriction Mapping; D012231:Rhizobium / Q000235:genetics; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000302:isolation & purification; D013045:Species Specificity