Transformation and genomic restriction mapping of Rochalimaea spp.
Transformation procedures using electroporation were established for Rochalimaea quintana. Several cosmid/plasmids possessing the RK2 or RSF1010 origin of replication were successfully inserted. Plasmid retention and replication were verified by antibiotic resistance and Southern blot analysis. The highest level of transformation was obtained at a voltage field strength of 12.5 kV/cm with a pulse time of 10 miliseconds. Transformation efficiency was low (0.3%) with approximately 10(5) transformants/microgram of DNA. One construct, designated pAG10, reached sufficient levels in R. quintana to be isolated by density gradient centrifugation. Analysis of this plasmid after several cycles of growth in R. quintana revealed no obvious modifications. Physical maps of Rochalimaea spp. chromosomal DNA using pulse-field electrophoresis are being developed. Digestion of R. vinsonii chromosomal DNA with NotI or SfiI resulted in three and one fragments, respectively. When R. quintana was digested in a similar manner, both NotI and SfiI produced four fragments. Double digestion of R. quintana DNA with NotI and SfiI yield seven fragments ranging in size from 11 to 925 kb. Summing the fragments indicate an approximate genome size of 2.1 x 10(6) bp for R. vinsonii and 1.7 x 10(6) bp for R. quintana chromosomal DNA.
Reschke
D K
DK
Department of Microbiology, Washington State University, Pullman 99164-4233.
Frazier
M E
ME
Mallavia
L P
LP
D016521:Electrophoresis, Gel, Pulsed-Field; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D012287:Rickettsiaceae / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*