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Transformation and genomic restriction mapping of Rochalimaea spp

Reschke, D.K.; Frazier, M.E.; Mallavia, L.P.

Acta Virologica 35(6): 519-525

1991

ISSN/ISBN: 0001-723X
PMID: 1687634
Document Number: 376359
Transformation procedures using electroporation were established for R. quintana [Bartonella 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 milliseconds. Transformation efficiency was low (0.3%) with approximately 105 transformants/ micro g of DNA. One construct, designated pAG10, reached sufficient levels in B. quintana to be isolated by density gradient centrifugation. Analysis of this plasmid after several cycles of growth in B. quintana revealed no obvious modifications. Physical maps of Rochalimaea spp. chromosomal DNA using pulse-field electrophoresis are being developed. Digestion of R. vinsonii [B. vinsonii] chromosomal DNA with NotI or SfiI resulted in 3 and 1 fragments, respectively. When B. quintana was digested in a similar manner, both NotI and SfiI produced 4 fragments. Double digestion of B. quintana DNA with NotI and SfiI yield 7 fragments ranging in size from 11 to 925 kb. Summing the fragments indicate an approximate genome size of 2.1x106 bp for B. vinsonii and 1.7x106 bp for B. quintana chromosomal DNA.

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