Transformation of Xenorhabdus nematophilus.
The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.
Xu
J
J
Department of Microbiology, Washington State University, Pullman 99164-4340.
Lohrke
S
S
Hurlbert
I M
IM
Hurlbert
R E
RE
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D004755:Enterobacteriaceae / Q000235:genetics* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D013696:Temperature; D014169:Transformation, Bacterial
D003470:Culture Media; D004269:DNA, Bacterial; D002118:Calcium