Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia. This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia. Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 105 arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio. This approach requires no special equipment that might complicate biocontainment. Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure. A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C. immitis and perhaps other fungi of medical importance.
Bibliographical noteFunding Information:
Received 13 December 1999; revised 14 February 2000; electronically published 5 June 2000. Presented in part: 100th annual meeting of the American Society for Microbiology, Los Angeles, 24 May 2000. Financial support: US Department of Veterans Affairs; California Health Care Foundation; Purdue University (grant U.S.D.A. Prime 96-34340-2711). a Present affiliation: College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates. Reprints or correspondence: Dr. John N. Galgiani, Valley Fever Center for Excellence (1-111), 3601 S. Sixth Ave., Tucson, AZ 85723 (spherule @u.arizona.edu).