Juxtaposition of an active promoter to vsp genes via site-specific DNA inversions generates antigenic variation in Mycoplasma bovis

Mycoplasma bovis, the most important etiological agent of bovine mycoplasmo sis, undergoes extensive antigenic variation of major and highly immunogeni c surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome of M. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal iso lates exhibiting phase transitions of VspA or of VspC (i.e., ON --> OFF --> ON) has shown that DNA inversions occur during Vsp phase variation. The up stream region of each vsp gene contains two sequence cassettes. The first ( cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhib its 98% homology among all vsp genes, while the second (cassette no. 2), up stream of cassette no. 1, ranges in size from 50 to 180 bp and is more dive rgent. Examination of the ends of the inverted fragments during VspA or Vsp C phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer e xtension and Northern blot analysis have shown that a specific cassette no. 2, designated A., is an active promoter and that juxtaposition of this reg ulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that p hase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present wit hin the conserved cassette no. 1. A model for the control of Vsp phase vari ation is proposed.