GAA trinucleotide repeat region regulates M9/pMGA gene expression in Mycoplasma gallisepticum

Mycoplasma gallisepticum, the cause of chronic respiratory infections in th e avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) as sociated with hemagglutination, Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlat ion between gene expression and the number of tandem GAA repeat motifs loca ted upstream of the putative promoter. In this study, transposon Tn4001 was used as a vector with the Escherichia coli lacZ gene as the reporter syste m to examine the role of the GAA repeats in M9/pMGA gene expression in M. g allisepticum, A 336-bp Mo gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligate d with a lacZ gene from E. coli, and inserted into the Tn4001-containing pl asmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-1-chloro -3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloni ng resulted in cell lineages in which lacZ gene expression alternated betwe en the On and Off states in successive generations of progeny clones. The p romoter regions of the M9-lacZ hybrid genes of individual progeny clones we re amplified by PCR and sequenced. The only differences between the promote r regions of the blue and white colonies were in the number of GAA repeats. Clones that expressed lacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M, gallis epticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data stro ngly indicate that changes in the length of the GAA repeat region are respo nsible for regulating M9/pMGA gene expression.