CONSTRUCTION OF TN4001LAC DERIVATIVES TO BE USED AS PROMOTER PROBE VECTORS IN MYCOPLASMAS

Studies of gene expression in mycoplasmas has been difficult, because the elements involved in gene expression remain relatively undefined. In order to be able to examine these regulatory elements in vivo, derivatives of Tn4001 containing the promoterless Escherichia coli lacZ reporter gene have been constructed. A Tn4001lac derivative, Tn4001.2062.21ac, transforms Mycoplasma gallisepticum and Acholeplasma strain ISM1499. Approximately 3% of the M. gallisepticum and 8% of the Acholeplasma ISM1499 transformants appeared to generate lacZ fusions based on blue colony formation on XGal-containing media. However, placement of lacZ into an IS256 arm of Tn4001 resulted in a derivative that transformed at a frequency about 3-7-fold lower than that of wild-type Tn4001. Another Tn4001lac derivative, Tn4001.2065, possesses a plasmid, as well as lacZ, in the IS256 arm. This construct was designed to permit the direct rescue of adjacent chromosomal sequences that are driving the expression of lacZ contained within the transposon These constructs should be useful in locating and defining the upstream elements involved in mycoplasma gene expression.