Design and construction of two genetic tools for use in mycoplasmas

Abstract

The members of the bacterial class Mollicutes, commonly referred to as mycoplasmas have a number of unusual characteristics that make them difficult research subjects. These include complex media requirements, a lack of cell walls, a high genomic AT/GC ratio and frequently a non-standard translational system. In most orders of the Mollicutes, the codon UGA, a stop codon in the universal genetic code, codes for the amino acid tryptophan. As a consequence, standard genetic tools frequently do not function properly in these mycoplasmas. The focus of the research described here has been on the construction of two tools with wide applicability for future mycoplasmal research. The first is a derivative of transposon Tn4001, mini-Tn4001, that can be used to irreversibly insert a variety of markers into mycoplasmal chromosomes. The second is a derivative of the popular genetic marker, green fluorescent protein (GFP). The DNA sequence of this construct, designated GFPmyco was modified to reflect a mycoplasma coding usage pattern. The results of preliminary expression studies have confirmed that mini-Tn400J tet is a functional mini-transposon in Mycoplasma gallisepticum and that GFPmyco can produce an observable fluorescent product in E. coli. Factors effecting GFP fluorescence and possible improvements in GFPmyco construction and expression are discussed. Both minitransposons and GFP markers have been extremely useful for both eucaryotic and nonmycoplasma bacterial studies. It is hoped that these constructs, or their descendants, will be equally useful for the study of mycoplasmas.