A series of transposons are described which contain the gusA gene, encoding B-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.Keywords : GUS transposons, microbial ecology, rhizobial competition, rhizosphere colonization
INTRODUCTIONReporter genes are powerful molecular biological tools with a diversity of applications. They may be used to substitute for a structural gene-of-interest and hence to report on regulation of gene expression through creation of a gene fusion. They are used in microbial ecology to facilitate the detection of individual marked strains of bacteria (Drahos, 1991 ;Wilson, 1995 Abbreviations : GUS, P-D-g I ucu ron idase ; X-G IcA, 5-bromo-4-ch I oro-3-indolyl p-D-glucuronide; pNPG, p-nitrophenyl P-D-glucuronide; PFGE, pulsed-field gel electrophoresis; RBS, ribosome-binding site; UAS, upstream activating sequence; YM, yeast-mannitol.The GenBank accession number for the sequence reported in this paper is M14641.The key advantage of reporter genes as tools in microbial ecology is that they enable closely related strains of bacteria to be readily distinguished, and provide a rapid means of identifying the strain of interest (Wilson, 1995 , 1994). We had earlier suggested its use as a marker for rhizobial competition studies (Wilson et al., 1991) and the initial GUS transposon developed by us proved useful for studying competition for nodulation of the common bean, Pbaseoltls vdgaris (Streit e t al., 1992(Streit e t al., , 1995. More recently, GUS has been used to look at the physical location of plant-associated bacteria (ChristiansenWeniger '& Vanderleyden, 1993;Hurek e t al., 1994). However, no comprehensive set of GUS transposons existed that could be used to study the ecology of a wide range of Gram-negative bacteria. In this paper we describe the construction and initial application of a set of gtlsAexpressing transposons for ecological studies. In addition, further tools for the manipula...
