Identification of three complementation units in the gerA spore germination locus of Bacillus subtilis

Abstract

The gerA locus, mutations in which affect the germination response of spores to L-alanine and related amino acids, is contained within a 6-kilobase region of DNA cloned in phage and plasmid vectors. Fragments from this region, subcloned in the shuttle vector pHV33, were introduced into Bacillus subtilis, and their ability to complement chromosomal gerA mutations in a recE4 background was examined. Although the plasmids were somewhat unstable, it was possible to score complementation within spore-containing colonies on nutrient agar by their ability to reduce 2,3,5-triphenyltetrazolium chloride in an overlay. These studies have assigned the 10 gerA mutations tested to three complementation groups. An analysis of Tn1000 insertions into the cloned DNA of two relatively stable plasmids that together encompass the entire gerA region has identified more precisely the location and extent of the complementation units; recombination studies and in vitro mutagenesis were used to further delineate the extents of two of the units. The evidence suggests that the three complementation units are adjacent and that they are probably capable of separate transcription.