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 p!asmid 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 examihed. Although the plasmids were somewhat unstable, it was possible to score complement!tion 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 Tn1l000 insertions into the cloned DNA of two relatively stabie 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'wer,e 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. * Corresponding author. 756Plasmids. Fragments of B. subtilis DNA were subcloned amphenicol at 3 ,ug ml-' and purified on the nutrient agar described above, again supplemented with chloramphenicol at 3 ,ug ml-'. All cultures were incubated at 37°C, unless otherwise specified.

The use of inhibitors to identify early events during Bacillus megaterium KM spore germination

“…A 10.2 kDa inner-membrane protein has been identified as the possible germinant receptor protein after acetic anhydride and germinant-analogue treatment of spores and vegetative cells of B. megaterium QMB1551 (Racine et al, 1981;Rossignol & Vary, 1979). More recently, cloning experiments have suggested that the B. subtilis L-alanine germination receptor is transcribed from the gerA operon containing three complementation groups (Zuberi et al, 1985). It will be of considerable interest to establish how similar the B. megaterium KM L-alanine receptor is to these two systems.…”

The trigger mechanism of spore germination: current concepts

“…This includes changes in anisotropy (Skomurski et al 1983) and fluidity (Janoff et al 1979) of isolated inner membranes of B. megaterium QMB155 1 by the germinant L-proline, specific labelling of a 10.2 kDa inner membrane protein in spores on B. megaterium QMB155 1 by the germinant analogue L-proline chloromethyl ketone (Rossignol and Vary 1979) (1993) ability of the germination inhibitor 203Hg2+ to label a lO.5-kDa inner membrane protein of spores of B. megaterium K M (Foster 1986). This view is also supported by sequence analysis of the cloned gerA genes of B. subtilis which suggests the presence of a membrane-bound multi subunit complex Zuberi et al 1985Zuberi et al , 1987. Definitive evidence for the location of the germinant receptor, however, remains to be established.…”

The genetic analysis of bacterial spore germination

“…The gerA locus contains three genes (gerAA, AB and AC), arranged in an operon Zuberi et al 1985;Zuberi et al 1987). The collection of known gerA mutants included mutations in each gene, indicating that all the GerA protein products are required for ALA germination.…”

“…The collection of known gerA mutants included mutations in each gene, indicating that all the GerA protein products are required for ALA germination. The gerA38 and A## mutations that require higher concentrations of alanine for germination are both located in the middle gene, gerAB, suggesting that the GerAB protein is likely to bind alanine (Zuberi et al 1985).…”