Role of the gerA operon in L-alanine germination of Bacillus licheniformis spores

Abstract

Background: The genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine.

Results: In this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid.

Conclusions: These results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate.

Figure 1

Germination of B. licheniformis with L-alanine. Germination is followed as a change in initial absorbance at 600 nm (A₆₀₀) of phase bright spores in K-phosphate buffer pH 7.2 at 30 °C after addition of 100 mM L-alanine. Complete germination (>99% phase dark spores as observed by phase contrast microscopy) was observed at ~40% of initial A₆₀₀. The results shown are representative of experiments performed in duplicate on two individual spore batches repeated at least twice.

Figure 2

Phase contrast images of B. licheniformis spores following L-alanine germination. Phase contrast images (100 x) showing B. licheniformis spores after 3 hours germination at 30 °C with 100 mM L-alanine or negative control (MQ) in K-phosphatebuffer pH 7.2. The displayed images are representative of experiments performed in duplicate on two individual spore batches repeated at least twice.

Figure 3

Germination of B. licheniformis with casein hydrolysate. Germination is followed as a change in initial absorbance at 600 nm (A₆₀₀) of phase bright spores in Tris HCl buffer pH 7.4 at 30 °C after addition of 1% (w/v) casein hydrolysate. Complete germination (>99% phase dark spores as observed by phase contrast microscopy) was observed at ~40% of initial A₆₀₀. The results shown are representative of experiments performed in duplicate on two individual spore batches repeated at least twice.