Cloning and characterization of gerM gene involved in germination in Bacillus thuringiensis

Abstract:

In Bacilli, gerM is a very conservative gene. Primers were designed according to the gerM gene sequence of Bacillus cereus, and a 640bp DNA fragment was obtained from Bacillus thuringiensis subsp. kustaki 1.175 by PCR. Using this fragment as a probe, a 4.5kb DNA fragment was cloned from the partial DNA library of Bacillus thuringiensis subsp. kustaki 1.175.Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 349-amino acid (aa) protein, which has high homology with GerM protein from Bacillus subtilis. This gene was designated gerM (GenBank Accession No. DQ537381). RT-PCR analysis showed that gerM gene was only expressed in the process of sporulation, suggesting gerM is not required for the vegetative growth. The function of the gerM gene was studied by a strategy of gene disruption, and the resulting gerM disruption mutant did show normal growth and sporulation. However, gerM disruption mutant spores germinate slower than wild-type spores when triggered by L-alanine or inosine, indicating that gerM is required for the spore normal germination initiated by L-alanine or inosine in Bacillus thuringiensis.