Transcriptional Regulation and Characteristics of a Novel N -Acetylmuramoyl- <scp>l</scp> -Alanine Amidase Gene Involved in Bacillus thuringiensis Mother Cell Lysis

In Bacillus thuringiensis , a novel N -acetylmuramoyl- l -alanine amidase gene (named cwlB ) was detected, and the CwlB protein was purified and characterized. Reverse transcription-PCR (RT-PCR) results indicated that cwlB and an upstream gene (named cwlA ) formed one transcriptional unit. 5′ rapid amplification of cDNA ends (5′-RACE)-PCR and transcriptional fusions with the lacZ gene indicated that transcription of the operon was directed by a promoter, P cwlA , which is located upstream from the cwlA gene and that the transcription start site is a single 5′-end nucleotide residue T located 25 nucleotides (bp) upstream from the cwlA translational start codon. Moreover, the activity of P cwlA was controlled by σ K . Morphological analysis suggested that the mutation of cwlB could delay spore release compared to the timing of spore release in the wild-type strain. Western blot assay demonstrated that purified CwlB bound to the B. thuringiensis cell wall. Observations with laser confocal microscopy and a green fluorescent protein-based reporter system demonstrated that the CwlB protein localizes to the cell envelope. All results suggest that the CwlB protein is involved in mother cell lysis in B. thuringiensis .