A vector for promoter trapping in Bacillus cereus

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacillus cereus is an important bacterium in various environments.
• Identifying regulatory sequences is crucial for understanding gene expression and bacterial behavior.
• Existing methods for screening large bacterial libraries can be inefficient.

Purpose of the Study:

• To construct and validate a promoter-trap plasmid, pAD123, for Bacillus cereus.
• To develop an efficient screening method for identifying B. cereus regulatory sequences.
• To demonstrate the utility of the developed tools for tracking bacterial populations.

Main Methods:

• Construction of the promoter-trap plasmid pAD123 containing a green fluorescent protein (GFP) gene.
• Generation of a B. cereus chromosomal DNA library in pAD123.
• Screening of the library using a 96-well microtiter dish assay and fluorimager.
• Characterization of positive clones via restriction analysis, sequencing, and fluorescence quantification.
• Utilizing flow cytometry and cell sorting for efficient separation of GFP-expressing cells.

Main Results:

• The pAD123 plasmid successfully functions in both Escherichia coli and B. cereus.
• A library of 29,000 clones was constructed, with 988 screened initially.
• 21 fluorescent clones were identified, with subsequent characterization confirming their utility.
• Flow cytometry and cell sorting demonstrated high efficiency in separating expressing from non-expressing cells.
• Selected clones served as effective markers for B. cereus populations on plant surfaces.

Conclusions:

• The pAD123 plasmid and GFP are effective tools for identifying regulatory sequences in Bacillus cereus.
• Flow cytometry and cell sorting provide an efficient method for screening large libraries in B. cereus.
• This approach facilitates the study of bacterial gene regulation and population dynamics.