Implementation of Fluorescent-Protein-Based Quantification Analysis in L-Form Bacteria.

Area of Science:

• Microbiology
• Synthetic Biology
• Cell Biology

Background:

• Cell-wall-less (L-form) bacteria present challenges for quantitative analysis due to their morphological complexity and heterogeneity.
• Efficient and stable fluorescent labeling is crucial for fluorescence-based quantitative cell analysis of L-forms during growth and proliferation.

Purpose of the Study:

• To evaluate the expression of multiple fluorescent proteins (FPs) under different promoters in *Bacillus subtilis* L-form strain LR2.
• To establish stable FP-labeled L-form bacterial strains for quantitative morphological studies.

Main Methods:

• Confocal microscopy and imaging flow cytometry were used to assess FP expression.
• Multiple fluorescent proteins (EGFP, mKO2) were tested under various promoters, including the P-derived *NBP3510* promoter.
• Expression was validated in *Bacillus subtilis* and *Escherichia coli* wild-type and L-form strains.

Main Results:

• The P-derived *NBP3510* promoter demonstrated superior performance for inducing EGFP and mKO2 expression in both wild-type and L-form strains.
• *NBP3510* activity was confirmed in both *Bacillus subtilis* and *Escherichia coli* L-form strains.
• Established FP-labeled strains allowed for the quantitative demonstration of distinct L-form bacterial morphologies.

Conclusions:

• The *NBP3510* promoter is a highly effective tool for stable fluorescent labeling of L-form bacteria.
• The generated FP-labeled cell lines provide valuable tools for quantitative analysis of L-form morphology.
• These cell lines can advance research utilizing L-forms as protocell and synthetic cell models.