Revealing Cell Envelope Heterogeneity in Two Stable Escherichia coli L-Forms

Long-term adapted cell wall-deficient (L-forms) bacteria show unique cell shapes and growth patterns compared to wild-type bacteria. However, quantitative analysis to assess the morphological heterogeneity of existing L-forms is limited. In this study, we validated that two stable L-form strains of Escherichia coli (NC-7 and LWF+) hold spherical or pleomorphic morphology in confocal and electron microscopy. Using imaging flow cytometry, we further reported that the variations in cell size distribution and cell viability between L-forms and walled cells are statistically significant. Moreover, freeze-fracture electron microscopy observations revealed a clear presence of an outer membrane in NC-7 but not in LWF+, suggesting that E. coli L-form strains could survive in both spheroplastic and protoplastic forms after adaptive evolution. Accordingly, the mutations in genes associated with cell envelope and outer membrane components are more prevalent in the LWF+ genome, potentially leading to outer membrane depletion. Notably, experimental evidence derived from E. coli LWF+ cells exhibiting a monoderm phenotype may support the diderm-to-monoderm transition hypothesis, implying the monoderm phenotype arose from the evolutionary loss of the outer membrane in diderm ancestors. Taken together, our findings offer insights into quantification analysis and the cell envelope status of two E. coli L-forms, facilitating future investigations into genotype-phenotype associations in these two L-form bacteria models.