Fluorescence-activated cell sorting of Escherichia coli L-forms reveals maintained synchronized nucleic acid synthesis dynamics
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L-form bacteria are wall-deficient variants that proliferate without an intact cell wall and independently of the canonical cell division machinery. Despite increasing insights into how L-forms survive and proliferate without a cell wall, fundamental questions regarding how cell cycle processes, including DNA and RNA synthesis, are coordinated in the wall-less state remain poorly understood. Here, we demonstrate that fluorescence-activated cell sorting (FACS) provides a robust approach for quantitative analysis of Escherichia coli L-forms. Flow cytometry revealed two reversible subpopulations differing in intracellular nucleic acid content, which rapidly re-established their heterogeneous distribution following sorting. Remarkably, cells with initially low nucleic acid content underwent a synchronized, population-wide increase in nucleic acid synthesis within 24 hours, a phenomenon that was independently confirmed by time-lapse fluorescence microscopy. Analysis of liquid cultures stained with SYTO and DAPI dyes showed that this transient increase occurred during early exponential growth and was driven predominantly by RNA rather than DNA synthesis. Consistent with observations in walled E. coli , RNA levels peaked before declining as cultures transitioned towards nutrient limitation. Together, these findings establish FACS as a powerful tool for studying L-form biology and reveal that, despite the absence of an intact cell wall and canonical cell division, L-forms retain coordinated, population-wide regulation of nucleic acid synthesis.
Importance
Wall-deficient, or L-form, bacteria are increasingly recognized as biologically and medically relevant. L-forms can survive without the cell wall structure targeted by many commonly used antibiotics, and have been associated with persistent and recurrent infections, including recurrent urinary tract infections caused by Escherichia coli (Mickiewicz et al., 2019). Understanding how these unusual bacterial forms survive, grow and organize essential cellular processes is important for both fundamental microbiology and for understanding their potential clinical relevance. However, studying these cells is challenging because of their fragile nature. Reliable technologies that can analyze individual cells and separate distinct subpopulation are therefore needed. In this study, we investigate wall-deficient forms of Escherichia coli and demonstrate the usefulness of flow cytometry as a tool to study these cells. Expanding the methods available for studying wall-deficient bacteria will help clarify their biology and improve our understanding of their role in infection and treatment failure.
