Fluorescence-activated cell sorting of Escherichia coli L-forms reveals maintained synchronized nucleic acid synthesis dynamics

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.