Biology subjects

Crooijmans, M. E.

Publications and source records attributed to Crooijmans, M. E..

3 recordsLinked to original sources

bioRxiv2026

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.

microbiology↗

bioRxiv2024

Enhanced extracellular matrix production provides protection to cell wall-deficient Escherichia coli

Escherichia coli-induced recurrent urinary tract infections (rUTIs) present a complicated challenge within the medical field. Most first-line antibiotic treatments primarily target cell-wall synthesis, which can lead to the formation of cell wall-deficient cells. To investigate how such cells can sustain, we obtained an E. coli strain capable of efficiently proliferating without its cell wall. One of the mutations lead to enhanced expression of rcsA, encoding an important regulator involved in responding to cell envelope stress. RNA sequencing demonstrated an upregulation of genes associated with the production of extracellular matrix components, and this increased extracellular matrix production was confirmed using various imaging techniques. Remarkably, a subsequent long-term evolution experiment on this strain revealed a further augmentation in extracellular matrix production, coinciding with an enhanced ability to withstand harsh conditions. These findings demonstrate how E. coli adapts to loss of its cell wall and that an increased synthesis of matrix constituents can compensate for the protective properties of the cell wall. SignificanceThe cell envelope is crucial for the protection of E. coli. During bacterial infections such as urinary tract infections, antibiotics that disrupt cell wall synthesis are commonly prescribed. However, this can stimulate the formation of wall-deficient bacteria that are still able to proliferate despite the presence of these drugs. Our findings reveal that loss of the cell wall in E. coli increases the production of extracellular matrix, a mechanism found in other unicellular organism too. This adaptation allows the bacteria to maintain their structural integrity and survive, highlighting a potential challenge in the successful treatment of bacterial infections.

microbiology↗

bioRxiv2020

Rapidemic, a versatile and label-free DNAzyme-based platform for visual nucleic acid detection

In the last three decades, there have been recurring outbreaks of infectious diseases, brought to light with the recent outbreak of coronavirus disease 2019 (COVID-19). Attempts to effectively contain the spread of infectious diseases have been hampered by the lack of rapidly adaptable, accurate, and accessible point-of-care diagnostic testing. In this study, we present a novel design of a label-free DNAzyme-based detection method called Rapidemic. This assay combines recombinase polymerase amplification (RPA) with linear strand-displacement amplification (LSDA) and guanine-quadruplex (GQ) DNAzyme-catalysed colour-changing reaction. The colorimetry basis of the signal readout omits the need for extensive instrumentation. Moreover, the primer-based sequence detection of RPA gives Rapidemic a potential to be rapidly adapted to target a new sequence. As a proof of concept, we developed the assay to detect isolated genomic DNA of Saccharomyces cerevisiae. The use of low-pH buffers and the optimization of the dilution rates from each preceding reaction to the next showed to be successful strategies to enable visible detection with this method. These findings demonstrate for the first time that a label-free DNAzyme-based detection method can be coupled to RPA and LSDA for nucleic acid detection.

molecular biology↗