Biology subjects

Alwardt, D.

Publications and source records attributed to Alwardt, D..

3 recordsLinked to original sources

bioRxiv2025

mAIcrobe: an open-source framework for high-throughput bacterial image analysis

Quantitative analysis in bacterial microscopy is often hindered by diverse cell morphologies, population heterogeneity, and the requirement for specialised computational expertise. To address these challenges, mAIcrobe is introduced as an open-source framework that broadens access to advanced bacterial image analysis by integrating a suite of deep learning models. mAIcrobe incorporates multiple segmentation algorithms, including StarDist, CellPose, and U-Net, alongside comprehensive morphological profiling and an adaptable neural network classifier, all within the napari ecosystem. This unified platform enables the analysis of a wide range of bacterial species, from spherical Staphylococcus aureus to rod-shaped Escherichia coli, across various microscopy modalities within a single environment. The biological utility of mAIcrobe is demonstrated through its application to antibiotic phenotyping in E. coli and the identification of cell cycle defects in S. aureus DnaA mutants. The modular design, supported by Jupyter notebooks, facilitates custom model development and extends AI-driven image analysis capabilities to the broader microbiology community. Building upon the foundation established by eHooke, mAIcrobe represents a substantial advancement in automated and reproducible bacterial microscopy.

microbiology↗

bioRxiv2025

The ParA-like ATPase PldP influences the TatA dynamics in Corynebacterium glutamicum

In bacterial cells, precise localization of protein complexes is achieved by unique positioning systems. One of the examples of such positioning systems is the ParAB-parS which is responsible for plasmid and chromosome segregation. In Corynebacterium glutamicum, a parAB deletion results in cell division and growth defects, while deletion of an orphan ParA-like protein pldP results only in a moderate cell division phenotype. Having confirmed a basal ATPase activity of PldP, we aimed to explore if the {Delta}pldP-related phenotype could be a consequence of the mislocalized secreted proteins, as the loss of extracellular proteins involved in cell wall metabolism results in a similar phenotype characterized by disrupted separation of daughter cells. Putative peptidoglycan hydrolase Rv2525c from Mycobacterium tuberculosis and Rv2525c-like glycoside hydrolase-like domain-containing protein Cg0955 from C. glutamicum were previously shown to be transported outside of the cell by twin-arginine protein translocation machinery (Tat). Here, we found that although the deletion of pldP did not lead to the altered secretion of the putative hydrolase Cg0955 by the Tat system, it resulted in the reduction of the Tat dynamics. Our findings highlight the interplay between the ParA-like ATPase PldP and the Tat translocon and contribute to the studies of ParA-like proteins being essential in positioning various cargos in the bacterial cells. ImportancePrecise spatio-temporal localization of protein complexes within a bacterial cell is essential for the survival and proliferation of bacteria. ParA-like ATPases play a crucial role in protein positioning, as well as chromosome and plasmid segregation. Here, we characterize a novel ParA-like ATPase PldP in Corynebacterium glutamicum, a model organism for the cell biology of Mycobacteriales and a biotechnological workhorse. Deletion of pldP results in the cell division phenotypes and impacts the intracellular dynamics of TatA, a component of the twin-arginine protein transport. We suggest that the mislocalization of the Tat-secreted putative peptidoglycan hydrolase caused by the indirect influence of pldP deletion might account for the observed cell separation defect.

microbiology↗

bioRxiv2022

A CRISPRi-based genetic resource to study essential Staphylococcus aureus genes

We have optimized a CRISPR interference system to facilitate gene knockdown in the gram-positive bacterial pathogen Staphylococcus aureus. Our approach used a CRISPRi system derived from Streptococcus pyogenes, which involves the co-expression of the dcas9 gene encoding a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In our system, dcas9 is expressed from a single copy in the chromosome of methicillin resistant S. aureus (MRSA) strains COL or JE2, under the control of a tightly regulated promoter, inducible by anhydrotetracycline. The sgRNAs are expressed from a replicative plasmid under the control of a constitutively active promoter. This system enables efficient, inducible, knockdown of both essential and non-essential genes. Using this approach, we constructed the Lisbon CRISPRi Mutant Library (LCML) comprising 261 strains, in the JE2 background, containing sgRNAs targeting 200 essential genes/operons. This library facilitates the study of the function of essential S. aureus genes and is complementary to the Nebraska Transposon Mutant Library which consists of nearly 2000 strains, each carrying a transposon insertion within a non-essential gene. The availability of these two libraries will facilitate the study of S. aureus pathogenesis and biology. Abstract ImportanceStaphylococcus aureus is an important clinical pathogen that causes a high number of antibiotic resistant infections. The study of S. aureus biology, and particularly of the function of essential proteins, is of particular importance to develop new approaches to combat this pathogen. We have optimized a CRISPRi system that allows efficient targeting of essential S. aureus genes. Furthermore, we have used that system to construct a library of 261 strains which allow the depletion of essential proteins encoded in 200 genes/operons. This library, which we have named Lisbon CRISPRi Mutant Library (LCML), should facilitate the study of S. aureus pathogenesis and biology.

microbiology↗