Biology subjects

Holowka, J.

Publications and source records attributed to Holowka, J..

4 recordsLinked to original sources

bioRxiv2026

FtsK with a unique N-terminal extension is involved in coordinating the final steps of chromosome segregation with asymmetrical division in mycobacterial cells

In this study, we identify and functionally characterize a previously unrecognized, conserved N-terminal extension of mycobacterial FtsK (nFtsK) that contributes to the coordination of chromosome segregation with asymmetric cell division. Although FtsK is broadly conserved in bacteria as a late-stage DNA translocase and divisome component, mycobacterial FtsK uniquely contains a long, positively charged, intrinsically disordered N-terminal region. We show that this extension is dispensable for core FtsK functions--including septal localization, DNA translocation, and completion of cell division--but instead modulates the spatial confinement, temporal regulation, and interaction specificity of these processes. Mechanistically, nFtsK mediates specific protein interaction, binds the anionic phospholipids cardiolipin and phosphatidic acid, and exhibits intrinsic affinity for negatively curved membrane. Together, our findings uncover a lineage-specific adaptation that fine-tunes a conserved molecular machine, enhancing the robustness of asymmetric cell division in mycobacteria.

molecular biology↗

bioRxiv2024

SpoIIDMP-driven peptidoglycan rearrangement is crucial for ribosome translocation into the spore.

In a spore-forming bacterium Bacillus subtilis transcription and translation are uncoupled and the translational machinery is located at the cell poles. During sporulation the cell undergoes morphological changes including asymmetric septation and chromosome translocation. However, the fate of translational machinery during sporulation has not been described. Here, using a combination of microscopic assays and mass spectrometry, we are tracking the ribosome localisation during sporulation in B. subtilis WT and mutants. We show that the ribosomes are associated with the asymmetric septum which is a functionally important organelle and that peptidoglycan rearrangement is essential for ribosome packing into the forespore. We also show that the feeding tube channel SpoIIIA-SpoIIQ is not required for the ribosome translocation, but is essential for maintaining the chromosome inside the spore. One-Sentence SummaryMovement of ribosomes into the spore of B. subtilis follows chromosome transport and is precisely orchestrated in the cell.

microbiology↗

bioRxiv2020

Lsr2 is a nucleoid-associated protein that exerts pleiotropic effects on mycobacterial cellular processes

Lsr2 is involved in maintaining chromosome structure in asymmetrically dividing mycobacteria and is essential in the tubercle bacillus (M. tuberculosis) during infection. Here, we report that a lack of Lsr2 profoundly impacts the mycobacterial cell morphology and the properties of the cell envelope resulting in the formation of smooth, short and antibiotics sensitive cells. Lsr2 forms large and dynamic nucleoprotein complexes in vivo and deletion of lsr2 gene exerts a profound effect on the replication time and replisome dynamics. We suggest that the Lsr2 nucleoprotein complexes may contribute to maintaining the proper organization of the newly synthesized DNA. Moreover, we demonstrate that the N-terminal oligomerization domain of Lsr2 is indispensable for the formation of nucleoprotein complexes in vivo. Collectively, our results indicate that Lsr2 exerts a pleiotropic effect on cellular processes and appears to be an attractive target for the development of a novel antitubercular drugs.

microbiology↗

bioRxiv2018

Watching antibiotics in action: Exploiting time-lapse microfluidic microscopy as a tool for target-drug interaction studies in Mycobacterium

Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria. The genus Mycobacterium encompasses both human and animal pathogens that cause severe diseases and have profound impacts on global health and the world economy. Here, we used a novel system of microfluidics, fluorescence microscopy and target-tagged fluorescent reporter strains of M. smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid, nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, and griselimycin caused replisome instability with subsequent over-initiation of chromosome replication and over-relaxation of the nucleoid. This work is an example of using a microscopy-based approach to evaluate the activity of potential replication inhibitors and provides mechanistic insights into their modes of action. Our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.). Because proteins involved in the DNA replication are well conserved among bacteria (including mycobacterial species), the properties of various replication inhibitors observed here in fast-growing M. smegmatis may be easily extrapolated to slow-growing pathogenic tubercle bacilli, such as M. tuberculosis.\n\nSignificanceThe growing problem of bacterial resistance to antibiotics and the emergence of new strains that are resistant to multiple drugs raise the need to explore new antibiotics and re-evaluate the existing options. Here, we present a system that allows the action of antibiotics to be monitored at the single-cell level. Such studies are important in the light of bacterial heterogeneity, which may be enhanced in unfavorable conditions, such as under antibiotic treatment. Moreover, our studies provide mechanistic insights into the action modes of the tested compounds. As combined therapies have recently gained increased interest, it is also notable that our described system may help researchers identify the best combination of antimicrobials for use against infections caused by a variety of bacteria.

microbiology↗