Biology subjects

Adebiyi, K. O.

Publications and source records attributed to Adebiyi, K. O..

5 recordsLinked to original sources

bioRxiv2026

A two-step model of FtsZ-ring disassembly in Bacillus subtilis

Bacillus subtilis grows and divides by binary fission, directed by medial localization of cell division protein FtsZ. Disruption of either the Min system or EzrA results in aberrant FtsZ positioning. Here we compare FtsZ dynamics in cells disrupted for either MinD or EzrA when grown in microfluidic channels. Here we show that cells lacking MinD or EzrA appear to be similarly defective in Z-ring disassembly after septation, but play different roles as simultaneous disruption results in a synergistic defect in division. Moreover, we account for a low frequency of minicell formation in the absence of EzrA, as MinD but not EzrA is necessary for removal of ZapA from polar Z-rings. Finally, overexpression of MinCD results inhibits division through pervasive Z-ring disassembly but appears to concentrate ZapA through localized sequestration. Combined, our results indicate a closer relationship between MinCD and ZapA than previously recognized and show that Z-ring disassembly can be genetically separated into discrete steps. We propose a two-step model for Z-ring disassembly that mirrors the assembly process, such that after and/or during septation, the Z-ring separately decondenses and protofilaments are disassembled to monomers for recycling.

microbiology↗

bioRxiv2026

MinJ is a conserved nine-pass transmembrane protein that contains a putative transmembrane β-sheet

The Min system disassembles FtsZ-rings after septation in Bacillus subtilis and is localized to the nascent division plane and cell poles by the protein MinJ. The N-terminal region of MinJ contains transmembrane segments while the C-terminal region of MinJ contains a PDZ domain but its topology and functional domains are poorly understood. Here we empirically test MinJ topology based on a variety of transmembrane prediction models and find that the data is most consistent with Alphafold3, which predicts a 9-pass transmembrane protein with an external N-terminus and internal C-terminus. Deletion analysis indicates that all regions of the protein tested are required for function but deletion of the PDZ domain alone preserves polar localization and interaction with both MinD and DivIVA. Moreover, Alphafold predicts that transmembrane segments 6 and 7 comprise staves of an unusual transmembrane {beta}-sheet and deletion of the putative {beta}-sheet in the absence of MinD results in a minicell frequency that exceeds mutation of MinD alone. Bioinformatic analysis indicates that MinJ is highly conserved within Firmicutes and is co-conserved with MinD and DivIVA with which it interacts. Our data clarify the structure of MinJ and support models in which MinJ has functions in addition to restricting the activity of the Min system. IMPORTANCEFaithful positioning of the bacterial division site is important for cell growth and is coordinated by the conserved Min system. Although the Min system of Bacillus subtilis has been extensively studied, MinJ, the membrane protein that links the division inhibitor MinCD to the polar determinant DivIVA, remains the least well-understood. Here we experimentally define the membrane topology of MinJ and show that our data are most consistent with a nine-pass transmembrane architecture predicted by AlphaFold3. We further provide genetic, cell biological, and evolutionary evidence supporting that two of the staves form a highly conserved putative transmembrane {beta}-sheet, a structure normally excluded from the plasma membrane. Our findings refine MinJ structural organization and provide a framework for understanding its conserved functions in bacterial cell division.

microbiology↗

bioRxiv2026

Efficient septum formation is essential for chromosome segregation in Bacillus subtilis when SMC function is impaired

Structural maintenance of chromosomes (SMC) complexes play conserved roles in chromosome organization, segregation, and repair in all domains of life. In Bacillus subtilis, SMC is required for segregation of newly replicated origins. To investigate whether other proteins function with SMC in this process, we performed a synthetic lethal screen with an smc hypomorphic allele (smc*) that is mildly defective in chromosome segregation. In addition to recovering previously reported interactions of smc with parB and spoIIIE, our screen identified minJ and divIVA as essential in the smc* background. We show that the synthetic lethality between smc* and{Delta} minJ or{Delta} divIVA arises from defects in segregating the replication terminus. Importantly, deletion of minD, which suppresses the cell division defects of{Delta} minJ and{Delta} divIVA, restored terminus segregation and viability in the smc* background. These findings support a model in which proper septum formation promotes chromosome terminus resolution and segregation by enabling SpoIIIE-mediated DNA clearance from the division septum during cytokinesis. These findings highlight the interdependence between chromosome segregation and cell division. ImportanceThe SMC complex plays a central role in chromosome organization and segregation in Bacillus subtilis, but the cellular functions that become important when SMC activity is reduced are not well understood. Using a hypomorphic smc allele, we discovered that mutations affecting cell division become essential when chromosome organization and segregation are impaired. Our findings support a model in which efficient septum formation enables proper localization of the SpoIIIE DNA translocase, which in turn resolves and segregates the chromosome terminus region. These results highlight the critical role of cell division in supporting chromosome segregation.

microbiology↗

bioRxiv2026

Flagellar toxicity: flagellar synthesis is lytic for Bacillus subtilis in the absence of PBP1

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in Gram positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. Mutation of penicillin binding protein 1 (PBP1/PonA), a cell wall repair protein thought to decrease peptidoglycan pore frequency and/or size, resulted in a severe growth defect and cell lysis in the ancestral strain of Bacillus subtilis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan hydrolases that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, our work indicates that cost of envelope integrity by flagellar synthesis may be underappreciated due to strain domestication, and suggests that specialized systems may compensate for the cost of assembly of transenvelope machines in general. SIGNIFICANCEBacteria assemble nanomachines through the cell envelope but how the machines transit the peptidoglycan is poorly understood. Here we find that assembly of trans-envelope flagella results in cell lysis of Bacillus subtilis when the peptidoglycan repair protein PBP1 is absent. Lysis was due to multiple peptidoglycan lyases expressed as a consequence of flagellar assembly, and lytic activity required another PBP homolog, PBPX. Our work indicates that flagella, not normally thought to impact cell viability, can be lethal at the level of cell envelope integrity.

microbiology↗

bioRxiv2026

Constitutive, endogenous, fluorescent membrane reporters for dynamic cell cycle analysis in Bacillus subtilis

Bacteria increase in biomass and divide, but determining precisely when cell division completes is technically challenging. To aid time-lapse imaging and cell-cycle tracking, we set out to identify a protein in Bacillus subtilis, which when fused with a fluorophore would cause the membrane to fluoresce in a manner that was constitutive, uniform, and bright. A forward genetic transposon-based approach combined with fluorescence-activated cell sorting was used to identify a fluorescent fusion to the glucose PTS transport transmembrane protein PtsG with all desired properties. Moreover, PtsG-GFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsG-GFP to track cell growth in microfluidic channels and determine when cytokinesis occurred, defined as when fluorescence reached a local maximum at the division plane. Simultaneous imaging with a compatible fluorescent fusion to the cell division protein FtsZ indicated that FtsZ peak intensity occurred midway through septum constriction and that Z-ring recycling coincided with cytokinesis. We conclude that PtsG-GFP is a powerful tool for membrane imaging and cell cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and antibiotic resistance cassettes to facilitate deployment in B. subtilis. IMPORTANCEBacterial cells are fully divided when new membrane separates the cytoplasm of each daughter. Reproducibly staining of bacterial membranes with exogenous labels for fluorescence microscopy can be challenging, particularly during chemostatic growth in microfluidic devices. Here, we report that fusion of a fluorescent protein to the glucose transport protein PtsG causes the membrane of Bacillus subtilis to give off bright and even fluorescence under a variety of conditions. We use PtsG-GFP to operationally define when cytokinesis occurs during growth, and we note that a fluorescent PtsG fusion would likely make fluorescent staining of the membrane more facile theoretically in any organism.

microbiology↗