Biology subjects

Wise, B. M.

Publications and source records attributed to Wise, B. M..

4 recordsLinked to original sources

bioRxiv2026

Stator pool size shapes the mechanosensitivity of the bacterial flagellar motor

The bacterial flagellar motor adapts to mechanical load by adding or removing stator units, which can either be inactive in the inner membrane (the stator pool) or attached to the motor in a torque-generating configuration. This adaptation is well understood, but what sets the load range over which the motor responds remains unclear. Building on a previous two-state model of stator binding, we derive how the on-rate depends on the diffusing stator pool and find that it increases in proportion to pool size. The model predicts that pool size governs motor occupancy at low load but not at high load, where the motor saturates regardless of how many stators are available. We confirmed this prediction in Escherichia coli: raising stator expression increased swimming speed and single-motor rotation rate at low load. This dependence on expression weakened as load rose and was undetectable at high load. The model further predicts that larger pools make the motor mechanosensitive at lower loads, which our measurements of load-induced occupancy change support. Mechanosensitivity is therefore not fixed by motor architecture alone, but also determined by stator abundance, which cells can set through expression to position their mechanosensory range. SignificanceUsing a statistical mechanics model of stator binding, we predict how flagellar motor output depends on mechanical load and on the number of stators a cell expresses. Combining the model with measurements of swimming cells and single motors, we find that the size of the stator pool sets the range of load over which the motor is mechanosensitive. Mechanosensitivity is therefore a property the cell can tune through stator expression, not a fixed feature of the motor, a principle that may extend to other force-responsive molecular complexes assembled from a limited pool of components.

biophysics↗

bioRxiv2025

Identification and characterization of host-modulating effectors encoded by the Cluster F1 mycobacteriophage NormanBulbieJr

NormanBulbieJr (NBJ) is a temperate siphovirus isolated on the host Mycobacterium smegmatis mc2155 that encodes 102 gene products, 60 of which have no known function (NKF). Based on gene content, NBJ is classified as a Cluster F, Subcluster F1 phage and shares 70% of its encoded gene phamilies with Girr, another F1 mycobacteriophage that was recently analyzed in a genome-wide overexpression screen and found to encode 29 diverse gene products capable of inhibiting growth of M. smegmatis. Similar functional screens in other mycobacteriophages have uncovered a growing repertoire of diverse, phage-encoded bacterial growth inhibitors, providing prime candidates for dissecting novel bacterial-phage interactions. An arrayed overexpression library encoding all 102 genes was constructed and systematically screened using a plate-based cytotoxicity assay, identifying 29 genes that inhibit mycobacterial growth. Because mycobacteriophage genomes are also known to encode systems involved in phage-phage competition, we conducted additional phage defense assays for a subset of NBJ genes in our library. This analysis confirmed homotypic immunity by the predicted immunity repressor and identified gp45 as a second defense factor that protects against Cluster F phages and promotes NBJ lysogen stability. Additional mechanistic analyses indicate that gp45 functions in a secretion-dependent manner, acting after phage adsorption to reduce productive infection and early phage transcript accumulation. Escape mutant and bacterial two-hybrid analyses implicate Cluster F tape measure proteins as the likely target of gp45, consistent with disruption of an early step in phage genome delivery. Finally, we extended our analysis to explore the essentiality of all identified host-modulating genes in the NBJ life cycle, using CRISPR-enhanced recombineering to generate phage deletion mutants, revealing two host modulators that are critical for lytic growth. Conducted as part of the SEA-GENES undergraduate research consortium, this study adds to a growing functional genomics resource and provides new insights into the complex interactions between phage gene products and the mycobacterial host cell.

microbiology↗

bioRxiv2025

Long-range coupling regulates stator dynamics in the bacterial flagellar motor

The bacterial flagellar motor generates torque through MotAB stator complexes which couple ion flux to rotation. Stators anchor in the peptidoglycan cell wall and dynamically remodel in response to changes in external conditions such as the mechanical load, yet how stator anchoring is regulated remains unknown. Here, we show that long-range allosteric interactions within the MotB periplasmic domain tune stator binding in Escherichia coli. Using coarse-grained elastic-network modeling and co-evolutionary analyses, we identified residues mechanically coupled to peptidoglycan-interacting loops of MotB. Targeted mutagenesis at these coupled sites produced distinct motility phenotypes in some mutants, exhibiting altered swimming speeds compared to wild-type and characteristic expression-dependent swimming trends, indicating mutation-specific effects on stator dynamics or torque. Single-motor measurements distinguished mutants with altered torque from those with altered stator dynamics. Molecular dynamics simulations revealed that mutations at distal positions reshape loop flexibility in ways that quantitatively correlate with swimming speeds. These results demonstrate that allosteric communication within MotB propagates across length scales to modulate the performance of the entire motor, revealing how local molecular changes can tune large-scale bacterial motion.

biophysics↗

bioRxiv2023

A genome-wide overexpression screen reveals Mycobacterium smegmatis growth inhibitors encoded by mycobacteriophage Hammy

During infection, bacteriophages produce diverse gene products to overcome bacterial anti-phage defenses, to outcompete other phages, and take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing 25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known anti-mycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Elucidation by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

microbiology↗