Biology subjects

Hughes, K. T.

Publications and source records attributed to Hughes, K. T..

7 recordsLinked to original sources

bioRxiv2026

The cost-benefit trade-off of peritrichous flagellation in bacteria

Many bacteria assemble multiple flagella, although building flagella imposes a substantial biosynthetic and energetic cost. We used the peritrichously flagellated model organism Salmonella enterica to quantify how flagellar abundance affects bacterial growth, proteome allocation, and motility. For this, we generated genetically modified strains with inducible or constitutive expression of the flagellar master regulator flhDC, resulting in a panel of strains ranging from nearly non-flagellated to hyperflagellated cells. We found that higher flagellar investment reduced growth rate and redirected proteome allocation, with an expansion of the flagellar sector occurring largely at the expense of the ribosomal sector. Growth analyses of flagellar assembly mutants, combined with cost modeling, suggested that flagellin biosynthesis dominated the energetic burden, whereas motor rotation contributed a smaller additional cost. Despite the associated cost, increased flagellation improved soft-agar spreading, single-cell swimming speed, effective diffusivity, and competitive fitness in spatially structured environments. A coarse-grained proteome-allocation model parametrized from these data reproduced the observed growth penalties, while simulations of navigation in dynamic chemical gradients predicted that motility benefits saturate near a flagellar investment of 3% of proteome mass. Beyond this point, rising biosynthetic costs outweigh diminishing motility gains. In summary, these results support a quantitative cost-benefit model in which heterogeneous, spatially structured environments favor an intermediate number of flagella by balancing motility benefits against the biosynthetic costs of building and operating multiple flagella.

microbiology↗

bioRxiv2026

Active removal of inhibitory components drives the flagellarType III Secretion Specificity Switch

Type III secretion (T3S) systems assemble bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. In both systems, completion of an early assembly intermediate triggers an irreversible switch from early to late substrate secretion. In the flagellar system, this switch is controlled by the secreted molecular ruler FliK acting on the core T3S component FlhB, but the molecular mechanism governing this transition has remained unclear. Here we show that removal of two components, Fluke and the cleaved C-terminal domain of FlhB (FlhBCCD), locks the secretion apparatus in a constitutive late secretion state. In these mutants, secretion specificity no longer requires completion of the hook-basal body or the FliK ruler, indicating that Fluke and FlhBCCD function to maintain the apparatus in early secretion mode. Consistent with this model, synchronized flagellar gene expression experiments reveal that FlhBCCD is retained during early assembly and is lost coincident with hook-basal body completion and activation of {sigma}28-dependent late gene expression of flagellin and chemosensory genes. Structural modeling of the FliK C-terminal switch domain and FlhBCCD supports a mechanism in which secretion of FliK promotes destabilization and ejection of FlhBCCD from the secretion apparatus. Disruption of a folded region within FliK switch domain uncouples secretion from switching, indicating that the timing of FliK unfolding during secretion is critical for activation of the specificity switch. These findings show that secretion specificity switching is driven by FliK-dependent removal of inhibitory components, rather than passive sensing of assembly completion. Significance StatementType III secretion (T3S) systems build complex bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. How these systems switch secretion specificity during assembly has remained a long-standing question. We demonstrate that the flagellar T3S specificity switch requires removal of two inhibitory components that actively maintain the apparatus in an early secretion state. Their FliK-dependent ejection irreversibly triggers the transition to late substrate export, revealing that secretion switching is controlled by active inhibitory regulation rather than passive sensing of assembly completion. These results define a molecular mechanism for hierarchical control in bacterial secretion systems and provide insights likely relevant to other T3S nanomachines.

microbiology↗

bioRxiv2025

Nanopore sequencing reveals operon-specific ribosome remodeling accompanying naphthyridone resistance in Staphylococcus aureus

Antimicrobial resistance (AMR) threatens global health; however, the molecular adaptations underlying resistance to emerging antibiotic classes remain poorly defined. Here, we applied long-read DNA and direct RNA nanopore sequencing and developed methods to deconvolute operon-specific epitranscriptomic changes. Together, this platform uncovered a previously unrecognized, operon-specific pathway of resistance in Staphylococcus aureus to the naphthyridone antibiotic A-692345. Genomic nanopore sequencing identified a single 23S rRNA mutation (T1732C) confined to one of the six rRNA operons (operon 2), which uniquely contains nine tRNA genes. RNA direct nanopore sequencing generated a comprehensive and updated rRNA modification map for S. aureus and revealed extensive remodeling of rRNA modifications in the resistant strain. Differentially incorporated modifications included pseudouridine, dihydrouridine, and 5-hydroxycytidine at functionally relevant positions within the ribosome. Upon mapping these epitranscriptomic changes, we noted they were operon specific. This likely gives rise to ribosome heterogeneity with potential for selective translation of stress-response genes that favor resistance. Collectively, these findings establish nanopore sequencing as a powerful platform for resolving coupled genomic and epitranscriptomic adaptations, providing molecular insight into how bacteria evolve resistance to antibiotics.

biochemistry↗

bioRxiv2024

FlhE functions as a chaperone to prevent formation of periplasmic flagella in Gram-negative bacteria

The bacterial flagellum is an organelle utilized by many Gram-negative bacteria to facilitate motility. The flagellum is composed of a several {micro}m long, extracellular filament that is connected to a cytoplasmic rotor-stator complex via a periplasmic rod. Composed of [~]20 structural proteins, ranging from a few subunits to several thousand building blocks, the flagellum is a paradigm of a complex macromolecular structure that utilizes a highly regulated assembly process. This process is governed by multiple checkpoints that ensure an ordered gene expression pattern coupled to the assembly of the various flagellar building blocks in order to produce a functional flagellum. Using epifluorescence, super-resolution STED and transmission electron microscopy, we discovered that in Salmonella, the absence of one periplasmic protein, FlhE, prevents proper flagellar morphogenesis and results in the formation of periplasmic flagella. The periplasmic flagella disrupt cell wall synthesis, leading to a loss of the standard cell morphology resulting in cell lysis. We propose a model where FlhE functions as a periplasmic chaperone to control assembly of the periplasmic rod to prevent formation of periplasmic flagella. Our results highlight that bacteria evolved sophisticated regulatory mechanisms to control proper flagellar assembly and minor deviations from this highly regulated process can cause dramatic physiological consequences.

microbiology↗

bioRxiv2020

Molecular structure of the intact bacterial flagellar basal body

Bacterial flagella self-assemble a strong, multi-component drive shaft that couples rotation in the inner membrane to the microns-long flagellar filament that powers bacterial swimming in viscous fluids. We here present structures of the intact Salmonella flagellar basal body, solved using cryo-electron microscopy to resolutions between 2.2 and 3.7 [A]. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the inner membrane rotor component, and at the other end passes through a molecular bearing that is anchored in the outer membrane via interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insight into the assembly process of this molecular machine.

microbiology↗

bioRxiv2020

Regulatory crosstalk between motility and interbacterial communication in Salmonella Typhimurium

FliA is a broadly conserved {sigma} factor that directs transcription of genes involved in flagellar motility. We previously identified FliA-transcribed genes in Escherichia coli and Salmonella enterica serovar Typhimurium, and we showed that E. coli FliA transcribes many unstable, non-coding RNAs from intragenic promoters. Here, we show that FliA in S. Typhimurium also directs transcription of large numbers of unstable, non-coding RNAs from intragenic promoters, and we identify two previously unreported FliA-transcribed protein-coding genes. One of these genes, sdiA, encodes a transcription factor that responds to quorum sensing signals produced by other bacteria. We show that FliA-dependent transcription of sdiA is required for SdiA activity, highlighting a regulatory link between flagellar motility and intercellular communication. IMPORTANCEInitiation of bacterial transcription requires association of a {sigma} factor with the core RNA polymerase to facilitate sequence-specific recognition of promoter elements. FliA is a widely conserved {sigma} factor that directs transcription of genes involved in flagellar motility. We previously showed that Escherichia coli FliA transcribes many unstable, non-coding RNAs from promoters within genes. Here, we demonstrate the same phenomenon in Salmonella Typhimurium. We also show that S. Typhimurium FliA directs transcription of the sdiA gene, which encodes a transcription factor that responds to quorum sensing signals produced by other bacteria. FliA-dependent transcription of sdiA is required for transcriptional control of SdiA target genes, highlighting a regulatory link between flagellar motility and intercellular communication.

microbiology↗

bioRxiv2019

Flagella methylation promotes bacterial adhesion and host cell invasion

The flagellum is the motility device of many bacteria and the long external filament is made of several thousand copies of a single protein, flagellin. While posttranslational modifications of flagellin are common among bacterial pathogens, the role of lysine methylation remained unknown. Here, we show that both flagellins of Salmonella enterica, FliC and FljB, are methylated at surface-exposed lysine residues. A Salmonella mutant deficient in flagellin methylation was outcompeted for gut colonization in a gastroenteritis mouse model. In support, methylation of flagellin promoted invasion of epithelial cells in vitro. Lysine methylation increased the surface hydrophobicity of flagellin and enhanced flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization and successful infection of the host.

microbiology↗