Biology subjects

Garner, E.

Publications and source records attributed to Garner, E..

10 recordsLinked to original sources

bioRxiv2026

Inherited flagellar structures coordinate the onset of swimming among related E. coli cells

Bacteria commit to costly behaviors before they can benefit from them. In Escherichia coli, flagellar synthesis is expensive, yet the genes are expressed in stochastic pulses and only a small minority of pulses result in motility. Despite this noise, we find that lineage-related cells begin swimming at similar times, in groups of two to eight cells. Rare, high-amplitude pulses commit a single cell within one generation, but more commonly, pulses rise slowly and fail to trigger swimming before division. Rather than decaying, their output accumulates as cells inherit hook-basal bodies (HBBs) and continue to build new ones. This structural inheritance acts as a physical integrator, summing ongoing transcriptional activity across generations, so that an incomplete flagellar cascade is carried forward at division rather than lost. HBB accumulation sets when cells swim, and the anti-sigma factor FlgM sets how sharply. By holding late flagellar genes off until several HBBs have accumulated, FlgM makes motility depend steeply on early flagellar gene expression, so that relatives inheriting similar sets of HBBs become motile together. Without FlgM, this sharp dependence is largely lost, and activations of the full cascade do not reliably produce swimming. Together, our results show how the accumulation of a molecular machine integrates fluctuations, and how a checkpoint on its assembly sharpens them into a decision, setting both the speed and heritability of a behavioral trait.

systems biology↗

bioRxiv2025

The interplay of membrane tension and FtsZ filament condensation on the initiation and progression of cell division in B. subtilis

The first step of cell division is deforming the planar cell membrane inward towards the cytoplasm. As deforming membranes is energetically costly, biology has developed various protein systems to accomplish this task. The mechanisms providing the force to deform bacterial membranes to initiate division remain unknown. In vivo studies have shown the condensation of FtsZ filaments into a sharp ring is required to initiate cell division, an observation mirrored in vitro with FtsZ filaments encapsulated inside liposomes. Similarly, the force for membrane deformation in many eukaryotic deforming systems arises from the local crowding of proteins on the membrane surface. As any membrane deforming system works against the membrane tension, here we modulated the amount of lipid synthesis and thus membrane tension in Bacillus subtilis to examine: 1) if the condensation of FtsZ filaments by FtsZ bundling proteins serves to overcome the cellular membrane tension to deform the membrane inward and 2) how changes to the membrane tension affect the subsequent invagination of the septum. First, we developed methods to simultaneously measure and modulate membrane tension in live cells. Next, we determined how altering the membrane tension affected the cells ability to initiate division with reduced levels of FtsZ bundling proteins. While cells depleted of 2 FtsZ bundling proteins were unable to divide, reducing membrane tension to a given threshold restored their ability to initiate division. Likewise, cells with intermediate levels of FtsZ bundling proteins required a lesser decrease in membrane tension to initiate division. We also found that reductions in membrane tension increase the rate of Z ring constriction, with the constriction rate scaling linearly with the membrane tension. Interestingly, while the constriction rate in wild-type B. subtilis is limited by FtsZ treadmilling, the rate of constriction becomes independent of FtsZs treadmilling rate when membrane tension is reduced. These experiments give two major insights: First, the filament condensation caused by FtsZ bundling proteins works to overcome membrane tension and deform the membrane inward to initiate division. Second, the rate of septal constriction is limited by membrane tension, suggesting that membrane fluctuations at the tip of the growing septa limit the rate of cell wall synthesis. Finally, our measurements allow the estimation of several physical values of cell division, such as the force required to bend the membrane, but also that the cell membrane provides only 0.1%, a small amount of surface tension relative to the entire cell envelope, indicating 99.9% of the pressure drop occurs across the cell wall. These calculations also indicate that cell division occurs via comparatively very small membrane tension fluctuations relative to the high turgor pressure that exists across the entire cell envelope.

microbiology↗

bioRxiv2021

Cell-envelope synthesis is required for surface-to-mass coupling, which determines dry-mass density in Bacillus subtilis

Cells must increase their volumes in response to biomass growth to maintain intracellular mass density, the ratio of dry mass to cell volume, within physiologically permissive bounds. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. Recently, we demonstrated that the Gram-negative bacterium Escherichia coli expands cell-surface area rather than volume in proportion to mass. Here, we investigate the regulation of cell-volume growth in the evolutionarily distant Bacillus subtilis. First, we demonstrate that the coupling of surface growth to mass growth is conserved in B. subtilis. Therefore, mass density changes with cell shape at the single-cell level. Interestingly, mass density varies by more than 30% when we systematically change cell width by modulation of cell-wall insertion, without any effect on mass-growth rate. Second, we demonstrate that the coupling of surface- and mass growth is broken if peptidoglycan or membrane synthesis are inhibited. Once transient perturbations are relieved, the surface-to-mass ratio is rapidly restored. In conclusion, we demonstrate that surface-to-mass coupling is a conserved principle for volume regulation in bacteria, and that envelope synthesis provides an important link between surface growth and biomass growth in B. subtilis.

microbiology↗

bioRxiv2021

Bacterial galactosemia is caused by cytoplasmic interference of an essential cell wall biosynthesis glycosyltransferase

Bacterial galactosemia or "galactose death," triggered by incomplete galactose metabolism, was first discovered in Escherichia coli and Salmonella six decades ago, and later in many other microorganisms, yet the mechanism for the toxicity and subsequent cell death remains unclear. In Bacillus subtilis, galactosemia is manifested by a buildup of uridine-diphosphate-galactose (UDP-Gal) and a strong toxicity phenotype characterized by cell shape abnormality and rapid cell lysis. Here we present evidence that in B. subtilis, the toxicity is due to inhibition of cell wall biosynthesis through interference of the essential glycosyltransferase MurG that carries out lipid II synthesis from lipid I and uridine-diphosphate-N-acetyl-glucosamine (UDP-GlcNAc). Single-molecule imaging reveals real-time inhibition of cell wall biosynthesis and MurG activities in cells exhibiting toxicity. We further show that in vitro, MurG is able to utilize UDP-Gal as a substrate generating a "toxic" lipid II, causing a potential poisoning effect on peptidoglycan crosslinking. Evidence also suggests a similar mechanism in Vibrio cholerae and Staphylococcus aureus. Lastly, a strong synergistic lethality was seen in S. aureus wild-type cells treated with both galactose and sub-lethal doses of cell-wall antibiotics. Our study provides mechanistic explanation of the toxicity associated with bacterial galactosemia and its potential application in antibacterial solutions. SignificanceGalactosemia is a potentially fatal genetic disorder due to incomplete galactose metabolism, found in both eukaryotic and prokaryotic organisms. The molecular mechanisms of galactosemia-associated toxicity remain unclear in all cases. Here we present evidence that in the bacterium Bacillus subtilis, the toxicity is due to interference of an essential glycosyltransferase, MurG, which concerts lipid I to lipid II during peptidoglycan biosynthesis, by a nucleotide sugar derived from galactose metabolism. This interference leads to a halt of cell wall biosynthesis and structural defects causing rapid cell lysis. Our evidence also suggests a similar mechanism in other bacteria such as Staphylococcus aureus and Vibrio cholerae. Our study may help solve the long-time puzzle of bacterial galactosemia first uncovered six decades ago.

microbiology↗

bioRxiv2019

Visualization and Analysis of Whole Depot Adipose Tissue Innervation

Little is known about the diversity and function of adipose tissue nerves due, in part, to the inability to effectively visualize the various nerve subtypes residing within these tissues. The tools currently available for researchers to image and quantify adipose tissue innervation are limited and dependent on optical clearing techniques and light sheet microscopy. Here we present a method of tissue processing that uses mechanical force to compress tissue to decrease tissue thickness in the z-axis by expanding it in the x and y-axes whilst leaving cells intact. This has been combined with autofluorescence quenching techniques to permit intact whole tissues to be mounted on slides and imaged on any microscope, with a complementary means to perform whole tissue neurite density quantification. We have included examples of how this technique can be used to further our current knowledge of adipose-nerve communication by characterizing the nerves, nerve-subtypes, and neurovascular interactions within subcutaneous white adipose tissue in mice.

neuroscience↗

bioRxiv2019

Whole genome sequence analysis reveals broad distribution of the RtxA type 1 secretion system and four novel type 1 secretion systems throughout the Legionella genus

Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, suggesting that this system is a conserved virulence factor in L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legonella repeat protein secretion system (LRPSS) shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1-3) secretion systems (LB1SS-LB3SS) T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p = 0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a genetic virulence determinant.

microbiology↗

bioRxiv2018

An activation pathway governs cell wall polymerization by a bacterial morphogenic machine

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall (CW) assembly. In Escherichia coli, alterations in a CW synthase component of the system called PBP2 were identified that overcome other inactivating defects. Rod system activity was stimulated in the suppressors in vivo, and purified synthase complexes with these changes showed more robust CW synthesis in vitro. Polymerization of the actin-like MreB component of the Rod system was also found to be enhanced in cells with the activated synthase. The results suggest an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both CW glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient CW assembly. An analogous activation pathway involving similar enzymatic components is likely responsible for controlling CW synthesis by the division machinery.

microbiology↗

bioRxiv2018

ARG-miner: A web platform for crowdsourcing-based curation of antibiotic resistance genes

Curation of antibiotic resistance gene (ARG) databases is a labor-intensive process that requires expert knowledge to manually collect, correct, and/or annotate individual genes. Correspondingly, updates to existing databases tend to be infrequent, commonly requiring years for completion and often containing inconsistences. Further, because of limitations of manual curation, most existing ARG databases contain only a small proportion of known ARGs (~5k genes). A new approach is needed to achieve a truly comprehensive ARG database, while also maintaining a high level of accuracy. Here we propose a new web-based curation system, ARG-miner, which supports annotation of ARGs at multiple levels, including: gene name, antibiotic category, resistance mechanism, and evidence for mobility and occurrence in clinically-important bacterial strains. To overcome limitations of manual curation, we employ crowdsourcing as a novel strategy for expanding curation capacity towards achieving a truly comprehensive, up-to-date database. We develop and validate the approach by comparing performance of multiple cohorts of curators with varying levels of expertise, demonstrating that ARG-miner is more cost effective and less time-consuming relative to traditional expert curation. We further demonstrate the reliability of a trust validation filter for rejecting confounding input generated by spammers. Crowdsourcing was found to be as accurate as expert annotation, with an accuracy >90% for the annotation of a diverse test set of ARGs. ARG-miner provides a public API and database available at http://bench.cs.vt.edu/argminer.

bioinformatics↗

bioRxiv2017

MreB Filaments Create Rod Shape By Aligning Along Principal Membrane Curvature.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion and increased glycan crosslinking. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

microbiology↗

bioRxiv2017

DeepARG: A deep learning approach for predicting antibiotic resistance genes from metagenomic data

Growing concerns regarding increasing rates of antibiotic resistance call for global monitoring efforts. Monitoring of environmental media (e.g., wastewater, agricultural waste, food, and water) is of particular interest as these media can serve as sources of potential novel antibiotic resistance genes (ARGs), as hot spots for ARG exchange, and as pathways for the spread of ARGs and human exposure. Next-generation sequence-based monitoring has recently enabled direct access and profiling of the total metagenomic DNA pool, where ARGs are identified or predicted based on the \"best hits\" of homology searches against existing databases. Unfortunately, this approach tends to produce high rates of false negatives. To address such limitations, we propose here a deep leaning approach, taking into account a dissimilarity matrix created using all known categories of ARGs. Two models, deepARG-SS and deepARG-LS, were constructed for short read sequences and full gene length sequences, respectively. Performance evaluation of the deep learning models over 30 classes of antibiotics demonstrates that the deepARG models can predict ARGs with both high precision (>0.97) and recall (>0.90) for most of the antibiotic resistance categories. The models show advantage over the traditional best hit approach by having consistently much lower false negative rates and thus higher overall recall (>0.9). As more data become available for under-represented antibiotic resistance categories, the deepARG models performance can be expected to be further enhanced due to the nature of the underlying neural networks. The deepARG models are available both in command line version and via a Web server at http://bench.cs.vt.edu/deeparg. Our newly developed ARG database, deepARG-DB, containing predicted ARGs with high confidence and high degree of manual curation, greatly expands the current ARG repository. DeepARG-DB can be downloaded freely to benefit community research and future development of antibiotic resistance-related resources.\n\nAbbreviations

bioinformatics↗