Biology subjects

Holt, J. D.

Publications and source records attributed to Holt, J. D..

4 recordsLinked to original sources

bioRxiv2025

Spatial constraint drives negative frequency dependent selection of phage weaponization

Biofilm growth and phage exposure are common to diverse bacterial species. Studying phage-host interaction and population dynamics in biofilms with cellular resolution remains a significant challenge, especially when attempting to recapitulate the natural environments that microbes occupy. Here we study the population dynamics of phage K139 lysogenized and non-lysogenized Vibrio cholerae when growing in biofilms on the surface of chitin particles in seawater, replicating key features of V. cholerae ecology in the marine environment. We find that lysogenized V. cholerae, via spontaneous lytic induction and phage release, kill and displace non-lysogenized bacteria on chitin surfaces. After lysogens become common, however, they can no longer displace remaining non-lysogenized cells. Using a combination of modeling approaches and microscopy experiments, we show that the lysogens capacity to displace non-lysogens depends on the ability of phages released by spontaneous induction to reach susceptible non-lysogens. Phage access to non-lysogenized bacterial hosts declines once lysogens become common, and this occurs due to the spatial constraints inherent to biofilm growth as well as to superinfection immunity, which neutralizes phage particles adsorbed to lysogens. Once lysogens comprise the majority of the host population, they can be selected against, because they still incur the cost of spontaneous induction without gaining the benefit of killing non-lysogen cells via phage release. The cost of lytic induction, phage-mediated killing of non-lysogens, and constraints on phage mobility within host bacterial biofilms together yield population dynamics that are consistent with negative frequency dependent selection for lysogenized cells under physiologically realistic growth conditions. Significance StatementBacteria often produce and live within biofilm communities in natural environments, where they also encounter many threats including bacteriophages. Here we show how temperate phages can confer a competitive advantage to their hosts via lytic induction and phage release within biofilms of V. cholerae on lab-grown marine snow particles. However, the spatial constraints of biofilm architecture and phage-neutralizing superinfection immunity place limits on the extent to which lysogens can competitively displace non-lysogens via phage release.

microbiology↗

bioRxiv2025

Vibrio cholerae interaction with predatory bacteria on chitin suggests an alternative mode of biofilm formation in marine snow conditions

Vibrio cholerae is a ubiquitous marine microbe that solubilizes and consumes chitin in the marine water column. In both the marine environment and the intestinal track, V. cholerae forms biofilms; a key question regarding the lifestyle of V. cholerae is how do the diverse substrates that it encounters influence its biofilm formation and, in turn, shape its ecological interactions. Here, we use the predator-prey interaction between Bdellovibrio bacteriovorus and V. cholerae as a model to explore how the environmental chitin substrate alters V. cholerae biofilm formation and predator-prey interactions. We find that glass-bound biofilms provide strong protection for V. cholerae against predation while also allowing a population of predatory B. bacteriovorus to remain in place. In contrast, chitin-bound biofilms offer less protection against B. bacteriovorus predation and do not maintain a stable population of B. bacteriovorus. Using percolation and population dynamics models, we predict that these changes in predator-prey dynamics can be mostly explained by alterations in biofilm architecture between the two conditions, which changes the fraction of prey available to B. bacteriovorus. Performing targeted biofilm matrix deletions, we confirm this prediction by recapitulating key features of the chitin predator-prey interactions on glass surfaces. Following on this observation, we show that V. cholerae biofilms grown on chitin produce much less of the canonical biofilm matrix components and instead rely on other extracellular structures. Overall, our experiments detail how growth substrate can alter biofilm matrix composition and how these changes in biofilm architecture and cellular arrangement can impact higher-order ecological interactions.

microbiology↗

bioRxiv2025

Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

Diverse bacterial species utilize surface appendages called type IV pili (T4P) to interact with their environment. These structures are dynamically extended and retracted from the cell surface, which is critical for diverse functions. Some T4P systems rely on two distinct motor ATPases, PilT and PilU, whose combined activities are required to power forceful T4P retraction. However, the mechanism by which these motors coordinate to facilitate T4P retraction has remained unclear. Here, we utilize the competence T4P in V. cholerae as a model system to elucidate the molecular basis for PilT-PilU coordination during T4P retraction. Specifically, we modeled the interactions between PilT and PilU using AlphaFold 3 and molecular dynamics (MD) simulations. We then empirically tested these models using a combination of cytological and high-resolution genetic approaches. Our results reveal that interactions between PilT and the PilU C-terminus are critical for these motors to coordinate to drive T4P retraction. Finally, we show that PilT-PilU interactions are broadly conserved in T4P systems from diverse bacterial species, and we experimentally validate that they are required for T4P retraction in Acinetobacter baylyi. Together, this work expands our fundamental understanding of T4P dynamics, and more broadly it provides mechanistic insight into how these ATPases coordinate to assemble some of the strongest biological motors in nature. SIGNIFICANCEDiverse bacterial species use filamentous surface appendages called type IV pili (T4P) to move along surfaces, take up DNA for horizontal gene transfer, and stick to biotic and abiotic surfaces. The forceful retraction of these filaments is often required for these behaviors. In many T4P systems, the combined activity of two distinct motor ATPase proteins is required for forceful retraction; however, a detailed understanding of how these motor proteins interact to promote forceful retraction is currently lacking. Here, we use an integrated approach to uncover the molecular mechanism for motor ATPase coordination. Furthermore, we show that this mechanism is broadly conserved in diverse T4P systems.

microbiology↗

bioRxiv2022

Computationally efficient assembly of a Pseudomonas aeruginosa gene expression compendium

Over the past two decades, thousands of RNA sequencing (RNA-seq) gene expression profiles of Pseudomonas aeruginosa have been made publicly available via the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). In the work we present here, we draw on over 2,300 P. aeruginosa transcriptomes from hundreds of studies performed by over seventy-five different research groups. We first developed a pipeline, using the Salmon pseudo-aligner and two different P. aeruginosa reference genomes (strains PAO1 and PA14), that transformed raw sequence data into a uniformly processed data in the form of sample-wise normalized counts. In this workflow, P. aeruginosa RNA-seq data are filtered using technically and biologically driven criteria with characteristics tailored to bacterial gene expression and that account for the effects of alignment to different reference genomes. The filtered data are then normalized to enable cross experiment comparisons. Finally, annotations are programmatically collected for those samples with sufficient meta-data and expression-based metrics are used to further enhance strain assignment for each sample. Our processing and quality control methods provide a scalable framework for taking full advantage of the troves of biological information hibernating in the depths of microbial gene expression data. The re-analysis of these data in aggregate is a powerful approach for hypothesis generation and testing, and this approach can be applied to transcriptome datasets in other species. SignificancePseudomonas aeruginosa causes a wide range of infections including chronic infections associated with cystic fibrosis. P. aeruginosa infections are difficult to treat and people with CF-associated P. aeruginosa infections often have poor clinical outcomes. To aid the study of this important pathogen, we developed a methodology that facilitates analyses across experiments, strains, and conditions. We aligned, filtered for quality and normalized thousands of P. aeruginosa RNA-seq gene expression profiles that were publicly available via the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). The workflow that we present can be efficiently scaled to incorporate new data and applied to the analysis of other species.

microbiology↗