Biology subjects

Alleman, A. B.

Publications and source records attributed to Alleman, A. B..

6 recordsLinked to original sources

bioRxiv2026

Life on leaves left a lasting mark on the Methylobacterium core genome

The extent to which core genomes reflect strictly vertical inheritance remains debated, particularly in ecologically diverse bacterial lineages. Here, we investigate the evolution of the core genome of Methylobacterium, a major leaf-associated bacterial genus, to assess how evolutionary signals vary across genes and whether they relate to functions involved in adaptation to life at the surface of plant leaves (the phyllosphere). Identifying 1,099 protein-coding core genes across 293 genomes, we combined gene tree comparisons, phylogenetic network reconstruction, nucleotide polymorphism patterns, and functional enrichment analyses. We found that most core genes show some deviation from the consensus phylogenetic tree. However, this discordance is not randomly distributed. Across all analytical approaches, genes showing the strongest deviations are consistently associated with functions linked to life in the phyllosphere, including anoxygenic photosynthesis, protection against UV and osmotic stress. These genes are often grouped into conserved clusters in the genome. In particular, a large cluster involved in anoxygenic photosynthesis, including bacteriochlorophyll and carotenoid biosynthesis, shows strong variation in its evolutionary history and organization across lineages, despite maintaining a conserved core structure. Similar patterns of phylogenetic incongruence and gene organization are observed across related Hyphomicrobiales inhabiting the phyllosphere, suggesting that these functions extend beyond Methylobacterium. Together, these results indicate that evolutionary heterogeneity within the core genome is concentrated in genes involved in key ecological functions. While alternative explanations cannot be excluded, these patterns are consistent with horizontal gene transfer contributing to the diversification of traits important for survival and adaptation in phyllosphere bacterial communities.

evolutionary biology↗

bioRxiv2025

Storage polymer polyhydroxybutyrate promotes recovery and limits phenotypic heterogeneity in regrowth from carbon starvation in Methylobacterium extorquens

Bacteria in the wild often encounter long periods of starvation. Evolutionary adaptation to cycles of nutrient deficiency has led to metabolic and physiological strategies that prevent cell death and promote rapid recovery from starvation. A cellular factor shaping outcomes during carbon starvation is polyhydroxybutyrate (PHB), a widespread carbon polymer that helps maintain membrane integrity and promotes regrowth upon reintroduction to nutrients. The response to starvation is often highly heterogeneous across isogenic populations and confounded by environmental variables that mask underlying physiological mechanisms. Prior work has shown that hyper- or hypo-osmolarity greatly increases cell death, but little is known about how PHB and osmolarity jointly influence both population viability and the emergence of heterogeneity in periods of nutrient limitation. To investigate this, we subjected populations of Methylobacterium extorquens to carbon starvation across a range of osmolarities and analyzed the phenotypes of individuals regrowing from the combined stress. By resolving the distributions of key traits such as lag time and growth rate, we found that populations partition into subpopulations that appear either significantly or minimally impaired by the stresses presented. Osmolarity affected total survival, and surprisingly, populations maintained viability in ultra-pure water. PHB metabolism was beneficial for population viability across all conditions, and unexpectedly reduced the variance of populations in lag time and growth rate. These findings show that PHB aids survival and regrowth from starvation, and suggest that the restructuring of phenotypes in long-term stationary phase depends on PHB even though total osmotic sensitivity does not.

microbiology↗

bioRxiv2025

Microbial Cell-Adaptive Segmentation in Quantitative Phase Imaging

Quantitative phase imaging (QPI) enables label-free microscopy with exceptional signal-to-background contrast and provides direct measurements of key biophysical parameters such as cell mass. Despite these advantages, QPI lacks a generalized, accurate, and computationally efficient segmentation method, limiting its use in high-throughput single-cell biology. Here, we introduce microbial Cell-Adaptive Segmentation (mCAS), a purpose-built framework for QPI. mCAS is an object-based, modular pipeline that leverages the robust phase-edge signal to achieve accurate segmentation without Fourier filtering, deconvolution, or deep learning. It runs efficiently on standard hardware, is readily implemented in common software platforms, and requires no curated training data. mCAS consistently outperformed both intensity-based and deep learning approaches, achieving over 98% accuracy with up to tenfold faster processing. By eliminating the segmentation bottleneck, mCAS enables scalable, high-fidelity analysis of QPI datasets and provides a broadly applicable foundation for label-free, high-throughput single-cell biology. We validate its generality across diverse bacterial species, morphologies, and intracellular complexities. Significance StatementQuantitative phase imaging (QPI) enables label-free, noninvasive measurement of cellular biophysics, yet its broader adoption has been limited by the absence of a generalized and computationally efficient segmentation strategy. We present microbial Cell-Adaptive Segmentation (mCAS), a framework purpose-built for QPI that achieves high accuracy without training, specialized hardware, or intensive preprocessing. mCAS generalizes across microbial species and morphologies, outperforming state-of-the-art deep learning approaches while processing thousands of single-cell images in minutes. By eliminating the segmentation bottleneck, mCAS makes QPI broadly accessible for high-throughput, label-free single-cell biology and expands the computational toolkit for quantitative imaging in microbiology.

systems biology↗

bioRxiv2025

Engineered Methylobacterium extorquens grows well on methoxylated aromatics due to its formaldehyde metabolism and stress response

Lignin is a vast yet underutilized source of renewable energy. The microbial valorization of lignin is challenging due to the toxicity of its degradation intermediates, particularly formaldehyde. In this study, we engineered Methylobacterium extorquens to metabolize lignin-derived methoxylated aromatics, vanillate (VA) and protocatechuate (PCA), by introducing the van and pca gene clusters. Compared to Pseudomonas putida, M. extorquens exhibited better formaldehyde detoxification, enabling robust growth on VA without accumulation of formaldehyde. Genetic analyses confirmed that formaldehyde oxidation and stress response systems, rather than C1 assimilation, were important for VA metabolism. Additionally, VA and PCA were found to disrupt membrane potential, contributing to their inherent toxicity. Our findings establish M. extorquens as a promising chassis for lignin valorization and provide a framework for engineering formaldehyde-resistant microbial platforms.

microbiology↗

bioRxiv2021

Low potential electron generating enzymes serve different functions during aerobic nitrogen fixation in Azotobacter vinelandii

Biological nitrogen fixation requires large amounts of energy in the form of ATP and low potential electrons to overcome the high activation barrier for cleavage of the dinitrogen triple bond. The model aerobic nitrogen-fixing bacteria, Azotobacter vinelandii, generates low potential electrons in the form of reduced ferredoxin (Fd) and flavodoxin (Fld) using two distinct mechanisms via the enzyme complexes Rnf and Fix. Both Rnf and Fix are expressed during nitrogen fixation, and deleting either rnf1 or fix genes has little effect on diazotrophic growth. However, deleting both rnf1 and fix eliminates the ability to grow diazotrophically. Rnf and Fix both use NADH as a source of electrons, but overcoming the energetics of NADHs endergonic reduction of Fd/Fld is accomplished through different mechanisms. Rnf harnesses free energy from the proton motive force, whereas Fix uses electron bifurcation to effectively couple the endergonic reduction of Fd/Fld to the exergonic reduction of quinone. Different stoichiometries and gene expression analyses indicate specific roles for the two reactions under different conditions. In this work, complementary physiological studies and thermodynamic modeling reveal how Rnf and Fix simultaneously balance redox homeostasis in various conditions. Specifically, the Fix complex is required for efficient growth under low oxygen concentrations, while Rnf sustains homeostasis and delivers sufficient reduced Fd to nitrogenase under standard conditions. This work provides a framework for understanding how the production of low potential electrons sustains robust nitrogen fixation in various conditions. ImportanceThe availability of fixed nitrogen is critical for life in many ecosystems, from hot springs to agriculture. Due to the energy demands of biological nitrogen fixation, organisms must tailor their metabolism during diazotrophic growth to deliver energy requirements to nitrogenase in the form of ATP and low potential electrons. Therefore, a complete understanding of diazotrophic energy metabolism and redox homeostasis is required to understand the impact on ecological communities or to promote crop growth in agriculture through engineered diazotrophs.

microbiology↗

bioRxiv2021

Metabolic model of nitrogen-fixing obligate aerobe Azotobacter vinelandii demonstrates adaptation to oxygen concentration and metal availability

There is considerable interest in promoting biological nitrogen fixation as a mechanism to reduce the inputs of nitrogenous fertilizers in agriculture, a problem of agronomic, economic, and environmental importance. For the potential impact of biological nitrogen fixation in agriculture to be realized, there are considerable fundamental knowledge gaps that need to be addressed. Biological nitrogen fixation or the reduction of N2 to NH3 is catalyzed by nitrogenase which requires a large amount of energy in the form of ATP and low potential electrons. Nitrogen-fixing organisms that respire aerobically have an advantage in meeting the energy demands of biological nitrogen fixation but face challenges of protecting nitrogenase from inactivation in the presence of oxygen. Here, we have constructed a genome-scale metabolic model of the aerobic metabolism of nitrogen-fixing bacteria Azotobacter vinelandii, which uses a complex electron transport system, termed respiratory protection, to consume oxygen at a high rate keeping intracellular conditions microaerobic. Our model accurately determines growth rate under high oxygen and high substrate concentration conditions, demonstrating the large flux of energy directed to respiratory protection. While respiratory protection mechanisms compensate the energy balance in high oxygen conditions, it does not account for all substrate intake, leading to increased maintenance rates. We have also shown how A. vinelandii can adapt under different oxygen concentrations and metal availability by rearranging flux through the electron transport system. Accurately determining the energy balance in a genome-scale metabolic model is required for future engineering approaches. ImportanceThe worlds dependence on industrially produced nitrogenous fertilizers has created a dichotomy of issues. Some parts of the globe lack access to fertilizers and associated poor crop yields, significantly limiting nutrition, contributing to disease and starvation. In contrast, in other parts of the world, abundant nitrogenous fertilizers and associated overuse result in compromised soil quality and downstream environmental issues. There is considerable interest in expanding the impacts of biological nitrogen fixation to promote improved crop yields in places struggling with access to industrial fertilizers and reducing fertilizers inputs in areas where overuse is resulting in the degradation of soil health and other environmental problems. A more robust and fundamental understanding of biological nitrogen fixations biochemistry and microbial physiology will enable strategies to promote new and more robust associations between nitrogen-fixing microorganisms and crop plants.

microbiology↗