Biology subjects

Newman, D. K.

Publications and source records attributed to Newman, D. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Genome-wide fitness profiling reveals flagellar rotation as an energetic liability during anaerobic maintenance in Pseudomonas aeruginosa

Bacteria in nature and disease frequently spend time in non-growing states, yet the genes that allow cells to survive without growing remain poorly understood. Using Pseudomonas aeruginosa strain PA14 as a model system to study non-growth powered by anaerobic phenazine cycling, we employed randomly barcoded transposon-insertion sequencing (RB-TnSeq) to identify the genes required for this maintenance state using a platform that sustains anaerobic survival via continuous phenazine reoxidation. We found 167 genes whose disruption altered survival, including genes involved in transcription, translation, protein quality control, and cell envelope maintenance. Comparing our results to published TnSeq data from other forms of energy-limited growth arrest (conditions where electron acceptor or carbon availability constrains energy conservation below that required for cell growth/division) in PA14 revealed that while a subset of genes are fitness determinants across distinct growth-arrested states, most are condition-specific. Notably, genes involved in flagellar regulation and assembly were broadly detrimental to survival under this maintenance condition. Leveraging a high-throughput electrochemical system that allows for quantitative and mechanistic dissection of the phenazine cycling-dependent maintenance state, we found that flagellar abundance influences cells survival, metabolic rate, and ATP levels. Moreover, removing the flagellar stator proteins MotAC, which are required for flagellar rotation but not assembly, reversed these defects, indicating that the energetic cost of flagella comes from their rotation rather than construction under these conditions. These results show that in a low-powered maintenance state, limiting energy-dissipating processes, such as proton-motive force loss through flagellar rotation, supports cell survival.

microbiology↗

bioRxiv2026

Redox-dependent lipophilicity of phenazine metabolites is modulated by intramolecular hydrogen bonds and controls their biological distribution

Phenazines are redox-active microbial metabolites produced and secreted in diverse ecological contexts from soils to chronic infections. In these disparate environments phenazines can function variously as antibiotics, extracellular electron shuttles, and nutrient scavengers. Key to understanding the impact of these functions is a robust expectation of phenazine retention or diffusion in a given context. But predicting phenazine fate and transport is difficult because of the chemical complexity of their local microenvironments. To address this challenge, we measured the octanol water distribution coefficient (LogD) as a proxy for lipophilicity of three naturally occurring phenazines produced by the opportunistic pathogen Pseudomonas aeruginosa: phenazine-1-carboxylic acid, phenazine-1-carboxamide, and pyocyanin. We investigated the behavior of both oxidized and reduced forms of these phenazines across broad ionic strength and pH conditions. While the ionic context exerts only small effects, the pH and redox state contribute strongly and independently to changes in phenazine lipophilicity. The pH trends are expected, but the observed redox dependence is generally missed by existing lipophilicity calculation methods. Additional LogD measurements with 1-hydroxyphenazine and unsubstituted phenazine, together with density functional theory modeling of phenazines in their reduced and oxidized forms, reveal that intramolecular hydrogen bonding contributes significantly to the increased lipophilicity of reduced phenazines that possess H-bond accepting substituents in the 1-position. These results explain phenazine behavior in a biological context: redox state alone significantly alters retention of pyocyanin in planktonic P. aeruginosa cells, with the reduced species being predominantly retained by membranes. We propose that the modulation of phenazine lipophilicity in response to the local redox environment has evolved to give a competitive advantage to bacteria by retaining or dispersing these bioactive molecules. Beyond improving our understanding of natural phenazine fate in diverse microbial contexts, our results emphasize an oft-overlooked theme relevant to rational drug and electrochemical shuttle design: redox state matters.

biochemistry↗

bioRxiv2026

Discovery of a phenazine thiol conjugase from sparse data using genome-informed machine learning

Machine learning has enabled powerful biological discoveries using models trained on large datasets. However, for many important biological questions, such as identifying enzymes that transform understudied substrates, sparsity of training data is often a major bottleneck. Here, using phenazine natural products as a case study, we show that integrating genome-informed data augmentation with contrastive learning in protein language space enables identification of phenazine-interacting proteins starting from only 14 known phenazine modifying sequences. Applying this framework led to the discovery of PTC (Phenazine-Thiol Conjugase), the first enzyme known to catalyze phenazine thioconjugation, a phenazine modification reaction long observed but previously presumed to occur only through non-enzymatic chemistry. In silico simulation and experimental measurements demonstrate that PTC binds to both phenazine and glutathione as substrates. Recombinant expression and biochemical characterization reveal that PTC promotes glutathione-dependent modification of phenazines, yielding distinct reaction outcomes that depend on substrate identity. Although thiol-conjugated phenazine products exhibit reduced toxicity to bacterial cells, deletion of the gene encoding PTC does not confer a strong fitness disadvantage, illustrating how direct learning of sequences can uncover relevant enzymes that might evade phenotype-based genetic screens. Together, these results demonstrate that coupling comparative genomics with protein machine learning can convert "small data" typically outside the scope of machine learning into actionable predictive power, thereby facilitating enzyme discovery. SignificanceMachine learning excels when large, well-labeled datasets are available, yet many biologically important problems lack sufficient experimental data to support such approaches to discovery. This limitation is particularly acute for identifying enzymes acting on rare or understudied substrates. Here, we show that genomic organization can be leveraged as an additional source of biological information to address data sparsity. Starting with only 14 enzymes experimentally shown to modify phenazines, we developed a model identifying phenazine-interacting enzymes by integrating genome-informed data augmentation with protein machine learning. Guided by the model, we discovered the first enzyme known to catalyze thioconjugation modifications of phenazines, demonstrating a simple yet powerful strategy for extracting predictive insight from sparse biological knowledge.

microbiology↗

bioRxiv2026

Starvation enhances bacterial survival through drying and rewetting at the single cell level

Soil bacteria are critical to agricultural productivity and play a central role in several biogeochemical cycles. These organisms frequently experience desiccation, which deprives them of access to both water and nutrients. Desiccation eliminates the aqueous connections between soil pores, removing pathways for nutrient diffusion. Well-studied yet resource intensive water stress responses like osmolyte synthesis may thus be impractical in unsaturated environments. Accordingly, we observed how the rhizobacterium Pseudomonas synxantha 2-79 responds to co-occurring water and nutrient limitation at the single-cell level to understand what role osmolyte synthesis may play in its desiccation response. We constructed a transcriptional reporter to track P. synxanthas osmolyte response and collected extensive morphological and reporter expression data through experiments designed to mimic different rates and extents of soil drying. Only actively growing cells responded to an osmotic shock by synthesizing osmolytes: this response was not observed when we pre-starved bacteria. Soluble nutrient diffusivity in soil is restricted even when there is sufficient water to keep bacterial cells hydrated, so this pre-starved condition reflects gradual drying in which starvation precedes water stress. Despite the lack of osmolyte synthesis, prior starvation enhanced P. synxanthas ability to recover from osmotic stress once water and nutrients were restored. These results suggest that cellular changes associated with the response to starvation that go beyond osmolyte synthesis play an important role in microbial desiccation tolerance. Significance StatementSoil bacteria play a vital role in agriculture by promoting plant productivity. Despite this, it is unclear how these organisms respond to desiccation, a common stress whose frequency is rising. Desiccation both dehydrates bacterial cells and eliminates the liquid water connections between soil pores that bacteria use to access nutrients. We studied how a model soil bacterium responds to simultaneous starvation and water stress at the single cell level, focusing on whether the bacteria synthesized osmolytes, small molecules that bacteria can accumulate to limit water loss. We found that starvation restricts osmolyte synthesis but enables bacteria to withstand more severe water stress. The starvation that accompanies desiccation may play a protective, rather than an antagonistic, role in microbial desiccation tolerance.

microbiology↗

bioRxiv2025

Phosphorus stress and spatial confinement lower the threshold for quorum-sensing activation of redox-active metabolite production in Pseudomonas synxantha

Bacteria often coordinate collective behaviors such as biofilm formation and secondary metabolite production through quorum sensing (QS), a regulatory system traditionally linked to high cell density. However, in environments like soil, where microbial populations are spatially fragmented, sparse, nutrient-limited, and subject to mass transport, the mechanisms that enable QS-dependent processes remain incompletely understood. Here, we investigate the regulation of a secreted redox-active metabolite, phenazine-1-carboxylic acid (PCA), in Pseudomonas synxantha 2-79, a model rhizobacterium, under phosphorus (P) limitation, a persistent stress in many soils. Using a combination of microscopy and molecular genetic approaches, we show that P limitation sensitizes the QS activation threshold by an order of magnitude, enabling phenazine induction at relatively low population densities in comparison to P replete conditions. This induction is abolished in QS-deficient mutants and restored by the addition of exogenous acyl-homoserine lactone (AHL), demonstrating that quorum sensing remains essential but its threshold becomes environmentally tuned. Under P limitation, spatial confinement and pore saturation levels further shape the timing and location of induction, illustrating how physical structure and nutrient stress can modulate bacterial activities. Moreover, phosphorus stress confers both collaborative and competitive advantages, enabling P. synxantha to undergo low-cell density AHL cross-talk between related Pseudomonas spp. and to suppress other rhizobacteria. Lastly, on plant roots, PCA genes are more predominantly induced under P limitation. These findings illustrate how the nutrient status of an environment can modulate the onset of quorum sensing, enabling quorum-regulated behaviors to activate at lower thresholds.

microbiology↗

bioRxiv2025

Nitric oxide tunes secreted metabolite bioactivity

The radical nitric oxide ({middle dot}NO) is short-lived but has imprinted itself on many aspects of physiology and disease. {middle dot}NOs rapid production and consumption, coupled with its intrinsic reactivity, drive its biological importance; thus, defining mechanisms and targets of {middle dot}NO reactivity is necessary to assess its fate and impact. Cellular small molecules are a major class of {middle dot}NO-reactive targets, possessing a variety of molecular functionalities that can react with {middle dot}NO. Yet the capacity for secreted small molecules to react with {middle dot}NO, as well as the biological consequences of such reactivity, have received little attention. Here, we explore the reactivity of {middle dot}NO with phenazine metabolites, microbially-derived secreted small molecules that possess antibiotic properties and can modulate their microenvironment. Using Pseudomonas aeruginosa as a model phenazine producer, we find that {middle dot}NO reacts with specific phenazines to yield stable, chemically-distinct products. These chemical transformations significantly attenuate phenazine antibiotic properties, including against the phenazine nonproducer Staphylococcus aureus, a competitor with P. aeruginosa for niches in the context of infection. By contrast, P. aeruginosa experiences rapid loss in viability when phenazines and {middle dot}NO react. This toxicity occurs even in the presence of S. aureus, which displays resistance to nitrosylated phenazines, implicating a specific toxicity dependent on the formation of the phenazine-NO adduct. These findings highlight the capacity of {middle dot}NO to transform metabolite activity and suggest that {middle dot}NO can tune microbial interactions in complex environments by a mechanism of action hitherto unappreciated.

microbiology↗

bioRxiv2025

Extracellular matrix chemistry tunes bacterial biofilm metabolism and optimizes fitness

Chemically complex extracellular matrices define cellular microenvironments and shape cell behavior. We hypothesized a composition-properties-function relationship in these natural living materials, where interactions among matrix components govern material properties and cellular physiology. Using Pseudomonas aeruginosa biofilms as a model system, we show that electrostatic interactions between the cationic polysaccharide Pel and extracellular DNA (eDNA) regulate retention of pyocyanin (PYO), a redox-active metabolite that supports anaerobic metabolism via extracellular electron transfer (EET). Biofilm-mimetic hydrogels and natural biofilms revealed that altering Pels charge via pH adjustment or chemical acetylation, or tuning the Pel:eDNA ratio, predictably modulates PYO retention and EET efficiency. Functionally, a lower Pel:eDNA ratio enhances metabolism under oxygen limitation, whereas a higher ratio promotes survival under antibiotic stress. These findings highlight how matrix chemistry encodes tunable material properties that confer biofilm fitness advantages and establish a materials-based framework for understanding extracellular matrices in multicellular communities.

microbiology↗

bioRxiv2025

The nematode symbiotic bacterium Xenorhabdus griffiniae can sense and respond to the presence of its host Steinernema hermaphroditum

The mutualistic symbiosis between Xenorhabdus bacteria and Steinernema nematodes has both ecological and practical importance. Their relationship co-evolved to make them dependent on each other in their life cycle where they kill, feed and reproduce within insect larvae together. This behavior has shed light on them as promising candidates to replace conventional pesticides. Despite the importance of the Xenorhabdus in insect-killing, the mechanisms by which the bacteria might sense and respond to the presence of their nematode host are not well understood. We performed an RNA-sequencing experiment on the bacterial partner, X. griffiniae, in close proximity, but not in direct contact with their host nematode, Steinernema hermaphroditum, to identify differentially regulated genes in these conditions, followed by genetic analysis to determine their functional significance. We show that X. griffiniae changes its transcriptomic profile in a small number of genes in the presence of axenic nematodes, but not in the presence of nematodes that are already colonized by X. griffiniae. We select the most differentially regulated gene, ymdA, for further investigation, and show that it plays a role in biofilm formation and affects host colonization efficiency. This work advances our understanding of bacterial sensing of nematodes and motivates future research in deepening our understanding of this underexplored ecological interaction. ImportanceInteractions between different organisms in the soil environment are enabled by the ability to sense and respond to organisms nearby. In many cases, the underlying sensing mechanisms that mediate these ecologically important interactions are not known. Filling in the knowledge gap of how these interactions occur can help us understand how microbial populations organize themselves, establish symbiotic relationships, or compete with other species in complex environments. Furthermore, elucidating these mechanisms may inform the development of new tools in agriculture, such as improved biological control agents. Here we use the bacterial symbiont Xenorhabdus griffiniae of the entomopathogenic nematode Steinernema hermaphroditum to study whether symbiotic bacteria of nematodes are able to sense their presence. Our results suggest that X. griffiniae can sense the presence of its nematode host and respond by modulating the gene expression level of several genes, where at least one, ymdA, has implications for host colonization.

microbiology↗

bioRxiv2024

Real-time high-resolution microscopy reveals how single-cell lysis shapes biofilm matrix morphogenesis

During development, multiscale patterning requires that cells organize their behavior in space and time. Bacteria in biofilms must similarly dynamically pattern their behavior with a simpler toolkit. Like in eukaryotes, morphogenesis of the extracellular matrix is essential for biofilm development, but how it is patterned has remained unclear. Here, we explain how the architecture of eDNA, a key matrix component, is controlled by single cell lysis events during Pseudomonas aeruginosa biofilm development. We extend single-cell imaging methods to capture complete biofilm development, characterizing the stages of biofilm development and visualizing eDNA matrix morphogenesis. Mapping the spatiotemporal distribution of single cell lysis events reveals that cell lysis is restricted to a specific biofilm zone. Simulations indicate that this patterning couples cell lysis to growth, more uniformly distributing eDNA throughout the biofilm. Finally, we find that patterning of cell lysis is organized by nutrient gradients that act as positioning cues.

microbiology↗

bioRxiv2024

Investigating 3D microbial community dynamics in the rhizosphere using complex-field and fluorescence microscopy

Microbial interactions in the rhizosphere contribute to soil health. Understanding these interactions thus has the potential to advance sustainable agriculture, ecosystem management, and environmental stewardship. Yet it is difficult to understand what we cannot see; amongst the limitations in rhizosphere imaging are challenges associated with rapidly and non-invasively imaging microbial cells over field depths relevant to plant roots. Here, we present a novel bimodal imaging technique called Complex-field and Fluorescence microscopy using the Aperture Scanning Technique (CFAST) that begins to address these limitations by integrating quantitative phase and 3D fluorescence imaging. We showcase CFASTs practicality and versatility in two ways. First, by harnessing its depth of field of more than 100 microns, we significantly reduce the number of captures required for 3D imaging of plant roots and bacteria in the rhizoplane, thereby minimizing potential photobleaching and phototoxicity. Second, by leveraging CFASTs phase sensitivity and fluorescence specificity, we track early bacterial aggregate development, bacterial competition, and gene expression under varying environmental conditions. Specifically, we resolve bacterial growth dynamics of mixed populations at the early stages of colonization without the need for genetically labeling environmental isolates. Moreover, we find that the expression of genes of interest to rhizosphere chemistry (e.g. representative genes involved in phosphorus-sensing and antibiotic production) varies spatiotemporally within microbial populations that are surface-attached and appears distinct from their expression in planktonic cultures. Together, CFASTs attributes overcome commercial imaging platform limitations and enable new insights to be gained into microbial behavioral dynamics in experimental systems of relevance to the rhizosphere.

microbiology↗

bioRxiv2024

The proteome is a terminal electron acceptor

Microbial metabolism is impressively flexible, enabling growth even when available nutrients differ greatly from biomass in redox state. E. coli, for example, rearranges its physiology to grow on reduced and oxidized carbon sources through several forms of fermentation and respiration. To understand the limits on and evolutionary consequences of metabolic flexibility, we developed a mathematical model coupling redox chemistry with principles of cellular resource allocation. Our integrated model clarifies key phenomena, including demonstrating that autotrophs grow slower than heterotrophs because of constraints imposed by intracellular production of reduced carbon. Our model further indicates that growth is improved by adapting the redox state of biomass to nutrients, revealing an unexpected mode of evolution where proteins accumulate mutations benefiting organismal redox balance. One sentence summaryMicrobial proteins adapt their composition on physiological and evolutionary timescales to ensure organismal redox balance.

microbiology↗

bioRxiv2024

Annotation-free prediction of microbial dioxygen utilization

Aerobes require dioxygen (O2) to grow; anaerobes do not. But nearly all microbes -- aerobes, anaerobes, and facultative organisms alike -- express enzymes whose substrates include O2, if only for detoxification. This presents a challenge when trying to assess which organisms are aerobic from genomic data alone. This challenge can be overcome by noting that O2 utilization has wide-ranging effects on microbes: aerobes typically have larger genomes, encode more O2-utilizing enzymes, and tend to use different amino acids in their proteins. Here we show that these effects permit high-quality prediction of O2 utilization from genome sequences, with several models displaying >70% balanced accuracy on a ternary classification task wherein blind guessing is only 33.3% accurate. Since genome annotation is compute-intensive and relies on many assumptions, we asked if annotation-free methods also perform well. We discovered that simple and efficient models based entirely on genome sequence content -- e.g. triplets of amino acids -- perform about as well as intensive annotation-based algorithms, enabling the rapid processing of global-scale sequence data to predict aerobic physiology. To demonstrate the utility of efficient physiological predictions we estimated the prevalence of aerobes and anaerobes along a well-studied O2 gradient in the Black Sea, finding strong quantitative correspondence between local chemistry (O2:sulfide concentration ratio) and the composition of microbial communities. We therefore suggest that statistical methods like ours can be used to estimate, or "sense," pivotal features of the environment from DNA sequencing data. ImportanceWe now have access to sequence data from a wide variety of natural environments. These data document a bewildering diversity of microbes, many known only from their genomes. Physiology -- an organisms capacity to engage metabolically with its environment -- may provide a more useful lens than taxonomy for understanding microbial communities. As an example of this broader principle, we developed algorithms that accurately predict microbial dioxygen utilization directly from genome sequences without first annotating genes, e.g. by considering only the amino acids in protein sequences. Annotation-free algorithms enabled rapid characterization of natural samples, demonstrating a quantitative correspondence between sequences and local O2 levels. These results suggest that DNA sequencing can be repurposed as a multi-pronged chemical sensor, estimating concentrations of O2 and other key facets of complex natural settings.

microbiology↗

bioRxiv2023

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa. Author summaryMany bacteria have extremely flexible metabolisms, and we are only beginning to understand how they manifest in the environment. Our study focuses on the role of phenazine-1-carboxylic acid (PCA), a molecule that some bacteria synthesize and secrete into their surroundings. PCA is an "extracellular electron shuttle," a molecule that readily transfers electrons between cells and oxidizing/reducing compounds or other cells. Until our investigation, the role of PCA electron-shuttling had only been studied in one direction: how it takes electrons away from cells, and the effect this has on their viability. Here we present a detailed account of the opposite process and its mechanism: what happens when PCA delivers electrons to cells? Our findings indicate that this previously underappreciated process is generalizable to any anaerobically respiring bacterium. Consequently, we expect that electron donation by PCA is widespread in environments where PCA is plentiful and oxygen is sparse, such as in some agricultural soils. The universality of the extracellular electron shuttle oxidation mechanism we describe for PCA suggests that it should also occur with similar small molecules, of which there are thousands, deepening the implication that this is a significant process in the environment and motivating further research into its consequences.

microbiology↗

bioRxiv2023

Uncultivated DPANN archaea are ubiquitous inhabitants of global oxygen deficient zones with diverse metabolic potential

Archaea belonging to the DPANN superphylum have been found within an expanding number of environments and perform a variety of biogeochemical roles, including contributing to carbon, sulfur, and nitrogen cycling. Generally characterized by ultrasmall cell sizes and reduced genomes, DPANN archaea may form mutualistic, commensal, or parasitic interactions with various archaeal and bacterial hosts, influencing the ecology and functioning of microbial communities. While DPANN archaea reportedly comprise 15-26% of the archaeal community within marine oxygen deficient zone (ODZ) water columns, little is known about their metabolic capabilities in these ecosystems. We report 33 novel metagenome-assembled genomes belonging to DPANN phyla Nanoarchaeota, Pacearchaeota, Woesarchaeota, Undinarchaeota, Iainarchaeota, and SpSt-1190 from pelagic ODZs in the Eastern Tropical North Pacific and Arabian Sea. We find these archaea to be permanent, stable residents of all 3 major ODZs only within anoxic depths, comprising up to 1% of the total microbial community and up to 25-50% of archaea. ODZ DPANN appear capable of diverse metabolic functions, including fermentation, organic carbon scavenging, and the cycling of sulfur, hydrogen, and methane. Within a majority of ODZ DPANN, we identify a gene homologous to nitrous oxide reductase. Modeling analyses indicate the feasibility of a nitrous oxide reduction metabolism for host-attached symbionts, and the small genome sizes and reduced metabolic capabilities of most DPANN MAGs suggest host-associated lifestyles within ODZs. ImportanceArchaea from the DPANN superphylum have diverse metabolic capabilities and participate in multiple biogeochemical cycles. While metagenomics and enrichments have revealed that many DPANN are characterized by ultrasmall genomes, few biosynthetic genes, and episymbiotic lifestyles, much remains unknown about their biology. We report 33 new DPANN metagenome-assembled genomes originating from the 3 global marine oxygen deficient zones (ODZs), the first from these regions. We survey DPANN abundance and distribution within the ODZ water column, investigate their biosynthetic capabilities, and report potential roles in the cycling of organic carbon, methane, and nitrogen. We test the hypothesis that nitrous oxide reductases found within several ODZ DPANN genomes may enable ultrasmall episymbionts to serve as nitrous oxide consumers when attached to a host nitrous oxide producer. Our results indicate DPANN archaea as ubiquitous residents within the anoxic core of ODZs with the potential to produce or consume key compounds.

microbiology↗

bioRxiv2023

Engineering the soil bacterium Pseudomonas synxantha 2-79 into a ratiometric bioreporter for phosphorus limitation

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty of ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally-motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2-79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25-100 {micro}M without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8-8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporters performance in shaken liquid culture predict its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally-aware design process provides an example of how laboratory bioengineering efforts can generate microbes with greater promise to function reliably in their applied contexts.

synthetic biology↗

bioRxiv2023

Widespread detoxifying NO reductases impart a distinct isotopic fingerprint on N2O under anoxia

Nitrous oxide (N2O), a potent greenhouse gas, can be generated by compositionally complex microbial populations in diverse contexts. Accurately tracking the dominant biological sources of N2O has the potential to improve our understanding of N2O fluxes from soils as well as inform the diagnosis of human infections. Isotopic "Site Preference" (SP) values have been used towards this end, as bacterial and fungal nitric oxide reductases produce N2O with different isotopic fingerprints. Here we show that flavohemoglobin, a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto N2O under anoxic conditions that correlates with typical environmental N2O SP measurements. We suggest a new framework to guide the attribution of N2O biological sources in nature and disease. One-Sentence SummaryDetoxifying nitric oxide reductases impart a distinct isotopic biosignature on nitrous oxide.

microbiology↗

bioRxiv2023

Intracellular Pseudomonas aeruginosa within the airway epithelium of Cystic Fibrosis lung tissues

RATIONALEPseudomonas aeruginosa (P.a.) is the major bacterial pathogen colonizing the airways of adult cystic fibrosis (CF) patients and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evades the immune system and antibiotic therapy. While the ability of P.a. to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. OBJECTIVESTo detect and characterize intracellular P.a. in CF airway epithelium from human lung explant tissues. METHODSWe sampled the lung explant tissues from CF and non-CF patients undergoing lung transplantation and analyzed lung tissue sections for the presence of intracellular P.a. by quantitative culture and microscopy, in parallel to histopathology and airway morphometry. MEASUREMENTS AND MAIN RESULTSP.a. was isolated from the lungs of 7 CF patients undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P.a. within airway epithelial cells in 3 out of the 7 lungs analyzed, at a varying but rare frequency. We observed those events occurring in lung regions with high bacterial burden. CONCLUSIONThis is the first study describing the presence of intracellular P.a. in CF lung tissues. While intracellular P.a. in airway epithelial cells are likely relatively rare events, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

microbiology↗

bioRxiv2022

Electrochemical disruption of extracellular electron transfer inhibits Pseudomonas aeruginosa cell survival within biofilms and is synergistic with antibiotic treatment

Survival of cells within oxygen-limited regions in Pseudomonas aeruginosa biofilms is enabled by using small redox active molecules as electron shuttles to access distal oxidants. This respiratory versatility makes P. aeruginosa biofilms common in chronic wound infections and recalcitrant to treatment. Here, we show that electrochemically controlling the redox state of these electron shuttles, specifically pyocyanin, can impact cell survival within anaerobic P. aeruginosa biofilms and can act synergistically with antibiotic treatment. We inhibited pyocyanin redox cycling under anoxic conditions by blocking its ability to be re-oxidized and thus serve as an electron shuttle via poising an electrode at a reductive potential that cannot regenerate oxidized pyocyanin (i.e. -400mV vs Ag/AgCl). This resulted in a decrease in CFUs within the biofilm of 100x compared to samples exposed to an electrode poised at an oxidizing potential that permits pyocyanin re-oxidation (i.e. +100mV vs Ag/AgCl). Phenazine-deficient {Delta}phz* biofilms were not affected by the redox potential of the electrode, but were re-sensitized by adding pyocyanin. The effect of EET disruption was exacerbated when biofilms were treated with sub-MICs of a range of antibiotics. Most notably, 4 g/ml of the aminoglycoside gentamicin in a reductive environment almost completely eradicated wild type biofilms but had no effect on the survival of {Delta}phz* biofilms, suggesting reduced phenazines are toxic, and combined with antibiotic treatment can lead to extensive killing. ImportanceBiofilms provide a protective environment but they also present challenges to the cells living within them, such as overcoming diffusion limitation of nutrients and oxygen. Pseudomonas aeruginosa overcomes oxygen limitation by secreting soluble redox active molecules as electron shuttles to access distal oxygen. Here, we show that electrochemically blocking the redox cycling of one of these electron shuttles, pyocyanin, decreases cell survival within biofilms and acts synergistically with gentamicin to kill cells. Our results highlight the importance of the role that the redox cycling of electron shuttles fulfills within P. aeruginosa biofilms.

microbiology↗