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.
\nSignificance Machine 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.
\nNitrous 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.
\n", "doi": "10.1101/2023.10.13.562248", "pmcid": "PMC10592819", "publication_date": "2023-10-14" }, { "id": "authors:skw34-qy914", "collection": "authors", "collection_id": "skw34-qy914", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230322-367168000.14", "type": "monograph", "title": "Electrochemical disruption of extracellular electron transfer inhibits Pseudomonas aeruginosa cell survival within biofilms and is synergistic with antibiotic treatment", "author": [ { "family_name": "Jim\u00e9nez Otero", "given_name": "Fernanda", "orcid": "0000-0003-1583-6495", "clpid": "Jim\u00e9nez-Otero-Fernanda" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Tender", "given_name": "Leonard M.", "orcid": "0000-0001-8784-991X", "clpid": "Tender-Leonard-M" } ], "abstract": "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. \u2212400mV 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 \u0394phz* 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 \u03bcg/ml of the aminoglycoside gentamicin in a reductive environment almost completely eradicated wild type biofilms but had no effect on the survival of \u0394phz* 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.", "doi": "10.1101/2022.09.15.508205", "publication_date": "2022-09-19" }, { "id": "authors:eaa21-b8008", "collection": "authors", "collection_id": "eaa21-b8008", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220810-751722000", "type": "monograph", "title": "Optical O\u2082 sensors also respond to redox active molecules commonly secreted by bacteria", "author": [ { "family_name": "Flamholz", "given_name": "Avi I.", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi-I" }, { "family_name": "Saccomano", "given_name": "Samuel", "orcid": "0000-0001-9105-2663", "clpid": "Saccomano-Sameul-C" }, { "family_name": "Cash", "given_name": "Kevin", "orcid": "0000-0002-8059-0922", "clpid": "Cash-Kevin-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "abstract": "From a metabolic perspective, molecular oxygen (O\u2082) is arguably the most significant constituent of Earth's atmosphere. Nearly every facet of microbial physiology is sensitive to the presence and concentration of O\u2082, which is the most favorable terminal electron acceptor used by biology and also a dangerously reactive oxidant. As O\u2082 has such sweeping implications for physiology, researchers have developed diverse approaches to measure O\u2082 concentrations in natural and laboratory settings. Recent improvements to phosphorescent O\u2082 sensors piqued our interest due to the promise of optical measurement of spatiotemporal O\u2082 dynamics. However, we found that our preferred bacterial model, Pseudomonas aeruginosa PA14, secretes more than one molecule that quenches such sensors, complicating O\u2082 measurements in PA14 cultures and biofilms. Assaying supernatants from cultures of 9 bacterial species demonstrated that this phenotype is common: all supernatants quenched a soluble O\u2082 probe substantially. Phosphorescent O\u2082 probes are often embedded in solid support for protection, but an embedded probe called O\u2082 NS was quenched by most supernatants as well. Measurements using pure compounds indicated that quenching is due to interactions with redox-active small molecules including phenazines and flavins. Uncharged and weakly-polar molecules like pyocyanin were especially potent quenchers of O\u2082 NS. These findings underscore that optical O\u2082 measurements made in the presence of bacteria should be carefully controlled to ensure that O2, and not bacterial secretions, is measured, and motivate the design of custom O\u2082 probes for specific organisms to circumvent sensitivity to redox-active metabolites.", "doi": "10.1101/2022.08.08.503264", "publication_date": "2022-08-10" }, { "id": "authors:tjwxh-wyn19", "collection": "authors", "collection_id": "tjwxh-wyn19", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220104-461622500", "type": "monograph", "title": "Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa", "author": [ { "family_name": "Magkiriadou", "given_name": "S.", "orcid": "0000-0001-7660-5012", "clpid": "Magkiriadou-Sofia" }, { "family_name": "Habel", "given_name": "A.", "clpid": "Habel-A" }, { "family_name": "Stepp", "given_name": "W. L.", "orcid": "0000-0001-9818-0377", "clpid": "Stepp-Willi-L" }, { "family_name": "Newman", "given_name": "D. K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Manley", "given_name": "S.", "orcid": "0000-0002-4755-4778", "clpid": "Manley-Suliana" }, { "family_name": "Racki", "given_name": "L. R.", "clpid": "Racki-L-R" } ], "abstract": "Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we characterize the motion of chromosomal loci and free tracer particles in the cytoplasm. In the absence of polyP and upon starvation, we observe an increase in mobility both for chromosomal loci and for tracer particles. Tracer particles reveal that polyP also modulates the partitioning between a 'more mobile' and a 'less mobile' population: small particles in cells unable to make polyP are more likely to be 'mobile' and explore more of the cytoplasm, particularly during starvation. We speculate that this larger freedom of motion may be a consequence of nucleoid decompaction, which we also observe in starved cells deficient in polyP. Our observations suggest that polyP limits cytoplasmic mobility and accessibility during nitrogen starvation, which may help to explain the pleiotropic phenotypes observed in the absence of polyP.", "doi": "10.1101/2021.12.23.473106", "publication_date": "2021-12-24" }, { "id": "authors:t2agr-dms53", "collection": "authors", "collection_id": "t2agr-dms53", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211210-238484000", "type": "monograph", "title": "The contrasting roles of nitric oxide drive microbial community organization as a function of oxygen presence", "author": [ { "family_name": "Wilbert", "given_name": "Steven A.", "clpid": "Wilbert-Steven-A" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "abstract": "Microbial assemblages are omnipresent in the biosphere, forming communities on the surfaces of roots, rocks, and within living tissues. These communities can exhibit strikingly beautiful compositional structures, with certain members reproducibly occupying particular spatiotemporal microniches. Yet often, we lack the ability to explain the spatial patterns we see within them. To test the hypothesis that certain spatial patterns in microbial communities may be explained by the exchange of redox-active metabolites whose biological function is sensitive to environmental gradients, here we developed a simple community consisting of synthetic Pseudomonas aeruginosa strains with a partitioned denitrification pathway: a strict consumer and strict producer of nitric oxide (NO), a key pathway intermediate. Because NO can be both toxic or beneficial depending on the amount of oxygen present, this system provided an opportunity to investigate whether dynamic oxygen gradients can tune metabolic cross-feeding in a predictable fashion. Using a combination of genetic analysis, different growth environments and imaging, we show that oxygen availability controls whether NO cross-feeding is commensal or mutually beneficial, and that this organizing principle maps to the microscale. More generally, this work underscores the importance of considering the double-edged roles redox-active metabolites can play in shaping microbial communities.", "doi": "10.1101/2021.12.09.472001", "publication_date": "2021-12-10" }, { "id": "authors:hbm1w-gfy69", "collection": "authors", "collection_id": "hbm1w-gfy69", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210419-152102811", "type": "monograph", "title": "Paraburkholderia edwinii protects Aspergillus sp. from phenazines by acting as a toxin sponge", "author": [ { "family_name": "Dahlstrom", "given_name": "Kurt M.", "orcid": "0000-0001-6590-6020", "clpid": "Dahlstrom-Kurt-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "abstract": "Many environmentally and clinically important fungi are sensitive to toxic, bacterially-produced, redox-active molecules called phenazines. Despite being vulnerable to phenazine-assault, fungi inhabit microbial communities that contain phenazine producers. Because many fungi cannot withstand phenazine challenge, but some bacterial species can, we hypothesized that bacterial partners may protect fungi in phenazine-replete environments. In the first soil sample we collected, we co-isolated several such physically associated pairings. We discovered the novel species Paraburkholderia edwinii and demonstrated it can protect a co-isolated Aspergillus species from phenazine-1-carboxylic acid (PCA) by sequestering it, acting as a toxin sponge; in turn, it also gains protection. When challenged with PCA, P. edwinii changes its morphology, forming aggregates within the growing fungal colony. Further, the fungal partner triggers P. edwinii to sequester PCA and maintains conditions that limit PCA toxicity by promoting an anoxic and highly reducing environment. A mutagenic screen revealed this program depends on the stress-inducible transcriptional repressor HrcA. We show that one relevant stressor in response to PCA challenge is fungal acidification and that acid stress causes P. edwinii to behave as though the fungus were present. Finally, we reveal this phenomenon as widespread among Paraburkholderia with moderate specificity among bacterial and fungal partners, including plant and human pathogens. Our discovery suggests a common mechanism by which fungi can gain access to phenazine-replete environments, and provides a tractable model system for its study. These results have implications for how rhizosphere microbial communities as well as plant and human infection sites are policed for fungal membership.", "doi": "10.1101/2021.03.28.437412", "publication_date": "2021-03-29" }, { "id": "authors:47nw6-c7553", "collection": "authors", "collection_id": "47nw6-c7553", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201124-104632686", "type": "monograph", "title": "Bidirectional redox cycling of phenazine-1-carboxylic acid by Citrobacter portucalensis MBL drives increased nitrate reduction", "author": [ { "family_name": "Tsypin", "given_name": "Lev M.", "orcid": "0000-0002-0642-8468", "clpid": "Tsypin-Lev-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "abstract": "Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. Phenazines are able to contribute to these activities due to their redox activity. The physiological consequences of cellular phenazine reduction have been extensively studied, but the counterpart phenazine oxidation has been largely overlooked. Phenazine-1-carboxylic acid (PCA) is common in the environment and readily reduced by its producers. Here, we describe its anaerobic oxidation by Citrobacter portucalensis strain MBL, which was isolated from topsoil in Falmouth, MA, and which does not produce phenazines itself. This activity depends on the availability of a suitable terminal electron acceptor, specifically nitrate or fumarate. When C. portucalensis MBL is provided reduced PCA and either nitrate or fumarate, it continuously oxidizes the PCA. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other \u03b3-proteobacteria with varying capacities to respire nitrate and/or fumarate. We found that PCA oxidation by these strains in a fumarate-or nitrate-dependent manner is decoupled from growth and correlated with their possession of the fumarate or periplasmic nitrate reductases, respectively. We infer that bacterial PCA oxidation is widespread and genetically determined. Notably, reduced PCA enhances the rate of nitrate reduction to nitrite by C. portucalensis MBL beyond the stoichiometric prediction, which we attribute to C. portucalensis MBL's ability to also reduce oxidized PCA, thereby catalyzing a complete PCA redox cycle. This bidirectionality highlights the versatility of PCA as a biological redox agent.", "doi": "10.1101/2020.11.23.395335", "publication_date": "2020-11-24" }, { "id": "authors:tvf44-tvw68", "collection": "authors", "collection_id": "tvf44-tvw68", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180927-114224832", "type": "monograph", "title": "Extended hopanoid lipids promote bacterial motility, surface attachment, and root nodule development in the Bradyrhizobium diazoefficiens-Aeschynomene afraspera symbiosis", "author": [ { "family_name": "Belin", "given_name": "Brittany J.", "clpid": "Belin-B-J" }, { "family_name": "Tookmanian", "given_name": "Elise T.", "clpid": "Tookmanian-E-T" }, { "family_name": "de Anda", "given_name": "Jaime", "orcid": "0000-0003-2129-0775", "clpid": "de-Anda-J" }, { "family_name": "Wong", "given_name": "Gerard", "orcid": "0000-0003-0893-6383", "clpid": "Wong-Gerard-C-L" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "abstract": "Hopanoids are steroid-like bacterial lipids that enhance membrane rigidity and promote bacterial growth under diverse stresses. Roughly 10% of bacteria contain genes involved in hopanoid biosynthesis, and these genes are particularly conserved in plant-associated organisms. We previously found that the extended class of hopanoids (C35) in the nitrogen-fixing soil bacterium Bradyrhizobium diazoefficiens promotes its root nodule symbiosis with the tropical legume Aeschynomene afraspera. By quantitatively modeling root nodule development, we identify independent roles for hopanoids in the initiation of root nodule formation and in determining the rate of root nodule maturation. In vitro studies demonstrate that extended hopanoids support B. diazoefficiens motility and surface attachment, which may correlate with stable root colonization in planta. Confocal microscopy of maturing root nodules reveals that root nodules infected with extended hopanoid-deficient B. diazoefficiens contain unusually low densities of bacterial symbionts, indicating that extended hopanoids are necessary for persistent, high levels of host infection. This work identifies extended hopanoids as regulators of the efficiency of Bradyrhizobia nitrogen-fixing symbioses, agriculturally and economically significant associations with growing importance in a changing climate.", "doi": "10.1101/423301", "publication_date": "2018-09-21" } ]