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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Using phage display for rational engineering of a higher-affinity humanized 3’ phosphohistidine-specific antibody

Abstract Histidine phosphorylation is a non-canonical post-translational modification (PTM), with 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis) isoforms, that is understudied due to a lack of robust reagents, including high-affinity pHis-specific antibodies. Engineering pHis antibodies is challenging due to the labile nature of its phosphoramidate (P-N) bond. We developed a strategy for in vitro engineering of antibodies for the detection of native 3-pHis targets, in which the rabbit SC44-8 anti-3-pTza mAb is humanized into a scaffold (hSC44) that is suitable for phage display. Six unique Fab phage-displayed hSC44 scaffold libraries were screened for antibodies that bound 3-pHis with higher affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32F L , the best engineered antibody, has ~10-fold higher affinity for 3-pHis than parental hSC44. Eleven new Fab structures, including the first antibody-pHis peptide structures, together with structural and quantum mechanical calculations, provided molecular insights into 3-pHis and 3-pTza discrimination by hSC44.20N32F L and the increased affinity obtained through engineering. We demonstrated the utility of these high-affinity 3-pHis-specific antibodies for the recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Our work describes a general method for engineering labile PTM-specific antibodies and provides novel antibodies for investigating the role of 3-pHis in cell biology.

Martyn, Gregory D.↗
DOE OSTI2025

Virocell Necromass Provides Limited Plant Nitrogen and Elicits Rhizosphere Metabolites That Affect Phage Dynamics

Bacteriophages impact soil bacteria through lysis, altering the availability of organic carbon and plant nutrients. However, the magnitude of nutrient uptake by plants from lysed bacteria remains unknown, partly because this process is challenging to investigate in the field. In this study, we extend ecosystem fabrication (EcoFAB 2.0) approaches to study plant-bacteria-phage interactions by comparing the impact of virocell (phage-lysed) and uninfected 15 N-labelled bacterial necromass on plant nitrogen acquisition and rhizosphere exometabolites composition. We show that grass Brachypodium distachyon derives some nitrogen from amino acids in uninfected Pseudomonas putida necromass lysed by sonication but not from virocell necromass. Additionally, the bacterial necromass elicits the formation of rhizosphere exometabolites, some of which (guanosine), alongside tested aromatic acids ( p -coumaric and benzoic acid), show bacterium-specific effects on bacteriophage-induced lysis when tested in vitro. The study highlights the dynamic feedback between virocell necromass and plants and suggests that root exudate metabolites can impact bacteriophage infection dynamics.

Brachypodium↗
NASA NTRS1970

Analysis of T4 phage proteins. I

T4 phage proteins radioisotopical examination, determining quantitative analysis and molecular weight by autoradiography

Cone, R.↗
NASA NTRS2012

Methods for generation of reporter phages and immobilization of active bacteriophages on a polymer surface

Novel reporter bacteriophages are provided. Provided are compositions and methods that allow bacteriophages that are used for specific detection or killing of E. coli 0157:H7 to be propagated in nonpathogenic E. coli, thereby eliminating the safety and security risks of propagation in E. coli 0157:H7. Provided are compositions and methods for attaching active bacteriophages to the surface of a polymer in order to kill target bacteria with which the phage comes into contact. Provided are modified bacteriophages immobilized to a surface, which capture E. coli 0157:H7 and cause the captured cells to emit light or fluorescence, allowing detection of the bacteria in a sample.

Applegate, Bruce Michael↗
DOE OSTI2025

CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Survey of Thirteen Novel Pseudomonas putida Bacteriophages

Bacteriophages have been widely investigated as a promising treatment of food, medical equipment, and humans colonized by antibiotic-resistant bacteria. Phages pose particular interest in combating those bacteria which form biofilms, such as the medically important human pathogen Pseudomonas aeruginosa and several plant pathogens, including P. syringae . In an undergraduate lab course, P. putida was used as the host to isolate novel anti-pseudomonal bacteriophages. Environmental samples of soil and water were collected, and purified phage isolates were obtained. After Illumina sequencing, genomes of these phages were assembled de novo and annotated. Assembled genomes were compared with known genomes in the literature and GenBank to identify taxonomic relations and to refine their functional annotations. The thirteen phages described are sipho-, myo-, and podoviruses in several families of Caudoviricetes , spanning several novel genera, with genomes ranging from 40,000 to 96,000 bp. One phage (DDSR119) is unique and is the first reported P. putida siphovirus. The remaining 12 can be clustered into four distinct groups. Six are highly related to each other and to previously described Autotranscriptaviridae phages: Waldo5, PlaquesPlease, and Laces98 all belong to the Waldovirus genus, whereas Stalingrad, Bosely, and Stamos belong to the Troedvirus genus. Zuri was previously classified as the founding member of a new genus Zurivirus within the family Schitoviridae . Ebordelon and Holyagarpour each represent different species within Zurivirus , whereas Meara is a more distantly related member of the Schitoviridae . Dolphis and Jeremy are similar enough to form a genus but have only a few distant relatives among sequenced phages and are notable for being temperate. We identified the lysis cassettes in all 13 phages, compared tail spike structures, and found auxiliary metabolic genes in several. Studies like these, which isolate and characterize infectious virions, enable the identification of novel proteins and molecular systems and also provide the raw materials for further study, evaluation, and manipulation of phage proteins and their hosts.

Pseudomonas putida↗
DOE OSTI2026

Synergy and antagonism in a genome-scale model of metabolic hijacking by bacteriophages

Bacteriophage auxiliary metabolic genes (AMGs) alter host metabolism by hijacking reactions, but previous studies mostly inferred their roles from annotations, ignoring system-wide impacts and phage production. Here we integrate AMGs and phage assembly into a genome-scale metabolic model of Prochloroccocus marinus MED4 infected by P-HM2. We show that 17 directly hijacked reactions substantially affect more than 30% of the reactions in MED4 metabolism, including carbon fixation, photosynthesis, and nucleotide synthesis, distinguishing these AMGs as either phage aligned—shifting feasible reaction velocities in accordance with maximal phage production—or phage antialigned. Pareto optimization reveals that phage-aligned reactions alter phage-host growth trade-offs, while phage-antialigned reactions do not. We experimentally validate our predictions of system-level AMG impacts by measuring the N-dependent effect of P-HM2 cp12 expression on growth in a model relative of the genetically intractable MED4, Synechococcus elongatus. We also show that AMGs’ indirect impacts are synergistically and antagonistically coupled, providing systems-level insight into AMG perturbations and highlighting how nontrivial cascading effects shape host metabolism.

Rozum, Jordan C. [Pacific Northwest National Labor↗
DOE OSTI2025

Identification of shared viral sequences in peat moss metagenomes reveals elements of a possible Sphagnum core virome

Viruses are an understudied component of plant microbiomes. Identifying viruses that are shared between individual plants, or members of the “core virome”, could reveal stable viral populations with the potential to modulate the composition and function of the microbiome. Here, we examined the virome associated with Sphagnum mosses, a keystone species that has direct influence over the fate of peatland carbon stores. We analyzed bulk metagenomes and metatranscriptomes generated from Sphagnum field samples collected over a ten-month period to identify virus-like sequences shared among plants. Individual Sphagnum samples harbored distinct DNA and RNA viromes where only a small percentage (< 1%) of the total number of identified viral contigs were shared among all samples. Based on taxonomic classification, the shared viral contigs represent bacterial viruses, or phage (Caudoviricetes), as well as viruses of eukaryotes, namely nucleocytoplasmic large DNA viruses (Nucleocytoviricota) and RNA viruses (Riboviria). We linked the shared phage-like contigs to viral regions within sequenced genomes of bacterial taxa that are members of the Sphagnum core microbiome, suggesting that these contigs represent temperate phage or degraded prophage. The putative nucleocytoplasmic large DNA viruses and RNA viruses were phylogenetically diverse and showed sequence similarity to viruses associated with a broad range of hosts and environmental sources. The identification of shared viral contigs suggested that, despite the compositional heterogeneity between samples, Sphagnum mosses may harbor a core virome. Future work validating the presence of the core virome is warranted as it may aid in understanding how persistent viruses impact microbiome ecology and symbiont evolution within this climatically relevant keystone species.

Metagenomics↗
DOE OSTI2025

Nanobody screening and machine learning guided identification of cross-variant anti-SARS-CoV-2 neutralizing heavy-chain only antibodies

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to persist, demonstrating the risks posed by emerging infectious diseases to national security, public health, and the economy. Development of new vaccines and antibodies for emerging viral threats requires substantial resources and time, and traditional development platforms for vaccines and antibodies are often too slow to combat continuously evolving immunological escape variants, reducing their efficacy over time. Previously, we designed a next-generation synthetic humanized nanobody (Nb) phage display library and demonstrated that this library could be used to rapidly identify highly specific and potent neutralizing heavy chain-only antibodies (HCAbs) with prophylactic and therapeutic efficacy in vivo against the original SARS-CoV-2. In this study, we used a combination of high throughput screening and machine learning (ML) models to identify HCAbs with potent efficacy against SARS-CoV-2 viral variants of interest (VOIs) and concern (VOCs). To start, we screened our highly diverse Nb phage display library against several pre-Omicron VOI and VOC receptor binding domains (RBDs) to identify panels of cross-reactive HCAbs. Using HCAb affinity for SARS-CoV-2 VOI and VOCs (pre-Omicron variants) and model features from other published data, we were able to develop a ML model that successfully identified HCAbs with efficacy against Omicron variants, independent of our experimental biopanning workflow. This biopanning informed ML approach reduced the experimental screening burden by 78% to 90% for the Omicron BA.5 and Omicron BA.1 variants, respectively. The combined approach can be applied to other emerging viruses with pandemic potential to rapidly identify effective therapeutic antibodies against emerging variants.

Antibodies↗
NASA NTRS1974

Arthrobacter globiformis and its bacteriophage in soil

An attempt was made to correlate bacteriophages for Arthrobacter globiformis with soils containing that bacterium. The phages were not detected unless the soil was nutritionally amended (with glucose or sucrose) and incubated for several days. Phage was continuously produced after amendment without the addition of host Arthrobacter. These results indicate that the bacteriophage is present in a masked state and that the bacteria are present in an insensitive form which becomes sensitive after addition of nutrient.

Casida, L. E., Jr.↗