Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “bacteriophage”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Host and nonhost bacteria support bacteriophage dissemination along mycelia and abiotic dispersal networks

Abstract Bacteriophages play a crucial role in shaping bacterial communities, yet the mechanisms by which nonmotile bacteriophages interact with their hosts remain poorly understood. This knowledge gap is especially pronounced in structured environments like soil, where spatial constraints and air-filled zones hinder aqueous diffusion. In soil, hyphae of filamentous microorganisms form a network of ‘fungal highways’ (FHs) that facilitate the dispersal of other microorganisms. We propose that FHs also promote bacteriophage dissemination. Viral particles can diffuse in liquid films surrounding hyphae or be transported by infectable (host) or uninfectable (nonhost) bacterial carriers coexisting on FH networks. To test this, two bacteriophages that infect Pseudomonas putida DSM291 (host) but not KT2440 (nonhost) were used. In the absence of carriers, bacteriophages showed limited diffusion on 3D-printed abiotic networks, but diffusion was significantly improved in Pythium ultimum-formed FHs when the number of connecting hyphae exceeded 20. Transport by both host and nonhost carriers enhanced bacteriophage dissemination. Host carriers were five times more effective in transporting bacteriophages, particularly in FHs with over 30 connecting hyphae. This study enhances our understanding of bacteriophage dissemination in nonsaturated environments like soils, highlighting the importance of biotic networks and bacterial hosts in facilitating this process.

Périat, Claire (ORCID:000000020825625X)↗
DOE OSTI2022

SourceFinder: a Machine-Learning-Based Tool for Identification of Chromosomal, Plasmid, and Bacteriophage Sequences from Assemblies

High-throughput genome sequencing technologies enable the investigation of complex genetic interactions, including the horizontal gene transfer of plasmids and bacteriophages. However, identifying these elements from assembled reads remains challenging due to genome sequence plasticity and the difficulty in assembling complete sequences. In this study, we developed a classifier, using random forest, to identify whether sequences originated from bacterial chromosomes, plasmids, or bacteriophages. The classifier was trained on a diverse collection of 23,211 chromosomal, plasmid, and bacteriophage sequences from hundreds of bacterial species. In order to adapt the classifier to incomplete sequences, each complete sequence was subsampled into 5,000 nucleotide fragments and further subdivided into k-mers. This three-class classifier succeeded in identifying chromosomes, plasmids, and bacteriophages using k-mer distributions of complete and partial genome sequences, including simulated metagenomic scaffolds with minimum performance of 0.939 area under the receiver operating characteristic curve (AUC). This classifier, implemented as SourceFinder, has been made available as an online web service to help the community with predicting the chromosomal, plasmid, and bacteriophage sources of assembled bacterial sequence data (https://cge.food.dtu.dk/services/SourceFinder/).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents

The interaction between gut bacterial and viral microbiota is thought to be important in human health. While fluctuations in female genital tract (FGT) bacterial microbiota similarly determine sexual health, little is known about the presence, persistence, and function of vaginal bacteriophages. We conducted shotgun metagenome sequencing of cervicovaginal samples from South African adolescents collected longitudinally, who received no antibiotics. We annotated viral reads and circular bacteriophages, identified CRISPR loci and putative prophages, and assessed their diversity, persistence, and associations with bacterial microbiota composition. Siphoviridae was the most prevalent bacteriophage family, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. Full-length siphoviruses targeting bacterial vaginosis (BV)-associated bacteria were identified, suggesting their presence in vivo. CRISPR loci and prophage-like elements were common, and genomic analysis suggested higher diversity among Gardnerella than Lactobacillus prophages. We found that some prophages were highly persistent within participants, and identical prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. The number of CRISPR loci and prophages were associated with vaginal microbiota stability and absence of BV. Our analysis suggests that (pro)phages are common in the FGT and vaginal bacteria and (pro)phages may interact.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Bacteriophage-Induced Lipopolysaccharide Mutations in Escherichia coli Lead to Hypersensitivity to Food Grade Surfactant Sodium Dodecyl Sulfate

Bacteriophages (phages) are considered as one of the most promising antibiotic alternatives in combatting bacterial infectious diseases. However, one concern of employing phage application is the emergence of bacteriophage-insensitive mutants (BIMs). Here, we isolated six BIMs from E. coli B in the presence of phage T4 and characterized them using genomic and phenotypic methods. Of all six BIMs, a six-amino acid deletion in glucosyltransferase WaaG likely conferred phage resistance by deactivating the addition of T4 receptor glucose to the lipopolysaccharide (LPS). This finding was further supported by the impaired phage adsorption to BIMs and glycosyl composition analysis which quantitatively confirmed the absence of glucose in the LPS of BIMs. Since LPSs actively maintain outer membrane (OM) permeability, phage-induced truncations of LPSs destabilized the OM and sensitized BIMs to various substrates, especially to the food-grade surfactant sodium dodecyl sulfate (SDS). This hypersensitivity to SDS was exploited to design a T4–SDS combination which successfully prevented the generation of BIMs and eliminated the inoculated bacteria. Collectively, phage-driven modifications of LPSs immunized BIMs from T4 predation but increased their susceptibilities as a fitness cost. The findings of this study suggest a novel strategy to enhance the effectiveness of phage-based food safety interventions.

60 APPLIED LIFE 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 OSTI2020

Structural basis of superinfection exclusion by bacteriophage T4 Spackle

A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages – a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Unravelling the dynamics of the maturation protein in MS2 bacteriophage via molecular simulations

The MS2 bacteriophage capsid serves as a model system for studying viral structure and function. Mature MS2 virus consists of 178 capsid proteins and a single maturation protein (MP), which is essential for host receptor binding and infection initiation. Despite its critical role, the dynamic behavior of the capsid with the MP remains poorly understood. To address this, we conducted 0.5 µs all-atom molecular dynamics (MD) simulations of the MS2 capsid with and without the MP, revealing key insights into its structural dynamics. Our simulations showed that MP exhibits high flexibility, particularly in the “tip” and “side-loop” regions, which undergo significant motions that likely enhance its ability to engage with the F-pilus receptor. Detailed analysis of MP conformational states revealed that loop rearrangements around H357 enable transient switching between “semi-closed” and “open” conformations, suggesting a conformational selection mechanism for pilus binding. Additionally, ion interaction analyses revealed distinct sodium and chloride binding patterns, where sodium ions were mostly found at the outer capsid shell, while chloride ions interacted with the basic residues on the RNA-facing side. We also found that the presence of the MP enhances salt-bridge interactions, contributing to increased capsid stability, yet it does not significantly alter the pore sizes of pentameric and hexameric units. Together, these findings provide new insights into the functional role of the MP, highlighting its contribution to capsid stability and host receptor engagement. This study offers a foundation for understanding capsid dynamics relevant to viral infectivity and may guide future rational strategies aimed at disrupting host-virus interactions.

Capsid stability↗
DOE OSTI2021

Enhancing transcription in Escherichia coli and Pseudomonas putida using bacteriophage lambda anti-terminator protein Q

Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli . Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli , it relies on the host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the P R ' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator t R '. In this study we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3 , from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli . In addition, we expand the use of anti-terminator λQ in Pseudomonas putida . The results show ~ fivefold higher expression of a fluorescent reporter locate ~ 12.5kbp downstream from the promoter, when the transcription is driven by P R ' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Expansion of the global RNA virome reveals diverse clades of bacteriophages

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Rapid design of bacteriophage cocktails to suppress the burden and virulence of gut-resident carbapenem-resistant Klebsiella pneumoniae

Antibiotic use can lead to the expansion of multi-drug-resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, in this work, we describe a Klebsiella PhageBank for the tailored design of bacteriophage cocktails to treat multi-drug-resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae, we identify host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank, we formulate phage combinations that eliminate K. pneumoniae with minimal phage resistance. Optimized cocktails selectively suppress the burden of K. pneumoniae in the mouse gut and drive the loss of key virulence factors that act as phage receptors. Phage-mediated diversification of bacterial populations in the gut leads to co-evolution of phage variants with higher virulence and broader host range. Altogether, the Klebsiella PhageBank charts a roadmap for phage therapy against a critical multidrug-resistant human pathogen.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model

Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immune responses elicited following administration of the phage cocktail. While minimal innate responses are observed after phage administration, two rounds of treatment induces phage-specific neutralizing antibodies and accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induce a more robust neutralizing antibody response than the siphophages. This anti-phage immunity reduces the effectiveness of the phage cocktail in our murine model. Collectively, this study shows phage-specific immune responses may be an important consideration in the development of phage cocktails for therapeutic use.

60 APPLIED LIFE SCIENCES↗
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 OSTI2020

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The first sequenced Sphaerotilus natans bacteriophage– characterization and potential to control its filamentous bacterium host

ABSTRACT Bacteriophages (phages) are ubiquitous entities present in every conceivable habitat as a result of their bacterial parasitism. Their prevalence and impact in the ecology of bacterial communities and their ability to control pathogens make their characterization essential, particularly of new phages, improving knowledge and potential application. The isolation and characterization of a new lytic phage against Sphaerotilus natans strain DSM 6575, named vB_SnaP-R1 (SnaR1), is here described. Besides being the first sequenced genome of a Sphaerotilus natans infecting phage, 99% of its 41507 bp genome lacks homology with any other sequenced phage, revealing its uniqueness and previous lack of knowledge. Moreover, SnaR1 is the first Podoviridae phage described infecting this bacterium. Sphaerotilus natans is an important filamentous bacterium due to its deleterious effect on wastewater treatment plants (WWTP) and thus, phages may play a role as novel biotechnological tools against filamentous overgrowth in WWTP. The lytic spectrum of SnaR1 was restricted to its host strain, infecting only one out of three S. natans strains and infection assays revealed its ability to reduce bacterial loads. Results suggest SnaR1 as the prototype of a new phage genus and demonstrates its potential as a non-chemical alternative to reduce S. natans DSM 6575 cells.

Ferreira, Rute↗
DOE OSTI2024

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

Abstract RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron–electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Structural and functional insights into the interaction between the bacteriophage T4 DNA processing proteins gp32 and Dda

Abstract Bacteriophage T4 is a classic model system for studying the mechanisms of DNA processing. A key protein in T4 DNA processing is the gp32 single-stranded DNA-binding protein. gp32 has two key functions: it binds cooperatively to single-stranded DNA (ssDNA) to protect it from nucleases and remove regions of secondary structure, and it recruits proteins to initiate DNA processes including replication and repair. Dda is a T4 helicase recruited by gp32, and we purified and crystallized a gp32–Dda–ssDNA complex. The low-resolution structure revealed how the C-terminus of gp32 engages Dda. Analytical ultracentrifugation analyses were consistent with the crystal structure. An optimal Dda binding peptide from the gp32 C-terminus was identified using surface plasmon resonance. The crystal structure of the Dda–peptide complex was consistent with the corresponding interaction in the gp32–Dda–ssDNA structure. A Dda-dependent DNA unwinding assay supported the structural conclusions and confirmed that the bound gp32 sequesters the ssDNA generated by Dda. The structure of the gp32–Dda–ssDNA complex, together with the known structure of the gp32 body, reveals the entire ssDNA binding surface of gp32. gp32–Dda–ssDNA complexes in the crystal are connected by the N-terminal region of one gp32 binding to an adjacent gp32, and this provides key insights into this interaction.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Metagenomic stable isotope probing reveals bacteriophage participation in soil carbon cycling

Soil viruses are important components of the carbon (C) cycle, yet we still know little about viral ecology in soils. We added diverse 13 C-labelled carbon sources to soil and we used metagenomic-SIP to detect 13 C assimilation by viruses and their putative bacterial hosts. These data allowed us to link a 13 C-labelled bacteriophage to its 13 C-labelled Streptomyces putative host, and we used qPCR to track the dynamics of the putative host and phage in response to C inputs. Following C addition, putative host numbers increased rapidly for 3 days, and then more gradually, reaching maximal abundance on Day 6. Viral abundance and virus:host ratio increased dramatically over 6 days, and remained high thereafter (8.42 ± 2.94). From Days 6 to 30, virus:host ratio remained high, while putative host numbers declined more than 50%. Putative host populations were 13 C-labelled on Days 3–30, while 13 C-labelling of phage was detected on Days 14 and 30. This dynamic suggests rapid growth and 13 C-labelling of the host fueled by new C inputs, followed by extensive host mortality driven by phage lysis. Furthermore, these findings indicate that the viral shunt promotes microbial turnover in soil following new C inputs, thereby altering microbial community dynamics, and facilitating soil organic matter production.

54 ENVIRONMENTAL SCIENCES↗
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↗