Phage Infection Research Articles

Bacteriophage Treatment Induces Phenotype Switching and Alters Antibiotic Resistance of ESBL Escherichia coli

Background/Objectives: Bacteriophage therapy represents a promising strategy to combat multidrug-resistant pathogens, such as Escherichia coli. In this study, we explored the effects of a bacteriophage infection on an Extended Spectrum Beta-Lactamase (ESBL) positive E. coli isolate. Methods: We used next generation sequencing, proteomics and phenotypic screens to investigate the effect of bacteriophage infections on E. coli metabolism and resistance phenotypes. Results: The bacteriophage infection led to notable alterations in colony morphology, indicating profound changes in bacterial metabolism. Proteomic analysis revealed significant shifts in protein expression, with 65 proteins upregulated and 246 downregulated post-infection. The downregulated proteins were involved in various metabolic pathways, including nucleic acid, protein and lipid metabolism, and iron acquisition. Bacteriophage treatment also led to increased bacterial membrane permeability. Altogether, these alterations in bacterial metabolism and membrane permeability may lead to a general reduction in antibiotic resistance. Indeed, the bacteriophage-infected E. coli exhibited increased sensitivity to various classes of antibiotics, including beta-lactams, fluoroquinolones, trimethoprim-sulfamethoxazole, and aminoglycosides. Conclusions: Our findings highlight the potential of bacteriophage therapy as an adjunct to existing antibiotics, enhancing their efficacy against resistant strains.

Prophage-DB: a comprehensive database to explore diversity, distribution, and ecology of prophages

BackgroundViruses that infect prokaryotes (phages) constitute the most abundant group of biological agents, playing pivotal roles in microbial systems. They are known to impact microbial community dynamics, microbial ecology, and evolution. Efforts to document the diversity, host range, infection dynamics, and effects of bacteriophage infection on host cell metabolism are extremely underexplored. Phages are classified as virulent or temperate based on their life cycles. Temperate phages adopt the lysogenic mode of infection, where the genome integrates into the host cell genome forming a prophage. Prophages enable viral genome replication without host cell lysis, and often contribute novel and beneficial traits to the host genome. Current phage research predominantly focuses on lytic phages, leaving a significant gap in knowledge regarding prophages, including their biology, diversity, and ecological roles.ResultsHere we develop and describe Prophage-DB, a database of prophages, their proteins, and associated metadata that will serve as a resource for viral genomics and microbial ecology. To create the database, we identified and characterized prophages from genomes in three of the largest publicly available databases. We applied several state-of-the-art tools in our pipeline to annotate these viruses, cluster them, taxonomically classify them, and detect their respective auxiliary metabolic genes. In total, we identify and characterize over 350,000 prophages and 35,000 auxiliary metabolic genes. Our prophage database is highly representative based on statistical results and contains prophages from a diverse set of archaeal and bacterial hosts which show a wide environmental distribution.ConclusionGiven that prophages are particularly overlooked and merit increased attention due to their vital implications for microbiomes and their hosts, we created Prophage-DB to advance our understanding of prophages in microbiomes through a comprehensive characterization of prophages in publicly available genomes. We propose that Prophage-DB will serve as a valuable resource for advancing phage research, offering insights into viral taxonomy, host relationships, auxiliary metabolic genes, and environmental distribution.

MS2 bacteriophage infectivity after exposure to RH, ozone, chlorine dioxide and solar radiation using an oxidation flow reactor and a rotating drum

MS2 bacteriophage infectivity after exposure to RH, ozone, chlorine dioxide and solar radiation using an oxidation flow reactor and a rotating drum

Streptomyces secretes a siderophore that sensitizes competitor bacteria to phage infection.

To overtake competitors, microbes produce and secrete secondary metabolites that kill neighbouring cells and sequester nutrients. This metabolite-mediated competition probably evolved in complex microbial communities in the presence of viral pathogens. We therefore hypothesized that microbes secrete natural products that make competitors sensitive to phage infection. We used a binary-interaction screen and chemical characterization to identify a secondary metabolite (coelichelin) produced by Streptomyces sp. that sensitizes its soil competitor Bacillus subtilis to phage infection in vitro. The siderophore coelichelin sensitized B. subtilis to a panel of lytic phages (SPO1, SP10, SP50, Goe2) via iron sequestration, which prevented the activation of B. subtilis Spo0A, the master regulator of the stationary phase and sporulation. Metabolomics analysis revealed that other bacterial natural products may also provide phage-mediated competitive advantages to their producers. Overall, this work reveals that synergy between natural products and phages can shape the outcomes of competition between microbes.

Adaptive loss of tRNA gene expression leads to phage resistance in a marine Synechococcus cyanobacterium

Synechococcus is a significant primary producer in the oceans, coexisting with cyanophages, which are important agents of mortality. Bacterial resistance against phage infection is a topic of significant interest, yet little is known for ecologically relevant systems. Here we use exogenous gene expression and gene disruption to investigate mechanisms underlying intracellular resistance of marine Synechococcus WH5701 to the Syn9 cyanophage. The restriction–modification and Gabija defence systems possessed by Synechococcus WH5701 did not contribute to resistance. Instead, resistance was primarily driven by insufficient levels of LeuTAA tRNA, preventing translation of key phage genes in a passive, intracellular mode of resistance. Restoring cellular tRNA expression rendered the cyanobacterium sensitive to infection. We propose an evolutionary scenario whereby changes in cell codon usage, acquisition of tRNAs by the phage and loss of cell and phage tRNA expression resulted in an effective means of resistance, highlighting the dynamic interplay between bacteria and phages in shaping their co-evolutionary trajectories.

Engineered Phage Enables Efficient Control of Gene Expression upon Infection of the Host Cell.

Recently, we developed a spatial phage-assisted continuous evolution (SPACE) system. This system utilizes chemotaxis coupled with the growth of motile bacteria during their spatial range expansion in soft agar to provide fresh host cells for iterative phage infection and selection pressure for preserving evolved genes of interest carried by phage mutants. Controllable mutagenesis activated only in a subpopulation of the migrating cells is essential in this system to efficiently generate mutated progeny phages from which desired individuals are selected during the directed evolution process. But, the widely adopted small molecule-dependent inducible system could hardly fulfill this purpose because it always affects all cells homogeneously. In this study, we developed a phage infection-induced gene expression system using modified Escherichia coli (E. coli) phage shock protein operon or sigma factors from Bacillus subtilis. Results showed that this system enabled efficient control of gene expression upon phage infection with dynamic output ranges from small to large using combinations of different engineered phages and corresponding promoters. This system was incorporated into SPACE to function as a phage infection-induced mutagenesis module and successfully facilitated the evolution of T7 RNA polymerase, which generated diverse mutants with altered promoter recognition specificity. We expect that phage infection-induced gene expression system could be further extended to more applications involving partial induction in a portion of a population and targeted induction in specific strains among a mixed bacterial community, which provides an important complement to small molecule-dependent inducible systems.

Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection.

The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.

Structure and mechanism of the Zorya anti-phage defense system.

Zorya is a recently identified and widely distributed bacterial immune system that protects bacteria from viral (phage) infections. Three Zorya subtypes have been discovered, each containing predicted membrane-embedded ZorAB complexes paired with soluble subunits that differ among Zorya subtypes, notably ZorC and ZorD in type I Zorya systems1,2. Here, we investigate the molecular basis of Zorya defense using cryo-electron microscopy, mutagenesis, fluorescence microscopy, proteomics, and functional studies. We present cryo-EM structures of ZorAB and show that it shares stoichiometry and features of other 5:2 inner membrane ion-driven rotary motors. The ZorA5B2 complex contains a dimeric ZorB peptidoglycan binding domain and a pentameric α-helical coiled-coil tail made of ZorA that projects approximately 70 nm into the cytoplasm. We also characterize the structure and function of the soluble Zorya components, ZorC and ZorD, finding that they harbour DNA binding and nuclease activity, respectively. Comprehensive functional and mutational analyses demonstrate that all Zorya components work in concert to protect bacterial cells against invading phages. We provide evidence that ZorAB operates as a proton-driven motor that becomes activated upon sensing of phage invasion. Subsequently, ZorAB transfers the phage invasion signal through the ZorA cytoplasmic tail to recruit and activate the soluble ZorC and ZorD effectors, which facilitate degradation of the phage DNA. In summary, our study elucidates the foundational mechanisms of Zorya function as an anti-phage defense system.

Genomic analysis, culturing optimization, and characterization of Escherichia bacteriophage OSYSP, previously studied as effective pathogen control on fresh produce

Advances in bacteriophage genome sequencing and regulatory approvals of some bacteriophages in various applications have renewed interest in these antibacterial viruses as a potential solution to persistent food safety challenges. Here, we analyzed in depth the genome of the previously studied Escherichia bacteriophage OSYSP (phage OSYSP), revealed its application-related characteristics, and optimized its enumeration techniques for facilitating industrial implementation. We previously sequenced phage OSYSP genome completely by combining results from Illumina Miseq and Ion Torrent sequencing platforms and completing the remaining sequence gaps using PCR. Based on the genomics analysis completed herein, phage OSYSP was confirmed as an obligate lytic phage of the Caudoviricetes class. The genome encodes 81 proteins of identifiable functions, including two endolysins and 45 proteins that support host-independent DNA replication, transcription, and repair. Despite its similarities to T5-like phages, unique genome arrangements confirm phage OSYSP’s novelty. The genomic analysis also confirmed the absence of DNA sequences encoding virulence or antibiotic resistance factors. For optimizing phage detection and quantification in the conventional plaque assay, it was observed that decreasing the concentration of agar or agarose, when used as a medium gelling agent, increased phage recovery (p < 0.05), but using agarose resulted in smaller plaque diameters (p < 0.05). Phage OSYSP inactivated pathogenic and non-pathogenic strains of E. coli and some Salmonella enterica serovars, with more pronounced effect against E. coli O157:H7. Phage titers remained fairly unchanged throughout a 24-month storage at 4°C. Incubation for 30 min at 4°C−47°C or pH 4–11 had no significant detrimental effect (p > 0.05) on phage infectivity. In vitro application of phage OSYSP against E. coli O157:H7 EDL933 decreased the pathogen’s viable population by >5.7-log CFU/mL within 80 min, at a multiplicity of infection as low as 0.01. The favorable genome characteristics, combined with improved enumeration methodology, and the proven infectivity stability, make phage OSYSP a promising biocontrol agent against pathogenic E. coli for food or therapeutic applications.

Free DNA partially clarifies discrepancies between qPCR and the conventional phage quantification method.

To use phages in a personalized therapy and industrial applications, an accurate quantification is needed. The gold standard method, namely the culture-based double agar overlay (DAO) method, provides an accurate estimate of the number of infectious phages but is laborious and time-intensive. Quantitative polymerase chain reaction (qPCR) can be used as a fast alternative but tends to overestimate the number of infectious phage particles. Here we describe the use of a DNase treatment before quantification of the Staphylococcus aureus phage ISP with qPCR to obtain a more accurate estimate of the number of infectious phage particles. We showed that DNase treatment results in a significant decrease of the concentration when measured with qPCR although for two out of three tested ISP phage stocks, there was still a significant difference with the DAO method. We also showed that the discrepancy between quantification with qPCR and the DAO method is dependent on the storage period of the phage stock, with a larger discrepancy for older stocks. Additionally, we used negative contrast immune electron microscopy to confirm the presence of DNA in the medium of the phage stock and the impact of the DNase treatment on the free DNA.

Genomic Characterization of Bacillus sp. THPS1: A Hot Spring-Derived Species with Functional Features and Biotechnological Potential.

Bacillus sp. THPS1 is a novel strain isolated from a high-temperature hot spring in Thailand, exhibiting distinctive genomic features that enable adaptation to an extreme environment. This study aimed to characterize the genomic and functional attributes of Bacillus sp. THPS1 to understand its adaptation strategies and evaluate its potential for biotechnological applications. The draft genome is 5.38 Mbp with a GC content of 35.67%, encoding 5606 genes, including those linked to stress response and sporulation, which are essential for survival in high-temperature conditions. Phylogenetic analysis and average nucleotide identity (ANI) values confirmed its classification as a distinct species within the Bacillus genus. Pangenome analysis involving 19 others closely related thermophilic Bacillus species identified 1888 singleton genes associated with heat resistance, sporulation, and specialized metabolism, suggesting adaptation to nutrient-deficient, high-temperature environments. Genomic analysis revealed 12 biosynthetic gene clusters (BGCs), including those for polyketides and non-ribosomal peptides, highlighting its potential for synthesizing secondary metabolites that may facilitate its adaptation. Additionally, the presence of three Siphoviridae phage regions and 96 mobile genetic elements (MGEs) suggests significant genomic plasticity, whereas the existence of five CRISPR arrays implies an advanced defense mechanism against phage infections, contributing to genomic stability. The distinctive genomic features and functional capacities of Bacillus sp. THPS1 make it a promising candidate for biotechnological applications, particularly in the production of heat-stable enzymes and the development of resilient bioformulations.

The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.

Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cAn) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA4 or cA6 to its CARF domain, Cad1 converts ATP to ITP, both invivo and invitro. Cryoelectron microscopy (cryo-EM) structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cAn during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation. Our findings reveal that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.

Conformational Plasticity of SpyCas9 Induced by AcrIIA4 and AcrIIA2: Insights from Molecular Dynamics Simulation

CRISPR-Cas9 systems constitute bacterial adaptive immune systems that protect against phage infections. Bacteriophages encode anti-CRISPR proteins (Acrs) that mitigate the bacterial immune response. However, the structural basis for their inhibitory actions from a molecular perspective remains elusive. In this study, through microsecond atomistic molecular dynamics simulations, we demonstrated the remarkable flexibility of Streptococcus pyogenes Cas9 (SpyCas9) and its conformational adaptability during interactions with AcrIIA4 and AcrIIA2. Specifically, we demonstrated that the binding of AcrIIA4 and AcrIIA2 to SpyCas9 induces a conformational rearrangement that causes spatial separation between the nuclease and cleavage sites, thus making the endonuclease inactive. This separation disrupts the transmission of signals between the protospacer adjacent motif recognition and nuclease domains, thereby impeding the efficient processing of double-stranded DNA. The simulation also reveals that AcrIIA4 and AcrIIA2 cause different structural variations of SpyCas9. Our research illuminates the precise mechanisms underlying the suppression of SpyCas9 by AcrIIA4 and AcrIIA2, thus presenting new possibilities for controlling genome editing with higher accuracy.

The two-component system ArlRS is essential for wall teichoic acid glycoswitching in Staphylococcus aureus.

Staphylococcus aureus is among the leading causes of hospital-acquired infections. Critical to S. aureus biology and pathogenesis are the cell wall-anchored glycopolymers wall teichoic acids (WTA). Approximately one-third of S. aureus isolates decorates WTA with a mixture of α1,4- and β1,4-N-acetylglucosamine (GlcNAc), which requires the dedicated glycosyltransferases TarM and TarS, respectively. Environmental conditions, such as high salt concentrations, affect the abundance and ratio of α1,4- and β1,4-GlcNAc WTA decorations, thereby impacting biological properties such as antibody binding and phage infection. To identify regulatory mechanisms underlying WTA glycoswitching, we screened 1,920 S. aureus mutants (Nebraska Transposon Mutant Library) by immunoblotting for differential expression of WTA-linked α1,4- or β1,4-GlcNAc using specific monoclonal antibody Fab fragments. Three two-component systems (TCS), GraRS, ArlRS, and AgrCA, were among the 230 potential hits. Using isogenic TCS mutants, we demonstrated that ArlRS is essential for WTA β1,4-GlcNAc decoration. ArlRS repressed tarM expression through the transcriptional regulator MgrA. In bacteria lacking arlRS, the increased expression of tarM correlated with the absence of WTA β1,4-GlcNAc, likely by outcompeting TarS enzymatic activity. ArlRS was responsive to Mg2+, but not Na+, revealing its role in the previously reported salt-induced WTA glycoswitch from α1,4-GlcNAc to β1,4-GlcNAc. Importantly, ArlRS-mediated regulation of WTA glycosylation affected S. aureus interaction with the innate receptor langerin and lysis by β1,4-GlcNAc-dependent phages. Since WTA represents a promising target for future immune-based treatments and vaccines, our findings provide important insight to align strategies targeting S. aureus WTA glycosylation patterns during infection.IMPORTANCEStaphylococcus aureus is a common colonizer but can also cause severe infections in humans. The development of antibiotic resistance complicates the treatment of S. aureus infections, increasing the need for antibiotic alternatives such as vaccines and therapies with bacterial viruses also known as phages. Wall teichoic acids (WTA) are abundant glycosylated structures of the S. aureus cell wall that have gained attention as a promising target for new treatments. Importantly, WTA glycosylation patterns show variation depending on environmental conditions, thereby impacting phage binding and interaction with host factors, such as antibodies and innate pattern-recognition receptors. Here, we show that the two-component system ArlRS is involved in the regulation of WTA glycosylation by responding to environmental changes in Mg2+ concentration. These findings may support the design of new treatment strategies that target WTA glycosylation patterns of S. aureus during infection.

Bacteriophage Therapy as a Promising Alternative for Antibiotic-Resistant Enterococcus faecium: Advances and Challenges.

Enterococcus faecium is a Gram-positive bacterium increasingly identified as a critical nosocomial pathogen that poses significant treatment challenges due to its resistance to multiple antibiotics, particularly vancomycin-resistant E. faecium (VRE) strains. The urgent need for alternative therapeutic strategies has renewed interest in bacteriophage (phage) therapy, given phages specificity and bactericidal potential. This review explores the advancements in phage therapy against antibiotic-resistant E. faecium, including phage morphological diversity, genomic characteristics, and infection mechanisms. The efficacy of phage therapy in in vitro, ex vivo, and in vivo models and the compassionate use in clinical settings are evaluated, highlighting the promising outcomes of phage-antibiotic synergies and biofilm disruption. Key challenges and future research directions are discussed, with a focus on improving therapeutic efficacy and overcoming bacterial resistance. This review emphasizes the potential of phage therapy as a viable solution for managing multidrug-resistant E. faecium infections and underscores the importance of future investigations to enhance clinical applications.

Inferring strain-level mutational drivers of phage-bacteria interaction phenotypes arising during coevolutionary dynamics.

The enormous diversity of bacteriophages and their bacterial hosts presents a significant challenge to predict which phages infect a focal set of bacteria. Infection is largely determined by complementary - and largely uncharacterized - genetics of adsorption, injection, cell take-over and lysis. Here we present a machine learning approach to predict phage-bacteria interactions trained on genome sequences of and phenotypic interactions amongst 51 Escherichia coli strains and 45 phage strains that coevolved in laboratory conditions for 37 days. Leveraging multiple inference strategies and without a priori knowledge of driver mutations, this framework predicts both who infects whom and the quantitative levels of infections across a suite of 2,295 potential interactions. We found that the most effective approach inferred interaction phenotypes from independent contributions from phage and bacteria mutations, accurately predicting of interactions while reducing the relative error in the estimated strength of the infection phenotype by . Feature selection revealed key phage and E. coli mutations that have a significant influence on the outcome of phage-bacteria interactions, corroborating sites previously known to affect phage infections, as well as identifying mutations in genes of unknown function not previously shown to influence bacterial resistance. The method's success in recapitulating strain-level infection outcomes arising during coevolutionary dynamics may also help inform generalized approaches for imputing genetic drivers of interaction phenotypes in complex communities of phage and bacteria.

AcrIIIA1 is a protein-RNA anti-CRISPR complex that targets core Cas and accessory nucleases.

Clustered regularly-interspaced short palindromic repeats (CRISPRs) andCRISPR-associated (Cas) proteinsprotect bacteria and archaea from their viruses, and anti-CRISPRs (Acrs) are small virus-encoded proteins that inhibit CRISPR-Cas immunity. Over 80 families of Acrs have been described to date; however, only three of these subvert Type III CRISPR-Cas immunity. Type IIIsystems employ a complex network ofCasand accessory nucleases to degrade viral nucleic acids. Here, we discover and characterize AcrIIIA1, the first Type III-A specific anti-CRISPR protein. We demonstrate that AcrIIIA1 binds to Csm2 within the Cas10-Csm effector complex and attenuates Cas10's DNase activity and second messenger production. Additionally, AcrIIIA1 associates with fragmented t(m)RNAs (acrIIIA1-RNAs), and we show that they co-purify with the Cas10-Csm complex during phage infection. Although the precise role(s) of acrIIIA1-RNAs remain unclear, we found that they bind stably to RNase R, a host-encoded nuclease known to bolster immunity, and RNase R has the capacity to degrade them. Altogether, our results support a model in which AcrIIIA1 and its associated RNAs target both core Cas and accessory nucleases to provide robust protection against Type III CRISPR-Cas immunity.

Adaptations in gut Bacteroidales facilitate stable co-existence with their lytic bacteriophages.

Bacteriophages (phages) and bacteria within the gut microbiome persist in long-term stable coexistence. These interactions are driven by eco-evolutionary dynamics, where bacteria employ a variety of mechanisms to evade phage infection, while phages rely on counterstrategies to overcome these defences. Among the most abundant phages in the gut are the crAss-like phages that infect members of the Bacteroidales, in particular Bacteroides. In this study, we explored some of the mechanisms enabling the co-existence of four phage-Bacteroidales host pairs in vitro using a multi-omics approach (transcriptomics, proteomics and metabolomics). These included three Bacteroides species paired with three crAss-like phages (Bacteroides intestinalis and ϕcrAss001, Bacteroides xylanisolvens and ϕcrAss002, and an acapsular mutant of Bacteroides thetaiotaomicron with DAC15), and Parabacteroides distasonis paired with the siphovirus ϕPDS1. We show that phase variation of individual capsular polysaccharides (CPSs) is the primary mechanism promoting phage co-existence in Bacteroidales, but this is not the only strategy. Alternative resistance mechanisms, while potentially less efficient than CPS phase variation, can be activated to support bacterial survival by regulating gene expression and resulting in metabolic adaptations, particularly in amino acid degradation pathways. These mechanisms, also likely regulated by phase variation, enable bacterial populations to persist in the presence of phages, and vice versa. An acapsular variant of B. thetaiotaomicron demonstrated broader transcriptomic, proteomic, and metabolomic changes, supporting the involvement of additional resistance mechanisms beyond CPS variation. This study advances our understanding of long-term phage-host interaction, offering insights into the long-term persistence of crAss-like phages and extending these observations to other phages, such as ϕPDS1. Knowledge of the complexities of phage-bacteria interactions is essential for designing effective phage therapies and improving human health through targeted microbiome interventions.

Metabolites from intact phage-infected Synechococcus chemotactically attract heterotrophic marine bacteria.

Chemical cues mediate interactions between marine phytoplankton and bacteria, underpinning ecosystem-scale processes including nutrient cycling and carbon fixation. Phage infection alters host metabolism, stimulating the release of chemical cues from intact plankton, but how these dynamics impact ecology and biogeochemistry is poorly understood. Here we determine the impact of phage infection on dissolved metabolite pools from marine cyanobacteria and the subsequent chemotactic response of heterotrophic bacteria using time-resolved metabolomics and microfluidics. Metabolites released from intact, phage-infected Synechococcus elicited strong chemoattraction from Vibrio alginolyticus and Pseudoalteromonas haloplanktis, especially during early infection stages. Sustained bacterial chemotaxis occurred towards live-infected Synechococcus, contrasted by no discernible chemotaxis towards uninfected cyanobacteria. High-throughput microfluidics identified 5'-deoxyadenosine and 5'-methylthioadenosine as key attractants. Our findings establish that, before lysis, phage-infected picophytoplankton release compounds that attract motile heterotrophic bacteria, suggesting a mechanism for resource transfer that might impact carbon and nutrient fluxes across trophic levels.

Investigation of mutagenicity of styrene in tumor target and non-target tissues of transgenic Big Blue® mice.

Styrene has been shown to induce lung tumors in mice, but not in rats. The current study investigated the potential role of genotoxicity as an initial key event in the mode of action for styrene-induced lung tumors in mice. Transgenic male B6C3F1 Big Blue® mice were treated by oral gavage for 28 consecutive days with 0 (corn oil), 75, 150, or 300 mg/kg/day of styrene. The 300 mg/kg/day represented the tumorigenic dose in the oral gavage carcinogenicity study conducted in B6C3F1 mice. Following a 28-day expression period, mutant frequencies were assessed at the cII locus of the transgene in the tumor target (lung) and non-target tissues (liver, glandular stomach, and duodenum). Mice treated with N-ethyl-N-nitrosourea (40 mg/kg/day) by oral gavage on Days 1, 2, and 3 of the study and sacrificed on Day 56 served as the positive control group. Genomic DNA was extracted from the selected tissues, processed for the recovery of the transgene into infectious phage, plated onto Escherichia coli strain G1250, and incubated at 37°C for titer determination or at 24°C for the selection of mutant plaques. There were no treatment-related increases in mutant frequency in any of the tissues. The positive control group had a significant increase in the frequency of cII mutants assuring the adequacy of the experimental conditions to detect induced mutations. To conclude, mutagenicity is not considered a plausible initial key event in the mode of action for styrene-induced mouse lung tumors as these data support that styrene is not an in vivo mutagen.