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  • 1
    Online Resource
    Online Resource
    Community Structure and Microbial Associations in Sediment-Free Methanotrophic Enrichment Cultures from a Marine Methane Seep
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 11 ( 2022-06-14)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 11 ( 2022-06-14)
    Abstract: Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b (“ Candidatus Methanocomedenaceae”) and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides , Anaerolineales , and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. IMPORTANCE Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.02109-21
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
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    detail.hit.zdb_id: 1478346-0
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  • 2
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    Online Resource
    Disruption of c-di-GMP Signaling Networks Unlocks Cryptic Expression of Secondary Metabolites during Biofilm Growth in Burkholderia pseudomallei
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 8 ( 2022-04-26)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 8 ( 2022-04-26)
    Abstract: The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei , a soilborne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at various temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypobiofilm- and hyperbiofilm-forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) 2, 11, 14 (syrbactin), and 15 (malleipeptin) in parental and ΔII2523 backgrounds also reveal the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani . Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under different environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions. IMPORTANCE Burkholderia pseudomallei is a saprophytic bacterium residing in the environment that switches to a pathogenic lifestyle during infection of a wide range of hosts. The environmental cues that serve as the stimulus to trigger this change are largely unknown. However, it is well established that the cellular level of c-di-GMP, a secondary signal messenger, controls the switch from growth as planktonic cells to growth as a biofilm. Disrupting the signaling mediated by c-di-GMP allows for a better understanding of the regulation and the contribution of the surface associated and secreted molecules that contribute to the various lifestyles of this organism. The genome of B. pseudomallei also encodes cryptic biosynthetic gene clusters predicted to encode small molecules that potentially contribute to growth as a biofilm, adaptation, and interactions with other organisms. A better understanding of the regulation of these molecules is crucial to understanding how this versatile pathogen alters its lifestyle.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.02431-21
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 3
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    Improved performance of nucleic acid-based assays for genetically diverse norovirus surveillance
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology
    Abstract: Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are employed widely as a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of the rapid evolution and genetic variation of viruses. The study analyzed clinical and wastewater samples using multiple PCR assays and found significant performance variation among the PCR assays for genetically diverse norovirus surveillance. This finding suggests that some PCR assays may miss detecting certain virus strains, leading to a compromise in detection sensitivity. To address this issue, we propose a metric called the probability of detection, which can be simply calculated in silico using a code developed in this study, to evaluate nucleic acid-based assays for genetically diverse virus surveillance. This new approach can help improve the sensitivity and accuracy of virus detection, which is crucial for effective infectious disease surveillance and control.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00331-23
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 223011-2
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  • 4
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    Online Resource
    A Novel Actinobacterial Cutinase Containing a Noncatalytic Polymer-Binding Domain
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 1 ( 2022-01-11)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 1 ( 2022-01-11)
    Abstract: The single putative cutinase-encoding gene from the genome of Kineococcus radiotolerans SRS30216 was cloned and expressed in Escherichia coli as a secreted fusion protein, designated YebF-KrCUT, where YebF is the extracellular carrier protein. The 294-amino-acid sequence of KrCUT is unique among currently characterized cutinases by having a C-terminal extension that consists of a short (Pro-Thr)-rich linker and a 55-amino-acid region resembling the substrate binding domain of poly(hydroxybutyrate) (PHB) depolymerases. Phylogenetically, KrCUT takes a unique position among known cutinases and cutinase-like proteins of bacterial and fungal origins. A modeled structure of KrCUT, although displaying a typical α/β hydrolase fold, shows some unique loops close to the catalytic site. The 39-kDa YebF-KrCUT fusion protein and a truncated variant thereof were purified to electrophoretic homogeneity and functionally characterized. The melting temperatures ( T m ) of KrCUT and its variant KrCUT206 devoid of the putative PHB-binding domain were established to be very similar, at 50 to 51°C. Cutinase activity was confirmed by the appearance of characteristic cutin components, C 16 and C 18 hydroxyl fatty acids, in the mass chromatograms following incubation of KrCUT with apple cutin as the substrate. KrCUT also efficiently degraded synthetic polyesters such as polycaprolactone and poly(1,3-propylene adipate). Although incapable of PHB depolymerization, KrCUT could efficiently bind PHB, confirming the predicted characteristic of the C-terminal region. KrCUT also potentiated the activity of pectate lyase in the degradation of pectin from hemp fibers. This synergistic effect is relevant to the enzyme retting process of natural fibers. IMPORTANCE To date, only a limited number of cutinases have been isolated and characterized from nature, the majority being sourced from phytopathogenic fungi and thermophilic bacteria. The significance of our research relates to the identification and characterization of a unique member of the microbial cutinases, named KrCUT, that was derived from the genome of the Gram-positive Kineococcus radiotolerans SRS30216, a highly radiation-resistant actinobacterium. Given the wide-ranging importance of cutinases in applications such as the degradation of natural and synthetic polymers, in the textile industry, in laundry detergents, and in biocatalysis (e.g., transesterification reactions), our results could foster new research leading to broader biotechnological impacts. This study also demonstrated that genome mining or prospecting is a viable means to discover novel biocatalysts as environmentally friendly and biotechnological tools.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01522-21
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
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  • 5
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    A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 1 ( 2022-01-11)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 1 ( 2022-01-11)
    Abstract: Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. IMPORTANCE Cellulosic biofuels are a promising approach to decarbonize the heavy-duty-transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of cellulose to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum , are promising candidates for cellulosic biofuel production; however, they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially relevant organism, which can reduce the time needed for adaptive evolution experiments.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01531-21
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 6
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    The Tomato Brown Rugose Fruit Virus Movement Protein Gene Is a Novel Microbial Source Tracking Marker
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology Vol. 89, No. 7 ( 2023-07-26)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 89, No. 7 ( 2023-07-26)
    Abstract: Microbial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker. IMPORTANCE Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of host-associated MST markers. Here, we designed and tested novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool and highly abundant in human stool and wastewater samples.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00583-23
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 223011-2
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  • 7
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    Detection of SARS-CoV-2 Variants Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in Wastewater Settled Solids Using Mutation-Specific Assays Is Associated with Regional Detection of Variants in Clinical Samples
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 8 ( 2022-04-26)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 8 ( 2022-04-26)
    Abstract: Changes in the circulation of SARS-CoV-2 variants of concern (VOCs) may require changes in the public health response to the COVID-19 pandemic, as they have the potential to evade vaccines and pharmaceutical interventions and may be more transmissive than other SARS-CoV-2 variants. As such, it is essential to track and prevent their spread in susceptible communities. We developed digital reverse transcription (RT)-PCR assays for mutations characteristic of VOCs and used them to quantify those mutations in samples of wastewater settled solids collected from a publicly owned treatment works (POTW) during different phases of the COVID-19 pandemic. Wastewater concentrations of single mutations characteristic of each VOC, normalized by the concentration of a conserved SARS-CoV-2 N gene, correlate with regional estimates of the proportion of clinical infections caused by each VOC. These results suggest that targeted RT-PCR assays can be used to detect variants circulating in communities and inform the public health response to the pandemic. IMPORTANCE Wastewater represents a pooled biological sample of the contributing community and thus a resource for assessing community health. Here, we show that emergence, spread, and disappearance of SARS-CoV-2 infections caused by variants of concern are reflected in the presence of variant genomic RNA in wastewater settled solids. This work highlights an important public health use case for wastewater.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00045-22
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
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  • 8
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    Liquid-Liquid Phase Separation of the DEAD-Box Cyanobacterial RNA Helicase Redox (CrhR) into Dynamic Membraneless Organelles in Synechocystis sp. Strain PCC 6803
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology Vol. 89, No. 4 ( 2023-04-26)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 89, No. 4 ( 2023-04-26)
    Abstract: Compartmentalization of macromolecules into discrete non-lipid-bound bodies by liquid-liquid phase separation (LLPS) is a well-characterized regulatory mechanism frequently associated with the cellular stress response in eukaryotes. In contrast, the formation and importance of similar complexes is just becoming evident in bacteria. Here, we identify LLPS as the mechanism by which the DEAD-box RNA helicase, cyanobacterial RNA helicase redox (CrhR), compartmentalizes into dynamic membraneless organelles in a temporal and spatial manner in response to abiotic stress in the cyanobacterium Synechocystis sp. strain PCC 6803. Stress conditions induced CrhR to form a single crescent localized exterior to the thylakoid membrane, indicating that this region is a crucial domain in the cyanobacterial stress response. These crescents rapidly dissipate upon alleviation of the stress conditions. Furthermore, CrhR aggregation was mediated by LLPS in an RNA-dependent reaction. We propose that dynamic CrhR condensation performs crucial roles in RNA metabolism, enabling rapid adaptation of the photosynthetic apparatus to environmental stresses. These results expand our understanding of the role that functional compartmentalization of RNA helicases and thus RNA processing in membraneless organelles by LLPS-mediated protein condensation performs in the bacterial response to environmental stress. IMPORTANCE Oxygen-evolving photosynthetic cyanobacteria evolved ~3 billion years ago, performing fundamental roles in the biogeochemical evolution of the early Earth and continue to perform fundamental roles in nutrient cycling and primary productivity today. The phylum consists of diverse species that flourish in heterogeneous environments. A prime driver for survival is the ability to alter photosynthetic performance in response to the shifting environmental conditions these organisms continuously encounter. This study demonstrated that diverse abiotic stresses elicit dramatic changes in localization and structural organization of the RNA helicase CrhR associated with the photosynthetic thylakoid membrane. These dynamic changes, mediated by a liquid-liquid phase separation (LLPS)-mediated mechanism, reveal a novel mechanism by which cyanobacteria can compartmentalize the activity of ribonucleoprotein complexes in membraneless organelles. The results have significant consequences for understanding bacterial adaptation and survival in response to changing environmental conditions.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00015-23
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 9
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    Back to the Roots: Agrobacterium -Specific Phages Show Potential to Disinfect Nutrient Solution from Hydroponic Greenhouses
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology Vol. 89, No. 4 ( 2023-04-26)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 89, No. 4 ( 2023-04-26)
    Abstract: Agrobacterium biovar 1 is a soilborne plant pathogen with the ability to colonize the irrigation system of greenhouses, causing hairy root disease (HRD). Currently, management focuses on using hydrogen peroxide to disinfect the nutrient solution, but due to the emergence of resistant strains, its efficacy and sustainability are questioned. Using a relevant collection of pathogenic Agrobacterium biovar 1 strains, OLIVR1 to 6, six phages specific to this pathogen and belonging to three different genera were isolated from Agrobacterium biovar 1-infected greenhouses. All phages were named OLIVR, referring to their location of isolation, O nze- Li eve- Vr ouwe-Waver, and were characterized by whole-genome analysis, confirming their strictly lytic lifestyle. They remained stable under greenhouse-relevant conditions. To assess the efficacy of the phages, their ability to disinfect greenhouse nutrient solution inoculated with agrobacteria was tested. Each of the phages infected their host, but their ability to decrease the bacterial concentration differed. For instance, OLIVR1 reduced the bacterial concentration with 4 log units without phage resistance emerging. While OLIVR4 and OLIVR5 were also infectious in nutrient solution, they did not always decrease the bacterial load below the limit of detection, and phage resistance emerged. Finally, the mutations causing phage resistance by receptor modification were identified. For OLIVR4-resistant Agrobacterium isolates, but not for OLIVR5-resistant isolates, motility decreased. Together, these data show the potential of some of these phages as disinfectant of nutrient solution, and they might be a valuable tool to tackle HRD. IMPORTANCE Hairy root disease, caused by rhizogenic Agrobacterium biovar 1 is a rapidly emerging bacterial disease worldwide. It affects tomatoes, cucumbers, eggplant, and bell pepper, causing high yield losses in hydroponic greenhouses. Recent findings suggest that the current management practices, mainly focusing on UV-C and hydrogen peroxide to disinfect contaminated water, have a questionable efficacy. Hence, we investigate the potential of phages as a biological means of preventing this disease. Using a diverse collection of Agrobacterium biovar 1, we isolated three different phage species that together infect 75% of the collection. Since these phages are strictly lytic, while remaining both stable and infectious under greenhouse-relevant conditions, they might be suitable candidates for biological control.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00215-23
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 10
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    Diverse Durham collection phages demonstrate complex BREX defense responses
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology Vol. 89, No. 9 ( 2023-09-28)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 89, No. 9 ( 2023-09-28)
    Abstract: Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00623-23
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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