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- Elbasani, E, et al.
(author)
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Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter
- 2020
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In: Viruses. - : MDPI AG. - 1999-4915. ; 12:9
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Journal article (peer-reviewed)abstract
- CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.
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- Kuil, Teun, et al.
(author)
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Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases
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Other publication (other academic/artistic)abstract
- The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and activity (Vmax > 156 U mgprotein-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and activity (14.5 U mgprotein-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.
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- Kuil, Teun, et al.
(author)
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Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases
- 2023
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 743
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Journal article (peer-reviewed)abstract
- The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and maximum activity (Vmax > 156 U mg-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and maximum activity (14.5 U mg-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.
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- Uhlig, E., et al.
(author)
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The live bacterial load and microbiota composition of prepacked “ready-to-eat” leafy greens during household conditions, with special reference to E. coli
- 2022
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In: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 377
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Journal article (peer-reviewed)abstract
- Ready-to-eat (RTE) leafy greens are popular products that unfortunately have been associated with numerous foodborne illness outbreaks. Since the influence of consumer practices is essential for their quality and safety, the objective of this study was to analyze the microbiota of RTE products throughout shelf life during simulated household conditions. Products from different companies were analyzed in terms of plate counts, and resealed and unopened packages were compared. High bacterial loads were found, up to a total plate count of 9.6 log10 CFU/g, and Enterobacteriaceae plate counts up to 6.0 CFU/g on the expiration date. The effect of consumer practice varied, thus no conclusions regarding resealed or unopened bags could be drawn. The tested products contained opportunistic pathogens, such as Enterobacter homaechei, Hafnia paralvei and Pantoea agglomerans. Amplicon sequencing revealed that the relative abundance of major taxonomic groups changed during shelf life; Pseudomonadaceae and Xanthomonadaceae decreased, while Flavobacteriaceae and Marinomonadaceae inceased. Inoculation with E. coli CCUG 29300T showed that the relative abundance of Escherichia-Shigella was lower on rocket than on other tested leafy greens. Inoculation with E. coli strain 921 indicate growth at the beginning of shelf-life time, while E. coli 731 increases at the end, seemingly able to adapt to cold storage conditions. The high levels of live microorganisms, the detection of opportunistic pathogens, and the ability of E. coli strains to grow at refrigeration temperature raise concerns and indicate that the shelf life may be shortened to achieve a safer product. Due to variations between products, further studies are needed to define how long the shelf-life of these products should be, to ensure a safe product even at the end of the shelf-life period.
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- Uhlig, E., et al.
(author)
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Use of bacterial strains antagonistic to Escherichia coli for biocontrol of spinach : A field trial
- 2021
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In: Innovative Food Science and Emerging Technologies. - : Elsevier BV. - 1466-8564. ; 74
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Journal article (peer-reviewed)abstract
- To counteract global food safety hazards related to raw consumption of ready-to-eat leafy vegetables, a method to improve bacterial status using antagonistic bacteria was studied under field conditions. This is the first study to identify potential Escherichia coli antagonists from the native microbiota on leafy green vegetables and evaluate their effect in an industrial field production setting. Bacterial strains were isolated from different types of leafy green vegetables and selected upon their effect against E. coli in vitro, and out of 295 tested bacterial strains, 37 showed an antagonistic effect. Four of those antagonistic strains were coated in separate treatments onto spinach seeds and planted in the field. Both seeds and plants were analyzed by Illumina MiSeq next generation sequencing (NGS), and it was seen that the microbiota of the plants contained lower relative abundance of plant and human pathogenic genera. Higher β-diversity was observed for the samples treated with Bacillus coagulans LMG P-32205 and B. coagulans LMG P-32206 compared to control, indicating that those strains have induced substantial changes in the native microbiota of the leaves. A reduction of Escherichia-Shigella was seen for two of the isolates (Pseudomonas cedrina LMG P-32207 and Pseudomonas punonenis LMG P-32204) as the seeds developed into plants. Seeds inoculated with two of the strains (B. coagulans LMG P-32205 and B. coagulans LMG P-32206) had increased levels of Lactobacillaceae, and treatment with B. coagulans LMG P-32206 resulted in lower levels of Pantoea (from 31.4 to 12.2%). These results encourage the usage of bacterial antagonists as part of a global solution to reduce the risk of human pathogens on leafy green vegetables.
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