Articoli di riviste: "Reporter strain"

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Reuscher, Carina Maria, Lisa Schmidt, Anette Netsch e Benjamin Lamp. "Characterization of a Cytopathogenic Reporter CSFV". Viruses 13, n. 7 (23 giugno 2021): 1209. http://dx.doi.org/10.3390/v13071209.

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Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

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Singh, Vandana, Rajesh Kumar Biswas e Bhupendra N. Singh. "Double Recombinant Mycobacterium bovis BCG Strain for Screening of Primary and Rationale-Based Antimycobacterial Compounds". Antimicrobial Agents and Chemotherapy 58, n. 3 (16 dicembre 2013): 1389–96. http://dx.doi.org/10.1128/aac.01301-13.

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ABSTRACTConventional antimycobacterial screening involves CFU analysis, which poses a great challenge due to slow growth of mycobacteria. Recombinant strains carrying reporter genes under the influence of constitutive promoters allow rapid and wide screening of compounds but without revealing their modes of action. Reporter strains using pathway-specific promoters provide a better alternative but allow a limited screening of compounds interfering with only a particular metabolic pathway. This reduces these strains to merely a second-line screening system, as they fail to identify even the more potent compounds if they are not inhibiting the pathway of interest. In this study, we have generated a double recombinantMycobacterium bovisBCG strain carrying firefly andRenillaluciferase genes as two reporters under the control of a constitutive and an inducible mycobacterial promoter. The presence of dual reporters allows simultaneous expression and analysis of two reporter enzymes within a single system. The expression profile of the firefly luciferase gene, rendered by a constitutive mycobacterial promoter, coincides with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression ofRenillaluciferase mirrors the selective induction of the reporter gene expression as a result of pathway-specific inhibition. Thus, the double recombinant strain allows the screening of both primary and rationally synthesized antimycobacterial compounds in a single assay. The inhibiting response of drugs was monitored with a dual-luciferase reporter assay which can be easily adapted in high-throughput mode.

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Achatz-Straussberger, Gertrude, Roland Geisberger, Iris Oberndorfer, Daniela Inführ, Elke Luger, Padraic Fallon, Marinus Lamers e Gernot Achatz. "Construction of an sIgE:FLAG-mIgE:GFP Reporter Mouse Strain". International Archives of Allergy and Immunology 130, n. 4 (2003): 280–87. http://dx.doi.org/10.1159/000070215.

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Olias, Philipp, e L. David Sibley. "Functional Analysis of the Role of Toxoplasma gondii Nucleoside Triphosphate Hydrolases I and II in Acute Mouse Virulence and Immune Suppression". Infection and Immunity 84, n. 7 (18 aprile 2016): 1994–2001. http://dx.doi.org/10.1128/iai.00077-16.

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Bioluminescent reporter assays have been widely used to study the effect ofToxoplasma gondiion host gene expression. In the present study, we extend these studies by engineering novel reporter cell lines containing a gamma-activated sequence (GAS) element driving firefly luciferase (FLUC). In RAW264.7 macrophages,T. gondiitype I strain (GT1) infection blocked interferon gamma (IFN-γ)-induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of theT. gondiienzyme nucleoside triphosphate hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPases I and II were not essential for tachyzoite growthin vitroor virulence in mice. Our study reveals that althoughT. gondiiNTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP-independentGaussialuciferase, which provides a more appropriate reporter for use withT. gondiiinfection studies.

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Mao, Xiaohong, Yuko Fujiwara, Aimée Chapdelaine, Haidi Yang e Stuart H. Orkin. "Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain". Blood 97, n. 1 (1 gennaio 2001): 324–26. http://dx.doi.org/10.1182/blood.v97.1.324.

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Abstract Reporter mouse strains are important tools for monitoring Cre recombinase-mediated excision in vivo. In practice, excision may be incomplete in a given population due to threshold level or variegated expression of Cre. Hence, it is desirable in many experimental contexts to isolate cells that have undergone excision to assess the consequences of gene ablation. To generate alternative reporter mice, an enhanced green fluorescent protein (EGFP) gene was targeted to the retroviral-trapped ROSA26 locus. Upon Cre-mediated excision of “Stop” sequences, EGFP was expressed ubiquitously during embryogenesis and in adult tissues (including T cells, B cells, and myeloid cells). Using this new reporter strain, separation of excised from nonexcised cells in vitro was achieved in thymocytes in a noninvasive manner based on activated EGFP expression. This new EGFP reporter strain should facilitate a variety of conditional gene-targeting experiments, including the functional studies of hematopoietic cells in lineage-specific knockout mice.

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Wallace, Joselynn, Nicholas O. Bowlin, Debra M. Mills, Panatda Saenkham, Steven M. Kwasny, Timothy J. Opperman, John D. Williams, Charles O. Rock, Terry L. Bowlin e Donald T. Moir. "Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay". Antimicrobial Agents and Chemotherapy 59, n. 9 (13 luglio 2015): 5775–87. http://dx.doi.org/10.1128/aac.00686-15.

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ABSTRACTNovel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain ofPseudomonas aeruginosaand based on the discovery that FASII genes inP. aeruginosaare coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis inP. aeruginosaand exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficientEscherichia coli(ΔtolC) strain but had no detectable MIC against efflux-proficientE. colior againstP. aeruginosa(efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII inE. coliandP. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors ofE. coliandP. aeruginosaFASII.

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Filareto, Antonio, Katie Maguire-Nguyen, Qiang Gan, Garazi Aldanondo, Léo Machado, Jeffrey S. Chamberlain e Thomas A. Rando. "Monitoring disease activity noninvasively in the mdx model of Duchenne muscular dystrophy". Proceedings of the National Academy of Sciences 115, n. 30 (9 luglio 2018): 7741–46. http://dx.doi.org/10.1073/pnas.1802425115.

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Duchenne muscular dystrophy (DMD) is a rare, muscle degenerative disease resulting from the absence of the dystrophin protein. DMD is characterized by progressive loss of muscle fibers, muscle weakness, and eventually loss of ambulation and premature death. Currently, there is no cure for DMD and improved methods of disease monitoring are crucial for the development of novel treatments. In this study, we describe a new method of assessing disease progression noninvasively in the mdx model of DMD. The reporter mice, which we term the dystrophic Degeneration Reporter strains, contain an inducible CRE-responsive luciferase reporter active in mature myofibers. In these mice, muscle degeneration is reflected in changes in the level of luciferase expression, which can be monitored using noninvasive, bioluminescence imaging. We monitored the natural history and disease progression in these dystrophic report mice and found that decreases in luciferase signals directly correlated with muscle degeneration. We further demonstrated that this reporter strain, as well as a previously reported Regeneration Reporter strain, successfully reveals the effectiveness of a gene therapy treatment following systemic administration of a recombinant adeno-associated virus-6 (rAAV-6) encoding a microdystrophin construct. Our data demonstrate the value of these noninvasive imaging modalities for monitoring disease progression and response to therapy in mouse models of muscular dystrophy.

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K, Kalaiselvi, Mangayarkarasi V, Gomathi Ns, Balaji S, Shivshankar R. Mane e Raja Shunmugam. "LUCIFERASE REPORTER MYCOBACTERIOPHAGES FOR EVALUATING NORBORNENE-BASED ANTITUBERCULOSIS DRUG SUSCEPTIBILITY TESTING ON MYCOBACTERIUM TUBERCULOSIS". Asian Journal of Pharmaceutical and Clinical Research 10, n. 9 (1 settembre 2017): 406. http://dx.doi.org/10.22159/ajpcr.2017.v10i9.19660.

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Objective: In 2015, 9.6 million people around the world became sick with tuberculosis (TB) disease and 1.5 million TB-related deaths worldwide. Recent increasing incidence of multidrug-resistant (MDR; resistance to at least rifampicin (RIF) and isoniazid [INH]) and extensively drug-resistant (MDR resistance plus resistance to a fluoroquinolone and an aminoglycoside) makes TB a serious concern. Lot of research is needed to deal with this infectious disease for a better alternative in treatment or modification of these older TB drugs. The present study aimed at evaluating antimycobacterial activity of norbornene (NOR) derived INH copolymer with poly ethylene glycol (NOR- polyethylene glycol [PEG]-INH) a novel nanocarrier along with the anti-TB drug using luciferase reporter phages (LRPs).Methods: NOR derived INH accounts for 74% of INH, 24% of NOR, and 2% of PEG. H37Rv control strain, a sensitive, and a resistant strain of Mycobacterium TB (MTB) used in this study. The in vitro activity of the drug was evaluated using absolute concentration method. The resistant strain was evaluated using LRP assay to observe the minimum inhibitory concentration of the drug.Results: Reduction in light units was observed for the resistant strain exposed to plain INH and NOR-PEG-INH separately. 24% of reduction was observed in strains exposed to plain INH whereas 37% of reduction was observed in strains exposed to NOR-PEG-INH.Conclusion: NOR-based INH had better antimycobacterial activity compared to plain INH and RIF. Antimycobacterial activity of INH and RIF increases even with very low dosage with NOR conjugate.

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Banfield, Bruce W., Jessica D. Kaufman, Jessica A. Randall e Gary E. Pickard. "Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications". Journal of Virology 77, n. 18 (15 settembre 2003): 10106–12. http://dx.doi.org/10.1128/jvi.77.18.10106-10112.2003.

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ABSTRACT The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.

10

Soriano, Philippe. "Generalized lacZ expression with the ROSA26 Cre reporter strain". Nature Genetics 21, n. 1 (gennaio 1999): 70–71. http://dx.doi.org/10.1038/5007.

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Weiland-Bräuer, Nancy, Nicole Pinnow e Ruth A. Schmitz. "Novel Reporter for Identification of Interference with Acyl Homoserine Lactone and Autoinducer-2 Quorum Sensing". Applied and Environmental Microbiology 81, n. 4 (19 dicembre 2014): 1477–89. http://dx.doi.org/10.1128/aem.03290-14.

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ABSTRACTTwo reporter strains were established to identify novel biomolecules interfering with bacterial communication (quorum sensing [QS]). The basic design of theseEscherichia coli-based systems comprises a gene encoding a lethal protein fused to promoters induced in the presence of QS signal molecules. Consequently, theseE. colistrains are unable to grow in the presence of the respective QS signal molecules unless a nontoxic QS-interfering compound is present. The first reporter strain designed to detect autoinducer-2 (AI-2)-interfering activities (AI2-QQ.1) contained theE. coliccdBlethal gene under the control of theE. colilsrApromoter. The second reporter strain (AI1-QQ.1) contained theVibrio fischeriluxIpromoter fused to theccdBgene to detect interference with acyl-homoserine lactones. Bacteria isolated from the surfaces of several marine eukarya were screened for quorum-quenching (QQ) activities using the established reporter systems AI1-QQ.1 and AI2-QQ.1. Out of 34 isolates, two interfered with acylated homoserine lactone (AHL) signaling, five interfered with AI-2 QS signaling, and 10 were demonstrated to interfere with both signal molecules. Open reading frames (ORFs) conferring QQ activity were identified for three selected isolates (Photobacteriumsp.,Pseudoalteromonassp., andVibrio parahaemolyticus). Evaluation of the respective heterologously expressed and purified QQ proteins confirmed their ability to interfere with the AHL and AI-2 signaling processes.

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Aldea, M. Ramona, Rodrigo A. Mella-Herrera e James W. Golden. "Sigma Factor Genes sigC, sigE, and sigG Are Upregulated in Heterocysts of the Cyanobacterium Anabaena sp. Strain PCC 7120". Journal of Bacteriology 189, n. 22 (14 settembre 2007): 8392–96. http://dx.doi.org/10.1128/jb.00821-07.

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ABSTRACT We used gfp transcriptional fusions to investigate the regulation of eight sigma factor genes during heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Reporter strains containing gfp fusions with the upstream regions of sigB2, sigD, sigI, and sigJ did not show developmental regulation. Time-lapse microscopy of sigC, sigE, and sigG reporter strains showed increased green fluorescent protein fluorescence in differentiating cells at 4 h, 16 h, and 9 h, respectively, after nitrogen step down.

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Layton, A. C., M. Muccini, M. M. Ghosh e G. S. Sayler. "Construction of a Bioluminescent Reporter Strain To Detect Polychlorinated Biphenyls". Applied and Environmental Microbiology 64, n. 12 (1 dicembre 1998): 5023–26. http://dx.doi.org/10.1128/aem.64.12.5023-5026.1998.

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ABSTRACT A bioluminescent reporter strain, Ralstonia eutrophaENV307(pUTK60), was constructed for the detection of polychlorinated biphenyls by inserting the biphenyl promoter upstream of the bioluminescence genes. In the presence of a nonionic surfactant, which enhances the solubility of chlorinated biphenyls, bioluminescence was induced three- to fourfold over background by biphenyl, monochlorinated biphenyls, and Aroclor 1242. The minimum detection limits for these compounds ranged from 0.15 mg/liter for 4-chlorobiphenyl to 1.5 mg/liter for Aroclor 1242.

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Chen, S., M. Bagdasarian, M. G. Kaufman e E. D. Walker. "Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System". Applied and Environmental Microbiology 73, n. 4 (22 dicembre 2006): 1089–100. http://dx.doi.org/10.1128/aem.01577-06.

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ABSTRACT We developed techniques for the genetic manipulation of Flavobacterium species and used it to characterize several promoters found in these bacteria. Our studies utilized Flavobacterium hibernum strain W22, an environmental strain we isolated from tree hole habitats of mosquito larvae. Plasmids from F. hibernum strain W22 were more efficiently (∼1,250-fold) transferred by electroporation into F. hibernum strain W22 than those isolated from Escherichia coli, thus indicating that an efficient restriction barrier exists between these species. The strong promoter, tac, functional in proteobacteria, did not function in Flavobacterium strains. Therefore, a promoter-trap plasmid, pSCH03, containing a promoterless gfpmut3 gene was constructed. A library of 9,000 clones containing chromosomal fragments of F. hibernum strain W22 in pSCH03 was screened for their ability to drive expression of the promoterless gfpmut3 gene. Twenty strong promoters were used for further study. The transcription start points were determined from seven promoter clones by the 5′ rapid amplification of cDNA ends technique. Promoter consensus sequences from Flavobacterium were identified as TAnnTTTG and TTG, where n is any nucleotide, centered approximately 7 and 33 bp upstream of the transcription start site, respectively. A putative novel ribosome binding site consensus sequence is proposed as TAAAA by aligning the 20-bp regions upstream of the translational start site in 25 genes. Our primary results demonstrate that at least some promoter and ribosome binding site motifs of Flavobacterium strains are unusual within the bacterial domain and suggest an early evolutionary divergence of this bacterial group. The techniques presented here allow for more detailed genetics-based studies and analyses of Flavobacterium species in the environment.

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Clark, Leann, Charlotte A. Perrett, Layla Malt, Caryn Harward, Suzanne Humphrey, Katy A. Jepson, Isabel Martinez-Argudo et al. "Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression". Microbiology 157, n. 7 (1 luglio 2011): 2072–83. http://dx.doi.org/10.1099/mic.0.048496-0.

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Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE+ and SopE− S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH–gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA–gfp reporter mirrored that of PprgH–gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.

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Valenzuela, Alicia, Karen Fancher, Stephen Rockwood e Cathleen Lutz. "Mouse Models for Immunology Research available from The Jackson Laboratory Repository". Journal of Immunology 202, n. 1_Supplement (1 maggio 2019): 130.10. http://dx.doi.org/10.4049/jimmunol.202.supp.130.10.

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Abstract The Jackson Laboratory Repository (JAX) serves as a centralized facility for the development, distribution and cryopreservation of mouse models of human biology and disease. JAX distributes more than 10,500 strains to the scientific community, many with applications in immunology research, including mouse models for autoimmune disease, infectious disease, immunodeficient platforms for PDX studies, and multi-purpose “tool strains” (such as CRISPR cas9-expressing lines), recombinase expressing strains, as well as conditional and inducible expression lines. One of our newest strains is a conditional knock-out (KO) of STING/MYPS, a pathogen sensor involved in the activation of IL6 and IFNb. Additions to our extensive set of interleukin alleles include a SMART-17A knock-in (KI) expressing human NGFR on IL17A expressing cells and a KI/KO of Il9-expressing iCre/EYFP. The latest reporter and cre-expressing strains include an inducible cre KO/KI to Lyz2, and a KI/KO strain with dual reporters of chemokine Ccr2RFP and Cx3cr1GFPactivity, and two strains expressing the INDIA apoptosis reporter, one with widespread conditional expression and the other with expression in germinal center B cells. KOMP mice, offered via the MMRRC portion of the JAX Mouse Repository, provide a growing number of targeted immunological mutations. Donating a strain to the Repository fulfills NIH’s requirements for sharing mice. Researchers wishing to have mice considered for inclusion in the Repository are encouraged to submit strains: www.jax.org/donate-a-mouse. This work is supported by NIH, HHMI and private foundations.

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Loh, John T., e Timothy L. Cover. "Requirement of Histidine Kinases HP0165 and HP1364 for Acid Resistance in Helicobacter pylori". Infection and Immunity 74, n. 5 (maggio 2006): 3052–59. http://dx.doi.org/10.1128/iai.74.5.3052-3059.2006.

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ABSTRACT In this study, we investigated a potential requirement of two-component signal transduction systems for acid resistance in Helicobacter pylori. In comparison to a wild-type strain, isogenic strains with null mutations in either HP0165 or HP1364 histidine kinases were impaired in their ability to grow at pH 5.0. The growth of complemented mutant strains was similar to that of the wild-type strain. H. pylori DNA array analyses and transcriptional reporter assays indicated that acid-responsive gene transcription was altered in the HP0165 and HP1364 null mutant strains compared to the parental wild-type strain. These results indicate that intact HP0165 and HP1364 histidine kinases are required for acid resistance in H. pylori.

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Alam, Ashfaqul, Vincent Tam, Elaine Hamilton e Michelle Dziejman. "vttRA and vttRB Encode ToxR Family Proteins That Mediate Bile-Induced Expression of Type Three Secretion System Genes in a Non-O1/Non-O139 Vibrio cholerae Strain". Infection and Immunity 78, n. 6 (12 aprile 2010): 2554–70. http://dx.doi.org/10.1128/iai.01073-09.

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ABSTRACT Strain AM-19226 is a pathogenic non-O1/non-O139 serogroup Vibrio cholerae strain that does not encode the toxin-coregulated pilus or cholera toxin but instead causes disease using a type three secretion system (T3SS). Two genes within the T3SS pathogenicity island, herein named vttR A (locus tag A33_1664) and vttR B (locus tag A33_1675), are predicted to encode proteins that show similarity to the transcriptional regulator ToxR, which is found in all strains of V. cholerae. Strains with a deletion of vttR A or vttR B showed attenuated colonization in vivo, indicating that the T3SS-encoded regulatory proteins play a role in virulence. lacZ transcriptional reporter fusions to intergenic regions upstream of genes encoding the T3SS structural components identified growth in the presence of bile as a condition that modulates gene expression. Under this condition, VttRA and VttRB were necessary for maximal gene expression. In contrast, growth in bile did not substantially alter the expression of a reporter fusion to the vopF gene, which encodes an effector protein. Increased vttR B reporter fusion activity was observed in a ΔvttR B strain background, suggesting that VttRB may regulate its own expression. The collective results are consistent with the hypothesis that T3SS-encoded regulatory proteins are essential for pathogenesis and control the expression of selected T3SS genes.

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Donnini, Claudia, Francesca Farina, Barbara Neglia, Maria Concetta Compagno, Daniela Uccelletti, Paola Goffrini e Claudio Palleschi. "Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis". Applied and Environmental Microbiology 70, n. 5 (maggio 2004): 2632–38. http://dx.doi.org/10.1128/aem.70.5.2632-2638.2004.

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ABSTRACT The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.

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Yin, Shixue, Mayuree Fuangthong, William P. Laratta e James P. Shapleigh. "Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers". Applied and Environmental Microbiology 69, n. 7 (luglio 2003): 3938–44. http://dx.doi.org/10.1128/aem.69.7.3938-3944.2003.

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ABSTRACT To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.

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De Mol, Maarten L., Victoria Marcoen, Isabelle Maryns, Nico Snoeck, Joeri J. Beauprez, Sofie L. De Maeseneire e Wim K. Soetaert. "Evaluation of Long-Term Fermentation Performance with Engineered Saccharomyces cerevisiae Strains". Fermentation 9, n. 8 (30 luglio 2023): 721. http://dx.doi.org/10.3390/fermentation9080721.

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The performance of a microbial fermentation on an industrial scale is subjected to the robustness of the strain. Such strains are genetically engineered to optimize the production of desired compounds in minimal time, but they often fail to maintain high productivity levels for many generations, hindering their effective application in industrial conditions. This study focused on assessing the impact of genomic instability in yeasts that were engineered to produce a fluorescent output by incorporating a reporter gene at one or more genomic locations. The fermentation performance of these strains was evaluated over 100 generations in a sequential batch set-up. In order to bridge the gap between strain engineering and industrial implementation, we proposed the use of novel, host-specific parameters to standardize the strain robustness and evaluate potential improvements. It was observed that yeasts carrying multiple copies of the reporter gene exhibited a more pronounced decrease in output, and the genomic integration site significantly influenced the production. By leveraging these new, host-specific parameters, it becomes possible to anticipate strain behavior prior to incurring substantial costs associated with large-scale production. This approach enhances the economic viability of novel microbial fermentation processes and narrows the divide between laboratory findings and industrial applications.

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Jiang, Yide, Michael J. Vasconcelles, Sharon Wretzel, Anne Light, Charles E. Martin e Mark A. Goldberg. "MGA2 Is Involved in the Low-Oxygen Response Element-Dependent Hypoxic Induction of Genes inSaccharomyces cerevisiae". Molecular and Cellular Biology 21, n. 18 (15 settembre 2001): 6161–69. http://dx.doi.org/10.1128/mcb.21.18.6161-6169.2001.

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ABSTRACT Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example,OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-inducedOLE1 expression, we examined OLE1expression under normoxia, hypoxia, and cobalt treatment conditions in Δmga2 or Δspt23 deletion strains. The results of OLE1promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Δmga2 strain but not in a Δspt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae Δmga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Δmga2 strain. These results indicate thatMGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.

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Miller, William G., Anne H. Bates, Sharon T. Horn, Maria T. Brandl, Marian R. Wachtel e Robert E. Mandrell. "Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp, andcfp Marker Plasmids". Applied and Environmental Microbiology 66, n. 12 (1 dicembre 2000): 5426–36. http://dx.doi.org/10.1128/aem.66.12.5426-5436.2000.

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ABSTRACT We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), orcfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacterpromoter sequence (Pc). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the Pc fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the Pc fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

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Summers, Michael L., James G. Elkins, Brian A. Elliott e Timothy R. McDermott. "Expression and Regulation of Phosphate Stress Inducible Genes in Sinorhizobium meliloti". Molecular Plant-Microbe Interactions® 11, n. 11 (novembre 1998): 1094–101. http://dx.doi.org/10.1094/mpmi.1998.11.11.1094.

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Sinorhizobium meliloti 104A14 was mutated with transposon Tn5B22, which creates lacZ transcriptional fusions when inserted in the correct orientation relative to the promoter. This promoter reporter allowed us to identify six phosphate stress inducible (psi) genes in S. meliloti that are up-regulated in response to inorganic phosphate (Pi) starvation. The transposon and flanking DNA were cloned from each psi::Tn5B22 reporter mutant and the junction DNA sequenced. High identity/similarity of the inferred peptides with those in major data bases allowed identification of the following genes: dnaK, expC, pssB, ackA, vipC, and prkA. The prkA homolog was also found to be upregulated in response to carbon starvation and when nitrate replaced ammonium as the nitrogen source. Through allele replacement techniques, PhoB¯ mutants were generated for the expC, ackA, vipC, and pssB reporter strains. Loss of a functional PhoB resulted in the absence of Pi-sensitive induction in all four genes. These experiments suggest the Pho regulon in S. meliloti includes genes that presumably are not directly linked to Pi acquisition or assimilation. The psi strains were tested for their symbiotic properties under growth conditions that were Pi-limiting or Pi-nonlimiting for the host plant. All were Nod+ and Fix+ except the reporter strain of dnaK transcription, which was less effective than the wild-type strain under both P treatments, indicating DnaK is required for optimum symbiotic function.

25

Doughty, D. M., E. G. Kurth, L. A. Sayavedra-Soto, D. J. Arp e P. J. Bottomley. "Evidence for Involvement of Copper Ions and Redox State in Regulation of Butane Monooxygenase in Pseudomonas butanovora". Journal of Bacteriology 190, n. 8 (15 febbraio 2008): 2933–38. http://dx.doi.org/10.1128/jb.01409-07.

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ABSTRACT Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C2-C9 alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of β-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O2 when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH4Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O2 (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O2, CuSO4 (0.5 μM) repressed 1-butanol-dependent induction of β-galactosidase activity. Under oxic conditions (20% O2 [vol/vol]), significantly higher concentrations of CuSO4 (2 μM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu2+ reducing agent Na ascorbate (100 μM) and CuSO4 (0.5 μM) repressed the induction of β-galactosidase activity under oxic conditions to the same extent that 0.5 μM CuSO4 alone repressed it under anoxic conditions. Under oxic conditions, 2 μM CuSO4 repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO4 repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

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Ferreira, Elisabeth, Landon B. Gatrell, Luke Childress, Hong Wu e Ryan M. Porter. "A Transgenic Rat for Noninvasive Assessment of Chondrogenesis in Vivo". CARTILAGE 13, n. 2_suppl (22 novembre 2021): 1720S—1733S. http://dx.doi.org/10.1177/19476035211057243.

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Objective To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity. Design A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as ex vivo measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis. Results Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification. Conclusions This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.

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Matsumoto, Mitsuru, Minoru Matsumoto, Ryuichiro Miyazawa, Junko Morimoto, Koichi Tsuneyama e Hitoshi Nishijima. "Characterization of Aire-expressing DCs using a novel Aire-reporter strain". Journal of Immunology 204, n. 1_Supplement (1 maggio 2020): 143.13. http://dx.doi.org/10.4049/jimmunol.204.supp.143.13.

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Abstract Aire is predominantly expressed by medullary thymic epithelial cells (mTECs), and it is essential for maintaining self-tolerance by controlling the expression of tissue-restricted self-antigens (TRAs). Besides mTECs, extra-thymic Aire-expressing cells (eTACs) have been reported. However, the exact nature of eTACs has been difficult to study because of the small numbers together with low expression levels of Aire within the cells. We have recently established a novel Aire reporter strain in which endogenous Aire was replaced by the human AIRE-GFP-Flag tag fusion gene (Aire/hAGF-knockin: J. Immunol. 2015). hAGF-reporter protein was efficiently produced and retained within the mTECs as authentic Aire nuclear dot protein. With this high-sensitivity and high-fidelity Aire-reporter strain, we found that DCs in the spleen and lymph nodes expressed Aire although their expression levels were rather low. Aire-expressing DCs showed high levels of MHC-II and CD80/CD86, and half of which expressed CD8. In the thymus, we found that there were two types of DCs expressing Aire: hAGF-low DCs and hAGF-high DCs. Interestingly, hAGF-low DCs had small hAGF dots within the nuclei, whereas hAGF-high DCs retained larger hAGF dots which were mainly present outside the nuclei, suggesting that hAGF-high DCs had acquired hAGF protein from mTECs. Acquisition of hAGF protein by hAGF-high DCs from mTECs was confirmed by the bone-marrow transfer experiment. Mechanisms underlying the transfer of nuclear protein (i.e., hAGF protein) from mTECs to thymic DCs, and immunological consequence of this event are now under investigation.

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Siragusa, Gregory R., Kevin Nawotka, Stanley D. Spilman, Pamela R. Contag e Christopher H. Contag. "Real-Time Monitoring of Escherichia coliO157:H7 Adherence to Beef Carcass Surface Tissues with a Bioluminescent Reporter". Applied and Environmental Microbiology 65, n. 4 (1 aprile 1999): 1738–45. http://dx.doi.org/10.1128/aem.65.4.1738-1745.1999.

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ABSTRACT A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r 2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.

29

Herbert, Silvia, Steven W. Newell, Chia Lee, Karsten-Peter Wieland, Bruno Dassy, Jean-Michel Fournier, Christiane Wolz e Gerd Döring. "Regulation of Staphylococcus aureus Type 5 and Type 8 Capsular Polysaccharides by CO2". Journal of Bacteriology 183, n. 15 (1 agosto 2001): 4609–13. http://dx.doi.org/10.1128/jb.183.15.4609-4613.2001.

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ABSTRACT Staphylococcus aureus expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO2. Here we show that CO2 reduces CP5 expression at the transcriptional level and that CO2regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO2 caused a significant decrease in CP8 expression in fourS. aureus strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine S. aureus strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of cap5 andcap8. To test whether these sequence variations are responsible for the different responses to CO2, promoter regions from selected strains were fused to the reporter genexylE in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO2 in all derivatives of strain Newman and was always positively regulated by CO2 in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO2 response. trans-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive S. aureus strains.

30

SabathÃ©, Fabrice, Christian Croux, Emmanuel Cornillot e Philippe Soucaille. "amyP, a reporter gene to study strain degeneration inClostridium acetobutylicumATCC 824". FEMS Microbiology Letters 210, n. 1 (aprile 2002): 93–98. http://dx.doi.org/10.1111/j.1574-6968.2002.tb11165.x.

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Kim, Seol-Hee, Parmvir K. Bahia, Mayur Patil, Sydney Sutton, Isobel Sowells, Stephen H. Hadley, Marian Kollarik e Thomas E. Taylor-Clark. "Development of a Mouse Reporter Strain for the Purinergic P2X2 Receptor". eneuro 7, n. 4 (luglio 2020): ENEURO.0203–20.2020. http://dx.doi.org/10.1523/eneuro.0203-20.2020.

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32

Ideguchi, Yamato, Yuta Oshikoshi, Masashi Ryo, Shogo Motoki, Takashi Kuwano, Takafumi Tezuka e Setsuyuki Aoki. "Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain". Archives of Microbiology 198, n. 1 (27 ottobre 2015): 35–41. http://dx.doi.org/10.1007/s00203-015-1165-5.

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Ohinata, Yasuhide, Mitsue Sano, Mayo Shigeta, Kaori Yamanaka e Mitinori Saitou. "A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter". REPRODUCTION 136, n. 4 (ottobre 2008): 503–14. http://dx.doi.org/10.1530/rep-08-0053.

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The ability to monitor the development of a given cell lineage in a non-invasive manner by fluorescent markers bothin vivoandin vitroprovides a great advantage for the analysis of the lineage of interest. To date, a number of transgenic or knock-in mouse strains, in which developing germ cells are marked with fluorescent reporters, have been generated. We here describe a novel double transgenic reporter mouse strain that expresses membrane-targeted Venus (mVenus), a brighter variant of yellow fluorescent protein (YFP), under the control ofPrdm1(Blimp1) regulatory elements and enhanced cyan fluorescent protein (ECFP) under the control ofDppa3(Stella/Pgc7). The double transgenic strain unambiguously markedPrdm1expression in the lineage-restricted precursors of primordial germ cells (PGCs) in the proximal epiblast at embryonic day (E) 6.25 and specifically illuminatedPrdm1- andDppa3-positive migrating PGCs after E8.5. The double transgenic reporter also precisely recapitulated dynamic embryonic expression ofPrdm1outside the germ cell lineage. Moreover, we derived ES cells that bore both transgenes. These cells made a robust contribution both to the germ and somatic cell lineages in chimeras with accuratePrdm1-mVenus andDppa3-ECFP expression. The transgenic strain and the ES cells will serve as valuable experimental materials not only for analyzing the origin and properties of the germ cell lineagein vivo, but also for establishing a culture system to efficiently induce proper germ cells with temporally coordinatedPrdm1andDppa3expressionin vitro.

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Amgarten, Beatrice, Rakesh Rajan, Nuria Martínez-Sáez, Bruno L. Oliveira, Inês S. Albuquerque, Roger A. Brooks, David G. Reid, Melinda J. Duer e Gonçalo J. L. Bernardes. "Collagen labelling with an azide-proline chemical reporter in live cells". Chemical Communications 51, n. 25 (2015): 5250–52. http://dx.doi.org/10.1039/c4cc07974d.

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Biosynthetic incorporation of an azide-proline chemical reporter into collagen allows selective imaging in live foetal ovine osteoblasts using a strain-promoted [3+2] azide–alkyne cycloaddition reaction.

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Takeda, Midori, Masanori Ikeda, Yasuo Ariumi, Takaji Wakita e Nobuyuki Kato. "Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines". Journal of General Virology 93, n. 7 (1 luglio 2012): 1422–31. http://dx.doi.org/10.1099/vir.0.040725-0.

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A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

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Crouch, Elizabeth E., Zhiyu Li, Makiko Takizawa, Stefan Fichtner-Feigl, Polyxeni Gourzi, Carolina Montaño, Lionel Feigenbaum et al. "Regulation of AID expression in the immune response". Journal of Experimental Medicine 204, n. 5 (23 aprile 2007): 1145–56. http://dx.doi.org/10.1084/jem.20061952.

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The B cell–specific enzyme activation-induced cytidine deaminase (AID) has been shown to be essential for isotype switching and affinity maturation of antibody genes during the immune response. Conversely, AID activity has also been linked to autoimmunity and tumorigenesis. Determining how AID expression is regulated in vivo is therefore central to understanding its role in health and disease. Here we use phylogenetic footprinting and high-resolution histone acetylation mapping to accurately demarcate AID gene regulatory boundaries. Based on this strategy, we identify a novel, positive regulatory element required for AID transcription. Furthermore, we generate two AID indicator mouse strains using bacterial artificial chromosomes that faithfully recapitulate endogenous AID expression. The first strain uses a green fluorescent protein reporter to identify B cells that actively express AID during the immune response. In the second strain, AID transcription affects the permanent expression of a yellow fluorescent protein reporter in post–germinal center and terminally differentiated lymphocytes. We demonstrate the usefulness of these novel strains by resolving recent contradictory observations on AID expression during B cell ontogeny.

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Pierson, Elizabeth A., Derek W. Wood, Jeffrey A. Cannon, Francoise M. Blachere e Leland S. Pierson. "Interpopulation Signaling via N-Acyl-Homoserine Lactones among Bacteria in the Wheat Rhizosphere". Molecular Plant-Microbe Interactions® 11, n. 11 (novembre 1998): 1078–84. http://dx.doi.org/10.1094/mpmi.1998.11.11.1078.

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The biological control bacterium Pseudomonas aureofaciens 30-84 utilizes an N-acyl-homoserine lactone (AHL) signal molecule to control phenazine antibiotic production in the wheat rhizosphere (D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). In this study, we demonstrate that naturally co-existing, non-isogenic bacterial populations interact with strain 30-84 at the level of gene expression via the exchange of diffusible signals on wheat roots. Wheat plants from three geographic locations were used to generate a random library of 700 rhizobacterial isolates. Roughly 8% of the isolates from each location restored phenazine gene expression to an AHL-deficient strain of 30-84 in vitro. Five of these isolates were further tested for their ability to influence gene expression of an AHL-deficient reporter of strain 30-84 on wheat roots. All five, isolated from different geographic locations, restored phenazine gene expression by the reporter to wild-type levels. This suggests that in vitro assays can identify bacterial isolates with the potential to influence phenazine expression in strain 30-84 via AHLs on wheat roots. The occurrence of such strains in all fields sampled suggests that AHL-mediated communication is a common occurrence in the wheat rhizosphere.

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Hemrajani, Cordula, Olivier Marches, Siouxsie Wiles, Francis Girard, Alison Dennis, Francis Dziva, Angus Best et al. "Role of NleH, a Type III Secreted Effector from Attaching and Effacing Pathogens, in Colonization of the Bovine, Ovine, and Murine Gut". Infection and Immunity 76, n. 11 (25 agosto 2008): 4804–13. http://dx.doi.org/10.1128/iai.00742-08.

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ABSTRACT The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC ΔnleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

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O'Rourke, Sean M., e Ira Herskowitz. "A Third Osmosensing Branch in Saccharomyces cerevisiae Requires the Msb2 Protein and Functions in Parallel with the Sho1 Branch". Molecular and Cellular Biology 22, n. 13 (1 luglio 2002): 4739–49. http://dx.doi.org/10.1128/mcb.22.13.4739-4749.2002.

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ABSTRACT Two Saccharomyces cerevisiae plasma membrane-spanning proteins, Sho1 and Sln1, function during increased osmolarity to activate a mitogen-activated protein (MAP) kinase cascade. One of these proteins, Sho1, utilizes the MAP kinase kinase kinase Ste11 to activate Pbs2. We previously used the FUS1 gene of the pheromone response pathway as a reporter to monitor cross talk in hog1 mutants. Cross talk requires the Sho1-Ste11 branch of the HOG pathway, but some residual signaling, which is STE11 dependent, still occurs in the absence of Sho1. These observations led us to propose the existence of another osmosensor upstream of Ste11. To identify such an osmosensor, we screened for mutants in which the residual signaling in a hog1 sho1 mutant was further reduced. We identified the MSB2 gene, which encodes a protein with a single membrane-spanning domain and a large presumptive extracellular domain. Assay of the FUS1-lacZ reporter (in a hog1 mutant background) showed that sho1 and msb2 mutations both reduced the expression of the reporter partially and that the hog1 sho1 msb2 mutant was severely defective in the expression of the reporter. The use of DNA microarrays to monitor gene expression revealed that Sho1 and Msb2 regulate identical gene sets in hog1 mutants. A role for MSB2 in HOG1 strains was also seen in strains defective in the two known branches that activate Pbs2: an ssk1 sho1 msb2 strain was more osmosensitive than an ssk1 sho1 MSB2 strain. These observations indicate that Msb2 is partially redundant with the Sho1 osmosensing branch for the activation of Ste11.

40

F., Mbeunkui, Richaud C., Etienne A.-L., Schmid R. e Bachmann T. "Bioavailable nitrate detection in water by an immobilized luminescent cyanobacterial reporter strain". Applied Microbiology and Biotechnology 60, n. 3 (1 novembre 2002): 306–12. http://dx.doi.org/10.1007/s00253-002-1139-9.

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41

Mao, X., Y. Fujiwara e S. H. Orkin. "Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice". Proceedings of the National Academy of Sciences 96, n. 9 (27 aprile 1999): 5037–42. http://dx.doi.org/10.1073/pnas.96.9.5037.

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Fukamachi, Katsumi, Hajime Tanaka, Yuto Sakai, David B. Alexander, Mitsuru Futakuchi, Hiroyuki Tsuda e Masumi Suzui. "A novel reporter rat strain that expresses LacZ upon Cre-mediated recombination". genesis 51, n. 4 (25 febbraio 2013): 268–74. http://dx.doi.org/10.1002/dvg.22371.

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Kaci, Ghalia, Omar Lakhdari, Joël Doré, S. Dusko Ehrlich, Pierre Renault, Hervé M. Blottière e Christine Delorme. "Inhibition of the NF-κB Pathway in Human Intestinal Epithelial Cells by Commensal Streptococcus salivarius". Applied and Environmental Microbiology 77, n. 13 (20 maggio 2011): 4681–84. http://dx.doi.org/10.1128/aem.03021-10.

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ABSTRACTStreptococcus salivariusexhibited an anti-inflammatory effect on intestinal epithelial cells (IECs) and monocytes. Strains were screened using a reporter clone, HT-29/kB-luc-E, induced by tumor necrosis factor alpha (TNF-α). Supernatant from each strain downregulated NF-κB activation. The two most efficient strains produced an active metabolite (<3 kDa) which was able to downregulate the secretion of the proinflammatory chemokine interleukin-8 (IL-8).

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Janus, Danielle, Birgit Hoff, Eckhard Hofmann e Ulrich Kï¿½ck. "An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene". Applied and Environmental Microbiology 73, n. 3 (1 dicembre 2006): 962–70. http://dx.doi.org/10.1128/aem.02127-06.

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ABSTRACT In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.

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Staib, Peter, Gary P. Moran, Derek J. Sullivan, David C. Coleman e Joachim Morschhäuser. "Isogenic Strain Construction and Gene Targeting inCandida dubliniensis". Journal of Bacteriology 183, n. 9 (1 maggio 2001): 2859–65. http://dx.doi.org/10.1128/jb.183.9.2859-2865.2001.

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ABSTRACT Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensiswild-type isolate by targeted gene deletion. The two URA3alleles were sequentially inactivated using theMPAR -flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that theCdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.

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Wang, Yan, Peter Setlow e Stanley Brul. "Genomic versus Plasmid-Borne Expression of Germinant Receptor Proteins in Bacillus cereus Strain 14579". Microorganisms 10, n. 9 (2 settembre 2022): 1774. http://dx.doi.org/10.3390/microorganisms10091774.

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Germinant receptors (GRs) are proteins in the spore-forming bacteria of Bacillus species that are crucial in triggering spore germination by sensing nutrients in the spores’ environment. In the Gram-positive bacterium Bacillus cereus strain ATCC 14579, the GerR GR initiates germination with L-alanine. While we have expressed GerR subunits fused to reporter proteins from genes under control of their native promoter on plasmids in this B. cereus strain, here we sought increased flexibility in this work by studying genome integration and plasmid-borne inducible high level (over) expression. However, construction of chromosomal integrants to visualize and localize the GerR B subunit fused to fluorescent reporter protein SGFP2 was not successful in this B. cereus strain using constructs with either shorter (~600 bp) or longer (~1200 bp) regions of homology to the gerR operon. This failure was in contrast to successful IPTG-inducible expression of GerRB-SGFP2 from plasmid pDG148 in vegetative cells and dormant spores, as fluorescent GerRB-SGFP2 foci were present in vegetative cells and the protein was detected by Western blot analysis. In dormant spores, the fluorescence intensity with IPTG-inducible expression from pDG148-gerRB-SGFP2 was significantly higher than in wild type spores. However, the full length GerRB-SGFP2 protein was not detected in spores using Western blots. Clearly, there are still challenges in the construction of B. cereus strains harboring fluorescent reporter proteins in which tagged proteins are encoded by genes incorporated in the chromosome or on extrachromosomal expression plasmids.

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Bakshi, Shlomo, Abraham Sztejnberg e Oded Yarden. "Isolation and Characterization of a Cold-Tolerant Strain of Fusarium proliferatum, a Biocontrol Agent of Grape Downy Mildew". Phytopathology® 91, n. 11 (novembre 2001): 1062–68. http://dx.doi.org/10.1094/phyto.2001.91.11.1062.

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A cold-tolerant strain of the mycoparasite Fusarium proliferatum was isolated following UV mutagenesis of the G6 strain, which is a biocontrol agent of grape downy mildew. The isolated strain (designated 1505) exhibited radial growth two to threefold that of the parent strain when grown at 13°C, which is generally suboptimal for growth of Fusarium spp., but desirable for its host, Plasmopara viticola. This rapid growth was correlated with improved biological control of P. viticola, determined by a detached-leaf assay. Even though radial growth of strain 1505 at higher temperatures was slower than that of G6 and the strain failed to conidiate, there was no reduction in biocontrol efficacy. Significantly higher levels of extracellular β-glucosidase and endo-1,4-β-glucanase activity were measured in the culture filtrate of strain 1505 relative to that of strain G6. A DNA-mediated transformation procedure that included the introduction of antibiotic resistance and a GUS reporter gene system was adapted for F. proliferatum. Using the GUS-engineered strains, we demonstrated that both G6 and 1505 exhibit the characteristic coiling and penetration of host structures.

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Hickey, M. J., T. M. Arain, R. M. Shawar, D. J. Humble, M. H. Langhorne, J. N. Morgenroth e C. K. Stover. "Luciferase in vivo expression technology: use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice." Antimicrobial Agents and Chemotherapy 40, n. 2 (febbraio 1996): 400–407. http://dx.doi.org/10.1128/aac.40.2.400.

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The development of new drugs and vaccines directed against Mycobacterium tuberculosis is severely impeded by the slow growth of this organism and the need to work under stringent biosafety conditions. These difficulties pose considerable obstacles when animal studies with M. tuberculosis are performed. We investigated whether a novel approach termed luciferase in vivo expression, using an enhanced luciferase-expressing mycobacterial strain, could be used to evaluate antimycobacterial activity in mice. Vectors that expressed firefly luciferase (lux gene) at high levels in the bacillus Calmette-Gu-erin (BCG) strain of Mycobacterium bovis were constructed for use in vivo. One recombinant BCG reporter strain (rBCG-lux) was selected for high-level expression of the lux gene product and for its ability to replicate in mice. Methodology to monitor in vivo growth of the rBCG-lux reporter strain in mice by direct assay of luciferase luminescence in organ homogenates was developed. The utility of this approach for assessing the in vivo efficacies of antimycobacterial compounds was evaluated. The activities of standard antimycobacterial drugs were directly apparent in mice infected with the rBCG-lux reporter strain by statistically significant reductions in spleen luminescence. In addition, antimycobacterial immunity was also evident in BCG-immunized mice, in which suppression of rBCG-lux growth in comparison with that in naive mice was clearly observed. The use of luciferase in vivo expression for the in vivo evaluation of antimycobacterial activity compared favorably with standard CFU determinations in terms of time, labor, expense, and statistical significance but permitted the evaluation of antimycobacterial drugs and immunity in mice in 7 days or less. Thus, the use of this technology can greatly accelerate the process of evaluation of antibiotics and immunogens in animal models for the slowly growing pathogenic mycobacteria.

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KABIR, Ahmad Humayan, Anindya GHOSH ROY, Mohammad FIROZ ALAM e Rafiul ISLAM. "Detection of Quorum Sensing Signals in Gram-Negative Bacteria by Using Reporter Strain CV026". Notulae Scientia Biologicae 2, n. 4 (5 dicembre 2010): 72–75. http://dx.doi.org/10.15835/nsb244863.

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Quorum sensing signals are referred to as acylated homoserine lactones (AHL) that are mainly found in Gram-negative bacteria. It implies the ability of certain bacteria of producing different AHL molecules. The bacteria Pseudomonas aureofaciens and Xenorhabdus nematophila were cultured in Luria-Bertani (LB10) media and CV026 was used as a reporter strain to detect the presence of AHLs produced by the cultured bacteria. In this study, the reporter strain has revealed the quorum sensing ability of P. aureofaciens and X. nematophila by producing the purple pigment violacein in the supply of external AHLs molecules. Thin layer chromatography (TLC) bioassay having four controls was conducted to detect specific AHL molecule supplied by P. aureofaciens and X. nematophila. The specific AHL molecule was observed to be migrated according to their polarity on the TLC plate.

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Tiedt, Ralph, Tibor Schomber, Hui Hao-Shen e Radek C. Skoda. "Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo". Blood 109, n. 4 (10 ottobre 2006): 1503–6. http://dx.doi.org/10.1182/blood-2006-04-020362.

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Abstract To generate transgenic mice that express Cre-recombinase exclusively in the megakaryocytic lineage, we modified a mouse bacterial artificial chromosome (BAC) clone by homologous recombination and replaced the first exon of the platelet factor 4 (Pf4), also called CXCL4, with a codon-improved Cre cDNA. Several strains expressing the transgene were obtained and one strain, Q3, was studied in detail. Crossing Q3 mice with the ROSA26-lacZ reporter strain showed that Cre-recombinase activity was confined to megakaryocytes. These results were further verified by crossing the Q3 mice with a strain containing loxP-flanked integrin β1. Excision of this conditional allele in megakaryocytes was complete at the DNA level, and platelets were virtually devoid of the integrin β1 protein. The Pf4-Cre transgenic strain will be a valuable tool to study megakaryopoiesis, platelet formation, and platelet function.

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