The Escherichia coli gus Operon: Induction and Expression of the gus Operon in E. coli and the Occurrence and Use of GUS in Other Bacteria

Wilson, Kate; Hughes, Steve; Jefferson, Richard A.

doi:10.1016/b978-0-12-274010-7.50007-0

Cited by 88 publications

(94 citation statements)

References 14 publications

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“…This organization has nothing in common with any other Gram-positive or Gram-negative PTS that has been described, nor with other b-glucuronidases previously studied, such as the E. coli uidA gene (Wilson et al, 1992) or the L. gasseri gusA gene (Russel & Klaenhammer, 2001). In E. coli, the uidA gene belongs to the uidRABC operon, in which uidR encodes the transcriptional repressor while uidB encodes the transporter, and a membrane-associated protein (uidC) is involved in the glucuronide transport system (Wilson et al, 1992;Liang et al, 2005). In L. gasseri, unlike in E. coli, the gusA gene is transcribed as a monocistronic unit (Russel & Klaenhammer, 2001).…”

Section: Discussionmentioning

confidence: 98%

Genetic characterization of the β-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus

2005

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b-Glucuronidase activity (encoded by the gus gene) has been characterized for the first time from Ruminococcus gnavus E1, an anaerobic bacterium belonging to the dominant human gut microbiota. b-Glucuronidase activity plays a major role in the generation of toxic and carcinogenic metabolites in the large intestine, as well as in the absorption and enterohepatic circulation of many aglycone residues with protective effects, such as lignans, flavonoids, ceramide and glycyrrhetinic acid, that are liberated by the hydrolysis of the corresponding glucuronides. The complete nucleotide sequence of a 4537 bp DNA fragment containing the b-glucuronidase locus from R. gnavus E1 was determined. Five ORFs were detected on this fragment: three complete ORFs (ORF2, gus and ORF3) and two partial ORFs (ORF4 and ORF5). The products of ORF2 and ORF3 show strong similarities with many b-glucoside permeases of the phosphoenolpyruvate : b-glucoside phosphotransferase systems (PTSs), such as Escherichia coli BglC, Bacillus subtilis BglP and Bacillus halodurans PTS Enzyme II. The product of ORF5 presents strong similarities with the amino-terminal domain of Clostridium acetobutylicum b-glucosidase (bglA). The gus gene product presents similarities with several known b-glucuronidase enzymes, including those of Lactobacillus gasseri (69 %), E. coli (61 %), Clostridium perfringens (59 %) and Staphylococcus aureus (58 %). By complementing an E. coli strain in which the uidA gene encoding the enzyme was deleted, it was confirmed that the R. gnavus gus gene encodes the b-glucuronidase enzyme. Moreover, it was found that the gus gene was transcribed as part of an operon that includes ORF2, ORF3 and ORF5.

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Section: Discussionmentioning

confidence: 98%

Genetic characterization of the β-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus

2005

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“…At various times, aliquots of cells were removed, pelleted, and resuspended in M63 medium containing 100 µg/mL chloramphenicol and stored on ice (Wilson et al 1992). The optical density of cells was measured at 600 nm.…”

Section: Assay Of ␤-Glucuronidase Activitymentioning

confidence: 99%

“…Aliquots of 50 µL of permeabilization buffer (100 mM Tris at pH 7.8, 32 mM sodium phosphate, 8 mM dithiothreitol, 8 mM CDTA, and 4% Triton X-100) containing 200 µg/mL polymyxin B (Schupp et al 1995) were placed in the wells of a microtiter plate, followed by the addition of 100 µL of cells. Cells were allowed to permeabilize at room temperature for 15 min before 50-µL aliquots of GUS assay buffer (0.5 mM dithiothreitol, 1 mM EDTA, 50 mM sodium phosphate at pH 7.0) containing 1.25 mM ␣-p-nitrophenyl ␤-D-glucuronide (PNPG) were added (Wilson et al 1992). The rate of ␤-glucuronide hydrolysis was determined at 405 nm at 37°C.…”

Section: Assay Of ␤-Glucuronidase Activitymentioning

confidence: 99%

DNA trajectory in the Gal repressosome

Semsey¹,

Tolstorukov²,

Virnik³

et al. 2004

Genes Dev.

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“…At various times, aliquots of the cells were washed in the same medium containing 100 µg/ml chloramphenicol and stored on ice (Wilson et al 1992). ␤-Glucuronidase or ␤-galactosidase activities were measured for all cultures simultaneously by the Softmax microplate spectrophotometer system.…”

Section: Bacterial Strains Plasmids and Bacteriophagementioning

confidence: 99%

GalR mutants defective in repressosome formation

Geanacopoulos¹,

Vasmatzis²,

Lewis³

et al. 1999

Genes & Development

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Transcription repression of the galactose operon of Escherichia coli requires (1) the binding of the GalR repressor to tandem operators flanking the promoters, (2) the binding of histone-like protein, HU, to a site between the GalR-binding sites, and (3) negatively supercoiled DNA. Under these conditions, protein-protein interactions mediate the formation of a nucleoprotein complex in the form of a DNA loop, which we have termed a repressosome. To analyze the structure of the repressosome, we have screened and isolated galR mutants in which single amino acid substitutions in GalR lead to defects in loop formation while the protein's operator-binding activity is retained. The mutant proteins were purified and their properties confirmed in vitro. We verified that in the case of the two stronger mutations, the proteins had secondary structures that were identical to that of wild-type GalR as reflected by circular dichroism spectroscopy. Homology-based modeling of GalR by use of the crystal structures of PurR and LacI has enabled us to place the three sites of mutation in a structural context. They occur in the carboxy-terminal subdomain of the GalR core, are surface exposed, and, therefore, may be involved in protein-protein interactions. On the basis of our model of GalR and its structural alignment with LacI and PurR, we have identified additional residues, the substitution of which leads to a specific defect in repression by looping. The effects of the mutations are the same in the presence of HMG-17, a eukaryotic protein unrelated to HU, which can also mediate GalR-dependent repression of the gal promoter. This observation suggests that the mutations define sites of GalR-GalR interaction rather than HU-GalR interaction in the repressosome.[Key Words: Repressosome; transcription; galR mutants; protein-protein interactions; DNA looping; GalR; repression] Received October 13, 1998; revised version accepted April 6, 1999.DNA looping is a general phenomenon in the control of transcription in which two or more DNA-binding proteins recognize DNA sequences flanking a promoter and, by contacting each other, cause a reversible transition to a nucleoprotein complex in which the intervening DNA is looped out and the promoter activity is altered (Adhya 1989; Martin et al. 1990). The relative simplicity of these complexes in prokaryotes compared with their counterparts in eukaryotes has facilitated the investigation by genetic and physicochemical methods of questions critical to the understanding of regulation by DNA looping. Some of these questions are structural in nature: What are the protein-protein and protein-DNA contacts that stabilize these loops? What are the symmetries, if any, of the protein complexes? Other questions are framed in terms of thermodynamics and seek to understand the origin of the negative-free energy of the looped structure.Critical mechanistic questions include how loop formation modulates transcription activity and by what means the stability of the loop is sensitive to environmental conditions.W...

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The Escherichia coli gus Operon: Induction and Expression of the gus Operon in E. coli and the Occurrence and Use of GUS in Other Bacteria

Cited by 88 publications

References 14 publications

Genetic characterization of the β-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus

Genetic characterization of the β-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus

DNA trajectory in the Gal repressosome

GalR mutants defective in repressosome formation

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