scholarly journals Identification of UreR binding sites in the Enterobacteriaceae plasmid-encoded and Proteus mirabilis urease gene operons

1999 ◽  
Vol 31 (5) ◽  
pp. 1417-1428 ◽  
Author(s):  
Venetta J. Thomas ◽  
Carleen M. Collins
Keyword(s):  
Proteus Mirabilis ◽  
Binding Sites ◽  
Urease Gene

scholarly journals H-NS Is a Repressor of the Proteus mirabilis Urease Transcriptional Activator GeneureR

2000 ◽  
Vol 182 (9) ◽  
pp. 2649-2653 ◽  
Author(s):  
Christopher Coker ◽  
Olubunmi O. Bakare ◽  
Harry L. T. Mobley
Keyword(s):  
Nucleotide Sequence ◽  
Proteus Mirabilis ◽  
Binding Sites ◽  
Recombinant Plasmid ◽  
Urease Activity ◽  
Transcriptional Activator ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Urease Gene ◽  
In Trans

ABSTRACT Expression of Proteus mirabilis urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A6TA2CA2TGGTA5GA6TGA5, is located 16 bp upstream of the ς70-likeureR promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured β-galactosidase activity of wild-typeEscherichia coli MC4100 (H-NS+) and its isogenic derivative ATM121 (hns::Tn10) (H-NS−) harboring a ureR-lacZ operon fusion plasmid (pLC9801). β-Galactosidase activity in the H-NS− host strain was constitutive and sevenfold greater (P < 0.0001) than that in the H-NS+ host. A recombinant plasmid containing cloned P. mirabilis hns was able to complement and restore repression of the ureR promoter in the H-NS−host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in β-galactosidase activity in the H-NS+ host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS−host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of P. mirabilis) was equivalent in both the H-NS−background and the H-NS+ background in the presence of urea but was eightfold greater (P = 0.0001) in the H-NS− background in the absence of urea. We conclude that H-NS represses ureR expression in the absence of urea induction.

scholarly journals Detection of Proteus mirabilis Urease Gene in Urinary Calculi by Polymerase Chain Reaction

1996 ◽  
Vol 3 (3) ◽  
pp. 202-206 ◽  
Author(s):  
Hideo Takeuchi ◽  
Shingo Yamamoto ◽  
Akito Terai ◽  
Hisao Kurazono ◽  
Yoshifumi Takeda ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Proteus Mirabilis ◽  
Urinary Calculi ◽  
Chain Reaction ◽  
Urease Gene ◽  
Polymerase Chain

scholarly journals UreR, the Transcriptional Activator of the Proteus mirabilis Urease Gene Cluster, Is Required for Urease Activity and Virulence in Experimental Urinary Tract Infections

2003 ◽  
Vol 71 (2) ◽  
pp. 1026-1030 ◽  
Author(s):  
Jonathan D. Dattelbaum ◽  
C. Virginia Lockatell ◽  
David E. Johnson ◽  
Harry L. T. Mobley
Keyword(s):  
Urinary Tract ◽  
Gene Cluster ◽  
Proteus Mirabilis ◽  
Urease Activity ◽  
Complicated Urinary Tract Infection ◽  
Wild Type ◽  
Urease Gene ◽  
Low Copy Number ◽  
Mutant Strains ◽  
Tract Infections

ABSTRACT Proteus mirabilis, a cause of complicated urinary tract infection, produces urease, an essential virulence factor for this species. UreR, a member of the AraC/XylS family of transcriptional regulators, positively activates expression of the ure gene cluster in the presence of urea. To specifically evaluate the contribution of UreR to urease activity and virulence in the urinary tract, a ureR mutation was introduced into P. mirabilis HI4320 by homologous recombination. The isogenic ureR::aphA mutant, deficient in UreR production, lacked measurable urease activity. Expression was not detected in the UreR-deficient strain by Western blotting with monoclonal antibodies raised against UreD. Urease activity and UreD expression were restored by complementation of the mutant strain with ureR expressed from a low-copy-number plasmid. Virulence was assessed by transurethral cochallenge of CBA mice with wild-type and mutant strains. The isogenic ureR::aphA mutant of HI4320 was outcompeted in the urine (P = 0.004), bladder (P = 0.016), and kidneys (P ≤ 0.001) 7 days after inoculation. Thus, UreR is required for basal urease activity in the absence of urea, for induction of urease by urea, and for virulence of P. mirabilis in the urinary tract.

scholarly journals Multiple proteins encoded within the urease gene complex of Proteus mirabilis.

1988 ◽  
Vol 170 (3) ◽  
pp. 1027-1033 ◽  
Author(s):  
S E Walz ◽  
S K Wray ◽  
S I Hull ◽  
R A Hull
Keyword(s):  
Proteus Mirabilis ◽  
Gene Complex ◽  
Urease Gene

Differential regulation of the Proteus mirabilis urease gene cluster by UreR and H-NS

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3383-3394 ◽  
Author(s):  
Carrie A. Poore ◽  
Harry L. T. Mobley
Keyword(s):  
Urinary Tract ◽  
Proteus Mirabilis ◽  
Intergenic Region ◽  
Urinary Stones ◽  
E Coli ◽  
Urease Gene ◽  
Genes Encoding ◽  
Gel Shift Assay ◽  
Dna Fragment ◽  
Hydrolysis Of

Proteus mirabilis, a cause of catheter-associated urinary tract infection, relies on several virulence factors to colonize the urinary tract. Among these, urease contributes to the development of urinary stones resulting from the increase in local pH due to urease-mediated hydrolysis of urea to NH3 and CO2. UreR, an AraC-like transcriptional activator, activates transcription of the genes encoding the urease subunits and accessory proteins (ureDABCEFG) in the presence of urea. UreR also initiates transcription of its own gene in a urea-inducible manner by binding to the intergenic region between ureR and ureD. The intergenic region contains poly(A) tracts that appear to be the target of H-NS. It has been shown that Escherichia coli and P. mirabilis H-NS acts to repress transcription of ureR in an E. coli model system. It was hypothesized that H-NS represses urease gene expression in the absence of UreR and urea by binding to the intergenic region. To demonstrate this the P. mirabilis hns gene was cloned and the 15·6 kDa H-NS was overexpressed and purified as a myc-His tail fusion. Using a gel shift assay, purified H-NS-myc-His bound preferentially to a 609 bp DNA fragment containing the entire ureR-ureD intergenic region. H-NS and UreR were able to displace each other from the ureR-ureD intergenic region. Circular permutation analysis revealed that the intergenic region is bent. Moreover, H-NS recognizes this curvature, binds the DNA fragment and induces further bending of the DNA as shown by a circular ligation assay. The effects of H-NS, urea and temperature (25 vs 37 °C) on urease expression were shown in E. coli containing an hns knockout and P. mirabilis where expression was increased at 37 °C. Increased transcription from pureR was seen in the E. coli hns knockout when temperature was increased from 25 to 37 °C. These findings suggest H-NS and UreR differentially regulate urease in a negative and positive manner, respectively.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

Sites of Insulin Action Using Ferritin Labeling

1971 ◽  
Vol 29 ◽  
pp. 540-541
Author(s):  
Burton B. Silver ◽  
Ronald S. Nelson
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Anterior Pituitary ◽  
Insulin Action ◽  
Diabetic Rats ◽  
Insulin Antibody ◽  
Fat Pad ◽  
Target Organs ◽  
Uranium Salts ◽  
Sugar Levels

Some investigators feel that insulin does not enter cells but exerts its influence in some manner on the cell surface. Ferritin labeling of insulin and insulin antibody was used to determine if binding sites of insulin to specific target organs could be seen with electron microscopy.Alloxanized rats were considered diabetic if blood sugar levels were in excess of 300 mg %. Test reagents included ferritin, ferritin labeled insulin, and ferritin labeled insulin antibody. Target organs examined were were diaphragm, kidney, gastrocnemius, fat pad, liver and anterior pituitary. Reagents were administered through the left common carotid. Survival time was at least one hour in test animals. Tissue incubation studies were also done in normal as well as diabetic rats. Specimens were fixed in gluteraldehyde and osmium followed by staining with lead and uranium salts. Some tissues were not stained.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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