scholarly journals Characterization of the Catalytic Activities of the PhoQ Histidine Protein Kinase of Salmonella entericaSerovar Typhimurium

2001 ◽  
Vol 183 (5) ◽  
pp. 1787-1791 ◽  
Author(s):  
Martin Montagne ◽  
Alexandre Martel ◽  
Hervé Le Moual
Keyword(s):  
Escherichia Coli ◽  
Phosphatase Activity ◽  
Divalent Cations ◽  
Histidine Protein Kinase ◽  
Catalytic Activities ◽  
Serovar Typhimurium ◽  
High Concentrations ◽  
Kinetics Of ◽  
Autokinase Activity

ABSTRACT Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2+ or Mn2+was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.

scholarly journals Characterization of the Two Mycobacterium tuberculosis recA Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 6005-6015 ◽  
Author(s):  
Krishna K. Gopaul ◽  
Patricia C. Brooks ◽  
Jean-François Prost ◽  
Elaine O. Davis
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
Promoter Activity ◽  
The Other ◽  
Expression Level ◽  
Promoter Elements ◽  
Kinetics Of

ABSTRACT The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ70 −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

Kinetics of the cellular intake of a gene expression inducer at high concentrations

2015 ◽  
Vol 11 (9) ◽  
pp. 2579-2587 ◽  
Author(s):  
Huy Tran ◽  
Samuel M. D. Oliveira ◽  
Nadia Goncalves ◽  
Andre S. Ribeiro
Keyword(s):  
Gene Expression ◽  
Single Event ◽  
Transcription Activity ◽  
High Concentrations ◽  
Kinetics Of

Characterization of the cellular intake kinetics of a lactose analogue fromin vivosingle-event measurements of transcription activity.

scholarly journals In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

2016 ◽  
Vol 31 (6) ◽  
pp. 540-550 ◽  
Author(s):  
Kevin A. Feeney ◽  
Marrit Putker ◽  
Marco Brancaccio ◽  
John S. O’Neill
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Circadian Gene ◽  
Cultured Fibroblasts ◽  
High Concentrations ◽  
Lower Temperature ◽  
Kinetics Of

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

scholarly journals Characterization of the Binding of Adenosine Diphosphate to Human Platelet Membranes

1977 ◽  
Author(s):  
C. Legrand ◽  
B. Bauvois ◽  
J. P. Caen
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Adenosine Diphosphate ◽  
Affinity Constant ◽  
Membrane Bound ◽  
High Concentrations ◽  
Kinetics Of

ADP-mediated platelet aggregation is a routinely employed test but its mechanism is poorly understood. The aim of this study was to compare the binding of ADP to plasma membranes isolated from normal platelets and thrombasthenic platelets (which do not aggregate with ADP). Binding of ADP to isolated membranes was assayed by incubation with 14C-ADP followed by Mill i pore filtration. In standard conditions, 14C-ADP was not transformed and non specific binding represented lessthan 3 % of the total binding. Using 1 μM 14C-ADP, the binding has been shown to be a rapid (t 1/2 = 2 mn 30 sec), saturable and reversible phenomenon at 37° C. The existence of a major population of binding sites, with an affinity constant Ka = 0.43 (+ 0.1) χ 106M-1, has been demonstrated. The kinetics of the binding was normal with membranes Tsolated from the platelets of 4 thrombasthenic patients and the affinity constant, when determined, was in the normal range. Dissociation of the membrane-bound 14C-ADP occurred rapidly at 37° C (t l/2c≃3mn) when samples were diluted enough (dilution 1 : 100 was currently employed) to avoid rebinding of the radioligand. Accelerated dissociation (t 1/2 ≃ 1 mn) was observed when the dilution was performed in the presence of an excess of unlabeled ADP, suggesting the existence of negatively cooperative site-site interactions among the ADP binding sites. This effect was only observed at high concentrations of ADP (> 10–5M) and its eventual role in vivo remains to be established. Two thrombasthenic membrane preparations studied in the same way dissociated as did the control membranes.

Inactivation Kinetics of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Ready-to-Eat Sliced Ham by Near-Infrared Heating at Different Radiation Intensities

2014 ◽  
Vol 77 (7) ◽  
pp. 1224-1228 ◽  
Author(s):  
JAE-WON HA ◽  
DONG-HYUN KANG
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Logistic Model ◽  
Near Infrared ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Inactivation Kinetics ◽  
Escherichia Coli O157 ◽  
Survival Curves ◽  
Serovar Typhimurium ◽  
Kinetics Of

The aim of this study was to investigate the inactivation kinetics of Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes on ready-to-eat sliced ham by near-infrared (NIR) heating as a function of the processing parameter, radiation intensity. Precooked ham slices inoculated with the three pathogens were treated at different NIR intensities (ca. 100, 150, and 200 μW/cm2/nm). An increase in the applied radiation intensity resulted in a gradual increase of inactivation of all pathogens. The survival curves of the three pathogens exhibited both shoulder and tailing behavior at all light intensities. Among nonlinear models, the Weibull distribution and log-logistic model were used to describe the experimental data, and the statistical results (mean square error and R2 values) indicated the suitability of the model for prediction. The log-logistic model more accurately described survival curves of the three pathogens than did the Weibull distribution at all radiation intensities. The output of this study and the proposed kinetics model would be beneficial to the deli meat industry for selecting the optimum processing conditions of NIR heating to meet the target pathogen inactivation on ready-to-eat sliced ham.

scholarly journals Biochemical Characterization of the POM-1 Metallo-β-Lactamase from Pseudomonas otitidis

2014 ◽  
Vol 59 (3) ◽  
pp. 1755-1758 ◽  
Author(s):  
Luisa Borgianni ◽  
Filomena De Luca ◽  
Maria Cristina Thaller ◽  
Yunsop Chong ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Content Type ◽  
Broad Substrate Specificity ◽  
Catalytic Activities

ABSTRACTThe POM-1 metallo-β-lactamase is a subclass B3 resident enzyme produced byPseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced inEscherichia coliBL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.

Functional characterization of X-prolyl aminopeptidase from Toxoplasma gondii

Parasitology ◽  
2016 ◽  
Vol 143 (11) ◽  
pp. 1443-1449 ◽  
Author(s):  
MINGFA YANG ◽  
JUN ZHENG ◽  
HONGLIN JIA ◽  
MINGXIN SONG
Keyword(s):  
Escherichia Coli ◽  
Toxoplasma Gondii ◽  
Divalent Cations ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Strong Activity ◽  
Inhibition Of Growth ◽  
Aminopeptidase P ◽  
Insight Into

SUMMARYIn the present study, a recombinant aminopeptidase P (rTgAPP) from Toxoplasma gondii was expressed in Escherichia coli to evaluate its enzyme parameters. The rTgAPP showed strong activity against a synthetic substrate for aminopeptidase P at pH 8·0 with a Km value of 0·255 µm and a kcat value of 35·6 s−1. The overall catalytic efficiency (kcat/Km) of the rTgAPP was 139·6 × 105 M−1 s−1. The activity of rTgAPP was enhanced by the addition of divalent cations and inhibited by bestatin. Deletion of TgAPP gene in the parasite through a CRISPR/Cas9 system resulted in inhibition of growth indicating the importance of TgAPP. Thus our findings reveal that TgAPP is an active enzyme in T. gondii and provide an insight into the function of TgAPP.

scholarly journals Characterization of Chloramphenicol and Florfenicol Resistance in Escherichia coli Associated with Bovine Diarrhea

2000 ◽  
Vol 38 (12) ◽  
pp. 4593-4598 ◽  
Author(s):  
David G. White ◽  
Charlene Hudson ◽  
John J. Maurer ◽  
Sherry Ayers ◽  
Shaohua Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
The United States ◽  
Cross Resistance ◽  
Respiratory Pathogens ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Efflux Mechanism

Florfenicol, a veterinary fluorinated analog of thiamphenicol, is approved for treatment of bovine respiratory pathogens in the United States. However, florfenicol resistance has recently emerged among veterinary Escherichia coli isolates incriminated in bovine diarrhea. The flo gene, which confers resistance to florfenicol and chloramphenicol, has previously been identified inPhotobacterium piscicida and Salmonella enterica serovar Typhimurium DT104. The flo gene product is closely related to the CmlA protein identified inPseudomonas aeruginosa. The cmlA gene confers nonenzymatic chloramphenicol resistance via an efflux mechanism. Forty-eight E. coli isolates recovered from calves with diarrhea, including 41 that were both chloramphenicol and florfenicol resistant, were assayed for the presence of both flo andcmlA genes. Forty-two of the 44 isolates for which florfenicol MICs were ≥16 μg/ml were positive via PCR for theflo gene. All E. coli isolates for which florfenicol MICs were ≤8 μg/ml were negative for the flogene (n = 4). Twelve E. coli isolates were positive for cmlA, and chloramphenicol MICs for all 12 were ≥32 μg/ml. Additionally, eight isolates were positive for bothflo and cmlA, and both florfenicol and chloramphenicol MICs for these isolates were ≥64 μg/ml. DNA sequence analysis of the E. coli flo gene demonstrated 98% identity to the published GenBank sequences of both serovar TyphimuriumfloSt and P. piscicida pp-flo. Theflo gene was identified on high-molecular-weight plasmids of approximately 225 kb among the majority of florfenicol-resistantE. coli isolates. However, not all of the florfenicol-resistant E. coli isolates tested contained the large flo-positive plasmids. This suggests that several of the E. coli isolates may possess a chromosomalflo gene. The E. coli flo gene specifies nonenzymatic cross-resistance to both florfenicol and chloramphenicol, and its presence among bovine E. coli isolates of diverse genetic backgrounds indicates a distribution much wider than previously thought.

scholarly journals Characterization of a Highly Hop-Resistant Lactobacillus brevis Strain Lacking Hop Transport

2006 ◽  
Vol 72 (10) ◽  
pp. 6483-6492 ◽  
Author(s):  
Jürgen Behr ◽  
Michael G. Gänzle ◽  
Rudi F. Vogel
Keyword(s):  
Cell Wall ◽  
Divalent Cations ◽  
Lactobacillus Brevis ◽  
Cell Wall Composition ◽  
Arginine Deiminase ◽  
Ethanol Stress ◽  
Beer Spoilage ◽  
High Concentrations ◽  
Altered Cell

ABSTRACT Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.

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