scholarly journals Discovery and characterization of a sulfoquinovose mutarotase using kinetic analysis at equilibrium by exchange spectroscopy

2018 ◽  
Vol 475 (7) ◽  
pp. 1371-1383 ◽  
Author(s):  
Palika Abayakoon ◽  
James P. Lingford ◽  
Yi Jin ◽  
Christopher Bengt ◽  
Gideon J. Davies ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Glucuronic Acid ◽  
Chemical Exchange ◽  
Sequence Analyses ◽  
Uncharacterized Gene ◽  
Exchange Spectroscopy ◽  
First Time ◽  
Better Than

Bacterial sulfoglycolytic pathways catabolize sulfoquinovose (SQ), or glycosides thereof, to generate a three-carbon metabolite for primary cellular metabolism and a three-carbon sulfonate that is expelled from the cell. Sulfoglycolytic operons encoding an Embden–Meyerhof–Parnas-like or Entner–Doudoroff (ED)-like pathway harbor an uncharacterized gene (yihR in Escherichia coli; PpSQ1_00415 in Pseudomonas putida) that is up-regulated in the presence of SQ, has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modeling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We overexpressed the homolog from the sulfo-ED operon of Herbaspirillum seropedicaea (HsSQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using nuclear magnetic resonance exchange spectroscopy, a method that allows the chemical exchange of magnetization between the two SQ anomers at equilibrium. HsSQM also catalyzed the mutarotation of various aldohexoses with an equatorial 2-hydroxy group, including d-galactose, d-glucose, d-glucose-6-phosphate (Glc-6-P), and d-glucuronic acid, but not d-mannose. HsSQM displayed only 5-fold selectivity in terms of efficiency (kcat/KM) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [kuncat/(kcat/KM)] for SQ was 17 000-fold better than for Glc-6-P, revealing that HsSQM preferentially stabilizes the SQ transition state.

scholarly journals Resistance Profiling and Molecular Characterization of Extended-Spectrum/Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli Isolated from Healthy Broiler Chickens in South Korea

2020 ◽  
Vol 8 (9) ◽  
pp. 1434 ◽  
Author(s):  
Hyun-Ju Song ◽  
Dong Chan Moon ◽  
Abraham Fikru Mechesso ◽  
Hee Young Kang ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phylogenetic Analysis ◽  
Resistance Gene ◽  
Broiler Chickens ◽  
Human Consumption ◽  
E Coli ◽  
Extended Spectrum ◽  
First Time ◽  
Pfge Profiles

We aimed to identify and characterize extended-spectrum β-lactamase (ESBL)-and/or plasmid-mediated AmpC β-lactamase (pAmpC)-producing Escherichia coli isolated from healthy broiler chickens slaughtered for human consumption in Korea. A total of 332 E. coli isolates were identified from 339 cloacal swabs in 2019. More than 90% of the isolates were resistant to multiple antimicrobials. ESBL/pAmpC-production was noted in 14% (46/332) of the isolates. Six of the CTX-M-β-lactamase-producing isolates were found to co-harbor at least one plasmid-mediated quinolone resistance gene. We observed the co-existence of blaCMY-2 and mcr-1 genes in the same isolate for the first time in Korea. Phylogenetic analysis demonstrated that the majority of blaCMY-2-carrying isolates belonged to subgroup D. Conjugation confirmed the transferability of blaCTX-M and blaCMY-2 genes, as well as non-β-lactam resistance traits from 60.9% (28/46) of the ESBL/pAmpC-producing isolates to a recipient E. coli J53. The ISECP, IS903, and orf477 elements were detected in the upstream or downstream regions. The blaCTX-M and blaCMY-2 genes mainly belonged to the IncI1, IncHI2, and/or IncFII plasmids. Additionally, the majority of ESBL/pAmpC-producing isolates exhibited heterogeneous PFGE profiles. This study showed that healthy chickens act as reservoirs of ESBL/pAmpC-producing E. coli that can potentially be transmitted to humans.

Cloning, Expression and Characterization of a Monooxygenase P450BM3 from Bacillus megaterium ALA2

2012 ◽  
Vol 518-523 ◽  
pp. 5533-5538
Author(s):  
Ting Wang ◽  
Liang Liang Wang ◽  
Xun Li
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Linoleic Acid ◽  
Bacillus Megaterium ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
Nadph Oxidation ◽  
First Time ◽  
Optimal Ph

Cytochrome P450 monooxygenases are enzymes which are capable of oxidising saturated and unsaturated substrates. P450BM3 from Bacillus megaterium is one of this family. For the first time, the cyp gene for coding P450BM3 from B. megaterium ALA2 has been cloned and expressed in Escherichia coli. The recombinant enzyme is 120 kDa, containing 1049 aa. The highest activity of purified enzyme is 14.8 U/mg towards palmitic acid by monitoring the NADPH oxidation. The optimal pH and temperature were 9.0 and 40°C. The enzyme has higher activity towards linoleic acid, and 2-Methyl-7-octadecene can also be catalyzed which is a precursor of displar.

Characterization of a mucoid Escherichia coli K12 strain, and chemical analysis of the exopolysaccharide

1972 ◽  
Vol 18 (7) ◽  
pp. 969-973 ◽  
Author(s):  
Stephen Shugar ◽  
Kenneth E. Sanderson
Keyword(s):  
Escherichia Coli ◽  
Chemical Analysis ◽  
Glucuronic Acid ◽  
Extracellular Polysaccharide ◽  
Spontaneous Mutant ◽  
Escherichia Coli K12 ◽  
Molar Ratios ◽  
Genetic Lesion ◽  
Long Form

A spontaneous mutant of Escherichia coli K12 C600 was isolated which is mucoid as a result of overproduction of an extracellular polysaccharide. This strain also forms filaments, is sensitive to ultraviolet irradiation, and has its genetic lesion closely linked to the lac gene. Its phenotype and map position are analogous to the lon mutation (long form). The exopolysaccharide contains fucose, glucose, galactose, and may contain glucuronic acid, but the molar ratios of these sugars differ from those previously reported.

scholarly journals The Expression Profile and Prognostic Significance of Metallothionein Genes in Colorectal Cancer

2019 ◽  
Vol 20 (16) ◽  
pp. 3849 ◽  
Author(s):  
Kuo-Chen Hung ◽  
Tsui-Chin Huang ◽  
Chia-Hsiung Cheng ◽  
Ya-Wen Cheng ◽  
Ding-Yen Lin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Prognostic Significance ◽  
Gene Signature ◽  
Mrna Levels ◽  
Aberrant Expression ◽  
Gene Markers ◽  
Highly Sensitive ◽  
First Time ◽  
Better Than

Colorectal cancer (CRC) is a heterogeneous disease resulting from the combined influence of many genetic factors. This complexity has caused the molecular characterization of CRC to remain uncharacterized, with a lack of clear gene markers associated with CRC and the prognosis of this disease. Thus, highly sensitive tumor markers for the detection of CRC are the most essential determinants of survival. In this study, we examined the simultaneous downregulation of the mRNA levels of six metallothionein (MT) genes in CRC cell lines and public CRC datasets for the first time. In addition, we detected downregulation of these six MT mRNAs’ levels in 30 pairs of tumor (T) and adjacent non-tumor (N) CRC specimens. In order to understand the potential prognostic relevance of these six MT genes and CRC, we presented a four-gene signature to evaluate the prognosis of CRC patients. Further discovery suggested that the four-gene signature (MT1F, MT1G, MT1L, and MT1X) predicted survival better than any combination of two-, three-, four-, five-, or six-gene models. In conclusion, this study is the first to report that simultaneous downregulation of six MT mRNAs’ levels in CRC patients, and their aberrant expression together, accurately predicted CRC patients’ outcomes.

scholarly journals First Characterization of CTX-M-15-Producing Escherichia coli Strains Belonging to Sequence Type (ST) 410, ST224, and ST1284 from Commercial Swine in South America

2016 ◽  
Vol 60 (4) ◽  
pp. 2505-2508 ◽  
Author(s):  
Ketrin C. Silva ◽  
Marina Moreno ◽  
Carlos Cabrera ◽  
Beny Spira ◽  
Louise Cerdeira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
South America ◽  
Insertion Sequence ◽  
Sequence Type ◽  
Content Type ◽  
First Time ◽  
Genetic Context ◽  
New Reservoir

ABSTRACTWe report for the first time the isolation of CTX-M-15-producingEscherichia colistrains belonging to sequence type (ST) 410, ST224, and ST1284 in commercial swine in Brazil. TheblaCTX-M-15gene was located on F-::A9::B1 and C1::A9::B1 IncF-type plasmids, surrounded by a new genetic context comprising the IS26insertion sequence truncated with the ISEcp1element upstream ofblaCTX-M-15. These results reveal that commercial swine have become a new reservoir of CTX-M-15-producing bacteria in South America.

scholarly journals Contribution of the Ler- and H-NS-Regulated Long Polar Fimbriae of Escherichia coli O157:H7 during Binding to Tissue-Cultured Cells

2008 ◽  
Vol 76 (11) ◽  
pp. 5062-5071 ◽  
Author(s):  
Alfredo G. Torres ◽  
Terry M. Slater ◽  
Shilpa D. Patel ◽  
Vsevolod L. Popov ◽  
Margarita M. P. Arenas-Hernández
Keyword(s):  
Escherichia Coli ◽  
Cultured Cells ◽  
Immunogold Labeling ◽  
Regulatory Sequence ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
E Coli ◽  
First Time ◽  
Better Than

ABSTRACT The expression of the long polar fimbriae (LPF) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by a tightly regulated process, and, therefore, the role of these fimbriae during binding to epithelial cells has been difficult to establish. We recently found that histone-like nucleoid-structuring protein (H-NS) binds to the regulatory sequence of the E. coli O157:H7 lpf1 operon and “silences” its transcription, while Ler inhibits the action of the H-NS protein and allows lpf1 to be expressed. In the present study, we determined how the deregulated expression of LPF affects binding of EHEC O157:H7 to tissue-cultured cells, correlating the adherence phenotype with lpf1 expression. We tested the adherence properties of EHEC hns mutant and found that this strain adhered 2.8-fold better than the wild type. In contrast, the EHEC ler mutant adhered 2.1-fold less than the wild type. The EHEC hns ler mutant constitutively expressed the lpf genes, and, therefore, we observed that the double mutant adhered 5.6-fold times better than the wild type. Disruption of lpfA in the EHEC hns and hns ler mutants or the addition of anti-LpfA serum caused a reduction in adhesion, demonstrating that the increased adherence was due to the expression of LPF. Immunogold-labeling electron microscopy showed that LPF is present on the surface of EHEC lpfA + strains. Furthermore, we showed that EHEC expressing LPF agglutinates red blood cells from different species and that the agglutination was blocked by the addition of anti-LpfA serum. Overall, our data confirmed that expression of LPF is a tightly regulated process and, for the first time, demonstrated that these fimbriae are associated with adherence and hemagglutination phenotypes in EHEC O157:H7.

scholarly journals An improved method for atmospheric <sup>14</sup>CO measurements

2021 ◽  
Vol 14 (3) ◽  
pp. 2055-2063
Author(s):  
Vasilii V. Petrenko ◽  
Andrew M. Smith ◽  
Edward M. Crosier ◽  
Roxana Kazemi ◽  
Philip Place ◽  
...  
Keyword(s):  
Sample Volume ◽  
Spatial And Temporal Variability ◽  
Low Latitude ◽  
Mauna Loa ◽  
Carbon 14 ◽  
Air Sample ◽  
Atmospheric 14Co ◽  
First Time ◽  
Better Than

Abstract. Important uncertainties remain in our understanding of the spatial and temporal variability of atmospheric hydroxyl radical concentration ([OH]). Carbon-14-containing carbon monoxide (14CO) is a useful tracer that can help in the characterization of [OH] variability. Prior measurements of atmospheric 14CO concentration ([14CO] are limited in both their spatial and temporal extent, partly due to the very large air sample volumes that have been required for measurements (500–1000 L at standard temperature and pressure, L STP) and the difficulty and expense associated with the collection, shipment, and processing of such samples. Here we present a new method that reduces the air sample volume requirement to ≈90 L STP while allowing for [14CO] measurement uncertainties that are on par with or better than prior work (≈3 % or better, 1σ). The method also for the first time includes accurate characterization of the overall procedural [14CO] blank associated with individual samples, which is a key improvement over prior atmospheric 14CO work. The method was used to make measurements of [14CO] at the NOAA Mauna Loa Observatory, Hawaii, USA, between November 2017 and November 2018. The measurements show the expected [14CO] seasonal cycle (lowest in summer) and are in good agreement with prior [14CO] results from another low-latitude site in the Northern Hemisphere. The lowest overall [14CO] uncertainties (2.1 %, 1σ) are achieved for samples that are directly accompanied by procedural blanks and whose mass is increased to ≈50 µgC (micrograms of carbon) prior to the 14C measurement via dilution with a high-CO 14C-depleted gas.

scholarly journals Function-Based Selection and Characterization of Base-Pair Polymorphisms in a Promoter of Escherichia coli RNA Polymerase-ς70

2001 ◽  
Vol 183 (9) ◽  
pp. 2866-2873 ◽  
Author(s):  
Jian Xu ◽  
Barbara C. McCabe ◽  
Gerald B. Koudelka
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Promoter Sequence ◽  
Kinetic Properties ◽  
Optimal Sequence ◽  
Sequence Requirements ◽  
Better Than

ABSTRACT We performed two sets of in vitro selections to dissect the role of the −10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-ς70forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus −10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.

scholarly journals Precise Identification and Characterization of Catalytically Active Sites on the Surface of γ-Alumina

2020 ◽  
Author(s):  
Konstantin Khivantsev ◽  
Nicholas R. Jaegers ◽  
Ja-Hun Kwak ◽  
János Szanyi ◽  
Libor Kovarik
Keyword(s):  
Active Sites ◽  
Large Scale ◽  
High Surface ◽  
High Surface Area ◽  
Carbonyl Complexes ◽  
True Nature ◽  
Catalytically Active ◽  
Active Research ◽  
First Time

<p>γ-alumina is one of the oldest and most important commercial catalytic materials with high surface area and stability. These attributes enabled its use as the first commercial large-scale heterogeneous catalyst for ethanol dehydration. Despite progress in materials characterization and over a hundred years of active research, the nature of the specific sites on the surface of γ-alumina which are responsible for its unique catalytic properties has remained obscure and controversial. Herein, we identify for the first time using combined infrared spectroscopy, electron microscopy and solid-state nuclear magnetic resonance measurements that the octahedral amphoteric (O)<sub>5</sub>Al(VI)-OH sites on the massively restructured (110) facets on typical rhombus-platelet γ-alumina as well as (100) segments of irrational surfaces (invariably always present in all γ-alumina samples) are largely responsible for its unique catalytic activity. Such (O)<sub>5</sub>Al(VI)-OH sites are also present on macroscopically defined (100) facets γ-alumina of different, more elongated/rod-like geometry. They anchor organometallic fragments and isolated metal atoms. The exact mechanism by which these sites lose -OH group upon thermal dehydroxylation is clarified, resulting in coordinatively unsaturated penta-coordinate Al<sup>+3</sup>O<sub>5</sub> sites that can activate nitrogen. These coordinatively unsaturated Al penta-coordinate sites demonstrate new chemistry, producing the first well-defined thermally stable Al-carbonyl complexes. Our findings reunite and successfully clarify the contradictory findings in the literature over the last century, illuminating the true nature and the relationship between the catalytically active and coordinatively unsaturated Al sites on the surface of γ-alumina.</p>

scholarly journals An improved method for atmospheric <sup>14</sup>CO measurements

2020 ◽  
Author(s):  
Vasilii V. Petrenko ◽  
Andrew M. Smith ◽  
Edward M. Crosier ◽  
Roxana Kazemi ◽  
Philip Place ◽  
...  
Keyword(s):  
Sample Volume ◽  
Spatial And Temporal Variability ◽  
Low Latitude ◽  
Mauna Loa ◽  
Carbon 14 ◽  
Air Sample ◽  
Atmospheric 14Co ◽  
First Time ◽  
Better Than

Abstract. Important uncertainties remain in our understanding of the spatial and temporal variability of atmospheric hydroxyl radical concentration ([OH]). Carbon-14-containing carbon monoxide (14CO) is a useful tracer that can help in the characterization of [OH] variability. Prior measurements of atmospheric 14CO concentration ([14CO] are limited in both their spatial and temporal extent, partly due to the very large air sample volumes that have been required for measurements (500–1000 liters at standard temperature and pressure, L STP) and the difficulty and expense associated with the collection, shipment and processing of such samples. Here we present a new method that reduces the air sample volume requirement to &amp;approx; 90 L STP while allowing for [14CO] measurement uncertainties that are on par with or better than prior work (&amp;approx; 3 % or better, 1 σ). The method also for the first time includes accurate characterization of the overall procedural [14CO] blank associated with individual samples, a key improvement over prior atmospheric 14CO work. The method was used to make measurements of [14CO] at the NOAA Mauna Loa Observatory, Hawaii, USA, between November 2017 and November 2018. The measurements show the expected [14CO] seasonal cycle (lowest in summer) and are in good agreement with prior [14CO] results from another low-latitude site in the Northern Hemisphere. The lowest overall [14CO] uncertainties (2.1 %, 1 σ) are achieved for samples that are directly accompanied by procedural blanks and whose mass is increased to &amp;approx; 50 micrograms of carbon (µgC) prior to the 14C measurement via dilution with a high-CO, 14C-depleted gas.

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