scholarly journals A new spectrophotometric assay for citrate synthase and its use to assess the inhibitory effects of palmitoyl thioesters

1988 ◽  
Vol 251 (3) ◽  
pp. 803-807 ◽  
Author(s):  
A J Else ◽  
S J Barnes ◽  
M J Danson ◽  
P D J Weitzman
Keyword(s):  
Escherichia Coli ◽  
Bacillus Megaterium ◽  
Citrate Synthase ◽  
Spectrophotometric Assay ◽  
Time Dependent ◽  
Inhibitory Effects ◽  
E Coli ◽  
Cell Extracts ◽  
Thioester Bond ◽  
High Concentrations

We have demonstrated that citrate synthase may be assayed by a simple, discontinuous, spectrophotometric procedure based on the measurement of oxaloacetate utilization with 2,4-dinitrophenylhydrazine. The assay is applicable both to the purified enzyme and to cell extracts, and has the advantage that it can be used in the presence of high concentrations of thiols and thioesters. We have used this new assay in part of our investigations into the inhibitory effects of palmitoyl thioesters on diverse citrate synthases. Both palmitoyl-CoA and palmitoyl thioglycollate inhibit citrate synthases from pig heart, Bacillus megaterium and Escherichia coli, the E. coli enzyme showing the greatest sensitivity to these effectors. With palmitoyl-CoA the extent of inhibition is time-dependent, but the enzymes can be protected from the effect by the substrates oxaloacetate and acetyl-CoA. Using the dinitrophenylhydrazine assay, we have shown that the thioester bond is essential for inhibition; that is, if the palmitoyl thioesters are cleaved to give a mixture of palmitate and a thiol compound, the inhibitions of pig heart and B. megaterium citrate synthases are eliminated and that of the E. coli enzyme is markedly decreased.

scholarly journals Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases

1988 ◽  
Vol 254 (2) ◽  
pp. 437-442 ◽  
Author(s):  
A J Else ◽  
M J Danson ◽  
P D J Weitzman
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Bacillus Megaterium ◽  
Predictive Models ◽  
Statistical Approach ◽  
Citrate Synthase ◽  
The Other ◽  
E Coli ◽  
High Concentrations

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.

Biofilm Growth of Escherichia coli Is Subject to cAMP-Dependent and cAMP-Independent Inhibition

2015 ◽  
Vol 25 (2-3) ◽  
pp. 209-225 ◽  
Author(s):  
Sarah L. Sutrina ◽  
Kia Daniel ◽  
Michael Lewis ◽  
Naomi T. Charles ◽  
Cherysa K.E. Anselm ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Ph Effects ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Phosphotransferase System ◽  
Biofilm Growth ◽  
E Coli ◽  
Low Concentrations ◽  
High Concentrations

We established that <i>Escherichia coli </i>strain 15 (ATCC 9723) produces both curli and cellulose, and forms robust biofilms. Since this strain is wild type with respect to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), it is an ideal strain in which to investigate the effects of the PTS on the biofilm growth of <i>E. coli</i>. We began by looking into the effects of PTS and non-PTS sugars on the biofilm growth of this strain. All the sugars tested tended to activate biofilm growth at low concentrations but to inhibit biofilm growth at high concentrations. Acidification of the medium was an inhibitory factor in the absence of buffer, but buffering to prevent a pH drop did not prevent the inhibitory effects of the sugars. The concentration at which inhibition set in varied from sugar to sugar. For most sugars, cyclic (c)AMP counteracted the inhibition at the lowest inhibitory concentrations but became ineffective at higher concentrations. Our results suggest that cAMP-dependent catabolite repression, which is mediated by the PTS in <i>E. coli</i>, plays a role in the regulation of biofilm growth in response to sugars. cAMP-independent processes, possibly including Cra, also appear to be involved, in addition to pH effects.

scholarly journals [13C]propionate oxidation in wild-type and citrate synthase mutant Escherichia coli: evidence for multiple pathways of propionate utilization

1993 ◽  
Vol 291 (3) ◽  
pp. 927-932 ◽  
Author(s):  
C T Evans ◽  
B Sumegi ◽  
P A Srere ◽  
A D Sherry ◽  
C R Malloy
Keyword(s):  
Escherichia Coli ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Spin Coupling ◽  
Wild Type ◽  
Acid Cycle ◽  
E Coli ◽  
Cell Extracts ◽  
In Cells

The metabolism of propionate was examined in wild-type Escherichia coli and cells lacking citrate synthase by high-resolution 13C n.m.r. Spectra of cell extracts from wild-type E. coli show that glutamate becomes highly enriched in 13C when 13C-enriched propionate is the sole carbon source. No glutamate labelling was detected when the tricarboxylic acid cycle was blocked either by deletion of citrate synthase or by inhibition of succinate dehydrogenase by malonate. The 13C fractional enrichment in glutamate C-2, C-3 and C-4 in wild-type cells was quantitatively and qualitatively different when [2-13C]propionate as opposed to [3-13C]propionate was supplied. Approximately equal labelling occurred in the C-2, C-3 and C-4 positions of glutamate when [3-13C]propionate was available, and multiplets due to carbon-carbon spin-spin coupling were observed. However, in cells supplied with [2-13C]propionate, very little 13C appeared in the glutamate C-4 position, and the remaining glutamate resonances all appeared as singlets. The unequal and non-identical labelling of glutamate in cells supplied with [2-13C]- as opposed to [3-13C]propionate is consistent with the utilization of propionate by E. coli via two pathways, oxidation of propionate to pyruvate and carboxylation of propionate to succinate. These intermediates are further metabolized to glutamate by the action of the tricarboxylic acid cycle. The existence of an organized tricarboxylic acid cycle is discussed as a consequence of the ability to block utilization of propionate in tricarboxylic acid-cycle-defective E. coli.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

Screening of Commercial and Pecan Shell–Extracted Liquid Smoke Agents as Natural Antimicrobials against Foodborne Pathogens

2012 ◽  
Vol 75 (6) ◽  
pp. 1148-1152 ◽  
Author(s):  
ELLEN J. VAN LOO ◽  
D. BABU ◽  
PHILIP G. CRANDALL ◽  
STEVEN C. RICKE
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Salmonella Enteritidis ◽  
Antioxidant Properties ◽  
Inhibitory Effects ◽  
Natural Antimicrobials ◽  
E Coli ◽  
Liquid Smoke ◽  
Water Based ◽  
Smoke Sample

Liquid smoke extracts have traditionally been used as flavoring agents, are known to possess antioxidant properties, and serve as natural alternatives to conventional antimicrobials. The antimicrobial efficacies of commercial liquid smoke samples may vary depending on their source and composition and the methods used to extract and concentrate the smoke. We investigated the MICs of eight commercial liquid smoke samples against Salmonella Enteritidis, Staphylococcus aureus, and Escherichia coli. The commercial liquid smoke samples purchased were supplied by the manufacturer as water-based or concentrated extracts of smoke from different wood sources. The MICs of the commercial smokes to inhibit the growth of foodborne pathogens ranged from 0.5 to 6.0% for E. coli, 0.5 to 8.0% for Salmonella, and 0.38 to 6% for S. aureus. The MIC for each liquid smoke sample was similar in its effect on both E. coli and Salmonella. Solvent-extracted antimicrobials prepared using pecan shells displayed significant differences between their inhibitory concentrations depending on the type of solvent used for extraction. The results indicated that the liquid smoke samples tested in this study could serve as effective natural antimicrobials and that their inhibitory effects depended more on the solvents used for extraction than the wood source.

scholarly journals UJI AKTIVITAS ANTIBAKTERI DAN ANTI-UV Phyllidiella nigra DAN BAKTERI SIMBIOTIKNYA DARI PERAIRAN TANJUNG MANDOLANG

2020 ◽  
Vol 8 (2) ◽  
pp. 61
Author(s):  
Tessalonica Dajoh ◽  
Robert A Bara ◽  
Esther Angkouw ◽  
Medy Ompi ◽  
Rosita A Lintang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Secondary Metabolites ◽  
Bacillus Megaterium ◽  
Absorption Maximum ◽  
Chemical Compounds ◽  
Inhibition Zone ◽  
Defense System ◽  
E Coli ◽  
Self Defense

Phyllidiella nigra is an organism that is suspected to have secondary metabolites because their ability to develop its self defense system by camouflage and using chemical compounds derived from their nature diet as deterrent against their predators. The purpose of this study was to isolate symbiotic bacterial derived from P. nigra, extracted and followed by, the antibacterial assays against Escherichia coli and Bacillus megaterium as well as the anti-UV assay. The results showed that the five isolates tested had an antibacterial activity with the highest average inhibition zone against E. coli DSM 498 bacteria, isolate 1 (14.67 mm), isolate 5 (14 mm), and against B. Megaterium DSM 32T bacteria, isolate 3 (13.33 mm). The three isolates which had the highest inhibition zone and P. nigra extract were tested for anti-UV assay using a UV-Vis Spectrophotometer. The results obtained isolate 3 has absorption of UV-A with the UV absorbtion maximum at λ 340 nm and P. nigra extract has absorption on UV-B radiation with UV absorption maximum at λ 290 nm. Key words: Nudibranchia, Bacteria, Anti-bacteial, Anti-UV Phyllidiella nigra merupakan organisme yang diduga memiliki metabolit sekunder karena mampu mengembangkan sistem pertahanan dirinya dengan cara kamuflase dan menggunakan senyawa kimia sebagai racun yang didapat dari makanannya. Tujuan dari penelitian ini yaitu mendapatkan isolat bakteri yang bersimbiosis dengan P. nigra, mendapatkan ekstrak dari baktri simbion, dan menguji antibakteri dan anti-UV ekstrak etil aseta bakteri simbion dengan metode difusi agar terhadap bakteri Escherichia coli dan Bacillus megaterium. Hasil penelitian didapatkan kelima isolat yang diuji memiliki aktivitas antibakteri dengan rerata zona hambat tertinggi terhadap bakteri E. coli DSM 498 yaitu isolat 1 (14,67 mm), isolat 5 (14 mm), dan terhadap baktri B. megaterium DSM 32T yaitu isolat 3 (13,33 mm). Ketiga isolat yang memiliki zona hambat tertinggi dan ekstrak P. nigra diujikan anti-UV menggunakan alat UV-Vis Spektrofotometer. Hasil yang didapat isolat 3 memiliki serapan terhadap radiasi sinar UV-A dengan puncak tertinggi pada λ 340 nm dan ekstrak P. nigra memiliki serapan terhadap radiasi sinar UV-B dengan puncak tertinggi berada pada λ 290 nm. Kata kunci: Nudibranchia, Bacteria, Anti-bacteial, Anti-UV

scholarly journals Strain improvement of Escherichia coli K-12 for recombinant production of deuterated proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vinardas Kelpšas ◽  
Claes von Wachenfeldt
Keyword(s):  
Escherichia Coli ◽  
Structural Biology ◽  
Strain Improvement ◽  
Growth Media ◽  
Genomic Changes ◽  
E Coli ◽  
Recombinant Production ◽  
High Concentrations ◽  
Neutron Protein Crystallography ◽  
K 12

AbstractDeuterium isotope labelling is important for structural biology methods such as neutron protein crystallography, nuclear magnetic resonance and small angle neutron scattering studies of proteins. Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affect growth of cells. The generation time for Escherichia coli K-12 in deuterated medium is substantially increased compared to cells grown in hydrogenated (protiated) medium. By using a mutagenesis plasmid based approach we have isolated an E. coli strain derived from E. coli K-12 substrain MG1655 that show increased fitness in deuterium based growth media, without general adaptation to media components. By whole-genome sequencing we identified the genomic changes in the obtained strain and show that it can be used for recombinant production of perdeuterated proteins in amounts typically needed for structural biology studies.

scholarly journals Effects of Essential Oils on Escherichia coli Inactivation in Cheese as Described by Meta-Regression Modelling

Foods ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 716
Author(s):  
Beatriz Nunes Silva ◽  
Vasco Cadavez ◽  
José António Teixeira ◽  
Ursula Gonzales-Barron
Keyword(s):  
Escherichia Coli ◽  
Essential Oils ◽  
Meta Analysis ◽  
Inhibitory Effects ◽  
Food Preservatives ◽  
E Coli ◽  
Log Reduction ◽  
Bactericidal Properties ◽  
Study Characteristics ◽  
Meta Regression

The growing intention to replace chemical food preservatives with plant-based antimicrobials that pose lower risks to human health has produced numerous studies describing the bactericidal properties of biopreservatives such as essential oils (EOs) in a variety of products, including cheese. This study aimed to perform a meta-analysis of literature data that could summarize the inactivation of Escherichia coli in cheese achieved by added EOs; and compare its inhibitory effectiveness by application method, antimicrobial concentration, and specific antimicrobials. After a systematic review, 362 observations on log reduction data and study characteristics were extracted from 16 studies. The meta-regression model suggested that pathogenic E. coli is more resistant to EO action than the non-pathogenic type (p < 0.0001), although in both cases the higher the EO dose, the greater the mean log reduction achieved (p < 0.0001). It also showed that, among the factual application methods, EOs’ incorporation in films render a steadier inactivation (p < 0.0001) than when directly applied to milk or smeared on cheese surface. Lemon balm, sage, shallot, and anise EOs showed the best inhibitory outcomes against the pathogen. The model also revealed the inadequacy of inoculating antimicrobials in cheese purposely grated for performing challenge studies, as this non-realistic application overestimates (p < 0.0001) the inhibitory effects of EOs.

scholarly journals Poikilothermic Animals as a Previously Unrecognized Source of Fecal Indicator Bacteria in a Backwater Ecosystem of a Large River

2018 ◽  
Vol 84 (16) ◽  
Author(s):  
Christina Frick ◽  
Julia Vierheilig ◽  
Rita Linke ◽  
Domenico Savio ◽  
Horst Zornig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clostridium Perfringens ◽  
Fecal Indicator Bacteria ◽  
Indicator Bacteria ◽  
Primary Sources ◽  
Fecal Indicator ◽  
Content Type ◽  
E Coli ◽  
High Concentrations ◽  
Intestinal Enterococci

ABSTRACTQuantitative information regarding the presence ofEscherichia coli, intestinal enterococci, andClostridium perfringensin poikilotherms is notably scarce. Therefore, this study was designed to allow a systematic comparison of the occurrence of these standard fecal indicator bacteria (SFIB) in the excreta of wild homeothermic (ruminants, boars, carnivores, and birds) and poikilothermic (earthworms, gastropods, frogs, and fish) animals inhabiting an alluvial backwater area in eastern Austria. With the exception of earthworms, the average concentrations ofE. coliand enterococci in the excreta of poikilotherms were equal to or only slightly lower than those observed in homeothermic excreta and were 1 to 4 orders of magnitude higher than the levels observed in the ambient soils and sediments. Enterococci reached extraordinarily high concentrations in gastropods. Additional estimates of the daily excreted SFIB (E. coliand enterococcus) loads (DESL) further supported the importance of poikilotherms as potential pollution sources. The newly established DESL metric also allowed comparison to the standing stock of SFIB in the sediment and soil of the investigated area. In agreement with its biological characteristics, the highest concentrations ofC. perfringenswere observed in carnivores. In conclusion, the long-standing hypothesis that only humans and homeothermic animals are primary sources of SFIB is challenged by the results of this study. It may be necessary to extend the fecal indicator concept by additionally considering poikilotherms as potential important primary habitats of SFIB. Further studies in other geographical areas are needed to evaluate the general significance of our results. We hypothesize that the importance of poikilotherms as sources of SFIB is strongly correlated with the ambient temperature and would therefore be of increased significance in subtropical and tropical habitats and water resources.IMPORTANCEThe current fecal indicator concept is based on the assumption that the standard fecal indicator bacteria (SFIB)Escherichia coli, intestinal enterococci, andClostridium perfringensmultiply significantly only in the guts of humans and other homeothermic animals and can therefore indicate fecal pollution and the potential presence of pathogens from those groups. The findings of the present study showed that SFIB can also occur in high concentrations in poikilothermic animals (i.e., animals with body temperatures that vary with the ambient environmental temperature, such as fish, frogs, and snails) in an alluvial backwater area in a temperate region, indicating that a reconsideration of this long-standing indicator paradigm is needed. This study suggests that poikilotherms must be considered to be potential primary sources of SFIB in future studies.

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