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.

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 Escherichia coli O157:H7 SURVIVAL IN TRADITIONAL AND LOW LACTOSE YOGURT DURING FERMENTATION AND COOLING PERIODS

2017 ◽  
Vol 18 (0) ◽  
Author(s):  
Camila Sampaio Cutrim ◽  
Raphael Ferreira de Barros ◽  
Robson Maia Franco ◽  
Marco Antonio Sloboda Cortez
Keyword(s):  
Escherichia Coli ◽  
The Other ◽  
Lactose Hydrolysis ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Gas Formation ◽  
Whole Milk ◽  
Milk Contamination ◽  
Different Types ◽  
Fermentation Period

Abstract The purpose of this study was to evaluate the behavior of E. coli O157:H7 during lactose hydrolysis and fermentation of traditional and low lactose yogurt. It also aimed to verify E. coli O157:H7 survival after 12 h of storage at 4 ºC ±1 ºC. Two different types of yogurts were prepared, two with whole milk and two with pre-hydrolyzed whole milk; in both groups one yogurt was inoculated with E. coli O157:H7 and the other one was not inoculated. The survival of E. coli and pH of yogurt were determined during fermentation and after 12-h refrigeration. The results showed that E. coli O157:H7 was able to grow during the fermentation period (from 4.34 log CFU.mL-1 to 6.13 log CFU.mL-1 in traditional yogurt and 4.34 log CFU.mL-1 to 6.16 log CFU.mL-1 in low lactose yogurt). The samples with E. coli O157:H7 showed gas formation and syneresis. Thus, E. coli O157:H7 was able to survive and grow during fermentation of traditional and low lactose yogurts affecting the manufacture technology. Moreover, milk contamination by E. coli before LAB addition reduces the growth of L. bulgaricus and S. thermophilus especially when associated with reduction of lactose content.

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  Faecal shedding of verotoxigenic Escherichia coli in cattle in the Czech Republic

2011 ◽  
Vol 56 (No. 4) ◽  
pp. 149-155 ◽  
Author(s):  
P. Alexa ◽  
L. Konstantinova ◽  
Z. Sramkova-Zajacova
Keyword(s):  
Escherichia Coli ◽  
Czech Republic ◽  
Dairy Cattle ◽  
Age Groups ◽  
Immunomagnetic Separation ◽  
The Other ◽  
Somatic Antigen ◽  
The Czech Republic ◽  
E Coli ◽  
One Year

A survey to estimate the prevalence of verotoxigenic E. coli (VTEC) or enterohaemorrhagic E. coli (EHEC) in rectal swabs from healthy dairy cattle aged three weeks, three months and one year was conducted in three herds from the Czech Republic. Screening for the presence of the stx1, stx2 and eaeA genes in faecal swab cultures was performed by PCR, and in positive samples, isolated colonies were examined. Immunomagnetic separation was used for the isolation of the VTEC serogroup O157 from samples. VTEC were detected in animals from all three herds under study. In the group of 3-week-old calves, VTEC were only detected in samples collected in the summer months. However, in the other age-groups, VTEC were detected in both the summer and winter months. EHEC shedding was observed in 30 to 100% of the total samples collected from cattle aged three months and one year in the summer months, and in 30 to 60% of samples taken in the winter months. EHEC strains of serogroup O157 were detected in two herds. The range of verotoxins shed by VTEC isolates of serogroup O157 differed between herds. Besides serogroup O157, additional EHEC belonging to the antigen groups O26, O103, O128 and O153 have been identified, and in some of them, no somatic antigen was detected.

scholarly journals Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli, Including Sequence Type 131 (ST131), from Retail Chicken Breasts in the United States in 2013

2017 ◽  
Vol 83 (6) ◽  
Author(s):  
James R. Johnson ◽  
Stephen B. Porter ◽  
Brian Johnston ◽  
Paul Thuras ◽  
Sarah Clock ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Virulence Genes ◽  
Meat Products ◽  
The United States ◽  
Sequence Type ◽  
The Other ◽  
Antibiotic Resistant ◽  
Content Type ◽  
E Coli

ABSTRACT Chicken meat products are hypothesized to be vehicles for transmitting antimicrobial-resistant and extraintestinal pathogenic Escherichia coli (ExPEC) to consumers. To reassess this hypothesis in the current era of heightened concerns about antimicrobial use in food animals, we analyzed 175 chicken-source E. coli isolates from a 2013 Consumer Reports national survey. Isolates were screened by PCR for ExPEC-defining virulence genes. The 25 ExPEC isolates (12% of 175) and a 2:1 randomly selected set of 50 non-ExPEC isolates were assessed for their phylogenetic/clonal backgrounds and virulence genotypes for comparison with their resistance profiles and the claims on the retail packaging label (“organic,” “no antibiotics,” and “natural”). Compared with the findings for non-ExPEC isolates, the group of ExPEC isolates had a higher prevalence of phylogroup B2 isolates (44% versus 4%; P < 0.001) and a lower prevalence of phylogroup A isolates (4% versus 30%; P = 0.001), a higher prevalence of multiple individual virulence genes, higher virulence scores (median, 11 [range, 4 to 16] versus 8 [range, 1 to 14]; P = 0.001), and higher resistance scores (median, 4 [range, 0 to 8] versus 3 [range, 0 to 10]; P < 0.001). All five isolates of sequence type 131 (ST131) were ExPEC (P = 0.003), were as extensively resistant as the other isolates tested, and had higher virulence scores than the other isolates tested (median, 12 [range, 11 to 13] versus 8 [range, 1 to 16]; P = 0.005). Organic labeling predicted lower resistance scores (median, 2 [range, 0 to 3] versus 4 [range, 0 to 10]; P = 0.008) but no difference in ExPEC status or virulence scores. These findings document a persisting reservoir of extensively antimicrobial-resistant ExPEC isolates, including isolates from ST131, in retail chicken products in the United States, suggesting a potential public health threat. IMPORTANCE We found that among Escherichia coli isolates from retail chicken meat products purchased across the United States in 2013 (many of these isolates being extensively antibiotic resistant), a minority had genetic profiles suggesting an ability to cause extraintestinal infections in humans, such as urinary tract infection, implying a risk of foodborne disease. Although isolates from products labeled “organic” were less extensively antibiotic resistant than other isolates, they did not appear to be less virulent. These findings suggest that retail chicken products in the United States, even if they are labeled “organic,” pose a potential health threat to consumers because they are contaminated with extensively antibiotic-resistant and, presumably, virulent E. coli isolates.

scholarly journals NikAB- or NikB-Dependent Intracellular Recombination between Tandemly Repeated oriT Sequences of Plasmid R64 in Plasmid or Single-Stranded Phage Vectors

2003 ◽  
Vol 185 (13) ◽  
pp. 3871-3877 ◽  
Author(s):  
Nobuhisa Furuya ◽  
Teruya Komano
Keyword(s):  
Escherichia Coli ◽  
Dna Transfer ◽  
The Other ◽  
E Coli ◽  
Core Sequence ◽  
Bacterial Plasmid ◽  
Plasmid R64 ◽  
Dna Processing ◽  
Origin Of Transfer

ABSTRACT The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.

Correlation of cefpodoxime susceptibility with cephalothin and cefuroxime for urinary tract isolates

2014 ◽  
Vol 63 (2) ◽  
pp. 218-221 ◽  
Author(s):  
David A. Bookstaver ◽  
Christopher M. Bland ◽  
Mitchell W. Woodberry ◽  
Karon B. Mansell
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Error Rate ◽  
Surrogate Marker ◽  
Error Rates ◽  
The Other ◽  
Colony Count ◽  
Major Error ◽  
E Coli ◽  
Microscan System

This study attempted to determine whether cefuroxime was superior to cephalothin as a surrogate marker for cefpodoxime among urinary tract isolates. The MicroScan system (Siemens) was used to determine susceptibility for cephalothin and cefuroxime on consecutive cultures with a colony count of ≥50 000 organisms. Simultaneously, an Etest (bioMérieux) for cefpodoxime was conducted. The cefpodoxime interpretation was compared to that of the other two agents, and the categorical agreement was calculated, defined as the percentage of identical susceptibility interpretations. Cefuroxime (83 %) had a significantly higher categorical agreement than cephalothin (63 %) among 300 isolates (P<0.01). The major error rate was 16 % for cephalothin and 3 % for cefuroxime. The very major error rate was 7 % for cephalothin and 14 % for cefuroxime among the 14 cefpodoxime-resistant isolates. For Escherichia coli, the major error rates were 15 % and 1 % for cephalothin and cefuroxime, respectively. Very major error rates were 9 % for both agents. Cefuroxime was a better predictor of cefpodoxime susceptibility than cephalothin, and appears to be the preferred surrogate agent for the MicroScan system, particularly for E. coli.

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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