scholarly journals Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli

2020 ◽  
Author(s):  
Bruno Motta Nascimento ◽  
Nikhil U. Nair
Keyword(s):  
Structural Information ◽  
List Type ◽  
Enzyme Complex ◽  
Batch Cultures ◽  
E Coli ◽  
Deep Well ◽  
Structural And Functional Properties ◽  
First Time ◽  
Enzymatic Complex

ABSTRACTPoly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in the Escherichia coli membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In E. coli, we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results elucidate provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.HIGHLIGHTSSuccessfully expressed active poly-γ-glutamate synthetase (PGS) in E. coli.Confirmed PGS localization at inner membrane of E. coli.Elucidated topology of PGS components in E. coli membrane.Culture and expression in microplates might allow future screening of a high number of samples.Faster production of poly-γ-glutamate in E. coli supernatant compared to B. subtilis.

scholarly journals Characterization of bacteriocin and chitinase producing bacterial isolates with broad-spectrum antimicrobial activities

2021 ◽  
Vol 3 (8) ◽  
Author(s):  
Muhammad Yasir ◽  
Basit Zeshan ◽  
Nur Hardy A. Daud ◽  
Izzah Shahid ◽  
Hafza Khalid
Keyword(s):  
Antimicrobial Activity ◽  
Inhibitory Activity ◽  
Antifungal Drugs ◽  
Proteinase K ◽  
Diffusion Method ◽  
List Type ◽  
Bacterial Isolates ◽  
E Coli ◽  
Inhibitory Potential

Abstract There is a need for more efficient and eco-friendly approaches to overcome increasing microbial infections. Bacteriocins and chitinases from Bacillus spp. can be powerful alternatives to conventional antibiotics and antifungal drugs, respectively. The purpose of this study was to assess the inhibitory potential of bacteriocins and chitinase enzymes against multiple resistant bacterial and fungal pathogens. Bacterial isolates were selected by growth on minimal salts medium and after that were morphologically and biochemically characterized. The physiochemical characterization of bacteriocins was carried out. The inhibitory potential of bacteriocins towards six pathogenic bacteria was determined by the well diffusion assay while chitinase activity towards three fungal strains was determined by the dual plate culture assay. Two bacterial strains (WW2P1 and WRE4P2), out of nine showed inhibition of K. pneumonia, P. aeruginosa, E. coli and MRSA while WW4P2 was positive against S. typhimurium and E. coli and WRE10P2 against P. aeruginosa, S. pneumoniae. Two bacterial isolates (WW3P1 and WRE10P2) were chosen for further study on the basis of their antifungal activities. Of these, WW3P1 isolate was more effective against A. fumigatus as well as A. niger. The proteinaceous nature of the bacteriocins was confirmed by treatment of the crude extract with proteinase K. It was found that the inhibitory activity of strain WW3P1 against E. coli was highest at 20 °C, and against S. pneumoniae it was at 20 °C and pH 10 after treatment with EDTA. Inhibition by strain the WRE10P2 against P. aeruginosa was highest at 20 °C and pH 14. It was found that EDTA increased the inhibitory activity of strain WW2P1 against P. aeruginosa, K. pneumoniae and E. coli by 2 ± 0.235, 3.5 ± 0.288, 2.5 ± 1.040 times, respectively, of strain WRE4P2 against P. aeruginosa and E. coli by 2.5 ± 0.763, 2.7 ± 0.5 times, respectively, and of strain WRE10P2 against S. pneumoniae by 3 ± 0.6236 times. The isolates have promising inhibitory activity, which should be further analyzed for the commercial production of antimicrobials. Article highlights The current study aimed to isolate the microbiome from wheat plant (Triticum aestivum L.), to screen for bacteriocin production and to assess its antimicrobial activity against human pathogens. Forty-one phenotypically different bacterial colonies were subjected to bacteriocin purification from which 25 colonies showed positive reactions. These 25 bacterial isolates were screened against six different human bacterial pathogens using the well diffusion method to check the antimicrobial activity. Out of nine bacterial isolates, WW3P1 and WRE10P2 were able to degrade the chitin and utilize it as their sole energy source. Strain WRE4P2 exhibited partial inactivation in its activity against MRSA after treatment with proteinase K.

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.

scholarly journals Characterization of ESBL-Producing Enterobacteria from Fruit Bats in an Unprotected Area of Makokou, Gabon

2020 ◽  
Vol 8 (1) ◽  
pp. 138 ◽  
Author(s):  
Pierre Philippe Mbehang Nguema ◽  
Richard Onanga ◽  
Guy Roger Ndong Atome ◽  
Jean Constant Obague Mbeang ◽  
Arsène Mabika Mabika ◽  
...  
Keyword(s):  
Resistant Bacteria ◽  
Fruit Bats ◽  
Antibiotic Resistant ◽  
Disk Diffusion Test ◽  
Beta Lactamases ◽  
E Coli ◽  
Terrestrial Mammals ◽  
Extended Spectrum Beta Lactamases ◽  
First Time

In Gabon, terrestrial mammals of protected areas have been identified as a possible source of antibiotic-resistant bacteria. Some studies on antibiotic resistance in bats have already been carried out. The main goal of our study was to detect extended-spectrum beta-lactamases (ESBLs) that are produced by enterobacteria from bats in the Makokou region in Gabon. Sixty-eight fecal samples were obtained from 68 bats caught in the forests located 1 km from the little town of Makokou. After culture and isolation, 66 Gram-negative bacterial colonies were obtained. The double-disk diffusion test confirmed the presence of ESBLs in six (20.69%) Escherichia coli isolates, four (13.79%) Klebsiella pneumoniae isolates, and one (3.45%) Enterobacter cloacae isolate. The analysis based on the nucleotide sequences of the ESBL resistance genes showed that all cefotaximase-Munichs (CTX-Ms) were CTX-M-15 and that all sulfhydryl variables (SHVs) were SHV-11: 41.67% CTX-M-15-producing E. coli, 16.67% CTX-M-15+SHV-11-producing E. coli, 8.33% CTX-M-15-producing K. pneumoniae, 25% CTX-M-15+SHV-11-producing K. pneumoniae, and 8.33% CTX-M-15-produced E. cloacae. This study shows for the first time the presence of multiresistant ESBL-producing enterobacteria in fruit bats in Makokou.

scholarly journals Isolation and Initial Characterization of a Bacterial Consortium Able To Mineralize Fluorobenzene

2002 ◽  
Vol 68 (1) ◽  
pp. 102-105 ◽  
Author(s):  
M.F. Carvalho ◽  
C.C.T. Alves ◽  
M.I.M. Ferreira ◽  
P. De Marco ◽  
P.M.L. Castro
Keyword(s):  
Sole Source ◽  
Bacterial Consortium ◽  
Aerobic Biodegradation ◽  
Fluoride Ions ◽  
16S Rrna Analysis ◽  
Batch Cultures ◽  
Selective Enrichment ◽  
Microbial Strains ◽  
First Time

ABSTRACT Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the β-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter−1 and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.

Phenotypic and genotypic quinolone resistance in Escherichia coli underlining GyrA83/87 mutations as a target to detect ciprofloxacin resistance

2020 ◽  
Vol 75 (9) ◽  
pp. 2466-2470
Author(s):  
Anaëlle Muggeo ◽  
Emmanuelle Cambau ◽  
Marlène Amara ◽  
Maïté Micaëlo ◽  
Béatrice Pangon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Bloodstream Infections ◽  
Blood Cultures ◽  
Quinolone Resistance ◽  
E Coli ◽  
Molecular Determinants ◽  
Ciprofloxacin Resistance ◽  
First Time

Abstract Background Quinolone resistance (QR) is one component of the MDR emerging in Escherichia coli and is of particular concern given the widespread use of fluoroquinolones. Objectives To characterize the QR phenotypes and genotypes in E. coli responsible for bloodstream infections and to propose molecular determinants that could be targeted to predict ciprofloxacin resistance. Methods E. coli isolates from blood cultures in three French hospitals were studied for quinolone MICs and characterization of genotypic QR determinants (QRg). Results Among 507 isolates tested for MICs, 148 (29.2%) were resistant to quinolones based on EUCAST breakpoints and 143 (28.2%) harboured at least one QRg. QRg were mainly mutations in the QRDR (138 isolates, 27.2%), with 55.8% of these isolates carrying at least three QRDR mutations. gyrA mutations predominated (92.8%) followed by parC (61.6%), parE (32.6%) and gyrB (1.4%) mutations. Only 4.7% of the isolates harboured a plasmid-mediated quinolone resistance (PMQR) gene: aac(6′)-Ib-cr (60.0%) or qnr (qnrS, qnrB) (32.0%). For the first time in France, we reported the qepA4 allele of the plasmid-encoded efflux pump QepA. Only five isolates carried PMQR without a QRDR mutation. The positive predictive value (PPV) for ciprofloxacin resistance was 100% for any QRg and 99.2% for gyrA mutations specifically. Conclusions QR observed in E. coli isolates involved in bloodstream infections is still mainly due to QRDR mutations, especially at codons GyrA83/87, which could be used as a molecular target to rapidly detect resistance.

Genetic Characterization of Escherichia coli Isolated from Cattle Carcasses and Feces in Mexico State

2015 ◽  
Vol 78 (4) ◽  
pp. 796-801 ◽  
Author(s):  
NYDIA E. REYES-RODRÍGUEZ ◽  
EDGARDO SORIANO-VARGAS ◽  
JEANNETTE BARBA-LEÓN ◽  
ARMANDO NAVARRO ◽  
MARTÍN TALAVERA-ROJAS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Characterization ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
E Coli ◽  
Uremic Syndrome ◽  
Human Illness ◽  
First Time ◽  
Mlva Typing

Meat of bovine origin is one of the major vehicles in the transmission of verotoxigenic Escherichia coli (VTEC) to human consumers. This pathogen can produce serious human illness, including bloody diarrhea and hemolytic uremic syndrome. The aim of the current study was to characterize E. coli isolates (mainly VTEC strains) belonging to several serotypes in samples from cattle carcasses and feces of three municipal slaughter plants from Mexico State. The genetic diversity and molecular relatedness among the isolates was evaluated with multiple-locus variable-number tandem repeat analysis (MLVA). To our knowledge, and with the exception of E. coli O157:H7, this is the first time that serotypes analyzed here have been subtyped by MLVA in Mexico. MLVA typing grouped the 37 strains from this study into 30 distinct genotypes, 26 of which were unique. These findings indicate that cattle carcasses and feces from slaughter plants in Mexico are a source of VTEC that are genetically diverse in terms of serotypes and virulence profiles. The presence of these pathogens in carcasses indicates the high probability of the spread of VTEC strains during slaughter and processing.

scholarly journals Molecular and Phenotypic Characterization of Escherichia coli O26:H8 among Diarrheagenic E. coli O26 Strains Isolated in Brazil

2013 ◽  
Vol 79 (22) ◽  
pp. 6847-6854 ◽  
Author(s):  
Roxane M. F. Piazza ◽  
Sabine Delannoy ◽  
Patrick Fach ◽  
Halha O. Saridakis ◽  
Margareth Z. Pedroso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ex Vivo ◽  
Effector Proteins ◽  
Phenotypic Characterization ◽  
Intestinal Biopsy ◽  
Content Type ◽  
E Coli ◽  
Flagellar Antigen ◽  
First Time

ABSTRACTEscherichia colistrains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenicE. coli(EPEC) and enterohemorrhagicE. coli(EHEC). Among the several genes related to type III secretion system-secreted effector proteins,espKwas found to be highly specific for EHEC O26:H11 and itsstx-negative derivative strains isolated in European countries.E. coliO26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lackstxgenes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive forfliCH11, and 11 werefliCH8positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive forespKand could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and itsstx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragmentsex vivoconfirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content ofstx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.

Cloning and Characterization of a Carotenoid Cleavage Dioxygenase from Artemisia Annua L

2011 ◽  
Vol 108 ◽  
pp. 274-281
Author(s):  
Shuo Qian Liu ◽  
Na Tian ◽  
Zhong Hua Liu ◽  
Jia Nan Huang ◽  
Juan Li
Keyword(s):  
Amino Acid ◽  
Artemisia Annua ◽  
Amino Acid Identity ◽  
Carotenoid Cleavage Dioxygenase ◽  
E Coli ◽  
Acid Protein ◽  
Rapid Amplification ◽  
First Time

In order to discover the formation mechanism of carotenoid derived aroma, which has been wildly used on protection of crop against insect attacks, the full-length cDNA of an Artemisia annua carotenoid cleavage dioxygenase (AaCCD1) was cloned by rapid amplification of cDNA ends. The function of AaCCD1 was characterized by expression of AaCCD1 in a strain of E. coli accumulating carotenoids and enzyme assay in vitro. The completed open read frame of AaCCD1 was 1629 bp and it encoded a 542-amino acid protein with a 77% amino acid identity to Arabidopsis thaliana CCD1, a predicted molecular mass of 61.04 kDa and a pI of 5.8. AaCCD1 efficiently cleaves carotenoids and regulate the formation of terpenoid compounds. This is the first time to report the cloning and identification of carotenoid cleavage dioxygenase from Atemisia annua, which will play a great role on understanding the regulation of volatile compounds.

scholarly journals Purification and characterization of GlcNAc-6-P 2-epimerase from Escherichia coli K92.

2007 ◽  
Vol 54 (2) ◽  
pp. 387-399 ◽  
Author(s):  
Miguel Angel Ferrero ◽  
Honorina Martínez-Blanco ◽  
Federico Felino Lopez-Velasco ◽  
Carlos Ezquerro-Sáenz ◽  
Nicolas Navasa ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Metabolic Flux ◽  
Maximum Activity ◽  
Logarithmic Phase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Inducible Protein ◽  
First Time

N-Acetylmannosamine (ManNAc) is the first committed intermediate in sialic acid metabolism. Thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. In prokaryotic organisms, UDP-N-acetylglucosamine (GlcNAc) 2-epimerase and GlcNAc-6-P 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc-6-P, respectively. We have purified for the first time native GlcNAc-6-P 2-epimerase from bacterial source to apparent homogeneity (1 200 fold) using Butyl-agarose, DEAE-FPLC and Mannose-6-P-agarose chromatography. By SDS/PAGE the pure enzyme showed a molecular mass of 38.4 +/- 0.2 kDa. The maximum activity was achieved at pH 7.8 and 37 degrees C. Under these conditions, the K(m) calculated for GlcNAc-6-P was 1.5 mM. The 2-epimerase activity was activated by Na(+) and inhibited by mannose-6-P but not mannose-1-P. Genetic analysis revealed high homology with bacterial isomerases. GlcNAc-6-P 2-epimerase from E. coli K92 is a ManNAc-inducible protein and is detected from the early logarithmic phase of growth. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes the biosynthesis of sialic acid, GlcNAc-6-P 2-epimerase plays a catabolic role. When E. coli grows using ManNAc as a carbon source, this enzyme converts the intracellular ManNAc-6-P generated into GlcNAc-6-P, diverting the metabolic flux of ManNAc to GlcNAc.

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