Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Expression System ◽  
High Yield ◽  
Gene Synthesis ◽  
Post Translational Modification ◽  
Iptg Induction ◽  
E Coli ◽  
Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

scholarly journals Dietary Inclusion of Colicin E1 Is Effective in Preventing Postweaning Diarrhea Caused by F18-Positive Escherichia coli in Pigs

2007 ◽  
Vol 51 (11) ◽  
pp. 3830-3835 ◽  
Author(s):  
S. A. Cutler ◽  
S. M. Lonergan ◽  
N. Cornick ◽  
A. K. Johnson ◽  
C. H. Stahl
Keyword(s):  
Escherichia Coli ◽  
Tumor Necrosis ◽  
E Coli ◽  
Colicin E1 ◽  
Young Pigs ◽  
Weaning Pigs ◽  
Conventional Antibiotics ◽  
Postweaning Diarrhea ◽  
Necrosis Factor

ABSTRACT With worldwide concern over the use of antibiotics in animal agriculture and their contribution to the spread of antibiotic resistance, alternatives to conventional antibiotics are needed. Previous research in our laboratories has shown that colicin E1 is effective against some Escherichia coli strains responsible for postweaning diarrhea (PWD) in vitro. In this study we examined the efficacy of the dietary inclusion of colicin E1 in preventing experimentally induced PWD caused by F18-positive enterotoxigenic E. coli in young pigs. Twenty-four weaned pigs (23 days of age), identified by genotyping to be susceptible to F18-positive E. coli infections, were individually housed and fed diets containing 0, 11, or 16.5 mg colicin E1/kg diet. Two days after the start of the trial, all animals were orally inoculated with 1 × 109 CFU of each of two F18-positive E. coli strains isolated from pigs with PWD. The dietary inclusion of colicin E1 decreased the incidence and severity of PWD caused by F18-positive enterotoxigenic E. coli and improved the growth performance of the piglets. Additionally, the reduced incidence of PWD due to dietary colicin E1, lowered the levels of expression of the genes for interleukin 1β and tumor necrosis factor beta in ileal tissues from these animals. The dietary inclusion of colicin E1 may be an effective alternative to conventional antibiotics in the diets of weaning pigs for the prevention of PWD caused by F18-positive enterotoxigenic E. coli.

scholarly journals Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evgeniya Schastnaya ◽  
Zrinka Raguz Nakic ◽  
Christoph H. Gruber ◽  
Peter Francis Doubleday ◽  
Aarti Krishnan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pentose Phosphate ◽  
Acetate Metabolism ◽  
Post Translational Modification ◽  
E Coli ◽  
In Vivo Experiments ◽  
Functional Relevance ◽  
Regulation Of Enzyme Activity

AbstractProtein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.

scholarly journals The yefM-yoeB Toxin-Antitoxin Systems of Escherichia coli and Streptococcus pneumoniae: Functional and Structural Correlation

2006 ◽  
Vol 189 (4) ◽  
pp. 1266-1278 ◽  
Author(s):  
Concha Nieto ◽  
Izhack Cherny ◽  
Seok Kooi Khoo ◽  
Mario García de Lacoba ◽  
Wai Ting Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Structural Features ◽  
E Coli ◽  
Modeled Structure ◽  
The Family ◽  
Bona Fide ◽  
K 12

ABSTRACT Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM Spn ) and toxin (YoeB Spn ) products. We showed that overproduction of YoeB Spn is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB Spn -mediated toxicity could be reversed by the cognate antitoxin, YefM Spn , but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

scholarly journals Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside

2020 ◽  
Vol 21 (16) ◽  
pp. 5752
Author(s):  
Wenju Shu ◽  
Hongchen Zheng ◽  
Xiaoping Fu ◽  
Jie Zhen ◽  
Ming Tan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Low Cost ◽  
Expression System ◽  
Steviol Glycosides ◽  
Fusion Partner ◽  
Uridine Diphosphate ◽  
Heterologous Proteins ◽  
E Coli ◽  
Novel Strategy

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

scholarly journals EcDBS1R4, an Antimicrobial Peptide Effective against Escherichia coli with In Vitro Fusogenic Ability

2020 ◽  
Vol 21 (23) ◽  
pp. 9104
Author(s):  
Marcin Makowski ◽  
Mário R. Felício ◽  
Isabel C. M. Fensterseifer ◽  
Octávio L. Franco ◽  
Nuno C. Santos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mechanism Of Action ◽  
Membrane Curvature ◽  
Permeability Barrier ◽  
Bacterial Cells ◽  
Gram Negative ◽  
E Coli ◽  
Spatial Reorganization ◽  
Conventional Antibiotics

Discovering antibiotic molecules able to hold the growing spread of antimicrobial resistance is one of the most urgent endeavors that public health must tackle. The case of Gram-negative bacterial pathogens is of special concern, as they are intrinsically resistant to many antibiotics, due to an outer membrane that constitutes an effective permeability barrier. Antimicrobial peptides (AMPs) have been pointed out as potential alternatives to conventional antibiotics, as their main mechanism of action is membrane disruption, arguably less prone to elicit resistance in pathogens. Here, we investigate the in vitro activity and selectivity of EcDBS1R4, a bioinspired AMP. To this purpose, we have used bacterial cells and model membrane systems mimicking both the inner and the outer membranes of Escherichia coli, and a variety of optical spectroscopic methodologies. EcDBS1R4 is effective against the Gram-negative E. coli, ineffective against the Gram-positive Staphylococcus aureus and noncytotoxic for human cells. EcDBS1R4 does not form stable pores in E. coli, as the peptide does not dissipate its membrane potential, suggesting an unusual mechanism of action. Interestingly, EcDBS1R4 promotes a hemi-fusion of vesicles mimicking the inner membrane of E. coli. This fusogenic ability of EcDBS1R4 requires the presence of phospholipids with a negative curvature and a negative charge. This finding suggests that EcDBS1R4 promotes a large lipid spatial reorganization able to reshape membrane curvature, with interesting biological implications herein discussed.

scholarly journals In vitro Antibacterial Activity of Mometasone Furoate, Fluticasone Propionate and Fluticasone Furoate Nasal Preparations Against Streptococcus pneumoniae, Hemophilus influenzae, Streptococcus viridans, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli

2010 ◽  
Vol 25 (1) ◽  
pp. 13-16
Author(s):  
Philip Lance A. Liu ◽  
Rose Lou Marie C. Agbay ◽  
Samantha S. Castañeda
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Streptococcus Pneumoniae ◽  
Fluticasone Propionate ◽  
Mometasone Furoate ◽  
Fluticasone Furoate ◽  
E Coli ◽  
Inhibition Zones

Objective: To test the antibacterial properties of three commercially available nasal corticosteroid preparations containing Mometasone Furoate (MF), Fluticasone Propionate (FP) and Fluticasone Furoate (FF) against S. pneumoniae, S. viridans, S. aureus, H. influenza, P. aeruginosa and E. coli. Methods:   Study Design:  Experimental in vitro study using the disc diffusion method.   Clinical isolates of Streptococcus pneumoniae, Hemophilus influenzae, Streptococcus viridans, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were inoculated on separate plates. 0.15 ml of nasal corticosteroid preparations containing MF, FP and FF were applied to blank paper discs, then placed on the plates, including an empty disc.  Following 24 and 48 hours of incubation, the inhibition zones were measured to the nearest mm from the point of abrupt inhibition of growth. Results: After 24 and 48 hours of incubation, S. pneumoniae, S. viridans, and S. aureus showed inhibition zones to all three preparations. S. aureus and S. viridans show the largest zones of inhibition at 24 and 48 hours respectively. H. influenza, P. aeruginosa and E. coli were negative. The inhibition zones of each bacteria were shown to be statistically different. The preparation containing FP had the largest zone of inhibition at 24 and 48 hours, although post hoc tests showed their difference was not significant. Conclusion: The present study demonstrates possible antimicrobial properties of commercially-available nasal corticosteroid preparations. However, it is unclear whether these can be attributed to the steroids, their excipients, or both. Further studies testing each component may offer better insights into their therapeutic use. Keywords: Mometasone Furoate, Fluticasone Propionate, Fluticasone Furoate, Antibacterial, Nasal Corticosteroids, Allergic Rhinitis, Acute Bacterial Rhinosinusitis

scholarly journals Improved production of the non-native cofactor F420 in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mihir V. Shah ◽  
Hadi Nazem-Bokaee ◽  
James Antoney ◽  
Suk Woo Kang ◽  
Colin J. Jackson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Carbon Sources ◽  
Fold Increase ◽  
High Yield ◽  
Antibiotic Biosynthesis ◽  
Bacterial Strains ◽  
E Coli ◽  
A Genome

AbstractThe deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

scholarly journals Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms

1993 ◽  
Vol 294 (1) ◽  
pp. 79-86 ◽  
Author(s):  
N F Brown ◽  
A Sen ◽  
D A Soltis ◽  
B Jones ◽  
D W Foster ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Expression System ◽  
Active Form ◽  
Carnitine Palmitoyltransferase ◽  
Coding Region ◽  
E Coli ◽  
Catalytically Active ◽  
Carnitine Palmitoyltransferase Ii

cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5′ end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3′ to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II.

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

2020 ◽  
Vol 24 (19) ◽  
pp. 2272-2282
Author(s):  
Vu Ngoc Toan ◽  
Nguyen Minh Tri ◽  
Nguyen Dinh Thanh
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Selenium Dioxide ◽  
Antibacterial And Antifungal Activity ◽  
E Coli ◽  
Antibacterial And Antifungal Activities ◽  
Alkyl Ethers ◽  
Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

scholarly journals 1671. Diagnosis and management of Escherichia coli bacteriuria at a Veterans Affairs hospital

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S821-S821
Author(s):  
Niyati H Shah ◽  
Brooke K Decker ◽  
Brooke K Decker ◽  
Gaetan Sgro ◽  
Monique Y Boudreaux-Kelly ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Treatment ◽  
High Yield ◽  
Comorbid Conditions ◽  
Provider Education ◽  
Transmitted Infection ◽  
Physician Practices ◽  
E Coli ◽  
Diagnosis And Management ◽  
Tract Infections

Abstract Background The IDSA recommends against screening for and treating ASB in all patients except for those pregnant or undergoing urologic procedures. Nevertheless, antibiotic treatment of ASB is widespread. We conducted a retrospective analysis of physician practices in diagnosis and management of Escherichia coli (E. coli) ASB in a male Veteran population, and compared outcomes in ASB patients treated or not treated with antibiotics. Methods Patients with an E. coli positive urine culture during an ED visit or inpatient admission from 01/2017 to 12/2017 were screened. Patients admitted to the intensive care unit or diagnosed with a sexually transmitted infection, pyelonephritis, prostatitis, or epididymitis/orchitis were excluded. A total of 163 patients were included. Demographics, clinical comorbidities and severity of illness, and outcomes were compared in ASB patients managed with or without antibiotics. ANOVA and Chi-square or Fisher’s exact tests were utilized for comparing measurements. Results ASB was present in 92/163 patients. The majority (74%) of these patients were given antibiotics. Regardless of qSOFA score or alternate infection, there were no significant differences in outcomes between ASB patients treated or not treated with antibiotics: 3-month mortality (15% vs 21%; p = 0.53), emergence of newly resistant bacterial pathogens (7% vs 13%; p = 0.43), recurrent urinary tract infections (61% vs 50%; p = 0.72), clearance of urinary pathogens (75% vs 58%; p = 0.45), length of hospital stay (7 vs 6 days, p = 0.67). Factors that were predictive of physician treatment of ASB included patient comorbid conditions such as benign prostatic hyperplasia, pyuria, and the absence of hematuria. The incidence of adverse events with antibiotic treatment of ASB was low. Conclusion The rate of antibiotic treatment of E. coli ASB in male veterans is high. Outcomes do not differ among ASB patients managed with or without antibiotics. Future studies examining outcomes in patients prescribed antibiotics for multiple episodes of ASB may yield differences, particularly in emergence of resistant pathogens. Focusing on patients with comorbid conditions who are not critically ill would be a high yield target for provider education to reduce ASB treatment. Disclosures All Authors: No reported disclosures

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