Escherichia Coli Strain Research Articles

Production of Antimicrobial Bioactive Compounds from Endophytic Fungi Against Escherichia coli Strain NRC1 Isolated from the African Catfish

Production of Antimicrobial Bioactive Compounds from Endophytic Fungi Against Escherichia coli Strain NRC1 Isolated from the African Catfish

Genomic Dissection of an Enteroaggregative Escherichia coli Strain Isolated from Bacteremia Reveals Insights into Its Hybrid Pathogenic Potential.

Escherichia coli is a frequent pathogen isolated from bloodstream infections. This study aimed to characterize the genetic features of EC092, an E. coli strain isolated from bacteremia that harbors enteroaggregative E. coli (EAEC) genetic markers, indicating its hybrid pathogenic potential. Whole-genome sequencing showed that EC092 belongs to phylogroup B1, ST278, and serotype O165:H4. Genes encoding virulence factors such as fimbriae, toxins, iron-uptake systems, autotransporter proteins (Pet, Pic, Sat, and SepA), and secretion systems were detected, as well as EAEC virulence genes (aggR, aatA, aaiC, and aap). EC092 was found to be closely related to the other EAEC prototype strains and highly similar in terms of virulence to three EAEC strains isolated from diarrhea. The genomic neighborhood of pet, pic, sat, sepA, and the EAEC virulence genes of EC092 and its three genetically related fecal EAEC strains showed an identical genomic organization and nucleotide sequences. Also, EC092 produced and secreted Pet, Pic, Sat, and SepA in the culture supernatant and resisted the bactericidal activity of normal human serum. Our results demonstrate that the strain EC092, isolated from bacteremia, is a hybrid pathogenic extraintestinal E. coli (ExPEC)/EAEC with virulence features that could mediate both extraintestinal and intestinal infections.

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

A holistic approach for process intensification of nicotinamide mononucleotide production via high cell density cultivation under exponential feeding strategy

Nicotinamide mononucleotide (NMN) subsists in all living organisms and has drawn tremendous attention as a nutraceutical and pharmaceutical product for several diseases such as Alzheimer’s, cancer, aging, and vascular dysfunction. Here, NMN was produced intracellularly in a high cell density bioreactor using an engineered Escherichiacoli strain via exponential feeding of co-substrates. Fed-batch culture via exponential feeding of co-substrate (glucose) and continuous feeding of substrate (nicotinamide) were performed using different cumulative nicotinamide concentrations. The highest concentration of 19.3 g/L NMN with a dry cell weight of 117 g/L was acquired from a cumulative nicotinamide concentration of 7.2 g/L with a conversion of 98 % from nicotinamide in 28 h. Further, liquid chromatography-mass spectrometry analysis validated the NMN production. This approach will be beneficial in achieving simultaneously low cost and ensuring high quality and quantity of NMN production.

Antibacterial Activity of Seagrass Extract Against Pathogen Bacteria of Escherichia coli Strain Multi Drug Resistance (MDR)

Seagrass is a flowering plant that can adapt to the sea which has the potential to be used as an antibacterial. The purpose of this study to analyze seagrass extract as an antibacterial Escherichia coli strain Multi Drug Resistance (MDR). This research was conducted in May-July 2023. Seagrass samples came from the waters of Ketapang Beach, Pesawaran Regency, Lampung Province. The seagrass samples were meserated into three different solvents, namely methanol, n-hexane and ethyl acetate. Antibacterial test was carried out with scratch paper dripped with seagrass extract against E.coli bacteria colonies. The results obtained from the collection of seagrass samples found at Ketapang Beach contained four species, namely Enhalus acoroides, Cymodocea rotundata, Thalassia hemprichii and Halodule pinifolia. The highest yield of seagrass extract was found in E.acoroides with 14% methanol. The zone diameters of seagrass extracts against E.coli strain MDR were higher in seagrass extract H.pinifolia with n-hexana of 6,13 ± 3,27 mm. The conclusion of this research is that H.pinifolia seagrass extract using n-hexana solvent has potential as an antibacterial of E.coli strains Multi Drug Resistance (MDR).

Impact of Elevated Levels of Dissolved CO2 on Performance and Proteome Response of an Industrial 2'-Fucosyllactose Producing Escherichia coli Strain.

Large-scale microbial industrial fermentations have significantly higher absolute pressure and dissolved CO2 concentrations than otherwise comparable laboratory-scale processes. Yet the effect of increased dissolved CO2 (dCO2) levels is rarely addressed in the literature. In the current work, we have investigated the impact of industrial levels of dCO2 (measured as the partial pressure of CO2, pCO2) in an Escherichia coli-based fed-batch process producing the human milk oligosaccharide 2′-fucosyllactose (2′-FL). The study evaluated the effect of high pCO2 levels in both carbon-limited (C-limited) and carbon/nitrogen-limited (C/N-limited) fed-batch processes. High-cell density cultures were sparged with 10%, 15%, 20%, or 30% CO2 in the inlet air to cover and exceed the levels observed in the industrial scale process. While the 10% enrichment was estimated to achieve similar or higher pCO2 levels as the large-scale fermentation it did not impact the performance of the process. The product and biomass yields started being affected above 15% CO2 enrichment, while 30% impaired the cultures completely. Quantitative proteomics analysis of the C-limited process showed that 15% CO2 enrichment affected the culture on the protein level, but to a much smaller degree than expected. A more significant impact was seen in the dual C/N limited process, which likely stemmed from the effect pCO2 had on nitrogen availability. The results demonstrated that microbial cultures can be seriously affected by elevated CO2 levels, albeit at higher levels than expected.

High diversity in the regulatory region of Shiga toxin encoding bacteriophages

BackgroundEnterohemorrhagic Escherichia coli (EHEC) is an emerging health challenge worldwide and outbreaks caused by this pathogen poses a serious public health concern. Shiga toxin (Stx) is the major virulence factor of EHEC, and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor which maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains.ResultsIn this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and three already described Erus, ten novel Erus (Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 are widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type.ConclusionThe classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain.

Innovative Perspectives on Biofilm Interactions in Poultry Drinking Water Systems and Veterinary Antibiotics Used Worldwide

Prudent use of antibiotics in livestock is widely considered to be important to prevent antibiotic resistance. This study aimed to evaluate the interactions between biofilms and veterinary antibiotics in therapeutic concentrations administrated via drinking water through a standardized experimental setup. In this context, two biofilms formed by pseudomonads (Pseudomonas (P.) aeruginosa or P. fluorescens) and a susceptible Escherichia (E.) coli strain were developed in a nutrient-poor medium on the inner surface of polyvinyl chloride pipe pieces. Subsequently, developing biofilms were exposed to sulfadiazine/trimethoprim (SDZ/TMP) or tylosin A (TYL A) in dosages recommended for application in drinking water for 5 or 7 days, respectively. Various interactions were detected between biofilms and antibiotics. Microbiological examinations revealed that only TYL A reduced the number of bacteria on the surface of the pipes. Additionally, susceptible E. coli survived both antibiotic treatments without observable changes in the minimum inhibitory concentration to 13 relevant antibiotics. Furthermore, as demonstrated by HPLC-UV, the dynamics of SDZ/TMP and TYL A in liquid media differed between the biofilms of both pseudomonads over the exposure period. We conclude that this approach represents an innovative step toward the effective evaluation of safe veterinary antibiotic use.

Transcriptional analysis reveals specific niche factors and response to environmental stresses of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents

BackgroundEnterohemorrhagic Escherichia coli (EHEC) are responsible for severe diseases in humans, and the ruminant digestive tract is considered as their main reservoir. Their excretion in bovine feces leads to the contamination of foods and the environment. Thus, providing knowledge of processes used by EHEC to survive and/or develop all along the bovine gut represents a major step for strategies implementation.ResultsWe compared the transcriptome of the reference EHEC strain EDL933 incubated in vitro in triplicate samples in sterile bovine rumen, small intestine and rectum contents with that of the strain grown in an artificial medium using RNA-sequencing (RNA-seq), focusing on genes involved in stress response, adhesion systems including the LEE, iron uptake, motility and chemotaxis. We also compared expression of these genes in one digestive content relative to the others. In addition, we quantified short chain fatty acids and metal ions present in the three digestive contents. RNA-seq data first highlighted response of EHEC EDL933 to unfavorable physiochemical conditions encountered during its transit through the bovine gut lumen. Seventy-eight genes involved in stress responses including drug export, oxidative stress and acid resistance/pH adaptation were over-expressed in all the digestive contents compared with artificial medium. However, differences in stress fitness gene expression were observed depending on the digestive segment, suggesting that these differences were due to distinct physiochemical conditions in the bovine digestive contents. EHEC activated genes encoding three toxin/antitoxin systems in rumen content and many gene clusters involved in motility and chemotaxis in rectum contents. Genes involved in iron uptake and utilization were mostly down-regulated in all digestive contents compared with artificial medium, but feo genes were over-expressed in rumen and small intestine compared with rectum. The five LEE operons were more expressed in rectum than in rumen content, and LEE1 was also more expressed in rectum than in small intestine content.ConclusionOur results highlight various strategies that EHEC may implement to survive in the gastrointestinal environment of cattle. These data could also help defining new targets to limit EHEC O157:H7 carriage and shedding by cattle.

Efficacy of Omadacycline against Escherichia coli in a Mouse Urinary Tract Infection Model.

In a mouse urinary tract infection model, omadacycline (OMC) was comparable to gentamicin and better than ciprofloxacin (CIP) against a tetracycline-susceptible (TET-S), CIP-resistant (CIP-R) Escherichia coli strain. Gentamicin showed better efficacy than OMC against a TET-R, CIP-R E. coli strain, and OMC again showed better efficacy than CIP against this strain. OMC may warrant further study as a potential option for urinary tract infection treatment against CIP-R E. coli strains.

Genomic characterisation of a multidrug-resistant Escherichia coli strain carrying the mcr-1 gene recovered from a paediatric patient in China

ObjectivesThe global spread of carbapenem-resistant Enterobacterales is a leading public-health threat. Lack of effective treatment has resulted in use of colistin as a last-resort therapeutic option for multidrug-resistant (MDR) bacterial infections. Here we report the complete genome sequence of a MDR Escherichia coli strain carrying a plasmid-mediated colistin resistance gene mcr-1 recovered from a Chinese paediatric patient. MethodsWhole-genome sequencing of E. coli strain 1506 was performed using both Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. De novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler. In silico multilocus sequence typing (MLST), antimicrobial resistance genes (ARGs) and plasmid replicons were identified from the genome sequence. Core genome multilocus sequence typing (cgMLST) analysis between E. coli 1506 and all of the ST48 E. coli strains retrieved from the NCBI GenBank database was performed using BacWGSTdb 2.0 server. ResultsThe complete genome sequence of E. coli 1506 consists of six contigs comprising 4 849 058 bp, including one chromosome and five plasmids, and was assigned to ST48. Fourteen ARGs were identified, including mcr-1 located on a 33 309-bp IncX4 plasmid. The closest relative of E. coli 1506 was another isolate originating from livestock in Australia, which differed by 614 cgMLST alleles. ConclusionOur study reports the genome sequence of a MDR E. coli carrying mcr-1 isolated from a Chinese paediatric patient. These data may help to understand the antimicrobial resistance mechanisms and genomic features and highlight the growing threat of antimicrobial resistance in children.

Analysis of bacterial etiology of the outpatients with infectious diarrhea in the Children′s Hospital Capital Institute of Pediatrics

Objective To analyze the bacterial etiology and drug resistance of pathogens from the outpatients with acute infectious diarrhea in the Children′s Hospital Capital Institute of Pediatrics. Methods The children with acute infectious diarrhea in the outpatient department of our hospital were randomly enrolled in the period of August 2017 to July 2018. Enteric pathogenic bacteria were cultured and isolated from the fresh stool specimens. The serum of Salmonella spp were identified while five types of diarrheagenic Escherichia coli were detected by PCR method. Antibiotic susceptibility of Salmonella spp and diarrheagenic Escherichia coli were tested by Kirby-Bauer disk diffusion method.Chi-square or Fisher exact test was done for statistical analysis. Results Of the 356 stool specimens, 103 strains of 6 pathogenic bacterial species were detected. The positive rate was 27.8% (99/356), and 2 strains were detected in four stool samples. The isolation rate of Salmonella spp, which was the highest in this study, was 10.7% (38/356). The isolation rate of Staphylococcus aureus, diarrheagenic Escherichia coli and Pseudomonas aeruginosa were 8.4% (30/356), 5.6% (20/356) and 3.7% (13/356), respectively. No Shigella spp strain was isolated. Salmonella enteritidis and Salmonella typhimurium were the dominant serotype for 38 Salmonella spp stains with the isolation rate of 63.2% (24/38). Each 10 Enteropathogenic Escherichia coli and 10 Enteroaggregative Escherichia coli isolates were detected among the diarrheagenic Escherichia coli. Enterohemorrhagic Escherichia coli, Enteroinvasive Escherichia coli and Enterotoxigenic Escherichia colistrain was not found in this study. The resistant rates of Salmonella spp to ampicillin and ceftriaxone were 52.6% (20/38) and 13.2% (5/38), while the diarrheagenic Escherichia coli were 11/20 and 1/20, respectively. Three diarrheagenic Escherichia coli isolates produced extended spectrum beta-lactamase. Conclusions Salmonella spp was the major bacterial pathogen among the outpatients with infectious diarrhea in the hospital. The study indicated that the infection ratios of Staphylococcus aureus, diarrheagenic Escherichia coli and Pseudomonas aeruginosa increased. Shigella spp was no long the main pathogenic agent. The resistant rates of Salmonella spp and diarrheagenic Escherichia coli to ampicillin were high, but low resistant rates to ceftriaxone were found among these two kinds of bacteria. The third-generation cephalosporins should be the first choice for the treatment of childhood bacterial diarrhea. Key words: Diarrhea; Salmonella; Escherichia coli; Drug resistance,Bacterial; Child

Utilization of Emulsiflex Prepared Escherichia coli Strain EC4 Oxyrase for Improved Cultivation of Anaerobic Bacteria by using Hungate Technique and Determination of its Kinetic Parameters

Utilization of Emulsiflex Prepared Escherichia coli Strain EC4 Oxyrase for Improved Cultivation of Anaerobic Bacteria by using Hungate Technique and Determination of its Kinetic Parameters

Complete Sequence of a F33:A-:B- Conjugative Plasmid Carrying the oqxAB, fosA3, and blaCTX-M-55 Elements from a Foodborne Escherichia coli Strain.

This study reports the complete sequence of pE80, a conjugative IncFII plasmid recovered from an Escherichia coli strain isolated from chicken meat. This plasmid harbors multiple resistance determinants including oqxAB, fosA3, blaCTX-M-55, and blaTEM-1, and is a close variant of the recently reported p42-2 element, which was recovered from E. coli of veterinary source. Recovery of pE80 constitutes evidence that evolution or genetic re-arrangement of IncFII type plasmids residing in animal-borne organisms is an active event, which involves acquisition and integration of foreign resistance elements into the plasmid backbone. Dissemination of these plasmids may further compromise the effectiveness of current antimicrobial strategies.

An invitro model to study interactions between Escherichia coli and lactic acid bacterial inoculants for silage in rumen fluid.

Previous studies have shown that silages treated with lactic acid bacteria (LAB) inoculants enhance ruminants' performance. The objective of the current experiments was to develop an invitro model to study interactions between LAB silage inoculants and inoculated silages and Escherichia coli (EC) in rumen fluid (RF). Our hypothesis was that some inoculants inhibit EC in RF. For that purpose buffered RF was incubated under anaerobic conditions at 39°C with commercial strains of LAB silage inoculants or with laboratory corn and wheat silages treated with these LAB, an EC strain and with various ruminant feed ingredients. The EC strain was originally isolated from cattle manure and tagged with a plasmid expressing the green fluorescence protein and kanamycin and streptomycin resistance. Results indicate that the LAB or the treated silages did not suppress EC numbers in the RF. When the pH of the RF decreased below 5·0 the EC disappeared. We conclude that both LAB inoculants for silage and EC survived in RF for several days; however, the inoculants and silages treated with such inoculants did not inhibit EC in RF invitro. Forage crops, silage and hay are initial stages of the food chain for humans. Cattle harbours and sheds enterobacteria regularly, some strains of which are pathogens. These can contaminate forage crops through field fertilization with cattle manure. The objective of this study was to develop an invitro model to test whether lactic acid bacteria, which are used in silage inoculants, alone or in treated silages can inhibit Escherichia coli in rumen fluid. This study presents safety aspects and it is also part of a broad research effort aimed at finding out how LAB silage inoculants and inoculated silages enhance ruminant performance or exert probiotic effects in ruminants.

Laboratory Dilutions of Thioridaxine with Potential to Enhance Antibiotic Sensitivity in a Multidrug Resistant Escherichia Coli Uropathogen

This research effort seeks to use doses of thioridaxine to enhance antibiotic sensitivity in a MDR Escherichia coli strain. Five axenic (pure) strains of Escherichia coli coded EC1 to EC5 were obtained from five infected midstream urine samples among several other urine samples inoculated on sterile Cystine Lactose Electrolyte Deficient (CLED) agar with appropriate labeling in the Microbiology and Biotechnology Laboratory of Western Delta University, Oghara, Nigeria and stocked on sterile Nutrient agar slants at 4oC in a refrigerator. Slant cultures were sub-cultured aseptically on fresh sterile CLED agar plates and incubated aerobically at 37oC for 24hrs to confirm Escherichia coli strains. Gram staining, indole production, methyl red test, voges praskaeur and citrate utilization tests were done on the resulting colonies to further confirm the strains as E.coli. Antibiotic susceptibility test was done by agar disc diffusion method on all confirmed strains on sterile Mueller- Hinton agar plates before and after treatment with laboratory dilutions of thioridaxine. Only E.coli strain 2 (EC2) was multidrug resistant as it resisted 4(44.4%) of the antibiotics used which were cefuroxime, nalidixic acid, augmentin and tetracycline. Other strains resisted 1-2 antibiotics. The highest (15.6±20.6mm) and least (2.0±8.1mm) zones of inhibition by all five strains were recorded for ofloxacin and cefuroxime respectively. Whereas all five uropathogen strains resisted augmentin, they were sensitive to ciprofloxacin, ofloxacin (both being fluoroquinolones), gentamicin, chloramphenicol and nitrofurantoin. After treatment with 2000-2240ug/ml laboratory dilutions of thioridaxine, ≤50.0% loss of resistance was recorded for 2040ug/ml, 2160ug/ml and 2240ug/ml dilutions. Thioridaxine dilution of 2040ug/ml induced 250% and 90% resistance losses of EC2 to ciprofloxacin and nitrofurantoin respectively with an overall mean±S.E loss of 68.0±24.4%. Resistance losses of 112.5%, 130.0% and 100.0% to ciprofloxacin, nitrofurantoin and chloramphenicol respectively were recorded after 2160ug/ml treatment and 68.5±16.3% overall loss of resistance. Thioridaxine dilutions of 2240ug/ml induced 55.6±25.0% overall loss of resistance with a corresponding 50.0%, 50.0%, 58.1%, 70.0% and 50.0% resistance losses with gentamicin, ciprofloxacin, ofloxacin, nitrofurantoin and chloramphenicol respectively. Less than 50% resistance losses were recorded for 2000, 2080, 2120 and 2200ug/ml dilutions. Minimum inhibitory concentration of chloramphenicol was lowered by 2080ug/ml, 2160ug/ml and 2240ug/ml dilutions by two-fold (15ug), two-fold (15ug) and four-fold (7.5ug) respectively. The medical/ chemotherapeutic implications of these findings are discussed.

Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270bp from LAP and 411bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.

Self-processing of a barley subtilase expressed in E. coli

The barley protease BAJ93208 belongs to the subtilase family of serine proteases. We have expressed BAJ93208 in the cytoplasm of the Escherichiacoli strain SHuffle C3030 using a rhamnose-inducible promoter. The expression construct included a (His)6-tag at the N-terminus and a strep-tag at the C-terminus. Western blot analysis revealed that the protein was processed at the N- and C-terminus. To exclude that this processing was due to contaminating E. coli proteases, a mutated BAJ93208 protease was constructed. This inactive mutant was not processed, demonstrating that the processing was an autocatalytic process. To define the exact cleavage sites mass spectrometry was used which detected four differently processed versions of the protease. At the N-terminus, the self-processing removed the internal inhibitor and an additional 19 amino acids. At the C-terminus there was a cleavage site after Ala765 which also removed the strep-tag. This explained the inability to detect the purified (His)6-BAJ93208-strep protease with an anti-strep-tag antibody. Finally, an additional alanine was removed either at the N-terminus (Ala119) or at the C-terminus (Ala764).

Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli.

While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain—using chromosomal gene knock-in techniques—that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors.

Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express lysY Escherichia coli strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Nav1.7 in a dose dependent manner (IC50 = 463-727 nM). In comparison to HwTx-IV(amide) (IC50 = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Nav1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IVG36(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Nav1.7 in comparison to HwTx-IV(acid) (IC50 = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.