The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.
- MeSH
- COVID-19 * MeSH
- lidé MeSH
- odpadní voda MeSH
- RNA virová MeSH
- SARS-CoV-2 * MeSH
- veřejné zdravotnictví MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: -43, -44, -49, and -50 bp. To facilitate measuring of the recombinant promoter relative strength, ß-glucuronidase gene was used as a reporter gene. Three mutations located in positions -49 and -50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.
- MeSH
- bakteriální geny MeSH
- biomasa MeSH
- biotechnologie MeSH
- kyselina hyaluronová chemie metabolismus MeSH
- mutace * MeSH
- mutageneze cílená metody MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- Streptococcus equi genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.
- MeSH
- bioreaktory MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- enteropeptidasa biosyntéza chemie genetika MeSH
- enzymy imobilizované chemie genetika metabolismus MeSH
- histidin genetika metabolismus MeSH
- klonování DNA MeSH
- lidé MeSH
- Pichia genetika metabolismus MeSH
- rekombinantní fúzní proteiny biosyntéza chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The purpose of this study is to determine the effect of the corresponding nucleotides from Streptococcus pyogenes on the has promoter strength in highly encapsulated strain S. equi subsp. zooepidemicus (SEZ) and detect an empowering mutations in SEZ. Eight different strains of SEZ carrying nucleotide mutations in the -73 to -38 region upstream of the has promoter were constructed. The significant activity decrease to 36-1% was observed after the introduction of mutations in the promoter region from -44 to -38 site. The exception was observed in mutation in -49 site when no significant decrease was observed. When nucleotides TTT were used in positions -73 the promoter became weaker, whereas no significant effect was observed after using nucleotides CCC (96%). Unfortunately, introduction of these mutations into chromosome SEZ has no empowering effect. Six strains, which carried nucleotide sequences of different lengths upstream from the transcription start of hasA promoter, were constructed to determine the minimum upstream region required for the maximum transcription efficiency of the has operon. No change of the activity of the has promoter constructs containing as few as 101 nucleotides upstream from the transcription start point was observed.
- MeSH
- bakteriální geny genetika MeSH
- bakteriální proteiny MeSH
- glukuronidasa MeSH
- klonování DNA MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- mutageneze MeSH
- operon MeSH
- plazmidy MeSH
- promotorové oblasti (genetika) genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- Streptococcus equi genetika MeSH
- Streptococcus pyogenes genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.
- MeSH
- glukuronidasa genetika MeSH
- koně MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční homologie nukleových kyselin MeSH
- shluková analýza MeSH
- Streptococcus equi genetika MeSH
- terciární struktura proteinů genetika MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 microM; for Staphylococcus aureus 2.31 microM, and for Enterococcus faecalis 5.54 microM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of beta-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.
- MeSH
- bakteriofág T7 genetika MeSH
- Candida albicans účinky léků MeSH
- DNA řízené RNA-polymerasy biosyntéza genetika MeSH
- Enterococcus faecalis účinky léků MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- kationické antimikrobiální peptidy biosyntéza genetika izolace a purifikace MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- promotorové oblasti (genetika) MeSH
- rekombinantní fúzní proteiny biosyntéza genetika izolace a purifikace MeSH
- Staphylococcus aureus účinky léků MeSH
- virové proteiny biosyntéza genetika MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Recently, a new gene encoding beta-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37 degrees C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52 degrees C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of beta-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of beta-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified beta-glucuronidase from SEZ glcHis was four times as low as beta-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal beta-glucuronidase from E. coli with E. coli beta-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.
- MeSH
- Acetobacter genetika MeSH
- plazmidy MeSH
