scholarly journals Semi-in vitro Reconstitution of Roseocin, a Two-Component Lantibiotic from an Actinomycete

2018 ◽  
Author(s):  
Mangal Singh ◽  
Dipti Sareen
Keyword(s):  
Genome Mining ◽  
Disulfide Bridge ◽  
Biosynthetic Gene Cluster ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
C Terminus ◽  
In Vitro Reconstitution ◽  
Two Component ◽  
Modified Peptides

ABSTRACTLantibiotics are lanthionine containing peptide natural products that belong to the class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Recent expansion in the availability of microbial genomic data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic roseocin was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Post-translationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in E. coli, and were in vitro reconstituted to their bioactive form. The two lanthipeptides displayed antimicrobial activity against Gram-positive bacteria only synergistically, a property reminiscent of two-component lantibiotics. Structural analysis of the α-component identified a disulfide bridge flanking two of its four thioether bridges and the β-component having six thioether bridges with its C-terminus extended than the previously known two-component lantibiotics.

scholarly journals Restoration of Bioactive Lantibiotic Suicin from a RemnantlanLocus of Pathogenic Streptococcus suis Serotype 2

2013 ◽  
Vol 80 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Jian Wang ◽  
Yong Gao ◽  
Kunling Teng ◽  
Jie Zhang ◽  
Shutao Sun ◽  
...  
Keyword(s):  
Gene Promoter ◽  
Streptococcus Suis ◽  
Gene Clusters ◽  
Disulfide Bridge ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
System A ◽  
Bactericidal Activities

ABSTRACTLantibiotics are ribosomally synthesized, posttranslationally modified antimicrobial peptides. Their biosynthesis genes are usually organized in gene clusters, which are mainly found in Gram-positive bacteria, including pathogenic streptococci. Three highly virulentStreptococcus suisserotype 2 strains (98HAH33, 05ZYH33, and SC84) have been shown to contain an 89K pathogenicity island. Here, on these islands, we unveiled and reannotated a putative lantibiotic locus designatedsuiwhich contains a virulence-associated two-component regulator,suiK-suiR. In silicoanalysis revealed that the putative lantibiotic modification genesuiMwas interrupted by a 7.9-kb integron and that other biosynthesis-related genes contained various frameshift mutations. By reconstituting the intactsuiMinEscherichia colitogether with a semi-in vitrobiosynthesis system, a putative lantibiotic named suicin was produced with bactericidal activities against a variety of Gram-positive strains, including pathogenic streptococci and vancomycin-resistant enterococci. Ring topology dissection indicated that the 34-amino-acid lantibiotic contained two methyllanthionine residues and one disulfide bridge, which render suicin in an N-terminal linear and C-terminal globular shape. To confirm the function ofsuiK-suiR, SuiR was overexpressed and purified.In vitroanalysis showed that SuiR could specifically bind to thesuiAgene promoter. Its coexpression withsuiKcould activatesuiAgene promoter inLactococcus lactisNZ9000. Conclusively, we obtained a novel lantibiotic suicin by restoring its production from the remnantsuilocus and demonstrated that virulence-associated SuiK-SuiR regulates its production.

scholarly journals Subforms and In Vitro Reconstitution of the NAD-Reducing Hydrogenase of Alcaligenes eutrophus

1998 ◽  
Vol 180 (5) ◽  
pp. 1023-1029 ◽  
Author(s):  
Christian Massanz ◽  
Silke Schmidt ◽  
Bärbel Friedrich
Keyword(s):  
Alcaligenes Eutrophus ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Monomeric Form ◽  
Structural Genes ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
In Vitro Reconstitution ◽  
Reducing Activity

The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer. HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation. A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most ofhoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide. While the hydrogenase dimer, produced by a strain deleted of hoxF andhoxU, displayed H2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H2, although nickel was incorporated into HoxH. Deletion ofhoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity. Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.

scholarly journals In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

2018 ◽  
Vol 115 (24) ◽  
pp. E5477-E5486 ◽  
Author(s):  
Chungyu Chang ◽  
Brendan R. Amer ◽  
Jerzy Osipiuk ◽  
Scott A. McConnell ◽  
I-Hsiu Huang ◽  
...  
Keyword(s):  
Cross Linking ◽  
Structural Elements ◽  
Gram Positive Bacteria ◽  
Activating Mutations ◽  
In Vitro Reconstitution ◽  
Isopeptide Bonds ◽  
Signature Sequence ◽  
Class C ◽  
Enzyme Intermediate

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2Msynthesizes pilus fibers with correct Lys–Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA–SrtA–SpaA polymerization intermediate depicts SrtA2Msandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2Mcan terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Screening of Antimicrobial Activity and Cytotoxic Effects of Two Cladonia Species

2013 ◽  
Vol 68 (5-6) ◽  
pp. 191-197 ◽  
Author(s):  
Birkan Açıkgöz ◽  
İskender Karaltı ◽  
Melike Ersöz ◽  
Zeynep M. Coşkun ◽  
Gülşah Çobanoğlu ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Activities ◽  
Chloroform Extract ◽  
Cytotoxic Effects ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Methanol Extracts ◽  
Gram Positive Bacteria

The present study explores the antimicrobial activity and cytotoxic effects in culture assays of two fruticose soil lichens, Cladonia rangiformis Hoffm. and Cladonia convoluta (Lamkey) Cout., to contribute to possible pharmacological uses of lichens. In vitro antimicrobial activities of methanol and chloroform extracts against two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), two Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), and the yeast Candida albicans were examined using the paper disc method and through determination of minimal inhibitory concentrations (MICs). The data showed the presence of antibiotic substances in the chloroform and the methanol extracts of the lichen species. The chloroform extracts exhibited more signifi cant antimicrobial activity than the methanol extracts. However, a higher antifungal activity was noted in the methanol extract of C. rangiformis. The maximum antimicrobial activity was recorded for the chloroform extract of C. convoluta against E. coli. The cytotoxic effects of the lichen extracts on human breast cancer MCF-7 cells were evaluated by the trypan blue assay yielding IC50 values of ca. 173 and 167 μg/ml for the extracts from C. rangiformis and C. convoluta, respectively.

scholarly journals Enhanced Prokaryotic Expression of Dengue Virus Envelope Protein

2013 ◽  
Vol 16 (4) ◽  
pp. 609 ◽  
Author(s):  
Advaita Ganguly ◽  
Ravindra B. Malabadi ◽  
Dipankar Das ◽  
Mavanur R. Suresh ◽  
Hoon H Sunwoo
Keyword(s):  
Dengue Virus ◽  
Envelope Protein ◽  
Vaccine Development ◽  
Virus Envelope ◽  
E Coli ◽  
C Terminus ◽  
Virus Envelope Protein ◽  
Biological Functionality ◽  
Dengue Diagnostics

Purpose. To highlight the expression and purification of the recombinant dengue virus type-1 antigen exploiting the codon optimized full length envelope for increased yield in E. coli. Methods. A 6x His tag was inserted at the C terminus to facilitate purification. The purified protein was recognized in Western blot by Monoclonal antibody specific for the tag. The in vitro refolded recombinant protein was used to immunize mice for the development of hybridomas and also analyzed for its biological functionality with heparan sulfate binding assay. Results. The polyclonal anti-sera from the immunized mice were found to recognize the envelope protein thereby establishing the immunogenicity of the protein. Conclusion. The purified envelope protein could potentially be used towards dengue diagnostics and vaccine development efforts. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity

2006 ◽  
Vol 387 (4) ◽  
pp. 451-460 ◽  
Author(s):  
Christina Sieber ◽  
Frank Plöger ◽  
Raphaela Schwappacher ◽  
Rolf Bechtold ◽  
Michael Hanke ◽  
...  
Keyword(s):  
Biological Activity ◽  
Biochemical Analysis ◽  
Gene Activation ◽  
Disulfide Bridge ◽  
Living Cells ◽  
Type I ◽  
E Coli ◽  
Interchain Disulfide Bond ◽  
Dimeric Form

Abstract Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.

scholarly journals Redox Signal Transduction by the ArcB Sensor Kinase of Haemophilus influenzae Lacking the PAS Domain

2001 ◽  
Vol 183 (24) ◽  
pp. 7206-7212 ◽  
Author(s):  
Dimitris Georgellis ◽  
Ohsuk Kwon ◽  
Edmund C. C. Lin ◽  
Sandy M. Wong ◽  
Brian J. Akerley
Keyword(s):  
Signal Transduction ◽  
Haemophilus Influenzae ◽  
Bacterial Species ◽  
Redox Conditions ◽  
Sensor Kinase ◽  
Pas Domain ◽  
Signal Transduction System ◽  
E Coli ◽  
Two Component

ABSTRACT The Arc (anoxic redox control) two-component signal transduction system of Escherichia coli, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the arcA and arcBgenes of Haemophilus influenzae specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of H. influenzae mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in E. coli, indicating a high degree of functional conservation of the signal transduction system. The H. influenzae ArcB, however, lacks the PAS domain present in the region of E. coli ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of H. influenzae mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.

scholarly journals Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

2000 ◽  
Vol 182 (19) ◽  
pp. 5539-5550 ◽  
Author(s):  
Paula S. Fritsch ◽  
Mark L. Urbanowski ◽  
George V. Stauffer
Keyword(s):  
Rna Polymerase ◽  
Random Mutagenesis ◽  
Complex Surface ◽  
Α Subunit ◽  
C Terminus ◽  
Distinct Cluster ◽  
In Vitro Reconstitution ◽  
The Difference ◽  
Α Subunits

ABSTRACT Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α2, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the αCTD on the β-associated α subunit at metH. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.

scholarly journals Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway

2022 ◽  
Author(s):  
Asfandyar Sikandar ◽  
Maria Lopatniuk ◽  
Andriy Luzhetskyy ◽  
Rolf Müller ◽  
Jesko Koehnke
Keyword(s):  
Biosynthetic Pathway ◽  
Protein Complexes ◽  
Downstream Processing ◽  
Leader Peptide ◽  
Oxidative Decarboxylation ◽  
Cancerous Cell ◽  
In Vitro Reconstitution ◽  
Modified Peptides ◽  
In Vitro Biosynthesis

Thioholgamides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes – ThoH/I and ThoC/D. Harnessing the power of AlphaFold we deduce that ThoD acts as a lyase and also propose putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation – a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.

scholarly journals Antimicrobial Effect Of Honey Distributed Around Pasar Minggu Jakarta Selatan

2017 ◽  
Vol 8 (2) ◽  
pp. 101-106
Author(s):  
Ruth Elenora Kristanty ◽  
Junie Suriawati ◽  
Priyanto Dwi Nugroho
Keyword(s):  
Infectious Diseases ◽  
Food Product ◽  
Antimicrobial Effect ◽  
Inhibition Zone ◽  
Diffusion Method ◽  
Bacteria Growth ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Almost All

Honey is a highly nutritious food product and consumed by almost all the population in the world. It has a function as an antimicrobial. Staphylococcus aureus (S. aureus) is a common Gram-positive bacteria in food and Escherichia coli (E. coli) is a Gram-negative bacteria that often appears in environmental sanitation issues that both can cause infectious diseases.  Some infectious diseases can be treated with antimicrobials such as honey. The purpose of this study was to test the antimicrobial effects on honey products distributed in Pasar Minggu area. The antimicrobial effect test was performed in vitro using agar diffusion method by measuring the inhibition zone formed where the bacteria growth was inhibited by the presence of sample. The concentration of samples were 25%, 50%, 75%, and 100% (not diluted) and as aquades control. The results showed that honey tested with various dilution concentrations resulted inhibition zone and. The higher concentration of the inhibited zone zone showed antimicrobial activity against S. aureus and E. coli.

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