Differentiating closely affiliated Dehalococcoides lineages by a novel genetic marker identified via computational pangenome analysis

2021 ◽  
Author(s):  
Siyan Zhao ◽  
Chen Zhang ◽  
Matthew J. Rogers ◽  
Xuejie Zhao ◽  
Jianzhong He
Keyword(s):  
Amino Acid ◽  
Microbial Communities ◽  
Genetic Marker ◽  
Genetic Markers ◽  
Unknown Function ◽  
Computational Approach ◽  
Amino Acid Sequences ◽  
Reductive Dehalogenation ◽  
Wide Range

As a group, Dehalococcoides dehalogenate a wide range of organohalide pollutants but the range of organohalide compounds that can be utilized for reductive dehalogenation differs among the Dehalococcoides strains. Dehalococcoides lineages cannot be reliably disambiguated in mixed communities using typical phylogenetic markers, which often confounds bioremediation efforts. Here, we describe a computational approach to identify Dehalococcoides genetic markers with improved discriminatory resolution. Screening core genes from the Dehalococcoides pangenome for degree of similarity and frequency of 100% identity found a candidate genetic marker encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function. This gene exhibits the fewest completely identical amino acid sequences and among the lowest average amino acid sequence identity in the core pangenome. Primers targeting BNR could effectively discriminate between 40 available BNR sequences ( in silico ) and 10 different Dehalococcoides isolates ( in vitro ). Amplicon sequencing of BNR fragments generated from 22 subsurface soil samples revealed a total of 109 amplicon sequence variants, suggesting a high diversity of Dehalococcoides distributed in environment. Therefore, the BNR gene can serve as an alternative genetic marker to differentiate strains of Dehalococcoides in complicated microbial communities. Importance The challenge of discriminating between phylogenetically similar but functionally distinct bacterial lineages is particularly relevant to the development of technologies seeking to exploit the metabolic or physiological characteristics of specific members of bacterial genera. A computational approach was developed to expedite screening of potential genetic markers among phylogenetically affiliated bacteria. Using this approach, a gene encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function was selected and evaluated as a genetic marker to differentiate strains of Dehalococcoides , an environmentally relevant genus of bacteria whose members can transform and detoxify a range of halogenated organic solvents and persistent organic pollutants, in complex microbial communities to demonstrate the validity of the approach. Moreover, many apparently phylogenetically distinct, currently uncharacterized Dehalococcoides were detected in environmental samples derived from contaminated sites.

scholarly journals In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

Application of the Innovative and Non-Invasive Technique, Molecular Music Therapy (MMT), Bio-Frequency Therapy, for the Treatment of a Wide Range of Disorders and Pathologies, with Consequent Verification of Molecular Parameters by Using Bi-Digital O-Ring Test (BDORT)

2020 ◽  
Vol 44 (3) ◽  
pp. 177-189
Author(s):  
Momir Dunjic ◽  
Stefano Turini ◽  
Dejan Krstic ◽  
Katarina Dunjic ◽  
Marija Dunjic ◽  
...  
Keyword(s):  
Amino Acid ◽  
Music Therapy ◽  
Amino Acid Sequences ◽  
Sirtuin 1 ◽  
Ring Test ◽  
Invasive Technique ◽  
Specific Gene ◽  
Radiofrequency Therapy ◽  
Wide Range ◽  
Clinical Pictures

Radiofrequency therapy is an unconventional method, already applied for some time, with numerous results in numerous clinical pictures. Our group has developed a software, later called SONGENPROT-SOLARIS, capable of directly converting nucleotide sequences (DNA and/or RNA) and amino acid sequences (polypeptides and proteins) into musical sequences, based on mathematic matrices, designed by the French physicist and musician Joel Sternheimer, which allows to associate a musical note with a nucleotide or an amino acid. Innovation in our software is that, in the algorithm that defines it, a variant is directly implemented that allows the reproduction of sounds, phase-shifted by 30 Hz, between one ear and another reproducing the phenomenon of Binaural Tones, capable of induce a specific brain activity and also the release of particles called solitons. Thanks to this software we have developed a technique called MMT (Molecular Music Therapy) and currently, we are in the phase of applying the technique on a cohort of 91 patients, with a high spectrum of clinical pictures, examining the same, using the technique Bi-Digital-ORing-Test (BDORT), before and after treatment with MMT. Aim of project is to stimulate the expression of a specific gene (the same genetic sequence that the patient listens to, translated into music), only through the use of sound sequences. We have concentrated our attention on three main molecules: Sirtuin-1, Telomers and TP-53. The results obtained with BDORT, after treatment with MMT, showed a significant increase in the values of the three molecules, on all the examined patients, demonstrating the operative efficacy of the technique and the its applicability to numerous diseases. In order to confirm the data obtained by BDORT, we propose, with the help of an accredited laboratory, to perform epigenetic tests on the three parameters listed above, paving the way to understanding how frequencies can influence gene expression.

scholarly journals Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
M. Adamczyk ◽  
E. Lewicka ◽  
R. Szatkowska ◽  
H. Nieznanska ◽  
J. Ludwiczak ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Coiled Coil ◽  
Amino Acid Sequences ◽  
Broad Host Range ◽  
Biophysical Properties ◽  
Dna Binding Sites ◽  
In Silico Studies ◽  
Wide Range

Abstract Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

scholarly journals Charge influences substrate recognition and self-assembly of hydrophobic FG sequences

2016 ◽  
Author(s):  
Wesley G. Chen ◽  
Jacob Witten ◽  
Scott C. Grindy ◽  
Niels Holten-Andersen ◽  
Katharina Ribbeck
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Amino Acid Sequences ◽  
Nuclear Pore ◽  
Selective Binding ◽  
Charged Amino Acids ◽  
Essential Components ◽  
Transport Receptors

AbstractThe nuclear pore complex controls the passage of molecules via hydrophobic phenylalanine-glycine (FG) domains on nucleoporins. Such FG-domains consist of repeating units of FxFG, FG, or GLFG sequences, which can be interspersed with highly charged amino acid sequences. Despite the high density of charge exhibited in certain FG-domains, if and how charge influences FG-domain self-assembly and selective binding of nuclear transport receptors is largely unexplored. Studying how individual charged amino acids contribute to nuclear pore selectivity is challenging with modern in vivo and in vitro techniques due to the complexity of nucleoporin sequences. Here, we present a rationally designed approach to deconstruct essential components of nucleoporins down to 14 amino acid sequences. With these nucleoporin-based peptides, we systematically dissect how charge type and placement of charge influences self-assembly and selective binding of FG-containing gels. Specifically, we find that charge type determines which hydrophobic substrates FG sequences recognize while spatial localization of charge tunes hydrophobic self-assembly and receptor selectivity of FG sequences.

scholarly journals Unevolved De Novo Proteins Have Innate Tendencies to Bind Transition Metals

Life ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 8 ◽  
Author(s):  
Michael S. Wang ◽  
Kenric J. Hoegler ◽  
Michael H. Hecht
Keyword(s):  
Amino Acid ◽  
Transition Metals ◽  
Metal Binding ◽  
Combinatorial Library ◽  
De Novo ◽  
Protein Sequences ◽  
Amino Acid Sequences ◽  
Ancestral Sequences ◽  
Wide Range ◽  
Catalytic Functions

Life as we know it would not exist without the ability of protein sequences to bind metal ions. Transition metals, in particular, play essential roles in a wide range of structural and catalytic functions. The ubiquitous occurrence of metalloproteins in all organisms leads one to ask whether metal binding is an evolved trait that occurred only rarely in ancestral sequences, or alternatively, whether it is an innate property of amino acid sequences, occurring frequently in unevolved sequence space. To address this question, we studied 52 proteins from a combinatorial library of novel sequences designed to fold into 4-helix bundles. Although these sequences were neither designed nor evolved to bind metals, the majority of them have innate tendencies to bind the transition metals copper, cobalt, and zinc with high nanomolar to low-micromolar affinity.

Effects of peptide hormone structure on H+ secretion by Necturus gastric mucosa

1976 ◽  
Vol 231 (2) ◽  
pp. 573-578 ◽  
Author(s):  
JM Berkowitz ◽  
M Praissman ◽  
ME LeFevre
Keyword(s):  
Amino Acid ◽  
Gastric Mucosa ◽  
Peptide Hormone ◽  
Amino Acid Sequences ◽  
Gastrointestinal Hormone ◽  
Gastric Tissue ◽  
The Common ◽  
Subsequent Addition ◽  
Lower Molar

The actions of human synthetic gastrin I(G), the C-terminal tetrapeptide of gastrin (T), and the C-terminal octapeptide of cholecystokinin (OP) on acid secretion and transepithelial potential difference (PD) of the isolated Necturus gastric mucosa were determined. All three peptides induced H+ secretion, but the maximum H+ output was less with OP than with G or T. G and OP produced their maximum H+ output at lower molar concentrations than T. G- and OP-stimulated secretion was long sustained, but T-stimulated secretion rapidly returned to basal levels. T- and G-stimulated secretion was partially inhibited by the addition of OP. Evidence is presented that T rapidly disappears from solutions exposed to gastric mucosa, whereas G does not. Washing sensitized the mucosa to subsequent addition of T. The results suggest that the action of the common C-terminal tetrapeptide of G, T, and OP is modified by the preceding amino acid sequences, and that T, the smallest of the three peptides, is rapidly degraded by gastric tissue in vitro. The implications of the work for the study of gastrointestinal hormone structure-function relationships in isolated tissue preparations are discussed.

scholarly journals Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

1999 ◽  
Vol 65 (8) ◽  
pp. 3279-3286 ◽  
Author(s):  
Qiaoping Yuan ◽  
James J. Pestka ◽  
Brandon M. Hespenheide ◽  
Leslie A. Kuhn ◽  
John E. Linz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Synthetic Peptide ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.

scholarly journals Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

2006 ◽  
Vol 72 (4) ◽  
pp. 2824-2828 ◽  
Author(s):  
Bettina Bäuerle ◽  
Željko Cokesa ◽  
Silvia Hofmann ◽  
Paul-Gerhard Rieger
Keyword(s):  
Amino Acid ◽  
Agrobacterium Tumefaciens ◽  
Heterologous Expression ◽  
Bacterial Strain ◽  
Amino Acid Sequences ◽  
Complexing Agent ◽  
Recombinant Enzymes ◽  
Gene Encoding ◽  
Degrading Bacteria

ABSTRACT Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl B and icl S, encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl B gene was chromosomal like the corresponding icl S gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.

scholarly journals Identification of amino acid sequences determining interaction between the cucumber mosaic virus-encoded 2a polymerase and 3a movement proteins

2007 ◽  
Vol 88 (12) ◽  
pp. 3445-3451 ◽  
Author(s):  
Min Sook Hwang ◽  
Kyung Nam Kim ◽  
Jeong Hyun Lee ◽  
Young In Park
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Critical Role ◽  
Amino Acid Sequences ◽  
Multiple Sequence ◽  
Movement Proteins ◽  
Gdd Motif

The cucumber mosaic virus (CMV)-encoded 3a movement protein (MP) is indispensable for CMV movement in plants. We have previously shown that MP interacts directly with the CMV-encoded 2a polymerase protein in vitro. Here, we further dissected this interaction and determined the amino acid sequences that are responsible for the MP and 2a polymerase protein interaction. Both the N-terminal 21 amino acids and the central GDD motif of the 2a polymerase protein were important for interacting with the MP. Although each of the regions alone was sufficient for the interaction with MP, quantitative yeast two-hybrid analyses showed that they acted synergistically to enhance the binding affinity. The MP N-terminal 20 amino acids were sufficient for interacting with the 2a polymerase protein, and the serine residue at position 14 played a critical role in the interaction. Multiple sequence alignment showed that the 2a protein interacting regions and the serine at position 14 in the MP are highly conserved among subgroup I and II CMV isolates.

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