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

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.

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Interacting Regions in Stat3 and c-Jun That Participate in Cooperative Transcriptional Activation

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.7138 ◽

1999 ◽

Vol 19 (10) ◽

pp. 7138-7146 ◽

Cited By ~ 149

Author(s):

Xiaokui Zhang ◽

Melissa H. Wrzeszczynska ◽

Curt M. Horvath ◽

James E. Darnell

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Point Mutations ◽

Coiled Coil ◽

Transient Transfection ◽

Amino Acid Sequences ◽

Physical Interaction ◽

Enhancer Function

ABSTRACT Independent but closely spaced DNA binding sites for Stat3 and c-Jun are required for maximal enhancer function in a number of genes, including the gene encoding the interleukin-6 (IL-6)-induced acute-phase response protein, α2-macroglobulin. In addition, a physical interaction of Stat3 with c-Jun, based on yeast two-hybrid interaction experiments, has been reported. Here we confirm the existence of an interaction between Stat3 and c-Jun both in vitro, with recombinant proteins, and in vivo, during transient transfection. Using fragments of both proteins, we mapped the interactive sites to the C-terminal region of c-Jun and to two regions in Stat3, within the coiled-coil domain and in a portion of the DNA binding domain distant from DNA contact sites. In transient-transfection experiments with the α2-macroglobulin enhancer, Stat3 and c-Jun cooperated to yield maximal enhancer function. Point mutations of Stat3 within the interacting domains blocked both physical interaction of Stat3 with c-Jun and their cooperation in IL-6-induced transcription directed by the α2-macroglobulin enhancer. While the amino acid sequences and the three-dimensional structures of Stat3 and Stat1 cores are very similar, fragments of Stat1 failed to bind c-Jun in vitro. Although Stat1 binds in vitro to the gamma interferon gene response (GAS) element in the α2-macroglobulin enhancer, Stat1 did not stimulate transcription, nor did Stat1 and c-Jun cooperate in driving transcription controlled by the α2-macroglobulin enhancer.

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Characterization of vB_Sau_S90, and vB_Sau_S165, a Broad Host Range Phages, and Multiple Phage Doses as a Potential Therapeutic Strategy to Eliminate Transient Phage Resistant Mutants in Staphylococcus Aureus

10.21203/rs.3.rs-1070956/v1 ◽

2021 ◽

Author(s):

Archana Loganathan ◽

Ramesh Nachimuthu

Keyword(s):

Staphylococcus Aureus ◽

Host Range ◽

Serial Passage ◽

Broad Host Range ◽

Bacterial Cells ◽

Bacterial Killing ◽

Methicillin Resistant ◽

Adsorption Time ◽

Wide Range

Abstract Methicillin-Resistant Staphylococcus aureus is a human pathogen. MRSA has acquired resistance to major antibiotics; thus, phage therapy has become a potential alternative treatment. In this work, two broad host range Staphylococcus phages were characterized for lifecycle, physio-chemical parameters and bacterial killing kinetics, and the in vitro behavior of phage insensitive bacterial cells to alternative serial passage and multiple phage doses were assessed by reduction in the bacterial turbidity and spot assay. Phage vB_Sau_S90 showed an absorption efficiency of 91 ± 0.6% with an adsorption time of 17 ± 1 min and vB_Sau_S165 of 95 ± 0.5% adsorption efficiency and 15 ± 2 min adsorption time. Both the phages were stable over a wide range of temperature (20 to 50 ℃) and pH (3 to 11). vB_Sau_S90 phage belonging to the family Siphoviridae [order Caudovirals] showed killing efficiency against 88% (181/205) of S. aureus isolates, and vB_Sau_S165 belonging to family Podoviridae [order Caudovirals] showed killing efficiency against 94% (192/205) of S. aureus isolates. The sensitive and transient phage-resistant cells that remained uninfected during the single dose of phage treatment were eliminated upon a minimum of five alternative serial passage and multiple phage doses. This study concludes that both the phages showed promising activity against methicillin-resistant Staphylococcus aureus. Our study revealed that despite phage auto-dosing and high therapeutic efficiency, both phages did not produce a complete bacterial clearance at a single phage dose; hence indicated that multiple phage doses were required to attain a successful and complete bacterial eradication.

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Chalcones: As potent α-amylase enzyme inhibitors; synthesis, in vitro, and in silico studies

Medicinal Chemistry ◽

10.2174/1573406416666200611103039 ◽

2020 ◽

Vol 16 ◽

Author(s):

Mahboob Ali ◽

Momin Khan ◽

Khair Zaman ◽

Abdul Wadood ◽

Maryam Iqbal ◽

...

Keyword(s):

In Silico ◽

Enzyme Inhibitors ◽

Biological Activities ◽

Condensation Reaction ◽

Type Ii Diabetes Mellitus ◽

Spectroscopic Techniques ◽

Chalcone Derivatives ◽

In Silico Studies ◽

Wide Range

: Background: The inhibition of α-amylase enzyme is one of the best therapeutic approach for the management of type II diabetes mellitus. Chalcone possesses a wide range of biological activities. Objective: In the current study chalcone derivatives (1-17) were synthesized and evaluated their inhibitory potential against α-amylase enzyme. Method: For that purpose, a library of substituted (E)-1-(naphthalene-2-yl)-3-phenylprop-2-en-1-ones was synthesized by ClaisenSchmidt condensation reaction of 2-acetonaphthanone and substituted aryl benzaldehyde in the presence of base and characterized via different spectroscopic techniques such as EI-MS, HREI-MS, 1H-, and 13C-NMR. Results: Sixteen synthetic chalcones were evaluated for in vitro porcine pancreatic α-amylase inhibition. All the chalcones demonstrated good inhibitory activities in the range of IC50 = 1.25 ± 1.05 to 2.40 ± 0.09 μM as compared to the standard commercial drug acarbose (IC50 = 1.34 ± 0.3 μM). Conclusion: Chalcone derivatives (1-17) were synthesized, characterized, and evaluated for their α-amylase inhibition. SAR revealed that electron donating groups in the phenyl ring have more influence on enzyme inhibition. However, to insight the participation of different substituents in the chalcones on the binding interactions with the α-amylase enzyme, in silico (computer simulation) molecular modeling analyses were carried out.

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Insights into the Antioxidant Mechanism of Newly Synthesized Benzoxazinic Nitrones: In Vitro and In Silico Studies with DPPH Model Radical

Antioxidants ◽

10.3390/antiox10081224 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1224

Author(s):

Stefania Marano ◽

Cristina Minnelli ◽

Lorenzo Ripani ◽

Massimo Marcaccio ◽

Emiliano Laudadio ◽

...

Keyword(s):

Antioxidant Activity ◽

Epr Spectroscopy ◽

Point Of View ◽

Uv Spectrophotometry ◽

Design And Synthesis ◽

Antioxidant Mechanism ◽

Primary Mechanism ◽

In Silico Studies ◽

Wide Range

Synthetic nitrone spin-traps are being explored as therapeutic agents for the treatment of a wide range of oxidative stress-related pathologies, including but not limited to stroke, cancer, cardiovascular, and neurodegenerative diseases. In this context, increasing efforts are currently being made to the design and synthesis of new nitrone-based compounds with enhanced efficacy. The most researched nitrones are surely the ones related to α-phenyl-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) derivatives, which have shown to possess potent biological activity in many experimental animal models. However, more recently, nitrones with a benzoxazinic structure (3-aryl-2H-benzo[1,4]oxazin-N-oxides) have been demonstrated to have superior antioxidant activity compared to PBN. In this study, two new benzoxazinic nitrones bearing an electron-withdrawing methoxycarbonyl group on the benzo moiety (in para and meta positions respect to the nitronyl function) were synthesized. Their in vitro antioxidant activity was evaluated by two cellular-based assays (inhibition of AAPH-induced human erythrocyte hemolysis and cell death in human retinal pigmented epithelium (ARPE-19) cells) and a chemical approach by means of the α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging assay, using both electron paramagnetic resonance (EPR) spectroscopy and UV spectrophotometry. A computational approach was also used to investigate their potential primary mechanism of antioxidant action, as well as to rationalize the effect of functionalization on the nitrones reactivity toward DPPH, chosen as model radical in this study. Further insights were also gathered by exploring the nitrone electrochemical properties via cyclic voltammetry and by studying their kinetic behavior by means of EPR spectroscopy. Results showed that the introduction of an electron-withdrawing group in the phenyl moiety in the para position significantly increased the antioxidant capacity of benzoxazinic nitrones both in cell and cell-free systems. From the mechanistic point of view, the calculated results closely matched the experimental findings, strongly suggesting that the H-atom transfer (HAT) is likely to be the primary mechanism in the DPPH quenching.

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

10.21203/rs.3.rs-366314/v2 ◽

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.

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The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes

Development ◽

10.1242/dev.110.1.141 ◽

1990 ◽

Vol 110 (1) ◽

pp. 141-149 ◽

Cited By ~ 1

Author(s):

F. Payre ◽

S. Noselli ◽

V. Lefrere ◽

A. Vincent

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Polytene Chromosomes ◽

Duplication Event ◽

Amino Acid Sequences ◽

Evolutionary Divergence ◽

Coding Region ◽

Protein Coding

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12–13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02181-21 ◽

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.

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A65 Characterization of endolysin gene of bacteriophages infecting Listeria spp. isolated from dairy industry wastewater

Virus Evolution ◽

10.1093/ve/vez002.064 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

Blanco Fernández ◽

M E Barrios ◽

R V Cammarata ◽

C Torres ◽

V A Mbayed

Keyword(s):

Host Range ◽

High Titer ◽

Dairy Industry ◽

Foodborne Pathogen ◽

Dairy Farm ◽

Restriction Enzymes ◽

Invasive Disease ◽

Broad Host Range ◽

Wide Range ◽

Industry Wastewater

Abstract Bacteriophages and their endolysins, enzymes that degrade the cell walls of bacteria, are emerging as alternative tools to detect and inhibit growth of pathogen bacteria. Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a serious invasive disease that affects both humans and a wide range of animals. Listeria spp. are ubiquitous in the dairy farm environment and could be present in dairy-processing plants and wastewater. All Listeria-specific bacteriophages found to date are members of the Caudovirales, of the Siphoviridae or Myoviridae families. Myophages infecting Listeria have been recently classified by the ICTV in the Spounavirinae subfamily, as well as in the P100 virus genus. The aim of this work was to isolate Listeria spp. bacteriophages and their endolysin codifying genes from wastewater of a dairy industry. Wastewater with and without treatment was sampled during the course of a year, and isolation of bacteriophages was performed after an enrichment step using as hosts L. innocua, L. ivanovii, and L. monocytogenes serotypes 1/2a, 1/2b, and 4b. Bacteriophages infecting L. innocua and L. ivanovii were isolated (n = 24) from 3 out of 12 samples. Bacteriophages were purified, and the host range was determined using spot test and EOP against five collection strains and several field isolates of Listeria spp. Two bacteriophages of narrow and broad host range, vB_Lino_VEfB7, and vB_Liva_VAfA18, were selected for further characterization. High titer stocks of bacteriophages were purified by centrifugation with ammonium acetate, and morphological information on the purified bacteriophages was obtained by negative staining and transmission electronic microscopy. Their morphology, size, and contractile tails indicated that these bacteriophages belonged to the Myoviridae family. Bacteriophage genomes were extracted using phenol-chloroform, followed by ethanol precipitation, and tested by digestion with RNAsa A and DNAse I. RFLP was performed, digesting genomes with restriction enzymes HindIII and NcoI. Consistent with the morphological findings, bacteriophages contained dsDNA genomes but showed different RFLP patterns. A PCR designed to amplify conserved domains of endolysins—PGRP and CwlA—was applied to characterize this gene. Another PCR was designed to amplify the complete endolysin gene, and the complete sequence of this gene was obtained and analyzed. Substitution model selection and a maximum likelihood phylogenetic tree of the endolysin gene was carried out using IQ-Tree software. The sequences of the endolysin gene indicated that the codified enzyme is an N-acetyl-muramoyl-L-alanine amidase, related to A511 and P100 species of the recently described P100virus genus. Further evolutionary analyses are needed to evaluate their belonging to this species or their taxonomy within this genus.

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Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

Journal of Virology ◽

10.1128/jvi.01799-16 ◽

2016 ◽

Vol 91 (4) ◽

Cited By ~ 13

Author(s):

Qiang Ding ◽

Markus von Schaewen ◽

Gabriela Hrebikova ◽

Brigitte Heller ◽

Lisa Sandmann ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Host Range ◽

Amino Acid Sequences ◽

Host Factors ◽

Narrow Host Range ◽

Junction Protein ◽

Mouse Hepatocytes

ABSTRACT Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo. IMPORTANCE At least 150 million individuals are chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can result in progressive liver disease and liver cancer. New antiviral treatments can cure HCV in the majority of patients, but a vaccine remains elusive. To gain a better understanding of the processes culminating in liver failure and cancer and to prioritize vaccine candidates more efficiently, small-animal models are needed. Here, we describe the characterization of a new mouse model in which the parts of two host factors that are essential for HCV uptake, CD81 and occludin (OCLN), which differ between mice and humans, were humanized. We demonstrate that such minimally humanized mice develop normally, express the modified genes at physiological levels, and support HCV uptake. This model is of considerable utility for studying viral entry in the three-dimensional context of the liver and to test approaches aimed at preventing HCV entry.

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Limits to the host range of the highly polyphagous tephritid fruit fly Anastrepha ludens in its natural habitat

Bulletin of Entomological Research ◽

10.1017/s0007485315000711 ◽

2015 ◽

Vol 105 (6) ◽

pp. 743-753 ◽

Cited By ~ 2

Author(s):

A. Birke ◽

E. Acosta ◽

M. Aluja

Keyword(s):

Host Range ◽

Natural Habitat ◽

Fruit Fly ◽

Natural Host ◽

Pupal Weight ◽

Anastrepha Ludens ◽

Broad Host Range ◽

Ripe Fruit ◽

Natural Conditions ◽

Wide Range

AbstractAnastepha ludens (Diptera: Tephritidae) is a highly polyphagous fruit fly that is able to develop in a wide range of hosts. Understanding the limits of this pest's host range could provide valuable information for pest management and plant breeding for pest resistance. Previous studies have shown that guavas (Psidium guajava (Myrtaceae) L.), are not attacked under natural conditions by A. ludens. To understand this phenomenon, guavas were exposed to natural infestation by A. ludens and to other fruit fly species that infest guavas in nature (Anastrepha striata Schiner, Anastepha fraterculus (Wiedemann), Anastepha obliqua (Macquart)). Once the susceptible phenological stage of guavas was determined, fruit infestation levels were compared between A. ludens and A. striata. Choice and non-choice tests were performed under field-cage conditions. Under field conditions, guavas were susceptible to A. striata and A. fraterculus attack all the way from when fruit was undeveloped to when fruit began to ripen. No infestation by A. ludens was recorded under natural conditions. Similar results were obtained when forced exposures were performed, indicating that unripe guavas were preferred by A. striata over ripe fruit, and that infestation rates were higher at early fruit maturity stages. Under forced oviposition conditions, A. ludens larvae were unable to develop in unripe guavas but did so in fully ripe fruit. However, A. ludens fitness parameters were dramatically affected, exhibiting reduced survival and reduced pupal weight compared to conspecifics that developed in a natural host, grapefruit. We confirm that P. guajava should not be treated as a natural host of this pestiferous species, and suggest that both behavioral aspects and the fact that larvae are unable to adequately develop in this fruit, indeed represent clear limits to A. ludens's broad host range.

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