Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms

International Journal of Molecular Sciences ◽

10.3390/ijms22020681 ◽

2021 ◽

Vol 22 (2) ◽

pp. 681

Author(s):

Vaishnavi Amarr Reddy ◽

Chunhong Li ◽

Kumar Nadimuthu ◽

Jessica Gambino Tjhang ◽

In-Cheol Jang ◽

...

Keyword(s):

Glandular Trichomes ◽

Functional Characterization ◽

Rna Seq ◽

Post Translational Modifications ◽

E Coli ◽

Sweet Basil ◽

Specific Regulation

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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Characterization of in vitro and in vivo murine models of human enteropathogenic E. coli (EPEC) infection

Gastroenterology ◽

10.1016/s0016-5085(03)82826-3 ◽

2003 ◽

Vol 124 (4) ◽

pp. A558

Author(s):

Suzana D. Savkovic ◽

Farol L. Tomson ◽

Michelle Muza ◽

Gail Hecht

Keyword(s):

Murine Models ◽

E Coli

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Omics profiling identifies MAPK/ERK pathway as a gatekeeper of nephron progenitor metabolism

10.1101/2021.09.27.461969 ◽

2021 ◽

Author(s):

Hyuk Nam Kwon ◽

Kristen Kurtzeborn ◽

Xing Jin ◽

Bruno Reversade ◽

Sunghyouk Park ◽

...

Keyword(s):

Functional Characterization ◽

Mitogen Activated Protein Kinase ◽

Metabolic Effects ◽

Erk Pathway ◽

Double Knockout ◽

Nephron Progenitor ◽

Progenitor Maintenance

Nephron endowment is defined by fetal kidney growth and it critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitor maintenance, propagation, and differentiation, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor regulation remain poorly studied. Here we have analyzed the metabolic effects that depend on and are triggered by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is an essential intracellular cascade required for nephron progenitor maintenance. Our combined approach utilizing LC/MS-based metabolomics and transcriptional profiling of MAPK/ERK-deficient cells identified 18 out of total 46 metabolites (38 untargeted and 8 targeted) that were down-regulated. These represent glycolysis, gluconeogenesis, pentose phosphate, glycine, and proline pathways among others. We focused our functional characterization of identified metabolites on pyruvate and proline. Use of in vitro kidney cultures revealed dosage-specific functions for pyruvate in not only controlling ureteric bud branching but also determining progenitor and differentiated (tip-trunk) cell identities. Our in vivo characterization of Pycr1/2 double knockout kidneys revealed functional requirement for proline metabolism in nephron progenitor maintenance. In summary, our results demonstrate that MAPK/ERK cascade regulates energy and amino acid metabolism in developing kidney where these metabolic pathways specifically regulate progenitor preservation.

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Characterization of MazFSa, an Endoribonuclease from Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.01272-07 ◽

2007 ◽

Vol 189 (24) ◽

pp. 8871-8879 ◽

Cited By ~ 57

Author(s):

Zhibiao Fu ◽

Niles P. Donegan ◽

Guido Memmi ◽

Ambrose L. Cheung

Keyword(s):

Staphylococcus Aureus ◽

Environmental Stress Response ◽

Binding Studies ◽

Cell Growth Arrest ◽

Consensus Sequences ◽

E Coli ◽

Endoribonuclease Activity

ABSTRACT The mazEF homologs of Staphylococcus aureus, designated mazEFsa , have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF Sa , as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF Sa leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF Sa is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF Sa cleaves single-strand RNA preferentially at the 5′ side of the first U or 3′ side of the second U residue within the consensus sequences VUUV′ (where V and V′ are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE Sa binds MazF Sa to form a complex to inhibit the endoribonuclease activity of MazF Sa . Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEFsa transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.

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Molecular and functional characterization of somatostatin-type signalling in a deuterostome invertebrate

Open Biology ◽

10.1098/rsob.200172 ◽

2020 ◽

Vol 10 (9) ◽

pp. 200172

Author(s):

Ya Zhang ◽

Luis Alfonso Yañez Guerra ◽

Michaela Egertová ◽

Cleidiane G. Zampronio ◽

Alexandra M. Jones ◽

...

Keyword(s):

Functional Characterization ◽

The Body ◽

Pharmacological Characterization ◽

Cardiac Stomach ◽

Physiological Processes ◽

Pharmacological Tests ◽

The Central Nervous System

Somatostatin (SS) and allatostatin-C (ASTC) are structurally and evolutionarily related neuropeptides that act as inhibitory regulators of physiological processes in mammals and insects, respectively. Here, we report the first molecular and functional characterization of SS/ASTC-type signalling in a deuterostome invertebrate—the starfish Asterias rubens (phylum Echinodermata). Two SS/ASTC-type precursors were identified in A. rubens (ArSSP1 and ArSSP2) and the structures of neuropeptides derived from these proteins (ArSS1 and ArSS2) were analysed using mass spectrometry. Pharmacological characterization of three cloned A. rubens SS/ASTC-type receptors (ArSSR1–3) revealed that ArSS2, but not ArSS1, acts as a ligand for all three receptors. Analysis of ArSS2 expression in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed stained cells/fibres in the central nervous system, the digestive system (e.g. cardiac stomach) and the body wall and its appendages (e.g. tube feet). Furthermore, in vitro pharmacological tests revealed that ArSS2 causes dose-dependent relaxation of tube foot and cardiac stomach preparations, while injection of ArSS2 in vivo causes partial eversion of the cardiac stomach. Our findings provide new insights into the molecular evolution of SS/ASTC-type signalling in the animal kingdom and reveal an ancient role of SS-type neuropeptides as inhibitory regulators of muscle contractility.

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Functional and Genomic Characterization of Ligilactobacillus salivarius TUCO-L2 Isolated From Lama glama Milk: A Promising Immunobiotic Strain to Combat Infections

Frontiers in Microbiology ◽

10.3389/fmicb.2020.608752 ◽

2020 ◽

Vol 11 ◽

Author(s):

Sandra Quilodrán-Vega ◽

Leonardo Albarracin ◽

Flavia Mansilla ◽

Lorena Arce ◽

Binghui Zhou ◽

...

Keyword(s):

Epithelial Cells ◽

Functional Characterization ◽

Comparative Genomic ◽

Cytokines And Chemokines ◽

Isolation And Characterization ◽

Intestinal Pathogens ◽

Lama Glama

Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama.

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Functional characterization of human Kindlin-2 core promoter identifies a key role of SP1 in Kindlin-2 transcriptional regulation

Cellular & Molecular Biology Letters ◽

10.2478/s11658-011-0028-6 ◽

2011 ◽

Vol 16 (4) ◽

Cited By ~ 1

Author(s):

Ammad Khan ◽

Takashi Shimokawa ◽

Staffan Strömblad ◽

Hongquan Zhang

Keyword(s):

Transcriptional Regulation ◽

Core Promoter ◽

Functional Characterization ◽

Ph Domain ◽

Interacting Protein ◽

Normal Tissues ◽

Functional Aspects

AbstractKindlin-2 is a recently identified FERM and PH domain containing integrin interacting protein. Kindlin-2 is ubiquitously expressed in normal tissues. So far, much effort has been spent exploring the functional aspects of Kindlin-2. However, the transcriptional regulation of Kindlin-2 has not yet been investigated. In this study we identified and functionally characterized the promoter of the human Kindlin-2 gene. We show that the core promoter of Kindlin-2 is a 39 base pair long GC rich fragment located −122/-83 upstream of the Kindlin-2 transcription start site. Functional characterization of this core promoter region by both in silico as well as in vitro/in vivo analysis shows that the transcription factor SP1 plays an important role in regulation of Kindlin-2 expression.

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