scholarly journals Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of Bacillus subtilis by Inclusion in a Model Membrane

2004 ◽  
Vol 186 (10) ◽  
pp. 3195-3201 ◽  
Author(s):  
Tim Searls ◽  
Xingyong Chen ◽  
Stephanie Allen ◽  
Michael D. Yudkin
Keyword(s):  
Bacillus Subtilis ◽  
Conformational Changes ◽  
Asymmetric Cell Division ◽  
Developmental Process ◽  
Model Membrane ◽  
In Vitro Assays ◽  
Kinetic Properties ◽  
Local Concentration ◽  
Phosphatase Domain

ABSTRACT Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor σF, and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of B. subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.

scholarly journals Mutational Analysis of Active Site Residues Essential for Sensing of Organic Hydroperoxides by Bacillus subtilis OhrR

2007 ◽  
Vol 189 (19) ◽  
pp. 7069-7076 ◽  
Author(s):  
Sumarin Soonsanga ◽  
Mayuree Fuangthong ◽  
John D. Helmann
Keyword(s):  
Hydrogen Bond ◽  
Bacillus Subtilis ◽  
Conformational Changes ◽  
Active Site ◽  
Mutational Analysis ◽  
High Sensitivity ◽  
Oxidative Modification ◽  
Active Site Residues

ABSTRACT Bacillus subtilis OhrR is the prototype for the one-Cys family of organic peroxide-sensing regulatory proteins. Mutational analyses indicate that the high sensitivity of the active site cysteine (C15) to peroxidation requires three Tyr residues. Y29 and Y40 from the opposing subunit of the functional dimer hydrogen bond with the reactive Cys thiolate, and substitutions at these positions reduce or eliminate the ability of OhrR to respond to organic peroxides. Y19 is also critical for peroxide sensing, and the Ala substitution mutant (OhrR Y19A) is less susceptible to oxidation at the active site C15 in vivo. The Y19A protein also displays decreased sensitivity to peroxide-mediated oxidation in vitro. Y19 is in van der Waals contact with two residues critical for protein function, F16 and R23. The latter residue makes critical contact with the DNA backbone in the OhrR-operator complex. These results indicate that the high sensitivity of the OhrR C15 residue to oxidation requires interactions with the opposed Tyr residues. Oxidative modification of C15 likely disrupts the C15-Y29′-Y40′ hydrogen bond network and thereby initiates conformational changes that reduce the ability of OhrR to bind to its operator site.

scholarly journals In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors

2016 ◽  
Vol 21 (10) ◽  
pp. 1034-1041 ◽  
Author(s):  
Scott P. Salowe ◽  
Lei Zhang ◽  
Hratch J. Zokian ◽  
Jennifer J. Gesell ◽  
Deborah L. Zink ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Resonance Energy Transfer ◽  
Density Lipoprotein ◽  
Resonance Energy ◽  
In Vitro Study ◽  
In Vitro Assays ◽  
Time Resolved

PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escherichia coli for the in vitro study of autoprocessing. Using automated Western analysis, we demonstrate that autoprocessing exhibits the anticipated first-order kinetics. A high-throughput time-resolved fluorescence resonance energy transfer assay for autocleavage has been developed using a PCSK9 monoclonal antibody that is sensitive to the conformational changes that occur upon maturation of the proprotein. Kinetic theory has been developed that describes the behavior of both reversible and irreversible inhibitors of autocleavage. The analysis of an irreversible lactone inhibitor validates the expected relationship between potency and the reaction end point. An orthogonal liquid chromatography–mass spectrometry assay has also been implemented for the confirmation of hits from the antibody-based assays.

Purification, Kinetic Properties, and Intracellular Concentration of SpoIIE, an Integral Membrane Protein That Regulates Sporulation in Bacillus subtilis

1999 ◽  
Vol 181 (10) ◽  
pp. 3242-3245 ◽  
Author(s):  
Isabelle Lucet ◽  
Rainer Borriss ◽  
Michael D. Yudkin
Keyword(s):  
Bacillus Subtilis ◽  
Kinetic Studies ◽  
Integral Membrane Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Bifunctional Protein ◽  
Molar Basis ◽  
Quantitative Immunoblotting

ABSTRACT SpoIIE is a bifunctional protein which controls ςFactivation and formation of the asymmetric septum in sporulatingBacillus subtilis. The spoIIE gene of B. subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated.

scholarly journals Natural Variability in S-Adenosylmethionine (SAM)-Dependent Riboswitches: S-Box Elements in Bacillus subtilis Exhibit Differential Sensitivity to SAM In Vivo and In Vitro

2007 ◽  
Vol 190 (3) ◽  
pp. 823-833 ◽  
Author(s):  
Jerneja Tomšič ◽  
Brooke A. McDaniel ◽  
Frank J. Grundy ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Structural Rearrangement ◽  
Natural Variability ◽  
Differential Sensitivity ◽  
In Vitro Assays ◽  
Leader Region ◽  
Structural Elements ◽  
Wide Range

ABSTRACT Riboswitches are regulatory systems in which changes in structural elements in the 5′ region of the nascent RNA transcript (the “leader region”) control expression of the downstream coding sequence in response to a regulatory signal in the absence of a trans-acting protein factor. The S-box riboswitch, found primarily in low-G+C gram-positive bacteria, is the paradigm for riboswitches that sense S-adenosylmethionine (SAM). Genes in the S-box family are involved in methionine metabolism, and their expression is induced in response to starvation for methionine. S-box genes exhibit conserved primary sequence and secondary structural elements in their leader regions. We previously demonstrated that SAM binds directly to S-box leader RNA, causing a structural rearrangement that results in premature termination of transcription at S-box leader region terminators. S-box genes have a variety of physiological roles, and natural variability in S-box structure and regulatory response could provide additional insight into the role of conserved S-box leader elements in SAM-directed transcription termination. In the current study, in vivo and in vitro assays were employed to analyze the differential regulation of S-box genes in response to SAM. A wide range of responses to SAM were observed for the 11 S-box-regulated transcriptional units in Bacillus subtilis, demonstrating that S-box riboswitches can be calibrated to different physiological requirements.

scholarly journals PK11195, a peripheral benzodiazepine receptor (pBR) ligand, broadly blocks drug efflux to chemosensitize leukemia and myeloma cells by a pBR-independent, direct transporter-modulating mechanism

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3584-3593 ◽  
Author(s):  
Roland B. Walter ◽  
Jason L. Pirga ◽  
Michelle R. Cronk ◽  
Sasha Mayer ◽  
Frederick R. Appelbaum ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ectopic Expression ◽  
Leukemia Cell ◽  
Benzodiazepine Receptor ◽  
In Vitro Assays ◽  
Drug Efflux ◽  
Peripheral Benzodiazepine Receptor ◽  
Cancer Resistance ◽  
Resistance Protein

AbstractThe peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux to chemosensitize cancer cells at least as well or better than the Pgp modulator, cyclosporine A (CSA). We now show that PK11195 broadly inhibits adenosine triphosphate (ATP)-binding cassette (ABC) transporters in hematologic cancer cell lines and primary leukemia-cell samples, including multidrug resistance protein (MRP), breast cancer resistance protein (BCRP), and/or Pgp. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but showed that pBR expression is unnecessary to PK11195-mediated efflux inhibition. PK11195 binds plasma-membrane sites in Pgp-expressing cells, stimulates Pgp-associated adenosine triphosphatase (ATPase) activity, and causes conformational changes in Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bind noncompetitively in Pgp-expressing cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites. Importantly, PK11195 concentrations that were effective in these in vitro assays can be safely achieved in patients. Because PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism and broadly blocks drug efflux by an apparently pBR-independent, ABC transporter-dependent mechanism, PK11195 may be a useful clinical chemosensitizer in cancer patients.

scholarly journals Biocontrollers in the management of yam dry rot nematodes

2021 ◽  
Vol 6 (1) ◽  
pp. 24-35
Author(s):  
Cecilia Ramirez ◽  
Alverlan da Silva Araújo ◽  
Gilson Moura Filho ◽  
Fernando da Silva Rocha ◽  
Marylia Gabriella Silva Costa ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Organic Carbon ◽  
Nematode Population ◽  
In Vitro Assays ◽  
Population Densities ◽  
Dry Rot ◽  
Seed Tubers ◽  
Bacillus Spp

ABSTRACT: One of the main diseases affecting yam crops (Dioscorea spp.) in Brazil is the dry rot caused by Scutellonema bradys, Pratylenchus brachyurus and P. coffeae nematodes. The use of biological control agents is an auspicious procedure which has been tested in order to reduce losses by pathogens. The objective of this work was to evaluate the nematicidal activity in vitro and in vivo of commercial biological products on yam dry rot nematodes. Products based on Trichoderma harzianum (2.0 x 109 conidia mL-1) at dosages of 1.5 and 2.0 L 200 L-1 of water; Bacillus subtilis 20% - 1.0 x 1011 cfu g-1 + B. licheniformis 20% - 1.0 x 1011 cfu g-1 at 100 and 150 g 100 L-1; B. subtilis 200 g kg-1 + B. licheniformis 200 g kg-1 at 130 and 200 g 100 L-1; combination of rhizobacteria including Bacillus spp. and organic carbon at 5L and 7L 100 L-1; and the control (distilled water), were tested in in vitro assays on S. bradys or Pratylenchus sp. In experiments performed under greenhouse conditions, healthy seed tubers were planted in sterilized soil and thirty days later the soil was infested with a suspension of 1,000 specimens of a mixed population of S. bradys and P. coffeae. Then, after 30 days products based on B. subtilis 20% + B. licheniformis 20% - 150 g 100 L-1; T. harzianum 2 L 200 L-1 and rhizobacteria + organic carbon 7 L 100 L-1, at 100 mL per pot, were applied to the soil. Three months after planting, the percentage of sprouting of the seed tubers was evaluated and in the fifth month, the nematode population densities were determined. The sprouting of seed tubers was of 100% in all treatments. Bacillus subtilis 20% + B. licheniformis 20% and T. harzianum caused 89% and 61% mortality in S. bradys respectively, at the highest concentrations. In specimens of Pratylenchus sp., rhizobacteria + organic carbon exhibited 51% and 45% mortality at higher and lower concentrations, respectively. Under greenhouse conditions, B. subtilis 20% + B. licheniformis 20% and rhizobacteria + organic carbon were more effective in reducing nematode population densities, compared to the control. KEYWORDS: Dioscorea spp.; Scutellonema bradys; Pratylenchus sp.

Self-stabilized fibronectin films at the air/water interface

MRS Advances ◽  
2019 ◽  
Vol 5 (12-13) ◽  
pp. 609-620
Author(s):  
Thanga Bhuvanesh ◽  
Rainhard Machatschek ◽  
Yue Liu ◽  
Nan Ma ◽  
Andreas Lendlein
Keyword(s):  
Conformational Changes ◽  
Cell Attachment ◽  
Loss Modulus ◽  
Local Concentration ◽  
Interfacial Rheology ◽  
Self Assembling ◽  
Sheet Structure ◽  
Enhance Cell Attachment ◽  
Biomaterial Surfaces

ABSTRACTFibronectin (FN) is a mediator molecule, which can connect cell receptors to the extracellular matrix (ECM) in tissues. This function is highly desirable for biomaterial surfaces in order to support cell adhesion. Controlling the fibronectin adsorption profile on substrates is challenging because of possible conformational changes after deposition, or due to displacement by secondary proteins from the culture medium. Here, we aim to develop a method to realize self-stabilized ECM glycoprotein layers with preserved native secondary structure on substrates. Our concept is the assembly of FN layers at the air-water (A-W) interface by spreading FN solution as droplets on the interface and transfer of the layer by the Langmuir-Schäfer (LS) method onto a substrate. It is hypothesized that 2D confinement and high local concentration at A-W interface supports FN self-interlinking to form cohesive films. Rising surface pressure with time, plateauing at 10.5 mN·m-1 (after 10 hrs), indicated that FN was self-assembling at the A-W interface. In situ polarization-modulation infrared reflection absorption spectroscopy of the layer revealed that FN maintained its native anti-parallel β-sheet structure after adsorption at the A-W interface. FN self-interlinking and elasticity was shown by the increase in elastic modulus and loss modulus with time using interfacial rheology. A network-like structure of FN films formed at the A-W interface was confirmed by atomic force microscopy after LS transfer onto Si-wafer. FN films consisted of native, globular FN molecules self-stabilized by intermolecular interactions at the A-W interface. Therefore, the facile FN self-stabilized network-like films with native anti-parallel β-sheet structure produced here, could serve as stable ECM protein coatings to enhance cell attachment on in vitro cell culture substrates and planar implant materials.

scholarly journals New Insights into Structural and Functional Roles of Indole-3-acetic acid (IAA): Changes in DNA Topology and Gene Expression in Bacteria

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 522 ◽  
Author(s):  
Defez ◽  
Valenti ◽  
Andreozzi ◽  
Romano ◽  
Ciaramella ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acetic Acid ◽  
Conformational Changes ◽  
Regulation Of Gene Expression ◽  
Dna Supercoiling ◽  
Dna Topology ◽  
In Vitro Assays ◽  
Bacterial Cells ◽  
Indole 3 Acetic Acid

: Indole-3-acetic acid (IAA) is a major plant hormone that affects many cellular processes in plants, bacteria, yeast, and human cells through still unknown mechanisms. In this study, we demonstrated that the IAA-treatment of two unrelated bacteria, the Ensifer meliloti 1021 and Escherichia coli, harboring two different host range plasmids, influences the supercoiled state of the two plasmid DNAs in vivo. Results obtained from in vitro assays show that IAA interacts with DNA, leading to DNA conformational changes commonly induced by intercalating agents. We provide evidence that IAA inhibits the activity of the type IA topoisomerase, which regulates the DNA topological state in bacteria, through the relaxation of the negative supercoiled DNA. In addition, we demonstrate that the treatment of E. meliloti cells with IAA induces the expression of some genes, including the ones related to nitrogen fixation. In contrast, these genes were significantly repressed by the treatment with novobiocin, which reduces the DNA supercoiling in bacterial cells. Taking into account the overall results reported, we hypothesize that the IAA action and the DNA structure/function might be correlated and involved in the regulation of gene expression. This work points out that checking whether IAA influences the DNA topology under physiological conditions could be a useful strategy to clarify the mechanism of action of this hormone, not only in plants but also in other unrelated organisms.

scholarly journals Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation

2016 ◽  
Vol 198 (24) ◽  
pp. 3345-3354 ◽  
Author(s):  
Víctor M. Ayala-García ◽  
Luz I. Valenzuela-García ◽  
Peter Setlow ◽  
Mario Pedraza-Reyes
Keyword(s):  
Bacillus Subtilis ◽  
Developmental Process ◽  
Specific Pattern ◽  
Dna Glycosylase ◽  
Regulatory Sequences ◽  
Regulation Of Expression ◽  
Content Type ◽  
Mutagenic Effects

ABSTRACTAag fromBacillus subtilishas been implicated inin vitroremoval of hypoxanthine and alkylated bases from DNA. The regulation of expression ofaaginB. subtilisand the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptionalaag-lacZfusion expressed low levels of β-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably,aag-lacZexpression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in asigG-deficient background, suggesting a forespore-specific mechanism ofaagtranscription. Two additional findings supported this suggestion: (i) expression of anaag-yfpfusion was observed in the forespore, and (ii)in vivomapping of theaagtranscription start site revealed the existence of upstream regulatory sequences possessing homology to σG-dependent promoters. In comparison with the wild-type strain, disruption ofaagsignificantly reduced survival of sporulatingB. subtiliscells following nitrous acid or methyl methanesulfonate treatments, and the Rifrmutation frequency was significantly increased in anaagstrain. These results suggest that Aag protects the genome of developingB. subtilissporangia from the cytotoxic and genotoxic effects of base deamination and alkylation.IMPORTANCEIn this study, evidence is presented revealing thataag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression duringB. subtilissporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.

scholarly journals A 15-base-pair element activates the SPS4 gene midway through sporulation in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (7) ◽  
pp. 3934-3944 ◽  
Author(s):  
S R Hepworth ◽  
L K Ebisuzaki ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Developmental Process ◽  
Regulatory Region ◽  
Spore Wall ◽  
In Vitro Assays ◽  
Flanking Sequence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Expression

Sporulation of the yeast Saccharomyces cerevisiae represents a simple developmental process in which the events of meiosis and spore wall formation are accompanied by the sequential activation of temporally distinct classes of genes. In this study, we have examined expression of the SPS4 gene, which belongs to a group of genes that is activated midway through sporulation. We mapped the upstream boundary of the regulatory region of SPS4 by monitoring the effect of sequential deletions of 5'-flanking sequence on expression of plasmid-borne versions of SPS4 introduced into a MATa/MAT alpha delta sps4/delta sps4 strain. This analysis indicated that the 5' boundary of the regulatory region was within 50 bp of the putative TATA box of the gene. By testing various oligonucleotides that spanned this boundary and the downstream sequence for their ability to activate expression of a heterologous promoter, we found that a 15-bp sequence sufficed to act as a sporulation-specific upstream activation sequence. This 15-bp fragment, designated UASSPS4, activated expression of a CYC1-lacZ reporter gene midway through sporulation and was equally active in both orientations. Extending the UAS fragment to include the adjacent 14-bp enhanced its activity 10-fold. We show that expression of SPS4 is regulated in a manner distinct from that of early meiotic genes: mutation of UME6 did not lead to vegetative expression of SPS4, and sporulation-specific expression was delayed by mutation of IME2. In vivo and in vitro assays suggested that a factor present in vegetative cells bind to the UASSPS4 element. We speculate that during sporulation this factor is modified to serve as an activator of the SPS4 gene or, alternatively, that it recruits an activator to the promoter.

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