scholarly journals Analysis of a DtxR-Like Metalloregulatory Protein, MntR, from Corynebacterium diphtheriae That Controls Expression of an ABC Metal Transporter by an Mn2+-Dependent Mechanism

2002 ◽  
Vol 184 (24) ◽  
pp. 6882-6892 ◽  
Author(s):  
Michael P. Schmitt
Keyword(s):  
Binding Site ◽  
Genetic System ◽  
Corynebacterium Diphtheriae ◽  
Dnase I ◽  
Transport Systems ◽  
Binding Studies ◽  
Metal Transporters ◽  
Metal Transporter ◽  
Multiple Promoters ◽  
Metalloregulatory Proteins

ABSTRACT The DtxR protein is a global iron-dependent repressor in Corynebacterium diphtheriae that regulates transcription from multiple promoters. A search of the partially completed C. diphtheriae genome identified a gene, mntR, whose predicted product has significant homology with the DtxR repressor protein. The mntR gene is the terminal gene in a five-gene operon that also carries the mntABCD genes, whose predicted products are homologous to ABC metal transporters. Transcription of this genetic system, as measured by expression of an mntA-lacZ reporter fusion, is strongly repressed by Mn2+. The divalent metals Fe2+, Cu2+, and Zn2+ did not repress expression of the mntA-lacZ construct. A mutation in the mntR gene abolished Mn2+-dependent repression of the mntA-lacZ fusion, demonstrating that MntR is essential for the Mn2+-dependent regulation of this promoter. Footprinting experiments showed that MntR protects from DNase I digestion an approximately 73-bp AT-rich region that includes the entire mntA promoter. This large region protected from DNase I suggests that as many as three MntR dimer pairs may bind to this region. Binding studies also revealed that DtxR failed to bind to the MntR binding site and that MntR exhibited weak and diffuse binding at the DtxR binding site at the tox promoter. A C. diphtheriae mntA mutant grew as well as the wild type in a low-Mn2+ medium, which suggests that the mntABCD metal transporter is not required for growth in a low-Mn2+ medium and that additional Mn2+ transport systems may be present in C. diphtheriae. This study reports the characterization of MntR, a Mn2+-dependent repressor, and the second member of the family of DtxR-like metalloregulatory proteins to be identified in C. diphtheriae.

Improving the phytoextraction capacity of plants to scavenge metal(loid)-contaminated sites

2015 ◽  
Vol 23 (1) ◽  
pp. 44-65 ◽  
Author(s):  
Muhammad Iqbal ◽  
Altaf Ahmad ◽  
M.K.A. Ansari ◽  
M.I. Qureshi ◽  
Ibrahim M. Aref ◽  
...  
Keyword(s):  
Metal Accumulation ◽  
Single Gene ◽  
Specific Protein ◽  
Transport Systems ◽  
Contaminated Sites ◽  
High Concentration ◽  
Metal Transporters ◽  
Factors Affecting ◽  
Metal Transporter ◽  
Genomics And Proteomics

Plants are able to extract metal(loid) contaminants from the soil or water through their roots and translocate them to harvestable aerial shoots. Of late, this plant potential has been used as a phytotechnology, termed as phytoextraction, for cleaning contaminated sites, and this process has successfully removed elements like As, Cd, Cu, Ni, and Pb, among others. Exploring plants with high metal-accumulation capacity, as well as engineering new hyperaccumulators, is a need of the hour. It is assumed that hyperaccumulators have a >1 shoot:root metal-accumulation ratio, which they achieve by way of (i) overexpression of transport systems for improved sequestration, (ii) tissue-specific protein expression, and (iii) high concentration of metal chelators. Unlike nonhyperaccumulators, the hyperaccumulating species normally bind metal ions to weak oxygen ligands and use strong ligands only for transient binding during transport to storage sites. Adequate understanding of genetics, biochemistry, and molecular biology of metal accumulation is a prelude to developing transgenics with improved phytoremediation capacity. Current research in plant breeding, genomics, and proteomics suggest promising leads to the creation of “remediation” cultivars. Several transporter genes associated with metal uptake, transport, and accumulation have been identified. Efforts are underway to enhance the phytoextraction capacity of relevant species, not only by using chelating agents but also by attempting hybridization, protoplast fusion, as well as genetic engineering through novel gene transfer, overexpression of genes, and (or) reverse gene insertion, to enhance (i) transpiration rate; (ii) uptake, translocation, and metabolism of metals; (iii) activity of enzymes related to rate-limiting steps; and (iv) transformation of accumulated metal to volatile forms, and (or) silencing gene(s) that encode proteases. Genome evolution in hyperaccumulators needs to be understood through a systematic study of ecological and molecular genomics. Sequencing of a complete genome of hyperaccumulators can help in identifying the promising functional noncoding regions in the genome, thus making the experimental analysis more accurate. In addition to the constitutive overexpression of a single gene, simultaneous expression of several genes in specific cellular components has to be focused. Other areas that require expert attention include identification of metal-transporter proteins and the introduction of genes encoding the metal transporters, overexpression of metallothioneins and phytochelatin synthase, and overproduction of nicotianamine and histidine in plants. A comprehensive study of transgenic gene frequency, covering several plant generations growing on polluted as well as nonpolluted soils, may assess the possibility of gene escape into the environment and its transfer to the microorganisms present in the surroundings. This review attempts not only to collect and collate information available on mechanisms of metal accumulation and detoxification in plants and on the factors affecting the tolerance and phytoextraction capacity of plants but also the strategies that have been or can be devised for raising novel plant genotypes with elevated capacity of metal accumulation and toxicity tolerance.

scholarly journals The Role of Aquaporin Overexpression in the Modulation of Transcription of Heavy Metal Transporters under Cadmium Treatment in Poplar

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Andrea Neri ◽  
Silvia Traversari ◽  
Andrea Andreucci ◽  
Alessandra Francini ◽  
Luca Sebastiani
Keyword(s):  
Heavy Metal ◽  
Transcriptional Response ◽  
Metal Transporters ◽  
Metal Transporter ◽  
Cadmium Treatment ◽  
Tolerance To Cadmium ◽  
Different Response ◽  
Cd Translocation ◽  
Hydroponic Conditions

Populus alba ‘Villafranca’ clone is well-known for its tolerance to cadmium (Cd). To determine the mechanisms of Cd tolerance of this species, wild-type (wt) plants were compared with transgenic plants over-expressing an aquaporin (aqua1, GenBank GQ918138). Plants were maintained in hydroponic conditions with Hoagland’s solution and treated with 10 µM of Cd, renewed every 5 d. The transcription levels of heavy metal transporter genes (PaHMA2, PaNRAMP1.3, PaNRAMP2, PaNRAMP3.1, PaNRAMP3.2, PaABCC9, and PaABCC13) were analyzed at 1, 7, and 60 d of treatment. Cd application did not induce visible toxicity symptoms in wt and aqua1 plants even after 2 months of treatment confirming the high tolerance of this poplar species to Cd. Most of the analyzed genes showed in wt plants a quick response in transcription at 1 d of treatment and an adaptation at 60 d. On the contrary, a lower transcriptional response was observed in aqua1 plants in concomitance with a higher Cd concentration in medial leaves. Moreover, PaHMA2 showed at 1 d an opposite trend within organs since it was up-regulated in root and stem of wt plants and in leaves of aqua1 plants. In summary, aqua1 overexpression in poplar improved Cd translocation suggesting a lower Cd sensitivity of aqua1 plants. This different response might be due to a different transcription of PaNRAMP3 genes that were more transcribed in wt line because of the importance of this gene in Cd compartmentalization.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

1991 ◽  
Vol 11 (2) ◽  
pp. 1048-1061
Author(s):  
I J Lee ◽  
L Tung ◽  
D A Bumcrot ◽  
E S Weinberg
Keyword(s):  
Binding Site ◽  
Sea Urchin ◽  
Transcriptional Start Site ◽  
Sodium Dodecyl ◽  
Nuclear Extract ◽  
Dnase I ◽  
Histone H4 ◽  
Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
H. Ronald Kaback
Keyword(s):  
Binding Site ◽  
Lactose Permease ◽  
Binding Studies ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Transport Cycle ◽  
Open Structures ◽  
Unrestricted Access ◽  
Tight Association ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

A crosslinked and ribosylated actin trimer does not interact productively with myosin

2019 ◽  
Vol 97 (2) ◽  
pp. 140-147 ◽  
Author(s):  
Navneet Sidhu ◽  
John F. Dawson
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Binding Protein ◽  
Dnase I ◽  
Protein Protein Interactions ◽  
Adp Ribosylation ◽  
Myosin Binding ◽  
Pointed End

A purified F-actin-derived actin trimer that interacts with end-binding proteins did not activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-ribosylation on all actin subunits of an F-actin-derived trimer inhibits myosin binding and that the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the myosin binding site. Taken together, this work highlights the need for a careful balance between modification of actin subunits and maintaining protein–protein interactions to produce a physiologically relevant short F-actin complex.

The Corynebacterium diphtheriae HbpA hemoglobin-binding protein contains a domain that is critical for hemoprotein-binding, cellular localization and function.

2021 ◽  
Author(s):  
Lindsey R. Lyman ◽  
Eric D. Peng ◽  
Michael P. Schmitt
Keyword(s):  
Cellular Localization ◽  
Binding Protein ◽  
Protein Localization ◽  
Iron Acquisition ◽  
Corynebacterium Diphtheriae ◽  
Terminal Region ◽  
Surface Proteins ◽  
Size Exclusion ◽  
Transport Systems ◽  
Iron Source

The acquisition of hemin-iron from hemoglobin-haptoglobin (Hb-Hp) by Corynebacterium diphtheriae requires the iron-regulated surface proteins HtaA, ChtA, ChtC, and the recently identified Hb-Hp binding protein HbpA. We previously showed that a purified form of HbpA (HbpA-S), lacking the C-terminal region, was able to bind Hb-Hp. In this study, we show that the C-terminal region of HbpA significantly enhances binding to Hb-Hp. A purified form of HbpA that includes the C-terminal domain (HbpA-FL) exhibits much stronger binding to Hb-Hp than HbpA-S. Size exclusion chromatography (SEC) showed that HbpA-FL as well as HtaA-FL, ChtA-FL, and ChtC-FL exist as high molecular weight complexes, while HbpA-S is present as a monomer, indicating that the C-terminal region is required for formation of large aggregates. Growth studies showed that expression of HbpA-FL in the Δ hbpA mutant restored wild-type levels of growth in low-iron medium that contained Hb-Hp as the sole iron source, while HbpA-S failed to complement the Δ hbpA mutant. Protein localization studies in C. diphtheriae showed that HbpA-FL is present in both in the supernatant and in the membrane fractions, and that the C-terminal region is required for membrane anchoring. Purified HbpA-FL was able to enhance growth of the Δ hbpA mutant when added to culture medium that contained Hb-Hp as a sole iron source, suggesting that secreted HbpA is involved in the use of hemin-iron from Hb-Hp. These studies extend our understanding of this novel Hb-Hp binding protein in this important human pathogen. IMPORTANCE Hemoproteins, such as Hb, are an abundant source of iron in humans and are proposed to be required by numerous pathogens to cause disease. In this report, we expand on our previous studies in further defining the role of HbpA in hemin-iron acquisition in C. diphtheriae . HbpA is unique to C. diphtheriae , and appears to function unlike any previously described bacterial iron-regulated Hb- or Hb-Hp-binding protein. HbpA is both secreted and present in the membrane, and exists as a large aggregate that enhances its ability to bind Hb-Hp and promote hemin-iron uptake. Current studies with HbpA will increase our understanding of iron transport systems in C. diphtheriae .

Local and Distant Elements Regulate Tissue-Specific Expression of ANK-1 Gene Transcripts

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2432-2432
Author(s):  
Ashley N. Owen ◽  
Karina Laflamme ◽  
Andre M. Pilon ◽  
Lisa J. Garrett ◽  
Patrick G. Gallagher ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Histone Acetylation ◽  
Binding Site ◽  
Consensus Sequence ◽  
Dnase I ◽  
Transient Transfection ◽  
Chromatin Loop ◽  
Erythroid Cells ◽  
Specific Expression

Abstract Fewer than 20,000 protein-coding genes in the human genome generate more than 100,000 proteins. This diversity results from the selective use of alternative promoters and alternative mRNA splicing. Ankyrins are multifunctional linker/adapter proteins with isoforms expressed in cell-, tissue-, and developmental stage-specific patterns. The ANK-1 gene, which encodes a series of proteins that connect the red blood cell (RBC) membrane to the RBC skeleton, is an excellent system to study how specific promoters are selected for expression and others suppressed. The human ANK-1 locus has two tissue-specific promoters/first exons (erythroid, 1E; brain/muscle, 1B) and one ubiquitous promoter/first exon (1A). We have previously shown that the ANK-1E promoter sequences are contained in the 300 base pairs (bp) immediately upstream of exon 1E (including a critical GATA-1 binding site) are necessary for erythroid-specific expression in transgenic mice. We have recently reported a novel 9 base consensus sequence ([G/T][G/C][G/C]GGTGAG) located between +7 and +15 that serves as a binding site for the transcription initiation complex. This consensus is present in the other ANK-1 promoters, 30% of all mammalian promoters, and is highly enriched in those that lack known consensus elements (i.e, TATA box; Laflamme et al. submitted). We hypothesized that variation within this consensus sequence controls the level of mRNA transcription. We evaluated altered consensus sequences in the ANK-1E promoter linked to luciferase or gamma-globin reporter genes in transient transfection assays in erythroid K562 cells or transgenic mice, respectively. In both assays, the GCGGGTGAG sequence generated 7-fold higher levels of expression than the wild type sequence (TGCGGTGAG; p<0.01), while other variations gave similar or lower levels of expression. We concluded that while erythroid specificity of the minimal ANK-1E promoter is conferred by GATA-1 binding, the level of expression is controlled by the ([G/T][G/C][G/C]GGTGAG) box. In transient transfection assays in vitro, where the constraints of chromatin are released, the sequences adjacent to ANK-1E and ANK-1A promoters directed equivalent levels of expression in both erythroid and non-erythroid cells. We hypothesized that the activity of the ANK-1E promoter in vivo is controlled by both the core promoter sequence and the local chromatin architecture. Transcriptionally active regions of chromatin show increased sensitivity to DNase I digestion, which we have analyzed across a 200 kb region encompassing all three ANK-1 promoters. A region between the ANK-1E and ANK-1A promoters was sensitive to DNase I digestion only in erythroid cells, while the upstream (1B) and downstream (1A) regions were DNase I resistant. The 1E to 1A region is flanked by DNase hypersensitive sites (HS): one immediately 5′ to 1E (5′HS), and two adjacent HS (3′HS1, 3′HS2) located ~6 kb downstream. Histone acetylation is also associated with active chromatin. Chromatin Immunoprecipitation (ChIP) of the ANK-1E region showed erythroid-specific histone acetylation of the 6kb region between 5′HS and 3′HS1&2, with hyperacetylation at all three HS in all cell types. Barrier elements are found at the boundary between open and condensed chromatin. 5′HS provides a barrier against transgene silencing in cell lines and transgenic mice (p<0.01). 3′HS2 contains barrier activity in transfected cells (p<0.01), while the combination of 3′HS1 and 3′HS2 prevents silencing in transgenic mice (p<0.02). ChIP, EMSA (Mobility Shift Assay) and in vitro DNase I footprinting demonstrated that 3′HS1 binds the erythroid transcription factor NF-E2. In transient assays in erythroid cells, 3′HS1 increased reporter gene activity 5-fold when adjacent to the ANK-1E promoter. We hypothesized that NF-E2 could be translocated to the ANK-1E promoter by the formation of an internal chromatin loop. Chromatin Conformation Capture (3C) demonstrated the formation of a loop structure in which 5′HS and 3′HS1&2 are brought into physical proximity in erythroid, but not non-erythroid cells. In agreement with the 3C results, ChIP demonstrated that both ends of the ANK-1E chromatin loop bind GATA-1, NF-E2 and RNA Pol II. Our current model predicts that the 5′ HS barrier allows the ANK-1E promoter to function in transgenic mice, but in the native locus, ANK-1E promoter activity requires the formation of a chromatin loop mediated by GATA-1 and NF-E2.

scholarly journals The Divergently Transcribed Streptococcus parasanguis Virulence-Associated fimA Operon Encoding an Mn2+-Responsive Metal Transporter and pepO Encoding a Zinc Metallopeptidase Are Not Coordinately Regulated

2002 ◽  
Vol 70 (10) ◽  
pp. 5706-5714 ◽  
Author(s):  
Joyce Oetjen ◽  
Paula Fives-Taylor ◽  
Eunice H. Froeliger
Keyword(s):  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Divalent Metal Ions ◽  
Metal Transporters ◽  
Metal Transporter ◽  
Gram Positive Bacteria ◽  
Growth Deficiency ◽  
Domain Mapping ◽  
Transcriptional Start Sites

ABSTRACT The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn2+ concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn2+. This deficiency was not alleviated by compensation with either Mg2+ or Fe3+. Wild-type S. parasanguis could take up Mn2+ and Fe3+, while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn2+ and Fe3+. FimA expression itself was shown to be responsive to Mn2+ concentration, but not to availability of Fe3+ or Mg2+.

scholarly journals Modification of Cys-837 identifies an actin-binding site in the beta-propeller protein scruin.

1997 ◽  
Vol 8 (3) ◽  
pp. 421-430 ◽  
Author(s):  
S Sun ◽  
M Footer ◽  
P Matsudaira
Keyword(s):  
Chemical Modification ◽  
Binding Site ◽  
Solvent Accessibility ◽  
The Other ◽  
Actin Binding ◽  
Binding Region ◽  
Binding Studies ◽  
Chemically Modified ◽  
Cross Links ◽  
Actin Binding Site

In the acrosomal process of Limulus sperm, the beta-propeller protein scruin cross-links actin into a crystalline bundle. To confirm that scruin has the topology of a beta-propeller protein and to understand how scruin binds actin, we compared the solvent accessibility of cysteine residues in scruin and the acrosomal process by chemical modification with (1,5-IAEDANS). In soluble scruin, the two most reactive cysteines of soluble scruin are C837 and C900, whereas C146, C333, and C683 are moderately reactive. This pattern of reactivity is consistent with the topology of a typical beta-propeller protein; all of the reactive cysteines map to putative loops and turns whereas the unreactive cysteines lie within the predicted interior of the protein. The chemical reactivities of cysteine in the acrosomal process implicate C837 at an actin-binding site. In contrast to soluble scruin, in the acrosomal process, C837 is completely unreactive while the other cysteines become less reactive. Binding studies of chemically modified scruin correlate the extent of modification at C837 with the extent of inhibition of actin binding. Furthermore, peptides corresponding to residues flanking C837 bind actin and narrow a possible actin-binding region to a KQK sequence. On the basis of these studies, our results suggest that an actin-binding site lies in the C-terminal domain of scruin and involves a putative loop defined by C837.

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