Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors

ABSTRACT Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis. We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro. During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniae cop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.

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Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

10.1101/676700 ◽

2019 ◽

Cited By ~ 2

Author(s):

Henrik O’Brien ◽

Joseph W. Alvin ◽

Sanjay V. Menghani ◽

Koenraad Van Doorslaer ◽

Michael D. L. Johnson

Keyword(s):

Gene Regulation ◽

Streptococcus Pneumoniae ◽

Dna Binding ◽

Consensus Sequence ◽

Copper Chaperone ◽

Enterococcus Hirae ◽

Bacterial Gene ◽

Operator Sequence ◽

Operator Site

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Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

Journal of Bacteriology ◽

10.1128/jb.00249-15 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2383-2391 ◽

Cited By ~ 7

Author(s):

Semen A. Leyn ◽

Irina A. Rodionova ◽

Xiaoqing Li ◽

Dmitry A. Rodionov

Keyword(s):

Carbon Dioxide ◽

Transcriptional Regulation ◽

Comparative Genomics ◽

Dna Binding ◽

Transcriptional Control ◽

Binding Assays ◽

Promoter Regions ◽

Content Type ◽

Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

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Synthesis and Characterization of Oxidovanadium(IV) Complexes of 2-((E)-(6-Fluorobenzo[d]thiazol-2-ylimino)methyl)-6-methoxyphenol and Their Antimicrobial, Antioxidant, and DNA-Binding Studies

Bioinorganic Chemistry and Applications ◽

10.1155/2018/2452869 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 9

Author(s):

K. Savithri ◽

H. D. Revanasiddappa

Keyword(s):

Dna Binding ◽

Mixed Ligand Complex ◽

Radical Scavenging ◽

Molar Conductance ◽

Diffusion Method ◽

Spectral Studies ◽

Binding Studies ◽

Ligand Complex ◽

Intercalative Mode

Two novel oxidovanadium(IV) complexes with a new bidentate (O- and N-) imine-based ligand 2-((E)-(6-fluorobenzo[d]thiazol-2-ylimino)methyl)-6-methoxyphenol (HL) were synthesized under in situ experimental condition where VOSO4 acts as a kinetic template in the ratio 2 : 1 (L : M) and mixed ligand complex using 1,10-phenanthroline (phen) in 1 : 1 : 1 (L : M : phen) ratio. The synthesized compounds were structurally characterized by microanalysis, magnetic susceptibility, FTIR, electronic spectra, TG/DTA, ESR, and molar conductance studies. Based on the spectral studies, the complexes have the general composition [VO(L)2] (C1) and [VO(L)phen] (C2) in a square pyramid geometrical fashion. The synthesized compounds were primarily screened for their in vitro growth inhibiting activity against different strains of bacteria, namely, E. coli, B. subtilis, S. aureus, and P. aeruginosa by the disc diffusion method. Also, the antifungal activity was determined against C. albicans and A. niger by the Bateman poisoned technique. The in vitro antioxidant activity of all the compounds was determined by DPPH free radical-scavenging assay. Intercalative mode of DNA-binding properties of the oxidovanadium(IV) complexes with calf-thymus DNA (CT-DNA) was investigated using UV, fluorescence spectra, and viscosity measurements.

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Synthesis, characterization, DNA binding studies and in vitro cytotoxicity of platinum(II)-dihalogenido complexes containing bidentate chelating N-donor ligands

Journal of Coordination Chemistry ◽

10.1080/00958972.2016.1199862 ◽

2016 ◽

Vol 69 (16) ◽

pp. 2422-2436 ◽

Cited By ~ 11

Author(s):

Radka Křikavová ◽

Ján Vančo ◽

Tomáš Šilha ◽

Jaromír Marek ◽

Zdeněk Trávníček

Keyword(s):

Dna Binding ◽

In Vitro Cytotoxicity ◽

Donor Ligands ◽

Binding Studies ◽

Dna Binding Studies

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IN VITRO DNA BINDING STUDIES OF SELECTED HETEROCYCLIC COMPOUNDS

INDIAN DRUGS ◽

10.53879/id.55.12.10994 ◽

2018 ◽

Vol 55 (12) ◽

pp. 24-26

Author(s):

C Akhila ◽

◽

P Lalitha

Keyword(s):

Dna Binding ◽

Heterocyclic Compounds ◽

Uv Spectroscopy ◽

Binding Mode ◽

Spectroscopic Technique ◽

Binding Studies ◽

Base Pairs ◽

Dna Base ◽

Dna Binding Studies

DNA binding studies of selected heterocyclic compounds belonging to the class of quinolinones, substituted quinolinones and thiones were carried out using ct-DNA. The binding nature of the compounds with DNA analyzed using UV-spectroscopy revealed the compounds to be DNA intercalators demonstrating the binding nature of compounds with DNA base pairs. This study is aimed at establishing a facile UV spectroscopic technique to arrive at the binding mode of DNA to ligands.

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Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.964-971.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 964-971

Author(s):

R M Gronostajski ◽

S Adhya ◽

K Nagata ◽

R A Guggenheimer ◽

J Hurwitz

Keyword(s):

Dna Binding ◽

Hela Cell ◽

Dna Sequences ◽

Binding Sites ◽

Consensus Sequence ◽

Nuclear Factor ◽

Site Specific ◽

Factor I ◽

Nuclear Factor I

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.

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trans activation of gene expression by v-myb

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2285-2293.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2285-2293 ◽

Cited By ~ 1

Author(s):

C E Ibanez ◽

J S Lipsick

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Transcriptional Activation ◽

Consensus Sequence ◽

Amino Terminal ◽

Multiple Copies ◽

Vp16 Fusion ◽

Acute Myelomonocytic Leukemia ◽

Simplex Virus

The v-myb oncogene causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its product, p48v-myb, is a short-lived nuclear protein which binds DNA. We demonstrate that p48v-myb can function as a trans activator of gene expression in transient DNA transfection assays. trans activation requires the highly conserved amino-terminal DNA-binding domain and the less highly conserved carboxyl-terminal domain of p48v-myb, both of which are required for transformation. Multiple copies of a consensus sequence for DNA binding by p48v-myb inserted upstream of a herpes simplex virus thymidine kinase promoter are strongly stimulatory for transcriptional activation by a v-myb-VP16 fusion protein but not by p48v-myb itself, suggesting that the binding of p48v-myb to DNA may not be sufficient for trans activation.

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Characterization of the Chromosome Dimer Resolution Site in Caulobacter crescentus

Journal of Bacteriology ◽

10.1128/jb.00391-19 ◽

2019 ◽

Vol 201 (24) ◽

Cited By ~ 1

Author(s):

Ali Farrokhi ◽

Hua Liu ◽

George Szatmari

Keyword(s):

Cell Division ◽

Caulobacter Crescentus ◽

Consensus Sequence ◽

Markov Modeling ◽

Control Of Gene Expression ◽

Site Specific ◽

Content Type ◽

Site Specific Recombination ◽

Resolution System

ABSTRACT Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia coli dif site in vitro. Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC, which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology. IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus. Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.

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The Bacterial DNA Binding Protein MatP Involved in Linking the Nucleoid Terminal Domain to the Divisome at Midcell Interacts with Lipid Membranes

mBio ◽

10.1128/mbio.00376-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 5

Author(s):

Begoña Monterroso ◽

Silvia Zorrilla ◽

Marta Sobrinos-Sanguino ◽

Miguel Ángel Robles-Ramos ◽

Carlos Alfonso ◽

...

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Lipid Membranes ◽

Binding Protein ◽

Dna Binding Protein ◽

Content Type ◽

E Coli ◽

Daughter Cells ◽

Z Ring

ABSTRACTDivision ring formation at midcell is controlled by various mechanisms inEscherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipidsin vitro. We found that, when encapsulated inside vesicles and microdroplets generated by microfluidics, MatP accumulates at phospholipid bilayers and monolayers matching the lipid composition in theE. coliinner membrane. MatP binding to lipids was independently confirmed using lipid-coated microbeads and biolayer interferometry assays, which suggested that the recognition is mainly hydrophobic. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein, but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the association of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.IMPORTANCEThe division of anE. colicell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integratedin vivoandin vitroanalysis provides evidence that MatP can interact with lipid membranes reproducing the phospholipid mixture in theE. coliinner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

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The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

mBio ◽

10.1128/mbio.00448-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Rogério F. Lourenço ◽

Saumya Saurabh ◽

Jonathan Herrmann ◽

Soichi Wakatsuki ◽

Lucy Shapiro

Keyword(s):

Dna Binding ◽

Caulobacter Crescentus ◽

Genetic Material ◽

Bacterial Cells ◽

Structural Elements ◽

Content Type ◽

Time Dynamic ◽

Associated Proteins ◽

And Function

ABSTRACT Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA. IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

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