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1.
J Biol Chem ; 293(24): 9496-9505, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29695505

RESUMO

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Nucleoproteínas/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Multimerização Proteica
2.
Proc Natl Acad Sci U S A ; 114(47): 12560-12565, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29109287

RESUMO

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.


Assuntos
DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteínas de Fímbrias/química , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Sequência de Aminoácidos , Sítios de Ligação , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise de Célula Única/métodos , Eletricidade Estática
3.
Nat Commun ; 8(1): 1587, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29138484

RESUMO

Unlike eukaryotes, bacteria undergo large changes in osmolality and cytoplasmic pH. It has been described that during acid stress, bacteria internal pH promptly acidifies, followed by recovery. Here, using pH imaging in single living cells, we show that following acid stress, bacteria maintain an acidic cytoplasm and the osmotic stress transcription factor OmpR is required for acidification. The activation of this response is non-canonical, involving a regulatory mechanism requiring the OmpR cognate kinase EnvZ, but not OmpR phosphorylation. Single cell analysis further identifies an intracellular pH threshold ~6.5. Acid stress reduces the internal pH below this threshold, increasing OmpR dimerization and DNA binding. During osmotic stress, the internal pH is above the threshold, triggering distinct OmpR-related pathways. Preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. These results further emphasize the advantages of single cell analysis over studies of population averages.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Osmorregulação/genética , Salmonella typhimurium/genética , Análise de Célula Única/métodos , Transativadores/genética , Ácidos/química , Ácidos/metabolismo , Proteínas de Bactérias/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Pressão Osmótica , Salmonella typhimurium/citologia , Salmonella typhimurium/metabolismo , Transativadores/metabolismo
4.
Methods Mol Biol ; 1624: 173-191, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28842884

RESUMO

Nucleoid-associated proteins (NAPs) are important factors in shaping bacterial nucleoid and regulating global gene expression. A great deal of insights into NAPs can be obtained through studies using single DNA molecule, which has been made possible owing to recent rapid development of single-DNA manipulation techniques. These studies provide information on modes of binding to DNA, which shed light on the mechanism underlying the regulatory function of NAPs. In addition, how NAPs organize DNA and thus their contribution to chromosomal DNA packaging can be determined. In this chapter, we introduce transverse magnetic tweezers that allows for convenient manipulation of long DNA molecules, and its applications in studies of NAPs as exemplified by the E. coli H-NS protein. We describe how transverse magnetic tweezers is a powerful tool that can be used to characterize the DNA binding and organization modes of NAPs and how such information leads to better understanding of its roles in DNA packaging of bacterial nucleoid and transcription regulation.


Assuntos
DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Algoritmos , Bacteriófago lambda/genética , Empacotamento do DNA , Proteínas de Ligação a DNA/química , Expressão Gênica
5.
Polymers (Basel) ; 9(2)2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30970752

RESUMO

Recent progress in single-molecule manipulation technologies has made it possible to exert force and torque on individual DNA biopolymers to probe their mechanical stability and interaction with various DNA-binding proteins. It was revealed in these experiments that the DNA structure and formation of nucleoprotein complexes by DNA-architectural proteins can be strongly modulated by an intricate interplay between the entropic elasticity of DNA and its global topology, which is closely related to the mechanical constraints applied to the DNA. Detailed understanding of the physical processes underlying the DNA behavior observed in single-molecule experiments requires the development of a general theoretical framework, which turned out to be a rather challenging task. Here, we review recent advances in theoretical methods that can be used to interpret single-molecule manipulation experiments on DNA.

6.
Biophys J ; 111(11): 2349-2357, 2016 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-27926836

RESUMO

Unfolded protein, a disordered structure found before folding of newly synthesized protein or after protein denaturation, is a substrate for binding by many cellular factors such as heat-stable proteins, chaperones, and many small molecules. However, it is challenging to directly probe such interactions in physiological solution conditions because proteins are largely in their folded state. In this work we probed small molecule binding to mechanically unfolded polyprotein using sodium dodecyl sulfate (SDS) as an example. The effect of binding is quantified based on changes in the elasticity and refolding of the unfolded polyprotein in the presence of SDS. We show that this single-molecule mechanical detection of binding to unfolded polyprotein can serve, to our knowledge, as a novel label-free assay with a great potential to study many factors that interact with unfolded protein domains, which underlie many important biological processes.


Assuntos
Elasticidade , Desnaturação Proteica , Redobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Guanidina/metabolismo , Guanidina/farmacologia , Modelos Moleculares , Desnaturação Proteica/efeitos dos fármacos , Redobramento de Proteína/efeitos dos fármacos , Dodecilsulfato de Sódio/metabolismo , Dodecilsulfato de Sódio/farmacologia , Ureia/metabolismo , Ureia/farmacologia
7.
Phys Rev E ; 94(3-1): 032404, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27739846

RESUMO

Recent development of single-molecule manipulation technologies has made it possible to exert constant force and torque on individual DNA biopolymers to probe their elastic characteristics and structural stability. It has been previously shown that depending on the nature of applied mechanical constraints, DNA can exist in several forms including B-, L-, and P-DNA. However, there is still a lack of understanding of how structural heterogeneity of DNA, which may naturally arise due to sequence-dependent DNA properties, protein binding, or DNA damage, influences local stability of the above DNA states. To provide a more complete and detailed description of the DNA mechanics, we developed a theoretical framework based on transfer-matrix calculations and demonstrated how it can be used to predict the DNA behavior upon application of a wide range of force and torque constraints. The resulting phase diagram shows DNA structural transitions that are in good agreement with previous experimental and theoretical studies. We further discuss how the constructed formalism can be extended to include local inhomogeneities in the DNA physical properties, thus making it possible to investigate the effect of DNA sequence as well as protein binding on DNA structural stability.


Assuntos
DNA/química , Modelos Biológicos , Polímeros/química , Fenômenos Biofísicos , Conformação de Ácido Nucleico , Torque
8.
Elife ; 52016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26880544

RESUMO

A common strategy by which bacterial pathogens reside in humans is by shifting from a virulent lifestyle, (systemic infection), to a dormant carrier state. Two major serovars of Salmonella enterica, Typhi and Typhimurium, have evolved a two-component regulatory system to exist inside Salmonella-containing vacuoles in the macrophage, as well as to persist as asymptomatic biofilms in the gallbladder. Here we present evidence that SsrB, a transcriptional regulator encoded on the SPI-2 pathogenicity-island, determines the switch between these two lifestyles by controlling ancestral and horizontally-acquired genes. In the acidic macrophage vacuole, the kinase SsrA phosphorylates SsrB, and SsrB~P relieves silencing of virulence genes and activates their transcription. In the absence of SsrA, unphosphorylated SsrB directs transcription of factors required for biofilm formation specifically by activating csgD (agfD), the master biofilm regulator by disrupting the silenced, H-NS-bound promoter. Anti-silencing mechanisms thus control the switch between opposing lifestyles.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Fatores de Transcrição/metabolismo , Ilhas Genômicas
9.
Biophys J ; 109(7): 1321-9, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26445432

RESUMO

A set of abundant nucleoid-associated proteins (NAPs) play key functions in organizing the bacterial chromosome and regulating gene transcription globally. Histone-like nucleoid structuring protein (H-NS) is representative of a family of NAPs that are widespread across bacterial species. They have drawn extensive attention due to their crucial function in gene silencing in bacterial pathogens. Recent rapid progress in single-molecule manipulation and imaging technologies has made it possible to directly probe DNA binding by H-NS, its impact on DNA conformation and topology, and its competition with other DNA-binding proteins at the single-DNA-molecule level. Here, we review recent findings from such studies, and provide our views on how these findings yield new insights into the understanding of the roles of H-NS family members in DNA organization and gene silencing.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia
10.
J Am Chem Soc ; 137(10): 3540-6, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25726700

RESUMO

The mechanical stability of force-bearing proteins is crucial for their functions. However, slow transition rates of complex protein domains have made it challenging to investigate their equilibrium force-dependent structural transitions. Using ultra stable magnetic tweezers, we report the first equilibrium single-molecule force manipulation study of the classic titin I27 immunoglobulin domain. We found that individual I27 in a tandem repeat unfold/fold independently. We obtained the force-dependent free energy difference between unfolded and folded I27 and determined the critical force (∼5.4 pN) at which unfolding and folding have equal probability. We also determined the force-dependent free energy landscape of unfolding/folding transitions based on measurement of the free energy cost of unfolding. In addition to providing insights into the force-dependent structural transitions of titin I27, our results suggest that the conformations of titin immunoglobulin domains can be significantly altered during low force, long duration muscle stretching.


Assuntos
Conectina/química , Imunoglobulinas/química , Fenômenos Mecânicos , Desdobramento de Proteína , Fenômenos Biomecânicos , Elasticidade , Estrutura Terciária de Proteína , Termodinâmica
11.
PLoS One ; 9(11): e112246, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25372370

RESUMO

Pseudomonas aeruginosa contains two distinct members of H-NS family of nucleoid-structuring proteins: MvaT and MvaU. Together, these proteins bind to the same regions of the chromosome and function coordinately in the regulation of hundreds of genes. Due to their structural similarity, they can associate to form heteromeric complexes. These findings left us wondering whether they bear similar DNA binding properties that underlie their gene-silencing functions. Using single-molecule stretching and imaging experiments, we found striking similarities in the DNA organization modes of MvaU compared to the previously studied MvaT. MvaU can form protective nucleoprotein filaments that are insensitive to environmental factors, consistent with its role as a repressor of gene expression. Similar to MvaT, MvaU filament can mediate DNA bridging while excessive MvaU can cause DNA aggregation. The almost identical DNA organization modes of MvaU and MvaT explain their functional redundancy, and raise an interesting question regarding the evolutionary benefits of having multiple H-NS paralogues in the Pseudomonas genus.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Pseudomonas aeruginosa/química , Proteínas Repressoras/química , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Repressoras/metabolismo
12.
J Biol Chem ; 289(20): 13739-50, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24668810

RESUMO

The locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) functions to activate transcription of virulence genes silenced by the histone-like nucleoid-structuring protein (H-NS). Despite its important role in the bacterial gene regulation, the binding mode of Ler to DNA and its mechanism in alleviating genes repressed by H-NS are largely unknown. In this study, we use magnetic tweezers to demonstrate that Ler binds extended DNA through a largely noncooperative process, which results in DNA stiffening and DNA folding depending on protein concentration. We also show that Ler can replace prebound H-NS on DNA over a range of potassium and magnesium concentrations. Our findings reveal the DNA binding properties of Ler and shed light to further understand the anti-silencing activity of Ler.


Assuntos
Proteínas de Bactérias/metabolismo , Ligação Competitiva , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Transativadores/metabolismo , DNA Bacteriano/química , Relação Dose-Resposta a Droga , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Enteropatogênica/genética , Concentração de Íons de Hidrogênio , Cloreto de Magnésio/farmacologia , Conformação de Ácido Nucleico , Cloreto de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos , Especificidade por Substrato , Temperatura
13.
Nucleic Acids Res ; 40(18): 8942-52, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22798496

RESUMO

MvaT from Pseudomonas aeruginosa is a member of the histone-like nucleoid structuring protein (H-NS) family of nucleoid-associated proteins widely spread among Gram-negative bacteria that functions to repress the expression of many genes. Recently, it was reported that H-NS from Escherichia coli can form rigid nucleoproteins filaments on DNA, which are important for their gene-silencing function. This raises a question whether the gene-silencing function of MvaT, which has only ∼18% sequence similarity to H-NS, is also based on the formation of nucleoprotein filaments. Here, using magnetic tweezers and atomic force microscopy imaging, we demonstrate that MvaT binds to DNA through cooperative polymerization to form a nucleoprotein filament that can further organize DNA into hairpins or higher-order compact structures. Furthermore, we studied DNA binding by MvaT mutants that fail to repress gene expression in P. aeruginosa because they are specifically defective for higher-order oligomer formation. We found that, although the mutants can organize DNA into compact structures, they fail to form rigid nucleoprotein filaments. Our findings suggest that higher-order oligomerization of MvaT is required for the formation of rigid nucleoprotein filaments that silence at least some target genes in P. aeruginosa. Further, our findings suggest that formation of nucleoprotein filaments provide a general structural basis for the gene-silencing H-NS family members.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/ultraestrutura , Transativadores/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Microscopia de Força Atômica , Mutação , Conformação de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Multimerização Proteica , Transativadores/genética , Transativadores/metabolismo
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