RESUMO
Bioinformatic analysis of lp_2714 from Lactobacillus plantarum WCFS1 demonstrates that it encodes an EAL-domain protein associated with a membrane targeting signal-sequence. Comparison of the predicted primary amino-acid sequence of Lp_2714 shows that it lacks critical catalytic residues and heterologous expression has determined that it does not encode a functional phosphodiesterase. We designate Lp_2714 as a class-3 EAL domain protein probably involved in regulating polysaccharide synthesis on the cell surface the cell.
Assuntos
Proteínas de Bactérias/metabolismo , Lactobacillus plantarum/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Genoma Bacteriano , Lactobacillus plantarum/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Polissacarídeos Bacterianos/biossíntese , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Análise de Sequência de ProteínaRESUMO
Analysis of the essential cell division protein FtsL demonstrates the partial conservation of a cysteine-pair within the trans-membrane region which itself is flanked by histidine-pairs in the cytosol and periplasm. Similar arrangements of such amino acids are seen in proteins known to transport/bind metal ions in biological systems. Heterologous expression of ftsL in Escherichia coli K12 confers a Zn(II)-sensitive phenotype and alteration of the candidate metal-ion binding residues cysteine or histidine substantially alters this phenotype. Whilst the cysteine/histidine replacement derivatives of ftsL were able to complement an otherwise ftsL-null strain, the derivative carrying ftsL lacking the cysteine pair was sensitive to raised metal-ion concentrations in the media. We show that ftsL can confer a metal-ion sensitive phenotype and that trans-membrane cysteine residues play a role in FtsL function in elevated metal-ion concentrations.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Zinco/farmacologia , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Sequência Consenso , Cisteína/química , Cisteína/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Histidina/química , Histidina/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Zinco/metabolismoRESUMO
BACKGROUND: Genome-wide profiling has allowed the regulatory interaction networks of many organisms to be visualised and the pattern of connections between genes to be studied. These networks are non-random, following a power-law distribution with a small number of well-connected 'hubs' and many genes with only one or a few connections. Theoretical work predicts that power-law networks display several unique properties. One of the most biologically interesting of these is an intrinsic robustness to disturbance such that removal of a random gene will have little effect on network function. Conversely, targeted removal of a hub gene is expected to have a large effect. RESULTS: We compared the response of Escherichia coli to environmental and mutational stress following disruption of random or hub genes. We found that disruption of random genes had less effect on robustness to environmental stress than did the targeted disruption of hub genes. In contrast, random disruption strains were slightly less robust to the effect of mutational stress than were hub disruption strains. When we compared the effect of each disruption on environmental and mutational stress, we found a negative relationship, such that strains that were more environmentally robust tended to be less robust to mutational stress. CONCLUSION: Our results demonstrate that mutant strains of E. coli respond differently to stress, depending on whether random or hub genes are disrupted. This difference indicates that the power-law distribution of regulatory interactions has biological significance, making random disruptions less deleterious to organisms facing environmental stress. That E. coli can reduce the effect of environmental stress without reducing the phenotypic effect of additional mutations, indicates that robustness and evolvability need not be antagonistic.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos , Antibacterianos/farmacologia , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Ácido Edético/farmacologia , Meio Ambiente , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/fisiologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Marcação de Genes , Genes Bacterianos/genética , Ácido Clorídrico/farmacologia , Concentração de Íons de Hidrogênio , Metais/farmacologia , Mutagênese Insercional , Mutação , Solução Salina Hipertônica/farmacologia , Hidróxido de Sódio/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids.
Assuntos
Biguanidas/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Reparo do DNA/genética , Desinfetantes/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Teste de Complementação Genética , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/análise , RNA Mensageiro/análise , Transcrição GênicaRESUMO
DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.
Assuntos
DNA Ligases/metabolismo , Medições Luminescentes/métodos , Hibridização de Ácido Nucleico/métodos , Staphylococcus aureus/enzimologia , Acridinas/química , Acridinas/metabolismo , Catálise , Clonagem Molecular , DNA Ligases/biossíntese , DNA Ligases/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Hidrólise , Cinética , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Projetos de Pesquisa/normas , Coloração e Rotulagem/métodos , Staphylococcus aureus/genética , Especificidade por SubstratoRESUMO
The interaction between the broad-spectrum antimicrobial agent, polyhexamethylene biguanide (PHMB), and various nucleic acids was investigated. Titration of either single- or double-stranded 100-bp DNA, or mixed-molecular weight marker DNA, or tRNA with PHMB caused precipitation of a complex between nucleic acid and PHMB in which the nucleotide/biguanide ratio was always close to unity. Binding of PHMB was highly cooperative, with apparent Hill coefficients 10.3-14.6. When a fluorescent derivative of PHMB was titrated with increasing amounts of nucleic acid, all four forms of nucleic acid caused strong polarisation of fluorescence, demonstrating the association with PHMB. The intensity and broad-spectrum binding of PHMB to all forms of nucleic acid has significant implications for the mechanism of action of this biocide.
Assuntos
Biguanidas/metabolismo , DNA/metabolismo , RNA de Transferência/metabolismo , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Biguanidas/química , Sítios de Ligação , Cátions , Precipitação Química , DNA/química , DNA/genética , Escherichia coli/genética , Polarização de Fluorescência , Cinética , RNA de Transferência/genética , Titulometria/métodosRESUMO
It is difficult to over-state the importance of Zn(II) in biology. It is a ubiquitous essential metal ion and plays a role in catalysis, protein structure and perhaps as a signal molecule, in organisms from all three kingdoms. Of necessity, organisms have evolved to optimise the intracellular availability of Zn(II) despite the extracellular milieu. To this end, prokaryotes contain a range of Zn(II) import, Zn(II) export and/or binding proteins, some of which utilise either ATP or the chemiosmotic potential to drive the movement of Zn(II) across the cytosolic membrane, together with proteins that facilitate the diffusion of this ion across either the outer or inner membranes of prokaryotes. This review seeks to give an overview of the systems currently classified as altering Zn(II) availability in prokaryotes.
Assuntos
Células Procarióticas/metabolismo , Zinco/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Bactérias/genética , Bactérias/metabolismo , Transporte de Íons , Modelos Biológicos , Chaperonas Moleculares , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
The ZntR protein from Escherichia coli is a member of the MerR-family of transcriptional regulatory proteins and acts as a hyper-sensitive transcriptional switch primarily in response to Zn(II) and Cd(II). The binding of metal-ions to ZntR initiates a mechanism that remodels the cognate promoter, increasing its affinity for RNA polymerase. We have introduced site-directed mutations into zntR and shown that cysteine and histidine residues are important for transcriptional control and have an effect on metal-ion preference, sensitivity and magnitude of induction. We propose a three-dimensional model of the N-terminal region of ZntR based upon the coordinates of the MerR-family regulator BmrR.
Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Genes Reporter , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Escherichia coli was adapted to grow in medium containing substantially elevated concentrations of either Zn(II), Cd(II), Co(II) or Ni(II). Whole-genome transcriptional profiles were generated from adapted strains and analysed for significant alteration in transcript abundance with reference to a wild-type strain. Similar alterations in specific message levels were observed for strains adapted to the four metal ions. One unexpected trend was the increase in transcript level of genes involved in transposition of IS elements, particularly insA. Subsequent expression of insA-7 from a heterologous promoter in E. coli conferred tolerance to Zn(II).