RESUMO
Azospirillum brasilense is one of the most studied species of diverse agronomic plants worldwide. The benefits conferred to plants inoculated with Azospirillum have been primarily attributed to its capacity to fix atmospheric nitrogen and synthesize phytohormones, especially indole-3-acetic acid (IAA). The principal pathway for IAA synthesis involves the intermediate metabolite indole pyruvic acid. Successful colonization of plants by Azospirillum species is fundamental to the ability of these bacteria to promote the beneficial effects observed in plants. Biofilm formation is an essential step in this process and involves interactions with the host plant. In this study, the tyrR gene was cloned, and the translated product was observed to exhibit homology to TyrR protein, a NtrC/NifA-type activator. Structural studies of TyrR identified three putative domains, including a domain containing binding sites for aromatic amino acids in the N-terminus, a central AAA+ ATPase domain, and a helix-turn-helix DNA binding motif domain in the C-terminus, which binds DNA sequences in promoter-operator regions. In addition, a bioinformatic analysis of promoter sequences in A. brasilense Sp7 genome revealed that putative promoters encompass one to three TyrR boxes in genes predicted to be regulated by TyrR. To gain insight into the phenotypes regulated by TyrR, a tyrR-deficient strain derived from A. brasilense Sp7, named A. brasilense 2116 and a complemented 2116 strain harboring a plasmid carrying the tyrR gene were constructed. The observed phenotypes indicated that the putative transcriptional regulator TyrR is involved in biofilm production and is responsible for regulating the utilization of D-alanine as carbon source. In addition, TyrR was observed to be absolutely required for transcriptional regulation of the gene dadA encoding a D-amino acid dehydrogenase. The data suggested that TyrR may play a major role in the regulation of genes encoding a glucosyl transferase, essential signaling proteins, and amino acids transporters.
Assuntos
Aspergillus , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas , Fatores de Transcrição , Aspergillus/química , Aspergillus/fisiologia , D-Aminoácido Oxidase/biossíntese , D-Aminoácido Oxidase/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Sequências Hélice-Volta-Hélice , Domínios Proteicos , Elementos de Resposta , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologiaRESUMO
The ability of bacteria to deal with diverse environmental changes depends on their repertoire of genes and their ability to regulate their expression. In this process, DNA-binding transcription factors (TFs) have a fundamental role because they affect gene expression positively and/or negatively depending on operator context and ligand-binding status. Here, we show an exhaustive analysis of winged helix-turn-helix domains (wHTHs), a class of DNA-binding TFs. These proteins were identified in high proportions and widely distributed in bacteria, representing around half of the total TFs identified so far. In addition, we evaluated the repertoire of wHTHs in terms of their partner domains (PaDos), identifying a similar trend, as with TFs, i.e. they are abundant and widely distributed in bacteria. Based on the PaDos, we defined three main groups of families: (i) monolithic, those families with little PaDo diversity, such as LysR; (ii) promiscuous, those families with a high PaDo diversity; and (iii) monodomain, with families of small sizes, such as MarR. These findings suggest that PaDos have a very important role in the diversification of regulatory responses in bacteria, probably contributing to their regulatory complexity. Thus, the TFs discriminate over longer regions on the DNA through their diverse DNA-binding domains. On the other hand, the PaDos would allow a great flexibility for transcriptional regulation due to their ability to sense diverse stimuli through a variety of ligand-binding compounds.
Assuntos
Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Variação Genética , Estresse Fisiológico , Fatores de Transcrição/genética , Análise por Conglomerados , DNA Bacteriano/metabolismo , Sequências Hélice-Volta-Hélice/genética , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
The Hsp70 family is one of the most important and conserved molecular chaperone families. It is well documented that Hsp70 family members assist many cellular processes involving protein quality control, as follows: protein folding, transport through membranes, protein degradation, escape from aggregation, intracellular signaling, among several others. The Hsp70 proteins act as a cellular pivot capable of receiving and distributing substrates among the other molecular chaperone families. Despite the high identity of the Hsp70 proteins, there are several homologue Hsp70 members that do not have the same role in the cell, which allow them to develop and participate in such large number of activities. The Hsp70 proteins are composed of two main domains: one that binds ATP and hydrolyses it to ADP and another which directly interacts with substrates. These domains present bidirectional heterotrophic allosteric regulation allowing a fine regulated cycle of substrate binding and release. The general mechanism of the Hsp70s cycle is under the control of ATP hydrolysis that modulates the low (ATP-bound state) and high (ADP-bound state) affinity states of Hsp70 for substrates. An important feature of the Hsp70s cycle is that they have several co-chaperones that modulate their cycle and that can also interact and select substrates. Here, we review some known details of the bidirectional heterotrophic allosteric mechanism and other important features for Hsp70s regulating cycle and function.
Assuntos
Proteínas de Choque Térmico HSP70/fisiologia , Regulação Alostérica , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/química , Sequências Hélice-Volta-Hélice , Humanos , Conformação ProteicaRESUMO
The ribbon-helix-helix (RHH) superfamily of transcription factors uses a conserved three-dimensional structural motif to bind to DNA in a sequence-specific manner. This functionally diverse protein superfamily regulates the transcription of genes that are involved in the uptake of metals, amino-acid biosynthesis, cell division, the control of plasmid copy number, the lytic cycle of bacteriophages and, perhaps, many other cellular processes. In this Analysis, the structures of different RHH transcription factors are compared in order to evaluate the sequence motifs that are required for RHH-domain folding and DNA binding, as well as to identify conserved protein-DNA interactions in this superfamily.
Assuntos
Sequências Hélice-Volta-Hélice , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Modelos Moleculares , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Fatores de Transcrição/genéticaRESUMO
Xylella fastidiosa is a plant pathogen that colonizes the xylem vessels, causing vascular occlusion due to bacterial biofilm growth. However, little is known about the molecular mechanisms driving biofilm formation in Xylella-plant interactions. Here we show that BigR (for "biofilm growth-associated repressor") is a novel helix-turn-helix repressor that controls the transcription of an operon implicated in biofilm growth. This operon, which encodes BigR, membrane proteins, and an unusual beta-lactamase-like hydrolase (BLH), is restricted to a few plant-associated bacteria, and thus, we sought to understand its regulation and function in X. fastidiosa and Agrobacterium tumefaciens. BigR binds to a palindromic AT-rich element (the BigR box) in the Xylella and Agrobacterium blh promoters and strongly represses the transcription of the operon in these cells. The BigR box overlaps with two alternative -10 regions identified in the blh promoters, and mutations in this box significantly affected transcription, indicating that BigR competes with the RNA polymerase for the same promoter site. Although BigR is similar to members of the ArsR/SmtB family of regulators, our data suggest that, in contrast to the initial prediction, it does not act as a metal sensor. Increased activity of the BigR operon was observed in both Xylella and Agrobacterium biofilms. In addition, an A. tumefaciens bigR mutant showed constitutive expression of operon genes and increased biofilm formation on glass surfaces and tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development.
Assuntos
Agrobacterium tumefaciens/crescimento & desenvolvimento , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/fisiologia , Xylella/crescimento & desenvolvimento , Agrobacterium tumefaciens/genética , Sequência de Aminoácidos , Fusão Gênica Artificial , Sítios de Ligação/genética , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Sequências Hélice-Volta-Hélice , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Nicotiana/microbiologia , Transcrição Gênica , Xylella/genéticaRESUMO
Morganella morganii produces an inducible, chromosomally encoded AmpC beta-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators' helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (k(cat)/K(m)) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC beta-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High k(cat)/K(m) values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants.
Assuntos
Proteínas de Bactérias/genética , Variação Genética , Morganella morganii/enzimologia , beta-Lactamases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Sítios de Ligação , DNA/análise , DNA/genética , Genes Bacterianos , Sequências Hélice-Volta-Hélice , Focalização Isoelétrica , Ponto Isoelétrico , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Morganella morganii/genética , Morganella morganii/crescimento & desenvolvimento , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , beta-Lactamases/química , beta-Lactamases/isolamento & purificaçãoRESUMO
Intestinal fatty acid binding protein (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms for the transfer of fatty acids (FAs) to acceptor membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP, transfer occurs by diffusion through the aqueous phase. Earlier, we had shown that the helical domain of IFABP is critical in determining its collisional FA transfer mechanism. In the study presented here, we have engineered a pair of chimeric proteins, one with the "body" (ligand binding domain) of IFABP and the alpha-helical region of LFABP (alphaLbetaIFABP) and the other with the ligand binding pocket of LFABP and the helical domain of IFABP (alphaIbetaLFABP). The objective of this work was to determine whether the change in the alpha-helical domain of each FABP would alter the rate and mechanism of transfer of FA from the chimeric proteins in comparison with those of the wild-type proteins. The fatty acid transfer properties of the FABP chimeras were examined using a fluorescence resonance transfer assay. The results showed a significant modification of the absolute rate of FA transfer from the chimeric proteins compared to that of the wild type, indicating that the slower rate of FA transfer observed for wild-type LFABP relative to that of wild-type IFABP is, in part, determined by the helical domain of the proteins. In addition to these quantitative changes, it was of great interest to observe that the apparent mechanism of FA transfer also changed when the alpha-helical domain was exchanged, with transfer from alphaLbetaIFABP occurring by aqueous diffusion and transfer from alphaIbetaLFABP occurring via protein-membrane collisional interactions. These results demonstrate that the alpha-helical region of LFABP is responsible for its diffusional mechanism of fatty acid transfer to membranes.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fígado/química , Fígado/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , Difusão , Proteínas de Ligação a Ácido Graxo , Sequências Hélice-Volta-Hélice , Membranas Artificiais , Concentração Osmolar , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Ácidos Esteáricos/químicaRESUMO
An open reading frame encoding a protein of 312 amino acids was identified in an essential region of the 90 MDa plasmid of Azospirillum brasilense. The putative protein shows similarity with RepA proteins of plasmids of Sphingomonas aromacitovorans, Rhodotermus marinus, Chlorobium limicola and Bifidobacterium asteroides and with the RepA protein of the broad host range plasmid pSa. A putative helix-turn-helix motif was identified in the central part of the A. brasilense RepA protein. Analysis of the upstream and downstream region of the A. brasilense repA gene revealed the presence of eight 15-bp repeats, which can possibly function as iterons.
Assuntos
Azospirillum brasilense/genética , Proteínas de Bactérias/genética , DNA Helicases , Replicação do DNA , DNA Bacteriano , Proteínas de Ligação a DNA , Plasmídeos , Proteínas/genética , Transativadores , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Sequência de Bases , Sequências Hélice-Volta-Hélice , Dados de Sequência Molecular , Proteínas/classificação , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Using a combination of several approaches we estimated and characterized a total of 314 regulatory DNA-binding proteins in Escherichia coli, which might represent its minimal set of transcription factors. The collection is comprised of 35% activators, 43% repressors and 22% dual regulators. Within many regulatory protein families, the members are homogeneous in their regulatory roles, physiology of regulated genes, regulatory function, length and genome position, showing that these families have evolved homogeneously in prokaryotes, particularly in E.coli. This work describes a full characterization of the repertoire of regulatory interactions in a whole living cell. This repertoire should contribute to the interpretation of global gene expression profiles in both prokaryotes and eukaryotes.
Assuntos
Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/química , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Sequências Hélice-Volta-Hélice , Fatores de Transcrição/químicaRESUMO
Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful tool for the study of enzyme structure and function. This article discusses the general principles of NMR and the potential information this technique can provide in the study of enzymes along with its limitations.
Assuntos
Ativação Enzimática , Ligantes , Espectroscopia de Ressonância Magnética , Peptídeos , Conformação Proteica , Sítios de Ligação , Sequências Hélice-Volta-Hélice , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful tool for the study of enzyme structure and function. This article discusses the general principles of NMR and the potential information this technique can provide in the study of enzymes along with its limitations