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1.
Nat Commun ; 10(1): 4086, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501441

RESUMO

The bacterial second messenger cyclic-di-GMP is a widespread, prominent effector of lifestyle change. An example of this occurs in the predatory bacterium Bdellovibrio bacteriovorus, which cycles between free-living and intraperiplasmic phases after entering (and killing) another bacterium. The initiation of prey invasion is governed by DgcB (GGDEF enzyme) that produces cyclic-di-GMP in response to an unknown stimulus. Here, we report the structure of DgcB, and demonstrate that the GGDEF and sensory forkhead-associated (FHA) domains form an asymmetric dimer. Our structures indicate that the FHA domain is a consensus phosphopeptide sensor, and that the ligand for activation is surprisingly derived from the N-terminal region of DgcB itself. We confirm this hypothesis by determining the structure of a FHA:phosphopeptide complex, from which we design a constitutively-active mutant (confirmed via enzyme assays). Our results provide an understanding of the stimulus driving DgcB-mediated prey invasion and detail a unique mechanism of GGDEF enzyme regulation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bdellovibrio/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Ligantes , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade
2.
Future Microbiol ; 14: 969-980, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31382783

RESUMO

Aim: A structural model of chorismate synthase (CS) from the pathogenic fungus Candida albicans was used for virtual screening simulations. Methods: Docking, molecular dynamics, cell growth inhibition and protein binding assays were used for search and validation. Results: Two molecules termed CS8 and CaCS02 were identified. Further studies of the minimal inhibitory concentration demonstrated fungicidal activity against Paracoccidioides brasiliensis with a minimal inhibitory concentration and minimal fungicidal concentration of 512 and 32 µg·ml-1 for CS8 and CaCS02, respectively. In addition, CaCS02 showed a strong synergistic effect in combination with amphotericin B without cytotoxic effects. In vitro studies using recombinant CS from P. brasiliensis showed IC50 of 29 µM for CaCS02 supporting our interpretation that inhibition of CS causes the observed fungicidal activity.


Assuntos
Antifúngicos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Paracoccidioides/efeitos dos fármacos , Fósforo-Oxigênio Liases/antagonistas & inibidores , Sequência de Aminoácidos , Anfotericina B/farmacologia , Animais , Antifúngicos/química , Antifúngicos/metabolismo , Candida albicans/enzimologia , Sinergismo Farmacológico , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Paracoccidioides/enzimologia , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Ligação Proteica , Células Vero
3.
EMBO J ; 38(18): e100948, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31418899

RESUMO

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.


Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Sirtuínas/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , GMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Análise Serial de Proteínas/métodos , Proteômica/métodos , Sistemas do Segundo Mensageiro
4.
PLoS Genet ; 15(4): e1008059, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31022167

RESUMO

The ubiquitous second messenger c-di-GMP promotes bacterial biofilm formation by playing diverse roles in the underlying regulatory networks. This is reflected in the multiplicity of diguanylate cyclases (DGC) and phosphodiesterases (PDE) that synthesize and degrade c-di-GMP, respectively, in most bacterial species. One of the 12 DGCs of Escherichia coli, DgcE, serves as the top-level trigger for extracellular matrix production during macrocolony biofilm formation. Its multi-domain architecture-a N-terminal membrane-inserted MASE1 domain followed by three PAS, a GGDEF and a degenerate EAL domain-suggested complex signal integration and transmission through DgcE. Genetic dissection of DgcE revealed activating roles for the MASE1 domain and the dimerization-proficient PAS3 region, whereas the inhibitory EALdeg domain counteracts the formation of DgcE oligomers. The MASE1 domain is directly targeted by the GTPase RdcA (YjdA), a dimer or oligomer that together with its partner protein RdcB (YjcZ) activates DgcE, probably by aligning and promoting dimerization of the PAS3 and GGDEF domains. This activation and RdcA/DgcE interaction depend on GTP hydrolysis by RdcA, suggesting GTP as an inhibitor and the pronounced decrease of the cellular GTP pool during entry into stationary phase, which correlates with DgcE-dependent activation of matrix production, as a possible input signal sensed by RdcA. Furthermore, DgcE exhibits rapid, continuous and processive proteolytic turnover that also depends on the relatively disordered transmembrane MASE1 domain. Overall, our study reveals a novel GTP/c-di-GMP-connecting signaling pathway through the multi-domain DGC DgcE with a dual role for the previously uncharacterized MASE1 signaling domain.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Proteínas de Escherichia coli/química , Matriz Extracelular/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Fósforo-Oxigênio Liases/química , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais
5.
mBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015332

RESUMO

Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera.


Assuntos
Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Vibrio cholerae/enzimologia , Vibrio cholerae/fisiologia , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Locomoção , Fósforo-Oxigênio Liases/genética , Vibrio cholerae/genética
6.
Pathology ; 51(3): 274-280, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30853107

RESUMO

Genetic defects on 6-pyruvoyl-tetrahydropterin synthase (PTPS) are the most prevalent cause of hyperphenylalaninaemia not due to phenylalanine hydrolyase deficiency (phenylketonuria). PTPS catalyses the second step of tetrahydrobiopterin (BH4) cofactor biosynthesis, and its deficiency represents the most common form of BH4 deficiency. Untreated PTPS deficiency results in depletion of the neurotransmitters dopamine, catecholamine and serotonin causing neurological symptoms. We archived reported missense variants of the PTS gene. Common in silico algorithms were used to predict the effects of such variants, and substantial proportions (up to 19%) of the variants were falsely classified as benign or uncertain. We have determined the crystal structure of the human PTPS hexamer, allowing another level of interpretation to understand the potential deleterious consequences of the variants from a structural perspective. The in silico and structure approaches appear to be complimentary and may provide new insights that are not available from each alone. Information from the protein structure suggested that the variants affecting amino acid residues required for interaction between monomeric subunits of the PTPS hexamer were those misclassified as benign by in silico algorithms. Our findings illustrate the important utility of 3D protein structure in interpretation of variants and also current limitations of in silico prediction algorithms. However, software to analyse mutation in the perspective of 3D protein structure is far less readily available than other in silico prediction tools.


Assuntos
Mutação , Fenilcetonúrias/genética , Fósforo-Oxigênio Liases/deficiência , Fósforo-Oxigênio Liases/genética , Humanos , Fenilcetonúrias/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Conformação Proteica
7.
Nucleic Acids Res ; 47(10): 5141-5154, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30916351

RESUMO

Bacillus subtilis diadenylate cyclase DisA converts two ATPs into c-di-AMP, but this activity is suppressed upon interaction with sites of DNA damage. DisA forms a rapid moving focus that pauses upon induction of DNA damage during spore development. We report that DisA pausing, however, was not observed in the absence of the RecO mediator or of the RecA recombinase, suggesting that DisA binds to recombination intermediates formed by RecA in concert with RecO. DisA, which physically interacts with RecA, was found to reduce its ATPase activity without competing for nucleotides or ssDNA. Furthermore, increasing DisA concentrations inhibit RecA-mediated DNA strand exchange, but this inhibition failed to occur when RecA was added prior to DisA, and was independent of RecA-mediated nucleotide hydrolysis or increasing concentrations of c-di-AMP. We propose that DisA may preserve genome integrity by downregulating RecA activities at several steps of the DNA damage tolerance pathway, allowing time for the repair machineries to restore genome stability. DisA might reduce RecA-mediated template switching by binding to a stalled or reversed fork.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Recombinases Rec A/metabolismo , Domínio Catalítico , Dano ao DNA , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Genoma Bacteriano , Proteínas de Fluorescência Verde/metabolismo , Hidrólise , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Fósforo-Oxigênio Liases/genética , Mapeamento de Interação de Proteínas , Recombinação Genética
8.
DNA Repair (Amst) ; 77: 45-57, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30877841

RESUMO

Bacillus subtilis radA is epistatic to disA and recA genes in response to methyl methane sulfonate- and 4-nitroquinoline-1-oxide-induced DNA damage. We show that ΔradA cells were sensitive to mitomycin C- and H2O2-induced damage and impaired in natural chromosomal transformation, whereas cells lacking DisA were not. RadA/Sms mutants in the conserved H1 (K104A and K104R) or KNRFG (K255A and K255R) motifs fail to rescue the sensitivity of ΔradA in response to the four different DNA damaging agents. A RadA/Sms H1 or KNRFG mutation impairs both chromosomal and plasmid transformation, but the latter defect was suppressed by inactivating RecA. RadA/Sms K255A, K255R and wild type RadA/Sms reduced the diadenylate cyclase activity of DisA, whereas RadA/Sms K104A and K104R blocked it. Single-stranded and Holliday junction DNA are preferentially bound over double-stranded DNA by RadA/Sms and its variants. Moreover, RadA/Sms ATPase activity was neither stimulated by a variety of DNA substrates nor by DisA. RadA/Sms possesses a 5´â†’3´ DNA helicase activity. The RadA/Sms mutants neither hydrolyze ATP nor unwind DNA. Thus, we propose that RadA/Sms has two activities: to modulate DisA and to promote RecA-mediated DNA strand exchange. Both activities are required to coordinate responses to replicative stress and genetic recombination.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Recombinases Rec A/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Hidrólise , Mutação , Motivos de Nucleotídeos , Recombinação Genética , Estresse Fisiológico/genética
9.
Nat Commun ; 10(1): 545, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710081

RESUMO

Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-D-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-D-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent.


Assuntos
Anabaena/crescimento & desenvolvimento , Antimetabólitos/farmacologia , Arabidopsis/crescimento & desenvolvimento , Cianobactérias/metabolismo , Heptoses/farmacologia , Redes e Vias Metabólicas , Ácido Chiquímico/metabolismo , Anabaena/efeitos dos fármacos , Antifúngicos/farmacologia , Arabidopsis/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Heptoses/isolamento & purificação , Herbicidas/toxicidade , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma , Fósforo-Oxigênio Liases/antagonistas & inibidores , Fósforo-Oxigênio Liases/metabolismo , Fotossíntese/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Synechococcus/metabolismo
10.
PLoS Pathog ; 15(1): e1007537, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668586

RESUMO

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.


Assuntos
Inibidores de Adenilil Ciclases/metabolismo , Adenilil Ciclases/metabolismo , Adenilil Ciclases/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Fosfatos de Dinucleosídeos/antagonistas & inibidores , Fosfatos de Dinucleosídeos/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Óperon/genética , Fosfoglucomutase/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Domínios Proteicos , Espalhamento a Baixo Ângulo , Sistemas do Segundo Mensageiro/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/fisiologia , Difração de Raios X/métodos
11.
Curr Protoc Microbiol ; 52(1): e74, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30489040

RESUMO

Bacterial biofilms are notorious for their deleterious effects on human health and industrial biofouling. Key processes in biofilm formation are regulated by the second messenger signal cyclic dimeric guanosine monophosphate (c-di-GMP); accumulation of c-di-GMP promotes biofilm formation, while lowering c-di-GMP promotes motility. Complex networks of modular enzymes are involved in regulating c-di-GMP homeostasis. Understanding how these enzymes function in bacterial cells can help enlighten how bacteria use environmental cues to modulate c-di-GMP and cell physiology. In this article, we describe a workflow that utilizes Escherichia coli as a heterologous host to allow the researcher to identify genes encoding potential c-di-GMP-metabolizing proteins, to express the gene of interest from an inducible plasmid, and to directly detect changes in intracellular c-di-GMP using ultra-performance liquid chromatography-tandem mass spectrometry. © 2018 by John Wiley & Sons, Inc.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Clonagem Molecular/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Espectrometria de Massas/métodos , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , GMP Cíclico/química , GMP Cíclico/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/genética , Plasmídeos/genética , Plasmídeos/metabolismo
12.
J Biol Chem ; 294(4): 1420-1427, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30514762

RESUMO

6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions. Human PTS has two cysteines: Cys-43 at the catalytic site and Cys-10 at the N terminus. PTS can be oxidized and consequently inactivated by H2O2 treatment, oxidized GSH, or S-nitrosoglutathione, and determining the oxidized modifications of PTS induced by each oxidant by MALDI-TOF MS, we show that PTS is S-glutathionylated in the presence of GSH and H2O2 S-Glutathionylation at Cys-43 protected PTS from H2O2-induced irreversible sulfinylation and sulfonylation. We also found that PTS expressed in HeLa and THP-1 cells is reversibly modified under oxidative stress conditions. Our findings suggest that PTS activity and S-glutathionylation is regulated by the cellular redox environment and that reversible S-glutathionylation protects PTS against oxidative stress.


Assuntos
Cisteína/química , Regulação Enzimológica da Expressão Gênica , Glutationa/química , Fósforo-Oxigênio Liases/metabolismo , Pterinas/metabolismo , Células HeLa , Humanos , Oxirredução , Estresse Oxidativo , Fósforo-Oxigênio Liases/química , Substâncias Protetoras
13.
Environ Microbiol ; 21(1): 81-97, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30252211

RESUMO

Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA-biofilm) or at the air-liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non-pellicle-forming cheY3-deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c-di-GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c-di-GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c-di-GMP binding protein MxdA.


Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/metabolismo , Shewanella/enzimologia , Proteínas de Bactérias/genética , Biofilmes , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Flagelos/genética , Flagelos/metabolismo , Óperon , Fósforo-Oxigênio Liases/genética , Shewanella/genética , Shewanella/crescimento & desenvolvimento , Shewanella/fisiologia , Transdução de Sinais
14.
Chembiochem ; 20(1): 7-19, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30320963

RESUMO

Heme-nitric oxide/oxygen binding (H-NOX) proteins are a family of gas-binding hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O2 ). In bacteria, H-NOXs are often associated with signaling partners, including histidine kinases (HKs), diguanylate cyclases (DGCs) or methyl-accepting chemotaxis proteins (MCPs), either as a stand-alone protein or as a domain of a larger polypeptide. H-NOXs regulate the activity of cognate signaling proteins through ligand-induced conformational changes in the H-NOX domain and protein/protein interactions between the H-NOX and the cognate signaling partner. This review summarizes recent progress toward deciphering the molecular mechanism of bacterial H-NOX activation and the subsequent regulation of H-NOX-associated cognate sensor proteins from a structural and biochemical point of view.


Assuntos
Proteínas de Bactérias/metabolismo , Hemeproteínas/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Histidina Quinase/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Ligação Proteica , Domínios Proteicos , Transdução de Sinais/fisiologia
15.
Chembiochem ; 20(3): 394-407, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30395379

RESUMO

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Fósforo-Oxigênio Liases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Caulobacter crescentus/enzimologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/metabolismo , Estrutura Molecular , Fósforo-Oxigênio Liases/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
16.
Microbiol Res ; 218: 108-117, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30454652

RESUMO

All three domains of life have an ordered plasma membrane which is pivotal in the selective fitness of primitive life. Like cholesterol in eukaryotes, hopanoids are important in bacteria to modulate membrane order. Hopanoids are pentacyclic triterpenoid lipids biosynthesised in many eubacteria, few ferns and lichens. Hopanoid modulates outer membrane order and hopanoid deficiency results in the weakened structural integrity of the membrane which may in turn affect the other structures within or spanning the cell envelope and contributing to various membrane functions. Hence, to decipher the role of hopanoid, genome-wide transcriptome of wild-type and Δshc mutant of Rhodopseudomonas palustris TIE-1 was studied which indicated 299 genes were upregulated and 306 genes were downregulated in hopanoid deficient mutant, representing ∼11.5% of the genome. Thirty-eight genes involved in chemotaxis, response to stimuli and signal transduction were differentially regulated and impaired motility in hopanoid deficient mutant showed that hopanoid plays a crucial role in chemotaxis. The docking study demonstrated that diguanylate cyclase which catalyses the synthesis of secondary messenger exhibited the capability to interact with hopanoids and might be confederating in chemotaxis and signal transduction. Seventy-four genes involved in membrane transport were differentially expressed and cell assays also explicit that the multidrug transport is compromised in Δshc mutant. Membrane transport is reliant on hopanoids which may explain the basis for previous observations linking hopanoids to antibiotic resistance. Disturbing the membrane order by targeting lipid synthesis can be a possible novel approach in developing new antimicrobials and hopanoid biosynthesis could be a potential target.


Assuntos
Transporte Biológico/genética , Membrana Celular/fisiologia , Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana Transportadoras/genética , Rodopseudomonas/genética , Rodopseudomonas/metabolismo , Triterpenos/metabolismo , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Transdução de Sinais/genética
17.
Plant Biotechnol J ; 17(2): 517-530, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30059608

RESUMO

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed.


Assuntos
Euphorbiaceae/enzimologia , Jatropha/enzimologia , Família Multigênica , Fósforo-Oxigênio Liases/metabolismo , Biocombustíveis , Mapeamento Cromossômico , Euphorbiaceae/genética , Euphorbiaceae/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Jatropha/genética , Jatropha/crescimento & desenvolvimento , Lipídeos/biossíntese , Anotação de Sequência Molecular , Ésteres de Forbol/metabolismo , Fósforo-Oxigênio Liases/genética , Filogenia , Melhoramento Vegetal , Óleos Vegetais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento
18.
J Bioinform Comput Biol ; 16(6): 1850027, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30567475

RESUMO

Shikimate pathway plays an essential role in the biosynthesis of aromatic amino acids in various plants and bacteria, which consists of seven key enzymes and they are all attractive targets for antibacterial agent development due to their absence in humans. The Staphylococcus aureus dehydroquinate synthase (SaDHQS) is involved in the second step of shikimate pathway, which catalyzes the NAD + -dependent conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate via multiple steps. The enzyme active site can be characterized by two spatially separated subpockets 1 and 2, which represent the reaction center of substrate adduct with NAD + nicotinamide moiety and the assistant binding site of NAD + adenine moiety, respectively. In silico virtual screening is performed against a biogenic compound library to discover SaDHQS subpocket-specific inhibitors, which were then tested against both antibiotic-sensitive and antibiotic-resistant S. aureus strains by using in vitro susceptibility test. The activity profile of hit compounds has no considerable difference between the antibiotic-sensitive and -resistant strains. The subpocket 1-specific inhibitors exhibit a generally higher activity than subpocket 2-specific inhibitors, and they also hold a strong selectivity between their cognate and noncognate subpockets. Dynamics and energetics analyses reveal that the SaDHQS active site prefers to interact with amphipathic and polar inhibitors by forming multiple hydrogen bonds and van der Waals packing at the complex interfaces of the two subpockets with their cognate inhibitors.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fósforo-Oxigênio Liases/química , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/métodos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , NAD/metabolismo , Fósforo-Oxigênio Liases/antagonistas & inibidores , Fósforo-Oxigênio Liases/metabolismo
19.
mBio ; 9(6)2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30401769

RESUMO

Mixed-species biofilms display a number of emergent properties, including enhanced antimicrobial tolerance and communal metabolism. These properties may depend on interspecies relationships and the structure of the biofilm. However, the contribution of specific matrix components to emergent properties of mixed-species biofilms remains poorly understood. Using a dual-species biofilm community formed by the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus, we found that whilst neither Pel nor Psl polysaccharides, produced by P. aeruginosa, affect relative species abundance in mature P. aeruginosa and S. aureus biofilms, Psl production is associated with increased P. aeruginosa abundance and reduced S. aureus aggregation in the early stages of biofilm formation. Our data suggest that the competitive effect of Psl is not associated with its structural role in cross-linking the matrix and adhering to P. aeruginosa cells but is instead mediated through the activation of the diguanylate cyclase SiaD. This regulatory control was also found to be independent of the siderophore pyoverdine and Pseudomonas quinolone signal, which have previously been proposed to reduce S. aureus viability by inducing lactic acid fermentation-based growth. In contrast to the effect mediated by Psl, Pel reduced the effective crosslinking of the biofilm matrix and facilitated superdiffusivity in microcolony regions. These changes in matrix cross-linking enhance biofilm surface spreading and expansion of microcolonies in the later stages of biofilm development, improving overall dual-species biofilm growth and increasing biovolume severalfold. Thus, the biofilm matrix and regulators associated with matrix production play essential roles in mixed-species biofilm interactions.IMPORTANCE Bacteria in natural and engineered environments form biofilms that include many different species. Microorganisms rely on a number of different strategies to manage social interactions with other species and to access resources, build biofilm consortia, and optimize growth. For example, Pseudomonas aeruginosa and Staphylococcus aureus are biofilm-forming bacteria that coinfect the lungs of cystic fibrosis patients and diabetic and chronic wounds. P. aeruginosa is known to antagonize S. aureus growth. However, many of the factors responsible for mixed-species interactions and outcomes such as infections are poorly understood. Biofilm bacteria are encased in a self-produced extracellular matrix that facilitates interspecies behavior and biofilm development. In this study, we examined the poorly understood roles of the major matrix biopolymers and their regulators in mixed-species biofilm interactions and development.


Assuntos
Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Interações Microbianas , Fósforo-Oxigênio Liases/metabolismo , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/genética , Staphylococcus aureus/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Fósforo-Oxigênio Liases/genética , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/genética
20.
Biochemistry ; 57(42): 6119-6127, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30299084

RESUMO

DNA polymerase θ (Pol θ) is a multifunctional enzyme with double-strand break (DSB) repair, translesion synthesis, and lyase activities. Pol θ lyase activity on ternary substrates containing a 5'-dRP that are produced during base excision repair of abasic sites (AP) is weak compared to that of DNA polymerase ß (Pol ß), a polymerase integrally involved in base excision repair. This led us to explore whether Pol θ utilizes its lyase activity to remove 5'-dRP and incise abasic sites from alternative substrates that might be produced during DNA damage and repair. We found that Pol θ exhibited lyase activity on abasic lesions near DSB termini and on clustered lesions. To calibrate the Pol θ activity, Pol ß reactivity was examined with the same substrates. Pol ß excised 5'-dRP from within a 5'-overhang 80 times faster than did Pol θ. Pol θ and Pol ß also incised AP within clustered lesions but showed opposite preferences with respect to the polarity of the lesions. AP lesions in 5'-overhangs were typically excised by Pol ß 35-50 times faster than those in a duplex substrate but 15-20-fold more slowly than 5'-dRP in a ternary complex. This is the first report of Pol θ exhibiting lyase activity within an unincised strand. These results suggest that bifunctional polymerases may exhibit lyase activity on a greater variety of substrates than previously recognized. A role in DSB repair could potentially be beneficial, while the aberrant activity exhibited on clustered lesions may be deleterious because of their conversion to DSBs.


Assuntos
Dano ao DNA , DNA Polimerase beta/química , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , Fósforo-Oxigênio Liases/química , DNA Polimerase beta/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Fósforo-Oxigênio Liases/metabolismo , Especificidade por Substrato
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