RESUMO
Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H+/Na+ pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure-function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.
Assuntos
Bactérias , Pirofosfatase Inorgânica , Pirofosfatase Inorgânica/metabolismo , Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/genética , Bactérias/enzimologia , Fungos/enzimologia , Difosfatos/metabolismo , Difosfatos/químicaRESUMO
The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.
Assuntos
Archaea , Células Eucarióticas , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Eucariotos/genética , Células Procarióticas , Evolução Molecular , FilogeniaRESUMO
Macromolecular cell-envelope-spanning structures such as the bacterial flagellum must traverse the cell wall. Lytic transglycosylase enzymes are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. In the periplasmic space, the assembly of the flagellar rod requires the scaffold protein FlgJ, which includes a muramidase domain in the canonical models Salmonella enterica and Escherichia coli. In contrast, in Rhodobacter sphaeroides, FlgJ and the dedicated flagellar lytic transglycosylase SltF are separate entities that interact in the periplasm. In this study, we show that sltF is expressed, along with the genes encoding the early components of the flagellar hierarchy that include the hook-basal body proteins, making SltF available during the rod assembly. Protein-protein interaction experiments demonstrated that SltF interacts with the rod proteins FliE, FlgB, FlgC, FlgF, and FlgG through its C-terminal region. A deletion analysis that divides the C terminus in two halves revealed that the interacting regions for most of the rod proteins are not redundant. Our results also show that the presence of the rod proteins FliE, FlgB, FlgC, and FlgF displace the previously reported SltF-FlgJ interaction. In addition, we observed modulation of the transglycosylase activity of SltF mediated by FlgB and FlgJ that could be relevant to coordinate rod assembly with cell wall remodeling. In summary, different mechanisms regulate the flagellar lytic transglycosylase, SltF, ensuring a timely transcription, a proper localization and a controlled enzymatic activity. IMPORTANCE Several mechanisms participate in the assembly of cell-envelope-spanning macromolecular structures. The sequential expression of substrates to be exported, selective export, and a specific order of incorporation are some of the mechanisms that stand out to drive an efficient assembly process. Here, we analyze how the structural rod proteins, the scaffold protein FlgJ and the flagellar lytic enzyme SltF, interact in an orderly fashion to assemble the flagellar rod into the periplasmic space. A complex arrangement of transient interactions directs a dedicated flagellar muramidase toward the flagellar rod. All of these interactions bring this protein to the proximity of the peptidoglycan wall while also modulating its enzymatic activity. This study suggests how a dynamic network of interactions participates in controlling SltF, a prominent component for flagellar formation.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Flagelos/genética , Rhodobacter sphaeroides/genéticaRESUMO
BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.
Assuntos
Corantes/química , Proteínas Luminescentes/química , Pigmentos Biológicos/química , Proteínas/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cor , Corantes/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Luz , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Oxirredução , Pigmentos Biológicos/metabolismo , Conformação Proteica , Desnaturação Proteica , Proteínas/metabolismo , Espectrofotometria , TemperaturaRESUMO
In this work, we have characterized the soluble lytic transglycosylase (SltF) from Rhodobacter sphaeroides that interacts with the scaffolding protein FlgJ in the periplasm to open space at the cell wall peptidoglycan heteropolymer for the emerging rod. The characterization of the genetic context of flgJ and sltF in alphaproteobacteria shows that these two separate genes coexist frequently in a flagellar gene cluster. Two domains of unknown function in SltF were studied, and the results show that the deletion of a 17-amino-acid segment near the N terminus does not show a recognizable phenotype, whereas the deletion of 47 and 95 amino acids of the C terminus of SltF disrupts the interaction with FlgJ without affecting the transglycosylase catalytic activity of SltF. These mutant proteins are unable to support swimming, indicating that the physical interaction between SltF and FlgJ is central for flagellar formation. In a maximum likelihood tree of representative lytic transglycosylases, all of the flagellar SltF proteins cluster in subfamily 1F. From this analysis, it was also revealed that the lytic transglycosylases related to the type III secretion systems present in pathogens cluster with the closely related flagellar transglycosylases.IMPORTANCE Flagellar biogenesis is a highly orchestrated event where the flagellar structure spans the bacterial cell envelope. The rod diameter of approximately 4 nm is larger than the estimated pore size of the peptidoglycan layer; hence, its insertion requires the localized and controlled lysis of the cell wall. We found that a 47-residue domain of the C terminus of the lytic transglycosylase (LT) SltF of R. sphaeroides is involved in the recognition of the rod chaperone FlgJ. We also found that in many alphaproteobacteria, the flagellar cluster includes a homolog of SltF and FlgJ, indicating that association of an LT with the flagellar machinery is ancestral. A maximum likelihood tree shows that family 1 of LTs segregates into seven subfamilies.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/enzimologia , Glicosiltransferases/metabolismo , Filogenia , Rhodobacter sphaeroides/enzimologia , Proteínas de Bactérias/genética , Flagelos/genética , Glicosiltransferases/genética , Funções Verossimilhança , Mutação , Peptidoglicano/metabolismo , Rhodobacter sphaeroides/genética , Deleção de Sequência , Sistemas de Secreção Tipo III/genéticaRESUMO
The photosynthetic bacterium R. sphaeroides expresses two flagellar systems that are encoded by two complete gene clusters that have distinct phylogenetic origins. The isolation and purification of the Filament-Hook Basal Body (F-HBB) or the Hook Basal Body (HBB) structure is a troublesome task given the complexity of this nano-machine that is composed of multiple loosely bound substructures that can be lost during the isolation and purification procedure. A successful procedure requires adjustments to the standard method established for Salmonella. In this chapter, we describe a detailed protocol to isolate and purify the Fla2 F-HBB and HBB from R. sphaeroides a photosynthetic bacterium that has a complex intracellular membrane system that frequently interferes with isolation of high-quality samples.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Rhodobacter sphaeroides/metabolismo , Corpos Basais/metabolismo , Fotossíntese/fisiologiaRESUMO
Rhodobacter sphaeroides is an alphaproteobacterium that has two complete sets of flagellar genes. The fla1 set was acquired by horizontal transfer from an ancestral gammaproteobacterium and is the only set of flagellar genes that is expressed during growth under standard laboratory conditions. The products of these genes assemble a single, subpolar flagellum. In the absence of the Fla1 flagellum, a gain-of-function mutation in the histidine kinase CckA turns on the expression of the fla2 flagellar genes through the response regulator CtrA. The rotation of the Fla1 and Fla2 flagella is controlled by different sets of chemotaxis proteins. Here, we show that the expression of the chemotaxis proteins that control Fla2, along with the expression of the fla2 genes, is coordinated by CtrA, whereas the expression of the chemotaxis genes that control Fla1 is mediated by the master regulators of the Fla1 system. The coordinated expression of the chemosensory proteins with their cognate flagellar genes highlights the relevance of integrating the expression of the horizontally acquired fla1 genes with a chemosensory system independently of the regulatory proteins responsible for the expression of fla2 and its cognate chemosensory system. IMPORTANCE Gene acquisition via horizontal transfer represents a challenge to the recipient organism to adjust its metabolic and genetic networks to incorporate the new material in a way that represents an adaptive advantage. In the case of Rhodobacter sphaeroides, a complete set of flagellar genes was acquired and successfully coordinated with the native flagellar system. Here we show that the expression of the chemosensory proteins that control flagellar rotation is dependent on the master regulators of their corresponding flagellar system, minimizing the use of transcription factors required to express the native and horizontally acquired genes along with their chemotaxis proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/genética , Quimiotaxia/genética , Histidina Quinase/genética , Histidina Quinase/metabolismo , Rhodobacter sphaeroides/genéticaRESUMO
BACKGROUND: Epidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholera serogroups O1 and O139. In recent years, specific lytic phages of V. cholera have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholera O1 strains. METHODS: Sixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry. RESULTS: Sixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function. CONCLUSIONS: ØVC8 is a lytic phage with specific activity against V. cholerae O1 strains and is grouped as a member of the VP2-like phage subfamily. The encoding of an Ig domain by ØVC8 makes this phage a good candidate for use in phage therapy and an alternative tool for monitoring V. cholerae populations.
Assuntos
Bacteriólise , Bacteriófagos/fisiologia , Vibrio cholerae O1/virologia , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Cólera/microbiologia , Ordem dos Genes , Genoma Viral , Humanos , México , Conformação de Ácido Nucleico , Filogenia , Análise de Sequência de DNA , Tropismo ViralRESUMO
UNLABELLED: The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N- and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium. IMPORTANCE: Despite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Rhodobacter sphaeroides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , DNA Bacteriano/genética , Dados de Sequência Molecular , Conformação ProteicaRESUMO
UNLABELLED: Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 µm and a diameter of 1.4 µm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. IMPORTANCE: R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1(-) mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/ultraestrutura , Flagelina/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Rhodobacter sphaeroides/ultraestrutura , Proteínas de Bactérias/genética , Flagelina/metabolismo , Polimorfismo Genético , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismoRESUMO
Macromolecular structures such as the bacterial flagellum in Gram-negative bacteria must traverse the cell wall. Lytic transglycosylases are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. We have previously shown that in Rhodobacter sphaeroides SltF, the flagellar muramidase, and FlgJ, a flagellar scaffold protein, are separate entities that interact in the periplasm. In this study we show that the export of SltF to the periplasm is dependent on the SecA pathway. A deletion analysis of the C-terminal portion of SltF shows that this region is required for SltF-SltF interaction. These C terminus-truncated mutants lose the capacity to interact with themselves and also bind FlgJ with higher affinity than does the wild-type protein. We propose that this region modulates the interaction with the scaffold protein FlgJ during the assembly process.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Muramidase/química , Muramidase/metabolismo , Rhodobacter sphaeroides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Parede Celular/metabolismo , Dados de Sequência Molecular , Peptidoglicano Glicosiltransferase/metabolismo , Canais de Translocação SEC , Proteínas SecA , Alinhamento de Sequência , Deleção de SequênciaRESUMO
Enteropathogenic Escherichia coli (EPEC) is an important cause of infectious diarrhoea. It colonizes human intestinal epithelial cells by delivering effector proteins into the host cell cytoplasm via a type III secretion system (T3SS) encoded within the chromosomal locus of enterocyte effacement (LEE). The LEE pathogenicity island also encodes a lytic transglycosylase (LT) homologue named EtgA. In the present work we investigated the significance of EtgA function in type III secretion (T3S). Purified recombinant EtgA was found to have peptidoglycan lytic activity in vitro. Consistent with this function, signal peptide processing and bacterial cell fractionation revealed that EtgA is a periplasmic protein. EtgA possesses the conserved glutamate characteristic of the LT family, and we show here that it is essential for enzymic activity. Overproduction of EtgA in EPEC inhibits bacterial growth and induces cell lysis unless the predicted catalytic glutamate is mutated. An etgA mutant is attenuated for T3S, red blood cell haemolysis and EspA filamentation. BfpH, a plasmid-encoded putative LT, was not able to functionally replace EtgA. Overall, our results indicate that the muramidase activity of EtgA is not critical but makes a significant contribution to the efficiency of the T3S process.
Assuntos
Escherichia coli Enteropatogênica/enzimologia , Escherichia coli Enteropatogênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Muramidase/metabolismo , Fatores de Virulência/metabolismo , Bacteriólise , Eritrócitos/efeitos dos fármacos , Expressão Gênica , Técnicas de Inativação de Genes , Hemólise , Humanos , Hidrólise , Peptidoglicano/metabolismo , Proteínas Periplásmicas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , VirulênciaRESUMO
The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac(32)P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.
Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Rhodobacter sphaeroides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Quimiotáticas Aceptoras de Metil , Dados de Sequência Molecular , Fosforilação , Rhodobacter sphaeroides/crescimento & desenvolvimentoRESUMO
In this work, we show evidence regarding the functionality of a large cluster of flagellar genes in Rhodobacter sphaeroides. The genes of this cluster, flgGHIJKL and orf-1, are mainly involved in the formation of the basal body, and flgK and flgL encode the hook-associated proteins HAP1 and HAP3. In general, these genes showed a good similarity as compared with those reported for Salmonella enterica. However, flgJ and flgK showed particular features that make them unique among the flagellar sequences already reported. flgJ is only a third of the size reported for flgJ from Salmonella; whereas flgK is about three times larger than any other flgK sequence previously known. Our results indicate that both genes are functional, and their products are essential for flagellar assembly. In contrast, the interruption of orf-1, did not affect motility suggesting that this sequence, if functional, is not indispensable for flagellar assembly. Finally, we present genetic evidence suggesting that the flgGHIJKL genes are expressed as a single transcriptional unit depending on the sigma-54 factor.