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1.
J Biophotonics ; : e202100136, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34761529

RESUMO

The first step in photosynthesis is an extremely efficient energy transfer mechanism that led to the debate to which extent quantum coherence may be involved in the energy transfer between the photosynthetic pigments. In search of such a coherent behavior, we have embedded living cyanobacteria between the parallel mirrors of an optical microresonator irradiated with low intensity white light. As a consequence, we observe vacuum Rabi splitting in the transmission and fluorescence spectra as a result of strong light matter coupling of the chlorophyll a molecules in the photosystems (PSs) and the cavity modes. The Rabi-splitting scales with the number of the PSs chlorophyll a pigments involved in strong coupling indicating a delocalized polaritonic state. Our data provide evidence that a delocalized polaritonic state can be established between the chlorophyll a molecule of the PSs in living cyanobacterial cells at ambient conditions in a microcavity.

2.
Bio Protoc ; 11(17): e4152, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34604457

RESUMO

Biolayer interferometry (BLI) is an emerging analytical tool that allows the study of protein complexes in real time to determine protein complex kinetic parameters. This article describes a protocol to determine the KD of a protein complex using a 6×His tagged fusion protein as bait immobilized on the NTA sensor chip of the FortéBio® Octet K2 System (Sartorius). We also describe how to determine the half maximal effective concentration (EC50, also known as IC50 for inhibiting effectors) of a metabolite. The complete protocol allows the determination of protein complex KD and small molecular effector EC50 within 8 h, measured in triplicates. Graphic abstract: Principle of the Biolayer interferometry measurement. (Middle, top) Exemplary result of the BLI measurement using Octet® (Raw Data). Wavelength shift (Δλ) against time. (A) Baseline 1. Baseline measurement. When the sensor is equilibrated in the kinetics buffer. The light is reflected with no difference. (B) Load. The his-tagged proteins (ligand) are loaded onto the sensor surface. The light is reflected with a shift of the wavelength. (C) Baseline 2. The loaded sensor is equilibrated in the kinetics buffer. No further wavelength shift appears. (D) Association. The loaded sensor is dipped into the analyte solution. The analyte binds to the immobilized ligand along with an increased wavelength shift. (E) Dissociation. Afterward, the sensor is dipped again into the kinetics buffer without the analyte. Some analyte molecules dissociate. The wavelength shift decreases. (Subfigures A-E) The left side shows the position of the sensor during the measurement seen in the representative BLI measurement, marked with the figure label. The right side shows the light path in the sensor. Black waves represent the light emitted to the sensor surface. The red waves show the light reflected from the sensor surface back to the detector.

3.
Braz J Microbiol ; 52(4): 2069-2073, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34342836

RESUMO

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.

4.
Front Microbiol ; 12: 692986, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248919

RESUMO

7-Deoxysedoheptulose (7dSh) is a bioactive deoxy-sugar actively excreted by the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) but also Streptomyces setonensis. In our previous publications we have shown that in S. elongatus, 7dSh is exclusively synthesized by promiscuous enzyme activity from an inhibitory by-product of radical SAM enzymes, without a specific gene cluster being involved. Additionally, we showed that 7dSh inhibits the growth of cyanobacteria, but also the growth of plants and fungi, presumably by inhibiting the 3-dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, as the substrate of this enzyme strongly accumulates in cells treated with 7dSh. In this study, by using purified DHQS of Anabaena variabilis ATCC 29413 (A. variabilis) we biochemically confirmed that 7dSh is a competitive inhibitor of this enzyme. By analyzing the effect of 7dSh on a subset of cyanobacteria from all the five subsections, we identified different species whose growth was inhibited by 7dSh. We also found that in some of the susceptible cyanobacteria import of 7dSh is mediated by structurally different and promiscuous transporters: 7dSh can be taken up by the fructose ABC-transporter in A. variabilis and via the glucose permease in Synechocystis sp. PCC 6803 (Synechocystis sp.). In both cases, an effective uptake and thereby intracellular enrichment of 7dSh was essential for the inhibitory activity. Importantly, spontaneous mutations in the sugar transporters of A. variabilis and Synechocystis sp. not only disabled growth of the two strains on fructose and glucose, respectively, but also almost abolished their sensitivity to 7dSh. Although we have clearly shown in these examples that the effective uptake plays an essential role in the inhibitory effect of 7dSh, questions remain about how 7dSh resistance works in other (cyano)bacteria. Also, the involvement of a putative ribokinase in 7dSh resistance in the producer strain S. elongatus remained to be further investigated. Overall, these data establish 7dSh as the first allelochemical targeting the shikimate pathway in other cyanobacteria and plants and suggest a role of 7dSh in niche competition.

5.
6.
Sci Rep ; 11(1): 12535, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131190

RESUMO

PII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed "Large BiT" and a 1.3 kDa peptide termed "Small BiT", which only weakly associate. When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais , Proteínas PII Reguladoras de Nitrogênio/isolamento & purificação , Fosfotransferases (Aceptor do Grupo Carboxila)/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Ácidos Cetoglutáricos/química , Nanotecnologia , Proteínas PII Reguladoras de Nitrogênio/química , Synechococcus/química
7.
Microb Physiol ; 31(3): 248-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34126623

RESUMO

5-Deoxyadenosine (5dAdo) is a by-product of many radical SAM enzyme reactions in all domains of life, and an inhibitor of the radical SAM enzymes themselves. Hence, pathways to recycle or dispose of this toxic by-product must exist but remain largely unexplored. In this review, we discuss the current knowledge about canonical and atypical 5dAdo salvage pathways that have been characterized in the last years. We highlight studies that report on how, in certain organisms, the salvage of 5dAdo via specific pathways can confer a growth advantage by providing either intermediates for the synthesis of secondary metabolites or a carbon source for the synthesis of metabolites of the central carbon metabolism. Yet, an alternative recycling route exists in organisms that use 5dAdo as a substrate to synthesize and excrete 7-deoxysedoheptulose, an allelopathic inhibitor of one enzyme of the shikimate pathway, thereby competing for their own niche. Remarkably, most steps of 5dAdo salvage are the result of the activity of promiscuous enzymes. This strategy enables even organisms with a small genome to synthesize bioactive compounds which they can deploy under certain conditions to gain a competitive growth advantage. We conclude emphasizing that, unexpectedly, 5dAdo salvage pathways seem not to be ubiquitously present, raising questions about the fate of such a toxic by-product in those species. This observation also suggests that additional 5dAdo salvage pathways, possibly relying on the activity of promiscuous enzymes, may exist. The future challenge will be to bring to light these "cryptic" 5dAdo recycling pathways.


Assuntos
Desoxiadenosinas
8.
Microb Physiol ; 31(2): 67-77, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33979794

RESUMO

Polyhydroxybutyrate (PHB) is a carbon polymer with diverse functions, varying greatly on the organism producing it. This microreview describes the current knowledge about PHB metabolism, structure, and different physiological roles with a special focus on cyanobacteria. Despite the physiological function of PHB in the cyanobacterial phylum still being unknown, these organisms provide the unique opportunity to directly convert atmospheric CO2 into bioplastic using a solar-based process. Recent research on PHB metabolism in the cyanobacterial model organism Synechocystis revealed a sophisticated control of PHB granule formation. Novel insights about the metabolic background of PHB synthesis resulted in the engineering of the first cyanobacterial superproducer strain.


Assuntos
Hidroxibutiratos , Synechocystis , Carbono , Dióxido de Carbono , Polímeros
9.
Microb Physiol ; 31(2): 99-108, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34010833

RESUMO

Predatory bacteria gained interest in the last 20 years. Nevertheless, only a few species are well characterized. The endobiotic predator Bdellovibrio bacteriovorus invades its prey to consume it from the inside, whereas Myxococcus xanthus hunts as a whole group to overcome its prey. Both species were described to prey on cyanobacteria as well. This minireview summarizes the findings of the last 20 years of predatory bacteria of cyanobacteria and is supplemented by new findings from a screening experiment for bacterial predators of the model organism Anabaena variabilis PCC 7937. Known predatory bacteria of cyanobacteria belong to the phyla Proteobacteria, Bacteroidetes, and Firmicutes and follow different hunting strategies. The underlying mechanisms are in most cases not known in much detail. Isolates from the screening experiment were clustered after predation behaviour and analyzed with respect to their size. The effect of predation in high nitrate levels and the occurrence of nitrogen-fixing cells, called heterocysts, are addressed.


Assuntos
Bdellovibrio bacteriovorus , Cianobactérias , Myxococcus xanthus , Animais , Comportamento Predatório
10.
Microb Physiol ; 31(2): 78-87, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33878759

RESUMO

Nitrogen starvation induces developmental transitions in cyanobacteria. Whereas complex multicellular cyanobacteria of the order Nostocales can differentiate specialized cells that perform nitrogen fixation in the presence of oxygenic photosynthesis, non-diazotrophic unicellular strains, such as Synechococcus elongatus or Synechocystis PCC 6803, undergo a transition into a dormant non-growing state. Due to loss of pigments during this acclimation, the process is termed chlorosis. Cells maintain viability in this state for prolonged periods of time, until they encounter a useable nitrogen source, which triggers a highly coordinated awakening process, termed resuscitation. The minimal set of cellular activity that maintains the viability of cells during chlorosis and ensures efficient resuscitation represents the organism's equivalent of the BIOS, the basic input/output system of a computer, that helps "booting" the operation system after switching on. This review summarizes the recent research in the resuscitation of cyanobacteria, representing a powerful model for the awakening of dormant bacteria.


Assuntos
Anemia Hipocrômica , Synechococcus , Synechocystis , Humanos , Nitrogênio
11.
J Biol Chem ; 296: 100621, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33811856

RESUMO

5-Deoxyadenosine (5dAdo) is the byproduct of many radical S-adenosyl-l-methionine enzyme reactions in all domains of life. 5dAdo is also an inhibitor of the radical S-adenosyl-l-methionine enzymes themselves, making it necessary for cells to construct pathways to recycle or dispose of this toxic metabolite. However, the specific pathways involved have long remained unexplored. Recent research demonstrated a growth advantage in certain organisms by using 5dAdo or intermediates as a sole carbon source and elucidated the corresponding salvage pathway. We now provide evidence using supernatant analysis by GC-MS for another 5dAdo recycling route. Specifically, in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus), the activity of promiscuous enzymes leads to the synthesis and excretion first of 5-deoxyribose and subsequently of 7-deoxysedoheptulose. 7-Deoxysedoheptulose is an unusual deoxy-sugar, which acts as an antimetabolite of the shikimate pathway, thereby exhibiting antimicrobial and herbicidal activity. This strategy enables organisms with small genomes and lacking canonical gene clusters for the synthesis of secondary metabolites, like S. elongatus, to produce antimicrobial compounds from primary metabolism and enzymatic promiscuity. Our findings challenge the view of bioactive molecules as sole products of secondary metabolite gene clusters and expand the range of compounds that microorganisms can deploy to compete for their ecological niche.


Assuntos
Proteínas de Bactérias/metabolismo , Desoxiadenosinas/metabolismo , Hidrolases/metabolismo , S-Adenosilmetionina/metabolismo , Metabolismo Secundário , Synechococcus/metabolismo , Proteínas de Bactérias/genética , Hidrolases/genética , Synechococcus/crescimento & desenvolvimento
12.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758091

RESUMO

Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-l-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it P II-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-PII interaction requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell.IMPORTANCE Cyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g., as major primary producers. Due to their photosynthetic lifestyle, cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis. Besides its role as a proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. Therefore, the obtained results will not only enhance our understanding of flux control in these organisms but also help to provide a scientific basis for targeted metabolic engineering and, hence, the design of photosynthesis-driven biotechnological applications.


Assuntos
Amônia/metabolismo , Ornitina/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Arginina/biossíntese , Arginina/metabolismo , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Transdução de Sinais
13.
Curr Biol ; 31(8): 1606-1615.e2, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33571435

RESUMO

The ability to resume growth after a dormant period is an important strategy for the survival and spreading of bacterial populations. Energy homeostasis is critical in the transition into and out of a quiescent state. Synechocystis sp. PCC 6803, a non-diazotrophic cyanobacterium, enters metabolic dormancy as a response to nitrogen starvation. We used Synechocystis as a model to investigate the regulation of ATP homeostasis during dormancy, and we unraveled a critical role for sodium bioenergetics in dormant cells. During nitrogen starvation, cells reduce their ATP levels and engage sodium bioenergetics to maintain the minimum ATP content required for viability. When nitrogen becomes available, energy requirements rise, and cells immediately increase ATP levels, employing sodium bioenergetics and glycogen catabolism. These processes allow them to restore the photosynthetic machinery and resume photoautotrophic growth. Our work reveals a precise regulation of the energy metabolism essential for bacterial survival during periods of nutrient deprivation.

14.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526690

RESUMO

Nitrogen limitation imposes a major transition in the lifestyle of nondiazotrophic cyanobacteria that is controlled by a complex interplay of regulatory factors involving the pervasive signal processor PII Immediately upon nitrogen limitation, newly fixed carbon is redirected toward glycogen synthesis. How the metabolic switch for diverting fixed carbon toward the synthesis of glycogen or of cellular building blocks is operated was so far poorly understood. Here, using the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 as model system, we identified a novel PII interactor, the product of the sll0944 gene, which we named PirC. We show that PirC binds to and inhibits the activity of 2,3-phosphoglycerate-independent phosphoglycerate mutase (PGAM), the enzyme that deviates newly fixed CO2 toward lower glycolysis. The binding of PirC to either PII or PGAM is tuned by the metabolite 2-oxoglutarate (2-OG), which accumulates upon nitrogen starvation. In these conditions, the high levels of 2-OG dissociate the PirC-PII complex to promote PirC binding to and inhibition of PGAM. Accordingly, a PirC-deficient mutant showed strongly reduced glycogen levels upon nitrogen deprivation, whereas polyhydroxybutyrate granules were overaccumulated compared to wild-type. Metabolome analysis revealed an imbalance in 3-phosphoglycerate to pyruvate levels in the pirC mutant, confirming that PirC controls the carbon flux in cyanobacteria via mutually exclusive interaction with either PII or PGAM.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , Proteínas PII Reguladoras de Nitrogênio/genética , Fosfoglicerato Mutase/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Cianobactérias/metabolismo , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Fosfoglicerato Mutase/metabolismo , Synechocystis/genética , Synechocystis/metabolismo
15.
ACS Sens ; 6(3): 703-708, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33496577

RESUMO

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19 , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , SARS-CoV-2/imunologia , COVID-19/sangue , COVID-19/imunologia , Humanos , Imunoglobulina G/imunologia , Fenômenos Magnéticos
16.
FEBS J ; 288(4): 1142-1162, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32599651

RESUMO

The PII-like protein CutA is annotated as being involved in Cu2+ tolerance, based on analysis of Escherichia coli mutants. However, the precise cellular function of CutA remains unclear. Our bioinformatic analysis reveals that CutA proteins are universally distributed across all domains of life. Based on sequence-based clustering, we chose representative cyanobacterial CutA proteins for physiological, biochemical, and structural characterization and examined their involvement in heavy metal tolerance, by generating CutA mutants in filamentous Nostoc sp. and in unicellular Synechococcus elongatus. However, we were unable to find any involvement of cyanobacterial CutA in metal tolerance under various conditions. This prompted us to re-examine experimentally the role of CutA in protecting E. coli from Cu2+ . Since we found no effect on copper tolerance, we conclude that CutA plays a different role that is not involved in metal protection. We resolved high-resolution CutA structures from Nostoc and S. elongatus. Similarly to their counterpart from E. coli and to canonical PII proteins, cyanobacterial CutA proteins are trimeric in solution and in crystal structure; however, no binding affinity for small signaling molecules or for Cu2+ could be detected. The clefts between the CutA subunits, corresponding to the binding pockets of PII proteins, are formed by conserved aromatic and charged residues, suggesting a conserved binding/signaling function for CutA. In fact, we find binding of organic Bis-Tris/MES molecules in CutA crystal structures, revealing a strong tendency of these pockets to accommodate cargo. This highlights the need to search for the potential physiological ligands and for their signaling functions upon binding to CutA. DATABASES: Structural data are available in Protein Data Bank (PDB) under the accession numbers 6GDU, 6GDV, 6GDW, 6GDX, 6T76, and 6T7E.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Proteínas de Bactérias/química , Metais Pesados/farmacologia , Nostoc/química , Synechococcus/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Calorimetria/métodos , Cobre/farmacologia , Cristalografia por Raios X , Modelos Moleculares , Mutação , Nostoc/genética , Nostoc/metabolismo , Conformação Proteica , Multimerização Proteica , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Synechococcus/genética , Synechococcus/metabolismo
17.
Microb Cell Fact ; 19(1): 231, 2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33353555

RESUMO

BACKGROUND: PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon-neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW). RESULTS: We have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic. CONCLUSION: The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.


Assuntos
Hidroxibutiratos/metabolismo , Engenharia Metabólica , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Cupriavidus necator/genética , Grânulos Citoplasmáticos/ultraestrutura , Hidroxibutiratos/química , Polímeros/metabolismo , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento , Synechocystis/ultraestrutura
18.
mSystems ; 5(6)2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144311

RESUMO

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status.IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

19.
Curr Microbiol ; 77(11): 3538-3545, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32803419

RESUMO

Lactic acid bacteria are widespread in various ecological niches with the excess of nutrients and have reduced capabilities to adapt to starvation. Among more than 280 Lactobacillus species known to the date, only five, including Lactobacillus hilgardii, carry in their genome the gene encoding for PII-like protein, one of the central regulators of cellular metabolism generally responding to energy- and carbon-nitrogen status in many free-living Bacteria, Archaea and in plant chloroplasts. In contrast to the classical PII encoding genes, in L. hilgardii genome the gene for PII homologue is located within the potABCD operon, encoding the ABC transporter for polyamines. Based on the unique genetic context and low sequence identity with genes of any other so-far characterized PII subfamilies, we termed this gene potN (Pot-protein, Nucleotide-binding). The second specific feature of L. hilgardii genome is that many genes encoding the proteins with similar function are present in two copies, while with low mutual identity. Thus, L. hilgardii LMG 7934 genome carries two genes of glutamine synthetase with 55% identity. One gene is located within classical glnRA operon with the gene of GlnR-like transcriptional regulator, while the second is monocistronic. Together with the relative large genome of L. hilgardii as compared to other Lactobacilli (2.771.862 bp vs ~ 2.2 Mbp in median), these data suggest significant re-arrangements of the genome and a wider range of adaptive capabilities of L. hilgardii in comparison to other bacteria of the genus Lactobacillus.


Assuntos
Lactobacillus , Óperon , Proteínas de Bactérias/genética , Sequência de Bases , Lactobacillus/genética
20.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140462, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32485238

RESUMO

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.


Assuntos
Acetilcoenzima A/metabolismo , Coenzima A/metabolismo , Malato Desidrogenase/metabolismo , Malatos/metabolismo , NADP/metabolismo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Malato Desidrogenase/genética , Fosfato Acetiltransferase/metabolismo
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