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Aquat Toxicol ; 245: 106121, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35180454


Cyanobacteria are oxygen-evolving photosynthetic autotrophs essential for nutrient cycling in the environment. They possess multiple control mechanisms for their cellular activities in order to adapt to the environment. While protein translation is essential for cell survival and adaptation, the regulation and the flexibility of this process are poorly understood in cyanobacteria. ß-N-methylamino-L-alanine (BMAA), an amino acid analog proposed as an environmental neurotoxin, is highly toxic to the filamentous diazotrophic cyanobacterium Anabaena PCC 7120. In this study, through genetic analysis of BMAA-resistant mutants, we demonstrate that the system responsible for modification of ANN-decoding tRNAs with N(6)-threonylcarbamoyl adenosine (t6A) is involved in BMAA sensitivity through the control of translation. Both BMAA and inactivation of the t6A biosynthesis pathway affect translational fidelity and ribosome assembly. However, the two factors display either additive effects on translational elongation, or attenuate each other over translational fidelity or the resistance/sensitivity to antibiotics that inhibit different steps of the translational process. BMAA has a broad effect on translation and transcription, and once BMAA enters the cells, the presence of the t6A biosynthesis pathway increases the sensitivity of the cells towards this toxin. BMAA-resistant mutants screening is an effective method for getting insight into the toxic mechanisms of BMAA. In addition, BMAA is a useful tool for probing translational flexibility of cyanobacteria, and the characterization of the corresponding resistant mutants should help us to reveal translational mechanism allowing cyanobacteria to adapt to changing environments.

Diamino Aminoácidos , Anabaena , Cianobactérias , Poluentes Químicos da Água , Adenosina/análogos & derivados , Diamino Aminoácidos/toxicidade , Anabaena/genética , Anabaena/metabolismo , Cianobactérias/metabolismo , Neurotoxinas/metabolismo , RNA de Transferência/metabolismo , Poluentes Químicos da Água/toxicidade
J Anal Methods Chem ; 2021: 6333989, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34513111


In this study, a method using ultrahigh-performance liquid chromatography with photodiode array (UHPLC-PDA) was established and validated for the simultaneous quantification of 10 active components, including eight macamides and two glucosinolates, in Lepidium meyenii (maca). A gas chromatographic mass spectroscopy (GC-MS) method was used to determine the levels of three benzyl isothiocyanates and two sterols in maca. Liquid chromatographic separation was achieved on a Waters Acquity UHPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) with gradient elution over 15 min. The mobile phase was (B) acetonitrile-(A) 10 mM aqueous ammonium phosphate, and the detection wavelength was 210 nm. The gas chromatographic separation was performed on an SH-Rxi-1 MS column, and the ionization mode was electron ionization (EI). Two methods were confirmed to have desirable precision (RSD < 1.58%), repeatability (RSD < 1.97%), stability (RSD < 1.76%), and good linearity (R 2 ≥ 0.999) within the test range. The recoveries were in the range of 96.79-109.99%, with an RSD below 2.39%. We applied the established methods and successfully analyzed 15 compounds in maca processed under different drying conditions, providing a comprehensive reference for maca processing method of development. In summary, this study provided two rapid and effective methods for the quantification of 15 active components, which contributed to the in-depth maca quality control and provided a reference for the development of maca products.

mBio ; 12(4): e0138221, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34253066


Bacterial cell division, with a few exceptions, is driven by FtsZ through a treadmilling mechanism to remodel and constrict the rigid peptidoglycan (PG) layer. Yet different organisms may differ in the composition of the cell division complex (divisome). In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, hetF is required for the initiation of the differentiation of heterocysts, cells specialized in N2 fixation under combined-nitrogen deprivation. In this study, we demonstrate that hetF is expressed in vegetative cells and necessary for cell division under certain conditions. Under nonpermissive conditions, cells of a ΔhetF mutant stop dividing, consistent with increased levels of HetF under similar conditions in the wild type. Furthermore, HetF is a membrane protein located at midcell and cell-cell junctions. In the absence of HetF, FtsZ rings are still present in the elongated cells; however, PG remodeling is abolished. This phenotype is similar to that observed with the inhibition of the septal PG synthase FtsI. We further reveal that HetF is recruited to or stabilized at the divisome by interacting with FtsI and that this interaction is necessary for HetF function in cell division. Our results indicate that HetF is a member of the divisome depending mainly on light intensity and reveal distinct features of the cell division machinery in cyanobacteria that are of high ecological and environmental importance. IMPORTANCE Cyanobacteria shaped the Earth's evolutionary history and are still playing important roles for elementary cycles in different environments. They consist of highly diverse species with different cell shapes, sizes, and morphologies. Although these properties are strongly affected by the process of cytokinesis, the mechanism remains largely unexplored. Using different approaches, we demonstrate that HetF is a new component of the cell division machinery under certain environmental conditions in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. The common and diverged characteristics of cell division in prokaryotes reflect the evolutionary history of different bacteria as an adaptive measure to proliferate under certain environmental conditions. As a protein for cell differentiation, the recruitment of HetF to the septum illustrates such an adaptive mechanism in cyanobacteria.

Anabaena/genética , Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Divisão Celular/genética , Anabaena/química , Proteínas de Bactérias/genética , Divisão Celular/fisiologia , Regulação Bacteriana da Expressão Gênica , Fenótipo
Toxins (Basel) ; 12(8)2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823543


Produced by cyanobacteria and some plants, BMAA is considered as an important environmental factor in the occurrence of some neurodegenerative diseases. Neither the underlying mechanism of its toxicity, nor its biosynthetic or metabolic pathway in cyanobacteria is understood. Interestingly, BMAA is found to be toxic to some cyanobacteria, making it possible to dissect the mechanism of BMAA metabolism by genetic approaches using these organisms. In this study, we used the cyanobacterium Anabaena PCC 7120 to isolate BMAA-resistant mutants. Following genomic sequencing, several mutations were mapped to two genes involved in amino acids transport, suggesting that BMAA was taken up through amino acid transporters. This conclusion was supported by the protective effect of several amino acids against BMAA toxicity. Furthermore, targeted inactivation of genes encoding different amino acid transport pathways conferred various levels of resistance to BMAA. One mutant inactivating all three major amino acid transport systems could no longer take up BMAA and gained full resistance to BMAA toxicity. Therefore, BMAA is a substrate of amino acid transporters, and cyanobacteria are interesting models for genetic analysis of BMAA transport and metabolism.

Sistemas de Transporte de Aminoácidos/genética , Diamino Aminoácidos/metabolismo , Aminoácidos/metabolismo , Anabaena/genética , Anabaena/metabolismo , Diamino Aminoácidos/farmacologia , Anabaena/efeitos dos fármacos , Genoma Bacteriano , Mutação , Neurotoxinas/metabolismo
Artigo em Inglês | MEDLINE | ID: mdl-30877893


A methionine methyl ester-modified coumarin derivative was designed and synthesized, which could discriminate Cu2+ from other metal ions in HEPES buffer (10 mM, pH 7.4)/CH3CN (40:60, V/V). The detection limit of WM toward Cu2+ was 1.84 × 10-7 M, which was lower than the concentration of Cu2+ in drinking water suggested by WHO and EPA. And the proposed coordination mode exhibiting the interaction between WM and Cu2+ was studied by UV-Vis, fluorescence spectrum, ESI-MS and FT-IR. Based on the fluorescent reversibility of WM, WM was considered as a molecular logic gate and molecular keypad lock. In addition, the test strips and the silica gel plates prepared from the solution of WM also demonstrate the favorable selectivity toward Cu2+.

Cobre/análise , Cumarínicos/síntese química , Água Potável/análise , Corantes Fluorescentes/síntese química , Metionina/análogos & derivados , Poluentes Químicos da Água/análise , Cátions Bivalentes/análise , Colorimetria/métodos , Cumarínicos/química , Corantes Fluorescentes/química , Metionina/síntese química , Metionina/química , Modelos Moleculares , Sílica Gel/química
ACS Synth Biol ; 8(1): 170-180, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30525474


CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a "two-spacers" strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.

Anabaena/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Genoma Bacteriano/genética , DNA Polimerase I/genética , Edição de Genes