Caracterização morfofisiológica e genética de bactérias endofíticas isoladas de raízes de diferentes genótipos de milho (Zea mays L.).

A cultura do milho (Zea mays L.) tem relevante expressão no cenário mundial e o Estado do Paraná desempenha importante papel como maior produtor de milho no Brasil. Assim, todas as estratégias que permitam otimizar a produção deste importante cultivo são importantes para a pesquisa aplicada. Bactérias endofíticas apresentam alto potencial na elevação dos índices de produtividade, por mecanismos como a fixação biológica do nitrogênio, a promoção do crescimento de plantas pela produção de fitohormônios, o controle de patógenos, entre outros. Objetivos: Isolar bactérias que se associam endofiticamente com diferentes genótipos de milho (linhagens e híbridos) e caracterizá-las quanto a diversas propriedades morfofisiológicas e genéticas. Métodos: Inicialmente foi estabelecida uma coleção de 217 isolados de bactérias endofíticas de raízes de milho e destes, 98 foram mantidos em condições de laboratório. Foram realizadas caracterizações morfofisiológicas, incluindo morfologia de colônias, diversos testes bioquímicos (crescimento em diferentes meios de cultura, redução do nitrato, urease, catalase, tolerância intrínseca a antibióticos) e avaliação da capacidade de fixação do nitrogênio in vitro. Como etapa subsequente, avaliou-se o perfil genético das bactérias através da amplificação do DNA com o primer BOX-PCR, relacionado a regiões repetitivas e não codificantes do DNA. Foi realizado, ainda, o sequenciamento parcial do gene 16S RNAr de bactérias representantes dos principais agrupamentos obtidos com os dados morfofisiológicos, sendo identificados os gêneros Pantoea, Bacillus, Burkholderia e Klebsiella. Resultados: Foi observada alta variabilidade entre os isolados obtidos em todos os parâmetros analisados, confirmando que populações com elevado grau de diversidade morfofisiológica e genética se estabelece endofiticamente com o milho. É interessante constatar que essa diversidade ocorre mesmo em linhagens e híbridos de milho obtidos em condições normais de melhoramento para a gramínea, que não consideram a capacidade de associação com bactérias endofíticas. Conclusão: O estabelecimento dessa importante coleção, com microrganismos pertencentes a gêneros pouco estudados com a cultura do milho no Brasil permitirá a condução de estudos para a avaliação da capacidade promotora de crescimento ou mesmo fixação biológica de nitrogênio nesses isolados bacterianos

Architecture and regulation of filamentous human cystathionine beta-synthase

\ua9 The Author(s) 2024. Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders

Identification and characterization of endophytic bacteria from corn (Zea mays L.) roots with biotechnological potential in agriculture.

Six endophytic bacteria of corn roots were identified as Bacillus sp. and as Enterobacter sp, by sequencing of the 16S rRNA gene. Four of the strains, CNPSo 2476, CNPSo 2477, CNPSo 2478 and CNPSo 2480 were positive for the nitrogen fixation ability evaluated through the acetylene reduction assay and amplification of nifH gene. Two Bacillus strains (CNPSo 2477 and CNPSo 2478) showed outstanding skills for the production of IAA, siderophores and lytic enzymes, but were not good candidates as growth promoters, because they reduced seed germination. However, the same strains were antagonists against the pathogenic fungi Fusarium verticillioides, Colletotrichum graminicola, Bipolaris maydis and Cercospora zea-maydis. As an indication of favorable bacterial action, Enterobacter sp. CNPSo 2480 and Bacillus sp. CNPSo 2481 increased the root volume by 44% and 39%, respectively, and the seed germination by 47% and 56%, respectively. Therefore, these two strains are good candidates for future testing as biological inoculants for corn.201

The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C-termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors

Functional Diversification of Cerato-Platanins in Moniliophthora perniciosa as Seen by Differential Expression and Protein Function Specialization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Cerato-platanins (CP) are small, cysteine-rich fungal-secreted proteins involved in the various stages of the host-fungus interaction process, acting as phytotoxins, elicitors, and allergens. We identified 12 CP genes (MpCP1 to MpCP12) in the genome of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao, and showed that they present distinct expression profiles throughout fungal development and infection. We determined the X-ray crystal structures of MpCP1, MpCP2, MpCP3, and MpCP5, representative of different branches of a phylogenetic tree and expressed at different stages of the disease. Structure-based biochemistry, in combination with nuclear magnetic resonance and mass spectrometry, allowed us to define specialized capabilities regarding self-assembling and the direct binding to chitin and N-acetyl-glucosamine (NAG) tetramers, a fungal cell wall building block, and to map a previously unknown binding region in MpCP5. Moreover, fibers of MpCP2 were shown to act as expansin and facilitate basidiospore germination whereas soluble MpCP5 blocked NAG6-induced defense response. The correlation between these roles, the fungus life cycle, and its tug-of-war interaction with cacao plants is discussed.261112811293Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)LNBio [D03B-MX1, W01B-MX2]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2010/51884-8, 2010/14504-2, 2010/51891-4]CNPq [400796/2012-0]LNBio [D03B-MX1, W01B-MX2

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose