RESUMO
Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.
Assuntos
Azospirillum brasilense , Microalgas , Microalgas/genética , Biocombustíveis , Transcriptoma , Ácidos Indolacéticos/metabolismo , Perfilação da Expressão Gênica , Adaptação Fisiológica/genética , Riboflavina/genética , Riboflavina/metabolismoRESUMO
Bifidobacterium longum subsp. infantis is a representative and dominant species in the infant gut and is considered a beneficial microbe. This organism displays multiple adaptations to thrive in the infant gut, regarded as a model for human milk oligosaccharides (HMOs) utilization. These carbohydrates are abundant in breast milk and include different molecules based on lactose. They contain fucose, sialic acid, and N-acetylglucosamine. Bifidobacterium metabolism is complex, and a systems view of relevant metabolic pathways and exchange metabolites during HMO consumption is missing. To address this limitation, a refined genome-scale network reconstruction of this bacterium is presented using a previous reconstruction of B. infantis ATCC 15967 as a template. The latter was expanded based on an extensive revision of genome annotations, current literature, and transcriptomic data integration. The metabolic reconstruction (iLR578) accounted for 578 genes, 1,047 reactions, and 924 metabolites. Starting from this reconstruction, we built context-specific genome-scale metabolic models using RNA-seq data from cultures growing in lactose and three HMOs. The models revealed notable differences in HMO metabolism depending on the functional characteristics of the substrates. Particularly, fucosyl-lactose showed a divergent metabolism due to a fucose moiety. High yields of lactate and acetate were predicted under growth rate maximization in all conditions, whereas formate, ethanol, and 1,2-propanediol were substantially lower. Similar results were also obtained under near-optimal growth on each substrate when varying the empirically observed acetate-to-lactate production ratio. Model predictions displayed reasonable agreement between central carbon metabolism fluxes and expression data across all conditions. Flux coupling analysis revealed additional connections between succinate exchange and arginine and sulfate metabolism and a strong coupling between central carbon reactions and adenine metabolism. More importantly, specific networks of coupled reactions under each carbon source were derived and analyzed. Overall, the presented network reconstruction constitutes a valuable platform for probing the metabolism of this prominent infant gut bifidobacteria.IMPORTANCEThis work presents a detailed reconstruction of the metabolism of Bifidobacterium longum subsp. infantis, a prominent member of the infant gut microbiome, providing a systems view of its metabolism of human milk oligosaccharides.
Assuntos
Fucose , Leite Humano , Lactente , Feminino , Humanos , Leite Humano/química , Fucose/análise , Lactose/análise , Oligossacarídeos/análise , Bifidobacterium/genética , Bifidobacterium longum subspecies infantis/metabolismo , Acetatos/análise , Carbono/análise , Lactatos/análiseRESUMO
Neither the Pseudomonas aeruginosa aldehyde dehydrogenase encoded by the PA4189 gene nor its ortholog proteins have been biochemically or structurally characterized and their physiological function is unknown. We cloned the PA4189 gene, obtained the PA4189 recombinant protein, and studied its structure-function relationships. PA4189 is an NAD+-dependent aminoaldehyde dehydrogenase highly efficient with protonated aminoacetaldehyde and 3-aminopropionaldehyde, which are much more preferred to the non-protonated species as indicated by pH studies. Based on the higher activity with aminoacetaldehyde than with 3-aminopropionaldehyde, we propose that aminoacetaldehyde might be the PA4189 physiological substrate. Even though at the physiological pH of P. aeruginosa cells the non-protonated aminoacetaldehyde species will be predominant, and despite the competition of these species with the protonated ones, PA4189 would very efficiently oxidize ACTAL in vivo, producing glycine. To our knowledge, PA4189 is the first reported enzyme that might metabolize ACTAL, which is considered a dead-end metabolite because its consuming reactions are unknown. The PA4189 crystal structure reported here suggested that the charge and size of the active-site residue Glu457, which narrows the aldehyde-entrance tunnel, greatly define the specificity for small positively charged aldehydes, as confirmed by the kinetics of the E457G and E457Q variants. Glu457 and the residues that determine Glu457 conformation inside the active site are conserved in the PA4189 orthologs, which we only found in proteobacteria species. Also is conserved the PA4189 genomic neighborhood, which suggests that PA4189 participates in an uncharacterized metabolic pathway. Our results open the door to future efforts to characterize this pathway.
Assuntos
Aldeídos , Pseudomonas aeruginosa , Aldeídos/química , Propilaminas , Oxirredutases , Cinética , Especificidade por SubstratoRESUMO
Escherichia coli exposed to tellurite shows augmented membrane lipid peroxidation and ROS content. Also, reduced thiols, protein carbonylation, [Fe-S] center dismantling, and accumulation of key metabolites occur in these bacteria. In spite of this, not much is known about tellurite effects on the E. coli electron transport chain (ETC). In this work, tellurite-mediated damage to the E. coli ETC's NADH dehydrogenases and terminal oxidases was assessed. Mutant lacking ETC components showed delayed growth, decreased oxygen consumption and increased ROS in the presence of the toxicant. Membranes from tellurite-exposed E. coli exhibited decreased oxygen consumption and dNADH/NADH dehydrogenase activity, showing an impairment of NDH-I but not of NDH-II activity. Regarding terminal oxidases, only the bo oxidase complex was affected by tellurite. When assaying NDH-I and NDH-II activity in the presence of superoxide, the NDH-I complex was preferentially damaged. The activity was partly restored in the presence of reducing agents, sulfide and Fe(2+) under anaerobic conditions, suggesting that damage affects NDH-I [4Fe-4S] centers. Finally, augmented membrane protein oxidation along with reduced oxidase activity was observed in the presence of the toxicant. Also, the increased expression of genes encoding alternative terminal oxidases probably reflects a cell's change towards anaerobic respiration when facing tellurite.