RESUMEN
Tetracycline destructases (TDases) are flavin monooxygenases which can confer resistance to all generations of tetracycline antibiotics. The recent increase in the number and diversity of reported TDase sequences enables a deep investigation of the TDase sequence-structure-function landscape. Here, we evaluate the sequence determinants of TDase function through two complementary approaches: (1) constructing profile hidden Markov models to predict new TDases, and (2) using multiple sequence alignments to identify conserved positions important to protein function. Using the HMM-based approach we screened 50 high-scoring candidate sequences in Escherichia coli, leading to the discovery of 13 new TDases. The X-ray crystal structures of two new enzymes from Legionella species were determined, and the ability of anhydrotetracycline to inhibit their tetracycline-inactivating activity was confirmed. Using the MSA-based approach we identified 31 amino acid positions 100% conserved across all known TDase sequences. The roles of these positions were analyzed by alanine-scanning mutagenesis in two TDases, to study the impact on cell and in vitro activity, structure, and stability. These results expand the diversity of TDase sequences and provide valuable insights into the roles of important residues in TDases, and flavin monooxygenases more broadly.
Asunto(s)
Antibacterianos , Tetraciclina , Tetraciclina/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Tetraciclinas/farmacología , Oxigenasas de Función Mixta , Escherichia coli/química , Farmacorresistencia Microbiana , FlavinasRESUMEN
Rapid microbial growth in the early phase of plant litter decomposition is viewed as an important component of soil organic matter (SOM) formation. However, the microbial taxa and chemical substrates that correlate with carbon storage are not well resolved. The complexity of microbial communities and diverse substrate chemistries that occur in natural soils make it difficult to identify links between community membership and decomposition processes in the soil environment. To identify potential relationships between microbes, soil organic matter, and their impact on carbon storage, we used sand microcosms to control for external environmental factors such as changes in temperature and moisture as well as the variability in available carbon that exist in soil cores. Using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) on microcosm samples from early phase litter decomposition, we found that protein- and tannin-like compounds exhibited the strongest correlation to dissolved organic carbon (DOC) concentration. Proteins correlated positively with DOC concentration, while tannins correlated negatively with DOC. Through random forest, neural network, and indicator species analyses, we identified 42 bacterial and 9 fungal taxa associated with DOC concentration. The majority of bacterial taxa (26 out of 42 taxa) belonged to the phylum Proteobacteria while all fungal taxa belonged to the phylum Ascomycota. Additionally, we identified significant connections between microorganisms and protein-like compounds and found that most taxa (12/14) correlated negatively with proteins indicating that microbial consumption of proteins is likely a significant driver of DOC concentration. This research links DOC concentration with microbial production and/or decomposition of specific metabolites to improve our understanding of microbial metabolism and carbon persistence.
RESUMEN
The gut microbiome can vary across differences in host lifestyle, geography, and host species. By comparing closely related host species across varying lifestyles and geography, we can evaluate the relative contributions of these factors in structuring the composition and functions of the microbiome. Here we show that the gut microbial taxa, microbial gene family composition, and resistomes of great apes and humans are more related by host lifestyle than geography. We show that captive chimpanzees and gorillas are enriched for microbial genera commonly found in non-Westernized humans. Captive ape microbiomes also had up to ~34-fold higher abundance and up to ~5-fold higher richness of all antibiotic resistance genes compared with wild apes. Through functional metagenomics, we identified a number of novel antibiotic resistance genes, including a gene conferring resistance to colistin, an antibiotic of last resort. Finally, by comparing our study cohorts to human and ape gut microbiomes from a diverse range of environments and lifestyles, we find that the influence of host lifestyle is robust to various geographic locations.
Asunto(s)
Gorilla gorilla/microbiología , Microbiota , Pan troglodytes/microbiología , Animales , Antibacterianos/farmacología , Bacterias/genética , Farmacorresistencia Microbiana , Microbioma Gastrointestinal/efectos de los fármacos , Geografía , Humanos , Estilo de Vida , MetagenómicaRESUMEN
The current extraction and use of fossil fuels has been linked to extensive negative health and environmental outcomes. Lignocellulosic biomass-derived biofuels and bioproducts are being actively considered as renewable alternatives to the fuels, chemicals, and materials produced from fossil fuels. A major challenge limiting large-scale, economic deployment of second-generation biorefineries is the insufficient product yield, diversity, and value that current conversion technologies can extract from lignocellulose, in particular from the underutilized lignin fraction. Rhodococcus opacus PD630 is an oleaginous gram-positive bacterium with innate catabolic pathways and tolerance mechanisms for the inhibitory aromatic compounds found in depolymerized lignin, as well as native or engineered pathways for hexose and pentose sugars found in the carbohydrate fractions of biomass. As a result, R. opacus holds potential as a biological chassis for the conversion of lignocellulosic biomass into biodiesel precursors and other value-added products. This review begins by examining the important role that lignin utilization will play in the future of biorefineries and by providing a concise survey of the current lignin conversion technologies. The genetic machinery and capabilities of R. opacus that allow the bacterium to tolerate and metabolize aromatic compounds and depolymerized lignin are also discussed, along with a synopsis of the genetic toolbox and synthetic biology methods now available for engineering this organism. Finally, we summarize the different feedstocks that R. opacus has been demonstrated to consume, and the high-value products that it has been shown to produce. Engineered R. opacus will enable lignin valorization over the coming years, leading to cost-effective conversion of lignocellulose into fuels, chemicals, and materials.
RESUMEN
Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho-cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner-Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus' metabolism for future lignin valorization.
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Proteínas Bacterianas , Evolución Molecular Dirigida , Redes y Vías Metabólicas , Fenol/metabolismo , Rhodococcus , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Biología de SistemasRESUMEN
Lignin utilization has been identified as a key factor in biorefinery profitability. However, lignin depolymerization generates heterogeneous aromatic mixtures that inhibit microbial growth and the conversion of lignocellulose to biochemicals. Rhodococcus opacus is a promising aromatic-catabolizing, oleaginous bacterium, but mechanisms for its aromatic tolerance and utilization remain undercharacterized. To better understand these mechanisms, we adaptively evolved R. opacus for improved utilization of 32 combinations of diverse aromatic compounds. Evolved R. opacus mutants showed up to 1900% growth improvement in the utilization of phenol, guaiacol, 4-hydroxybenzoate, vanillate, and benzoate compared to the wild-type strain. Whole genome sequencing revealed several redox-related genes with mutations shared across multiple adapted mutants. PVHG6, the mutant with the most improved growth on a mixture of multiple aromatic compounds, showed 56% lower superoxide dismutase activity than the wild-type strain, suggesting that redox reactions are important for aromatic tolerance and utilization. Comparative transcriptomics revealed by-product detoxification pathways and five aromatic funneling pathways that were upregulated in response to specific aromatic compounds. Gene knockout experiments confirmed the two degradation routes of the ß-ketoadipate pathway for five aromatic compounds. These results provide an improved understanding of aromatic bioconversion and facilitate development of R. opacus as a biorefinery host.