Genome-Wide Analyses of Proteome and Acetylome in Zymomonas mobilis Under N2-Fixing Condition

Zymomonas mobilis, a promising candidate for industrial biofuel production, is capable of nitrogen fixation naturally without hindering ethanol production. However, little is known about the regulation of nitrogen fixation in Z. mobilis. We herein conducted a high throughput analysis of proteome and protein acetylation in Z. mobilis under N2-fixing conditions and established its first acetylome. The upregulated proteins mainly belong to processes of nitrogen fixation, motility, chemotaxis, flagellar assembly, energy production, transportation, and oxidation–reduction. Whereas, downregulated proteins are mainly related to energy-consuming and biosynthetic processes. Our acetylome analyses revealed 197 uniquely acetylated proteins, belonging to major pathways such as nitrogen fixation, central carbon metabolism, ammonia assimilation pathway, protein biosynthesis, and amino acid metabolism. Further, we observed acetylation in glycolytic enzymes of central carbon metabolism, the nitrogenase complex, the master regulator NifA, and the enzyme in GS/GOGAT cycle. These findings suggest that protein acetylation may play an important role in regulating various aspects of N2-metabolism in Z. mobilis. This study provides new knowledge of specific proteins and their associated cellular processes and pathways that may be regulated by protein acetylation in Z. mobilis.

Metabolic control of nitrogen fixation in rhizobium-legume symbioses

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD+ ratio than sugars. Modeling and 13C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses.

Studying the Microbiome of Cyanobacterial Biocrusts From Drylands and Its Functional Influence on Biogeochemical Cycles

Background: Drylands are areas under continuous degradation and desertification largely covered by cyanobacterial biocrusts. Several studies have already shown that soil microorganisms play a fundamental role in the correct soil functioning. Nevertheless, little is known about the relationship taxonomy-function in arid soils and, in particular, in cyanobacterial biocrusts. An in-depth study of the taxonomic composition and the functions carried out by soil microorganisms in biogeochemical cycles was here carried out by using a shotgun metagenomic approach. Results: Metagenomic analysis carried out in this study showed a high taxonomic and functional similarity in both incipient and mature cyanobacterial biocrusts types with a dominance of Proteobacteria, Actinobacteria and Cyanobacteria. The predominant functional categories related to soil biogeochemical cycles were “carbon metabolism” followed by “phosphorus, nitrogen, sulfur, potassium and iron metabolism”. Reads involved in the metabolism of carbohydrates and respiration were the most abundant functional classes. In the N cycle dominated “ammonia assimilation” and “Nitrate and nitrite ammonification”. The major taxonomic groups also seemed to drive phosphorus and potassium cycling by the production of organic acids and the presence of extracellular enzymes and specialised transporters. Sulfur assimilation was also predominantly led by actinobacteria via the acquisition of sulfur from organosulfonated compounds. The main strategy followed for iron uptake seemed to be the synthesis and release of siderophores, mostly derived from representatives of the genus Pseudomonas. Conclusions: The absence of significant differences between both type of biocrusts was suggested to be due to the identical habitat-specific characteristics, the inherent variability associated with metagenomic sampling and experimental design limitations. There is metabolic diversity of respiration processes over the photosynthesis, but a diverse group of microorganisms, predominantly Actinobacteria and Proteobacteria were also involved in CO2 fixation metabolism. A preferential uptake of ammonium over nitrate as an economic strategy to avoid the high consumption of ATP was confirmed. Moreover, the functional redundancy presented by the microbial community was interpreted as a strategy to maintain the correct functioning of the soil biogeochemical cycles and therefore of the ecosystem in general. Evidence of sythrophic growth was nevertheless observed. Biotechnological potential as plant growth promoters was also identified.

Metabolic constraints on nitrogen fixation by rhizobia in legume nodules

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which use plant-derived dicarboxylates as energy and electron sources for reduction of atmospheric N2 into ammonia for secretion to plants. Using heterogeneous genome-scale datasets, we reconstructed a model of bacteroid metabolism to investigate the effects of varying dicarboxylate and oxygen supply on carbon and nitrogen allocation. Modelling and 13C metabolic flux analysis in bacteroids indicate that microaerobiosis restricts the decarboxylating arm of the TCA cycle and limits ammonia assimilation into glutamate. Catabolism of dicarboxylates induces a higher oxygen demand but also a higher NADH/NAD+ ratio compared to sugars. Carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules with alanine secretion increasing as oxygen tension decreases. Our results provide a framework for understanding fundamental constraints on rhizobial metabolism during symbiotic nitrogen fixation.

Improving sewage sludge compost process and quality by carbon sources addition

In present study, the effects of carbon sources on compost process and quality were evaluated in the lab-scale sewage sludge (SS) composting. The composting experiments were performed for 32 days in 5 L reactors. The results showed that carbon sources could change the nitrogen conversion and improve the compost quality. Especially, the readily degradable carbon source could promote organic matter degradation, improve nitrogen conversion process and accelerate compost maturation. The addition of glucose and sucrose could increase dissolved organic carbon, CO2 emission, dehydrogenase activity, nitrification and germination index during the SS composting. That's because glucose and sucrose could be quickly used by microbes as energy and carbon source substance to increase activity of microbes and ammonia assimilation. What's more, the NH3 emission was reduced by 26.9% and 32.1% in glucose and sucrose treatments, respectively. Therefore, the addition of readily degradable carbon source could reduce NH3 emission and improve compost maturity in the SS composting.

Improved ammonia production from soybean residues by cell surface-displayed l-amino acid oxidase on yeast

ABSTRACT Ammonia is critical for agricultural and chemical industries. The extracellular production of ammonia by yeast (Saccharomyces cerevisiae) using cell surface engineering can be efficient approach because yeast can avoid growth deficiencies caused by knockout of genes for ammonia assimilation. In this study, we produced ammonia outside the yeast cells by displaying an l-amino acid oxidase with a wide substrate specificity derived from Hebeloma cylindrosporum (HcLAAO) on yeast cell surfaces. The HcLAAO-displaying yeast successfully produced 12.6 m m ammonia from a mixture of 20 proteinogenic amino acids (the theoretical conversion efficiency was 63%). We also succeeded in producing ammonia from a food processing waste, soybean residues (okara) derived from tofu production. The conversion efficiency was 88.1%, a higher yield than reported in previous studies. Our study demonstrates that ammonia production outside of yeast cells is a promising strategy to utilize food processing wastes.

Seasonal and Nutrient Supplement Responses in Rumen Microbiota Structure and Metabolites of Tropical Rangeland Cattle

This study aimed to characterize the rumen microbiota structure of cattle grazing in tropical rangelands throughout seasons and their responses in rumen ecology and productivity to a N-based supplement during the dry season. Twenty pregnant heifers grazing during the dry season of northern Australia were allocated to either N-supplemented or un-supplemented diets and monitored through the seasons. Rumen fluid, blood, and feces were analyzed before supplementation (mid-dry season), after two months supplementation (late-dry season), and post supplementation (wet season). Supplementation increased average daily weight gain (ADWG), rumen NH3–N, branched fatty acids, butyrate and acetic:propionic ratio, and decreased plasma δ15N. The supplement promoted bacterial populations involved in hemicellulose and pectin degradation and ammonia assimilation: Bacteroidales BS11, Cyanobacteria, and Prevotella spp. During the dry season, fibrolytic populations were promoted: the bacteria Fibrobacter, Cyanobacteria and Kiritimatiellaeota groups; the fungi Cyllamyces; and the protozoa Ostracodinium. The wet season increased the abundances of rumen protozoa and fungi populations, with increases of bacterial families Lachnospiraceae, Ruminococcaceae, and Muribaculaceae; the protozoa Entodinium and Eudiplodinium; the fungi Pecoramyces; and the archaea Methanosphera. In conclusion, the rumen microbiota of cattle grazing in a tropical grassland is distinctive from published studies that mainly describe ruminants consuming better quality diets.