Biological and genomic analysis of a symbiotic nitrogen fixation defective mutant in Medicago truncatula.

Medicago truncatula has been selected as one of the model legume species for gene functional studies. To elucidate the functions of the very large number of genes present in plant genomes, genetic mutant resources are very useful and necessary tools. Fast Neutron (FN) mutagenesis is effective in inducing deletion mutations in genomes of diverse species. Through this method, we have generated a large mutant resource in M. truncatula. This mutant resources have been used to screen for different mutant using a forward genetics methods. We have isolated and identified a large amount of symbiotic nitrogen fixation (SNF) deficiency mutants. Here, we describe the detail procedures that are being used to characterize symbiotic mutants in M. truncatula. In recent years, whole genome sequencing has been used to speed up and scale up the deletion identification in the mutant. Using this method, we have successfully isolated a SNF defective mutant FN007 and identified that it has a large segment deletion on chromosome 3. The causal deletion in the mutant was confirmed by tail PCR amplication and sequencing. Our results illustrate the utility of whole genome sequencing analysis in the characterization of FN induced deletion mutants for gene discovery and functional studies in the M. truncatula. It is expected to improve our understanding of molecular mechanisms underlying symbiotic nitrogen fixation in legume plants to a great extent.

Removal of Mg2+ inhibition benefited the growth and isolation of ammonia-oxidizing bacteria: An inspiration from bacterial interaction.

Ammonia-oxidizing bacteria (AOB) play an important role in the global nitrogen cycle and have broad applications in the nitrogen removal from wastewater. However, the AOB species are sensitive to environmental factors and usually form tight relationships with other microbes, making the AOB isolation and maintenance are difficult and time-consuming. In this study, the relationship that occurred between AOB and their bacterial partners was found to be able to improve the ammonia oxidation; during the co-cultivation, the magnesium ions (Mg2+) with removal rate as high as 36.7% was removed from culture medium by the concomitant bacterial species, which was regarded as the main reason for improving ammonia oxidation. During the pure cultivation of AOB isolate, when the concentration of Mg2+ reduced to low levels, the ammonia-oxidizing activity was more than 5 times and the amoA gene expression was more than 12 times higher than that grown in the initial culture medium. Based on a newly designed culture medium, the ammonia oxidation of AOB isolate grown in liquid culture was significantly promoted and the visible AOB colonies with much more number and larger diameter were observed to form on agar plates. With the addition of high concentration of calcium carbonate (CaCO3), AOB colonies could be easily and specifically identified by following the hydrolytic zones that formed around AOB colonies. Another AOB isolates were successively obtained from different samples and within a short time, suggesting the feasibility and effectivity of this culture medium and strategy on the AOB isolation from environments.