Analysis and interpretation of next generation sequencing data from the nitrogen fixing bacterium Klebsiella oxytoca under different environmental conditions

• Κύριος συγγραφέας: Γκουβέλης, Μιχαλάκης
• Άλλοι συγγραφείς: Gkouvelis, Michalakis
• Γλώσσα: English
• Έκδοση: 2023
• Θέματα: Bioinformatics; Nitrogen fixation; Νext generation sequencing data analysis; Βιοπληροφορική; Αζωτοδέσμευση; Ανάλυση δεδομένων αλληλούχησης νέας γενιάς

Nitrogen fixation is employed by diazotrophic bacteria to alleviate the lack of fixed nitrogen compounds in their environment. Excess fixed nitrogen is released in their surroundings, in a form that can be used by plants. This makes free living diazotrophic bacteria potential candidates for biosynthetic strains that would provide nitrogen to crops instead of chemical fertilizers, which cause significant environmental pollution. For this to happen, the metabolic regulation that allows the bacteria to invest high amounts of energy, translational machinery, and space in the proteome for the nitrogenase, which is used for nitrogen fixation, must be fully understood. Klebsiella oxytoca is a model organism for the study of nitrogen fixation, and its nitrogen fixation mechanism has been successfully used by non-diazotrophic biosynthetic strains for the conduction of nitrogen fixation. In this project we used next generation sequencing data by K. oxytoca wild-type and gene knockout samples that were grown in low and high nitrogen conditions, to examine how its genes and pathways are regulated during nitrogen stress. The data consisted of transcriptomic reads and the analysis performed included quality control, preprocessing, alignment, creation of count tables, differential gene expression, and enrichment pathway analysis. The findings of this study show a significant upregulation of nitrogen fixation and nitrogen metabolism related genes during diazotrophic conditions, and at the same time downregulation of carbon metabolism energy consumption related genes. To a lesser extent, there was a down regulation of genes related to translation and growth of the cell. These phenomena were also evident in the enrichment of pathways, where activation of nitrogen compensation processes, and deactivation of energy consumption and growth processes were observed in diazotrophic conditions. In the gene knockout samples that were not able to fix atmospheric nitrogen and were grown in a low nitrogen environment, an increase in stress related process was observed. From these findings we conclude that during nitrogen stress the cells activate their nitrogen fixation machinery, and to accommodate it they decrease their translation and energy consumption, so the nitrogenase will have plenty of energy and space to act as long as it is needed. When there is no nitrogen fixation, and the nitrogen stress accumulates over time, the cells activate stress related processes that help them adapt in extreme conditions as a last line of defense.