| The overuse of chemical nitrogen fertilizers and its negative effects seriously restrict the sustainable development of agriculture in China.The development and utilization of characteristics of nitrogen-fixing bacteria could effectively reduce the use of chemical nitrogen fertilizers.The free-living nitrogen-fixing bacteria have the advantages of strong stress resistance and no limitation by the host.Therefore,the excavation and utilization of free-living nitrogen-fixing bacteria resources will play an important role in ensuring food security and developing ecological agriculture.To solve the problem that the activity of nitrogen-fixing bacteria was not high enough,in this paper,we collected different rhizosphere soil of different plants from all over the country for the isolation and screening of free-living nitrogen-fixing bacteria,and analyzed the nitrogen-fixing characteristics.The nitrogen-fixing activity was improved by adding rhamnolipid,using LC-MS/MS technology and biological information analysis to explore the mechanism to improve nitrogen-fixing activity.The main results were as follows:（1）A.chroococcum HR1 was isolated from the rhizosphere soil of Rosa omeiensis Rolfe in Shuozhou City,whose nitrogenase activity was 3.22 times higher than that of the control strain（A.chroococcum CICC 20603）.The strain was sensitive to most antibiotics,and its suitable growth temperature was about 35℃,with a wide p H range（p H 7.0-11.0）.The optimal nitrogen fixation conditions were as follows:temperature was 32.9℃,sucrose concentration was 14.6 g/L,shaking speed was 156 rpm,and nitrogen-fixing activity reached1228.69[nmol C2H4（mg protein h）-1],which was 1.34 times of before optimization.（2）A.chroococcum HR1 contained a circular chromosome（4,636,127 bp）,four circular plasmids（307,780 bp,76,351 bp,47,454 bp and 8,012 bp）and encoding 5,057 CDSs with complete genome size of 5,075,724 bp and 66.07%GC content.The HR1 contains the molybdenum and vanadium nitrogenase systems.In strain HR1,the nif region encode proteins for the structural of nitrogenase（nif HDK）,biosynthesis of Fe Mo-co（nif BEN）,in protection against oxidation（fes I）,iron-sulfur clusters（nif US,isc A,cys A）,a homocitrate synthase（nif V）,accessory proteins（nif TYXPWZMOQ,naf Y）,regulatory proteins for nif expression（nif AL）and flavodoxin（nif F）.The vnf genes（vnf YKGDFH and vnf UAENX）were scattered at both ends of nif genes.（3）Comparing the effects of different types of surfactants on A.chroococcum HR1,it was found that after 250 mg/L rhamnolipid treatment,the nitrogen-fixing activity of HR1increased by 39.7%.Real time PCR analysis showed that rhamnolipid could significantly increase the expression of nif A,nif H,nif D and nif K genes.Western blotting was used to detect the post-translational modifications（PTMs）levels in HR1,and it was found that succinylated modification was the main type of PTMs under nitrogen-fixing condition,and rhamnolipid affected its nitrogen-fixing activity by alleviating the decrease of protein succinylation modification level.The analysis showed that the differential succinylated modified proteins caused by rhamnolipid were mainly enriched in metabolic pathways such as TCA cycle,oxidative phosphorylation,flavodoxin（Nif F）and Mo-Fe protein（Nif DK）.The research results provide an experimental basis for the further development and utilization of nitrogen-fixing microorganisms,and also provide reference for the analysis of nitrogen-fixing mechanism. |
