Functional Analysis Of OxyR-Like And Crp Protein In Magnetospirillum Gryphiswaldense MSR-1

Magnetotactic bacteria (MTB) is a kind of specialized microorganism, the common characteristics of those microorganisms are that they synthesze intracellular membrane enveloped nanoparticles, which are composed of magnetite or greigite, called magnetosomes. Most of the studies about MTB are focused on the formation of magnetosome and the biological regulatory mechanism of this process. It has been proved that the formation of magnetosome needs the conditions of low dissolve oxygen and high ferric concentration. However, under thus condition, the intracellular reactive oxygen species (ROS) will increase and the energy metabolism process will be inhibited. It was confirmed previously that there are some relationships between magnetosome formation and ROS eliminating. Meanwhile, some early reports support that the ferric ion act as the terminal electron acceptor instead of oxygen under low oxygen condition.To confirm the speculations described above and reveal the regulatory mechanism of magnetosome synthesis. The existed oxyR-Like and crp single mutant strains of magnetospirillum gryphiswaldense MSR-1, a kind of model organism for MTB research, were used to study the functions of these two genes and their roles during the formation of magnetosome. In most other microorganisms, the protein homologous to OxyR-Like functions as a globel regulator for ROS eliminating, and Crp mainly in charge of carbon and energy metabolism.The results show that ferromagnetism and intracellular iron content in the oxyR-Like-deficient mutant cells decreased dramatically. Transmission electron microscopy revealed that the oxyiR-deficient mutants have increased number of disfigured magnetosomes compared to the wild type, whose crystal diameter was only ～-50% of the wild type. High resolution-transmission electron microscopy analysis further showed that these disfigured particles are composed of Fe34,ε-Fe2O3 and α-Fe2O3. Hinting α-Fe2O3, ε-Fe2O3 are intermediates for magnetosome maturation.Study about the component of magnetactic particles at the initial stage of magnetosomes biosynthesis further indicates that α-Fe2O3 and ε-Fe2O3 are intermedias during magnetosome maturation in MTB. Expression profile results proved that most genes strongly affected by the disruption of oxyR-Like are related to oxidative phosphorylation, metal ion binding and two-component system.Moreover, Studies about the Crp indicate that after the deletion of crp, growth and iron absorption of the mutant were inhibited. Cells lost their magnetism completely and magnetosome synthesis was strongly impaired by the disruption of crp, the magnetosomes formed by the mutant were no longer arranged in a line and their diameter and amount were decreased dramatically. The expression profile data show that by the affection of crp disruption, genes related to peptidoglycan and NADH-quinone oxidoreductase synthesis are down-regulated obviously. On the contrary, genes related to succinate/fumarate redox and F-type ATPase biosynthesis were up-regulated, these up-regulated genes may be used to make up for the mutation of crp. Further more, it was also indicated by qRT-PCR that a lot of magnetosome island genes are down-regulated obviously by the disruption of crp, these genes are crucial for the formation of magnetosomes.Collectively, this study indicated that in M. gryphiswaldense MSR-1, mature magnetite are formed via iron multiple oxide phases, inclouding α-Fe2O3 and ε-Fe2O3. Both oxidative stress resistance system and energy/carbon source metabolism system are closely related to magnetosome formation, and both regulators, OxyR-Like and Crp, can take part in the regulation of magnetosomes synthesis in direct or indirect ways. In summary, this study not only enriched the regulation mechanism of biological control of magnetite biomineralization, but also further clarify the chemical route of this process.