Study On The Denitrification Pathway And DNA Damage Repair System In Magnetospirillum Magneticum AMB-1and Their Influences On Magnetsome Formation As Well As Its Genetic Stability

Magnetotactic bacteria (MTBs) are kinds of facultative anaerobic prokaryote which are able to synthesis membrane-bounded, nano-sized magnetic particles, magnetosomes, which make them capable to response to magnetic field. MTBs can be identified in the phylum of α-, γ-,γ-Proteobacteia and Nitrospira. They can grow rigorously in oxic-anoxic transition zone of seawater or fresh water. Magnetosomes are composed by crystal nucleus, which are derived from crystallization of Fe3O4or Fe3S4under complicated genetic controlling, and membrane structure, which originated from cell inner membrane. Magnetosomes can be arranged in chains in parallel to long axis of the cell in order to maximize the cell magnetic dipolemoment. The uniformity, stable property and dispersibility of magnetosomes make them applicable in Biology, Physics and Material science.As kinds of species specific structure, the function and synthesis procedures of magnetosome having been studied for decades. So far, the results proved that the magnetosome production was an intricate process which regulated by dozens of genes and proteins. The step-wise procedures are composed of iron assimilation, transportation, oxidoreduction, magnetosome membrane origin, nucleus crystallization and magnetosome chain formation, etc,al.. Genomic analysis showed that the genes involved in magnetosome formation were arranged in a large genomic island, which called magnetosome island (MAI), and the horizontal gene transfer of the MAI between species may provide evidence for the original of MTBs.The procedures of magnetosome formation can be influenced by several metabolic pathways. Considering the iron metabolism involved in magnetsome formation, including iron transportation and oxidation state, it may coupled with denitrification pathway. Nitrate can be used in biomass synthesis by reducing to ammonium, which called assimilation. MTBs can grow with nitrate as sole nitrogen source. Two components of denitrification pathway were identified in Magnetospirillum magnetotacticum MS-1. It showed that nitrate reductase (Nar) may have function in energy supplement when cells were cultured in microaerobic condition or in magnetsome formation process. Besides, a novel nitrite reductase (Nir) with Fe(II):nitrite oxidoreductase was identified in MS-1. It was considered that the Fe(II) oxidase activity of Nir may contribute the iron metabolism which involved in magnetosome formation. Enzymology research of the two components revealed that they may participate in the process of magnetosome formation, but with non-critical function. Strains with inactivated enzymes can grow normally, but with a tiny weak in magnetosome formation. MS-1cannot grow with nitrate as sole nitrogen source without oxygen, so it is hard to confirm that there is independent denitrification pathway worked as energy supplement process under micro-or anaerobic condition.As kinds of facultative anaerobic prokaryote, MTBs can grow aerobically, with reactive oxygen species (ROS) causing damage to protein or nucleic acid. It was reported that three peroxiredoxin components were identified in Magnetospirillum magneticum AMB-1, which play a role in ROS elimination and genetic stability maintenance. The deficient of peroxiredoxin components may cause instability of the magnetosome synthesis capacity during subculture. Antioxidant mechanism of MTBs can take effect in maintaining the genetic stability of MAI. In Magnetospirillum giyphiswaldense, a recombinase, which mediate the frequent loss of MAI, RecA was identified. Strains lacking RecA showed a high stability of MAI during subculture.Different to MS-1, AMB-1can grow with nitrate as sole nitrogen source without oxygen. In this article, we intend to do two piece of work using AMB-1as material.1, we attempt to characterize the members involved in denitrification pathway, and to study the function of the pathway to the field of energy supplement and magnetosome formation process.2, we make effort in genetic screening of AMB-1to obtain some mutant with abnormal capacity in magnetosome synthesis. We obtained a mutant with defective in the function of DNA damage repair system, and learned more information of the relationship between DNA repair system and magnetosome synthesis process. The major results are follows:1. Function of Nir in denitrification pathway and magnetosome synthesis processThree Nir coding genes (nir) were predeterminated by analyzing in bioinformatics. The main Nir activity component was purified on the basis of enzyme activity assay, and then characterized by MOLDI-TOF and sequence blast. The Nir activity measurement results showed that the activity was higher in periplasmic space than cytoplasm, higher in static condition than aerobic condition.Transcript analysis of nir showed that the transcript level of nir in micro-or anaerobic condition is higher than aerobic condition, and it would increased in magnetosome synthesis phase in the time-course of life-cycle. We studied the function of nir by disrupting the coding sequence. Growth and magnetosome synthesis of the nir deficient mutant were not obviously influenced by the inactivated Nir. The results showed that Nir may have a hand but is not significant in energy supplement or magnetosome synthesis process.2. AMB-1can grow anaerobically by virtue of nitrate-dependent energy supplement pathwayAMB-1can grow with nitrate as sole nitrogen source. Ammonium used for biomass synthesis can be generated by nitrate reduction. AMB-1can grow with ammonium as sole nitrogen source under aerobic condition, but ammonium cannot support AMB-1's growth under anaerobic condition, which means the bacteria need an energy supplement pathway depending on nitrate. The Nar coding genes (nar) were predeterminated by analyzing in bioinformatics. The function of nar was studied by disrupting the coding sequence. Growth and magnetosome synthesis of nar deficient mutant in anaerobic condition were obviously inhibited by the inactivated Nar. The phenotype of the mutant was the same as wildtype AMB-1when oxygen was existed. The results suggested that energy supplement pathway depending on nitrate, which was denitrification pathway also, may existed in AMB-1, and may have function in anaerobic condition, but it may not involved in magnetosome synthesis process.3. DNA damage repair system UvrABC complex had function in the maintenance of MAI genetic stability.To learn more information about the genetic factors of magnetosome synthesis process, a genetic screen was carried out by transposon mutagenesis to obtain nonmagnetic mutants of AMB-1. A mutant with defect in UvrABC system was obtained. Growth, magnetosome synthesis capacity and MAI stability were further found to be influenced by inactivited UvrA. Site-specific insertion of uvrA led to weak magnetosome formation under aerobic condition. Only38%of the mutant cells can synthesis magnetosome, while the ratio in wildtype AMB-1is94%. The magnetosome number which produced by mutant cells is1-4, while8-20in wildtype AMB-1cells. Quantitative PCR analysis showed that the deficiency in UvrA also leads to lower MAI stability under aerobic conditions. These results indicated that the DNA repair system guarded against the instability of the MAI. Studies on the transcript level of a recombinase RecA showed that, the transcript level of recA in the mutant is11.9times higher than that in wildtype AMB-1, indicating the recombinase may involved in the loss of MAI. The insertion mutagenesis of recA in uvrA mutant rescues the phenotype of the MAI instability. These results suggested that the accumulation of unrepaired mutations caused by the absence of UvrA may account for the elevated recombination activity, which then led to the high instability level of MAI.