| As a kind of prokaryotes that can orient or swim towards the geomagnetic field, magnetotactic bacteria can synthesize magnetosomes, which are unique organelles comprising membrane-enveloped, nano-sized crystals of a magnetic iron mineral that are aligned in intracellular chains. Their perfect magnetic and crystalline properties, the presence of an membrane, the uniform size and their large surface-to-volume ratio make magnetosomes potentially useful in a number of fields, e.g., in the biology, medicine and industry.The formation of magnetosome is a process involved in uptake of iron, formation of magnetosome vesicle, magnetosomal iron transport and biomineralization of magnetic crystals, which is under strict genetic control. The establishment of genetic systems for several magnetotactic bacteria species made the study of magnetosome formation on the molecular level available. The sequence of several magnetotactic bacteria genomes and the identification of a large genomic island have promoted the research on the mechanism of magnetosome formation. It was shown that magnetosomes were assembled in a step-wise manner in which membrane invagination, magnetosome protein localization, and biomineralization are placed under discrete genetic control.We studied the characteristic of the biomineralization protein Mms6 in Magnetospirillum magneticum AMB-1 and the relationship between the the transcription of gene mms6 and magnetosome formation. Also, we obtained a nonmagnetic mutant via transposon mutagenesis and carried out more work to study the mechanism how the mutated gene affected the magnetosome formation. The major results are as follows:1. The relationship between the transcription of MAI gene mms6 and magnetosome formation in AMB-1The tightly bound to magnetic particles protein Mms6 can control the morphology of the crystal in AMB-1, which may function on the process of biomineralization in the formation of magnetosomes. On the genetic level, the transcription of gene mms6 may be related to the magnetosome formation in vivo. We studied the transcriptional level of gene mms6 under different culture conditions via Real Time PCR and reverse transcription PCR. Under the aerobic conditions AMB-1 cannot synthesize magnetosomes, the transcriptional level of mms6 kept constant with the growth of AMB-1 but was far below that under liquid static conditions. Under liquid static conditions the transcriptional level of mms6 kept fluctuant with the growth of AMB-1, which became increased as the Cmag value raised; however, the transcriptional level of mms6 decreased obviously when the Cmag value no more raised. These results indicated that the transcription of gene mms6 was closely associated with the magnetosome formation. The transcription of mms6 promoter maybe reflected via the observation on the fluorescent of GFP fused with the gene mms6 promoter. The results indicated that the fluorescent signal was observed with the appearance of Cmag value and the fluorescent signal became stronger with the increase of Cmag value. However, the fluorescent signal decreased when the Cmag value became steady. These results were in accord with that of Real Time PCR and indicated the relationship between the transcription of mms6 and magnetosome formation.2. The purification and characterization of the protein Mms6The recombinant protein GST-Mms6 and His-Mms6 were purified via affinity chromatography and the characteristic of His-Mms6 was studied in vitro. The recombinant protein His-Mms6 exited as a multi-polymer under native conditions, and CD spectrum indicated lots of a-helical secondary structure in His-Mms6. The observation of TEM showed the morphology of protein His-Mms6 resembled a loop via self-aggregation. The characteristic of binding iron in His-Mms6 could not be observed via staining for iron-binding proteins; however, the conformation of the protein became irregular with the present of iron ion. Also, the gene mms6 was mutated in AMB-1 and the Cmag value of the mutant decreased about 50% compared with wild type. This was consistent with the results of GFP and Real Time PCR, which both showed that the transcription of gene mms6 had a close relationship with the magnetosome formation in AMB-1.3. Two nonmagnetic mutants results from the mutated gene outside of "magnetosome island" (MAI) were obtained via TnSC189 transposon mutagenesisAlthough many works for magnetosome formation on the genetic level were reported, most of which focused on the MAI internal genes. There were few reports on the genes outside MAI that could affect magnetosome formation. To identify factors involved in magnetosome synthesis, a genetic screen with TnSC189 transposon was performed. After screening through about 2000 colonies, six mutants with defects in magnetite formation were obtained. Among the six mutants, three were found to result from the loss of entire MAI. One mutant was determined to result from the insertion in the mamAB gene cluster that has been shown to be implicated in magnetosome formation. Insertions outside MAI were identified in other two mutants, including the mutant AMB01 resulted from the disruption of gene amb2469 (excinuclease ABC subunit A) and AMB06 resulted from the disruption of gene amb2944 (nitric oxide reductase large subunit). Under the liquid static conditions, AMB01 displayed the similar growth rate as the wild type but had little response to magnetic fields; AMB06 had a clear growth defect and could not respond to magnetic fields.4. The denitrification pathway in AMB-1 involved in the growth and magnetosome formation of cells under anaerobic conditionsThe analysis on the genome indicated an entire denitrification pathway consist of nitrate reductases, nitrite reductases (Nir), nitric oxide reductases (Nor) and nitrous oxide reductases may well function in AMB-1. The Nir was mutated in mutant AMB06 (Nor mutated) and the double mutant AMB0602 was obtained. The strains AMB-1, AMB06 and AMB0602 were cultured under different conditions with the nitrate as the sole nitrogen source, the results indicated the denitrification in AMB-1 displayed different effect on the growth and magnetosome formation of cells under different oxygen pressure conditions:Under the liquid static conditions, wild type AMB-1 could grow normally and synthesize magnetosomes. AMB06 in which denitrification pathway was blocked had a growth defect and could not synthesize intracellular magnetosomes, however, the growth and magntosome formation were partially restored in the double mutant AMB0602. It was indicated that the mutation of Nir could alleviate the defects resulted from the mutation of Nor under the liquid static conditions. Under anaerobic conditions, AMB0602 as well as AMB06 displayed more severe growth defect compared with wild type and could synthesize shorter chains of only 2-8 magnetite crystals as compared with more than 20 magnetite crystals per wild type cell. The results suggested that the denitrification pathway could support AMB-1 to grow normally and synthesize magnetosomes under anaerobic conditions; the mutation of Nor did not cause AMB06 to loss the ability to synthesize magnetosomes. However, the capacity of AMB06 in growth and magnetosome formation under anaerobic conditions was severely affected because of the block of denitrification pathway. Under the conditions suitable for both growth and magnetosome formation (there were enough oxygen to support the growth of AMB-1, but the dissolved oxygen in the medium could decrease to the level suitable for magnetosome formation), all the strains of AMB-1, AMB06 and AMB0602 displayed little difference on the growth and magnetosome formation. Also, all the three strains had the similar growth rate and could not synthesize magnetosomes under aerobic conditions. On the other hand, AMB-1 maybe not utilize nitrate as terminal electron acceptors to perform denitrification in the presence of enough oxygen.5. AMB-1 could not utilize ammonium salt under anaerobic conditionsAmmonium salt was used as the sole nitrogen source to culture AMB-1 and AMB06. It was shown that both strains could utilize ammonium salt to growth under aerobic or liquid static conditions. However, both strains displayed little growth rate under anaerobic conditions. When the medium was added with nitrate or bubbling with air, AMB-1 performed growth rate under anaerobic conditions. These suggested that AMB-1 could not utilize ammonium salt as the sole nitrogen source under anaerobic conditions. On the other hand, AMB-1 could not utilize ammonium salt to perform denitrification. |
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