Mapping Protein Interactions Of Mam Proteins And Functional Analysis Of MamA In Magnetospirillum Gryphiswaldense MSR-1

Magnetotactic bacteria (MTB) refer to a class of prokaryotes which can sense geomagnetic field through linearly arranged magnetosome in cells and perform oriented movement to find suitable habitat. Magnetosome of MTB is Fe3O4 or Fe3O4 crystal coated by biofilm. Synthesis of magnetosome belongs to biomineralization process which is strictly controlled by cells, and molecular mechanism of magnetosome synthesis is now a hotspot in the MTB research. Currently, two kinds of research strategies are employed for studying the synthesis mechanism of magnetosome:1. One or several genes possibly associated with magnetosome synthesis were knocked out to screen mutants unable to synthesize magnetosome so as to identify functions of the genes.2. Proteins having physical contact with magnetosome are purified so as to study functions thereof.20-40 kinds of specific proteins are confirmed to be located on the magnetosome membrane (MM) and named as Mam proteins which play important roles in magnetosome synthesis. However, are these proteins interconnected with one another during magnetosome synthesis? Which proteins are in key positions of the process? To solve the questions, this research took Magnetospirillum gryphiswaldense MSR-1 as the original strain, constructed interaction networks among 17 selected Mam proteins, then found three proteins, namely MamA, MamS and MamT, which played important roles in magnetosome synthesis from the networks, determined interactions thereamong, and further studied how the three corresponding genes involved in magnetosome synthesis.This paper is divided into four parts. In the first part, in order to screen interactions among the Mam proteins, vectors, used for screening protein interactions in a bacterial two-hybrid system, for the 17 proteins were constructed. Preliminary interaction screening was performed through ampicillin resistance screening and totally 62 pairs of possibly interacted proteins were screened out. Beta-galactosidase activity assay was carried out for the 62 pairs of possibly interacted proteins in a host strain XL1-Blue. Enzyme activity assay results showed that enzyme activity of MamA and MamS and enzyme activity of MamA and MamT were nearly twice the enzyme activity of a positive control, which indicated that MamA had close link with MamS and MamT. Cytoscope was utilized to analyze protein interaction networks after the enzyme activity assay, and a cluster analysis showed that MamA, MamS, MamT, MamO and MamK were clustered into one group, indicating that MamA, MamS, MamT, MamO and MamK had close link and playing important roles in magnetosome synthesis.In the second part, in order to search positions where MamA interacted with MamS and MamA interacted with MamT, N-terminal truncation mutation was carried out on the MamA protein and point mutation was carried out on two amino acids. Through beta-galactosidase activity assay of the bacterial two-hybrid system and pull-down in E. coli, it was confirmed that interactions exited between MamA and MamS and between MamA and MamT. The key position where MamA interacted with MamS was aspartate at 159th site of MamA, and the interaction between MamA and MamT was related to the first 26 amino acids at the N-terminal of MamA. Through beta-galactosidase activity assay of the bacterial two-hybrid system and protein negative staining, it was confirmed that arginine at 50th site of MamA was the key position for self-interaction of MamA.In the third part, in order to further determine interactions between MamA and MamS and between MamA and MamT in MSR-1, gene deletion mutation was performed on mamS and mamT, and fusion expression was carried out for MamA and green fluorescent protein in the mutants. By using homologous double crossing over, mamS and mamT were subjected to gene deletion mutation and complementation, and phenotypes of the mutants were preliminarily analyzed. Transmission electron microscopy (TEM) observation results showed that in the mamS deletion mutant, the number of magnetosome was reduced, maturity was lowered, but crystal form was still complete; in the mamT deletion mutant, the crystal structure of magnetosome was destroyed but magnetosomes were still linearly arranged along the longitudinal direction of the cells. MamA and the green fluorescent protein were fused and expressed in wild MSR-1 and the two gene deletion mutants respectively, and we found that MamA-GFP were intensively expressed in the center of the cells of the wild MSR-1, while expressed all over the cells of the mamS deletion mutant, showing that deletion of mamS influences the positioning of MamA in MTB.In the fourth part, another important factor for magnetosome synthesis is Fe. In order to ensure the relationship between Fe absorption of MTB and Mam proteins,5 proteins, Bfr1, Bfr2, TolQ, FeoA1 and FeoB1, related to transportation and storage of Fe were constructed to be used for vectors for screening protein interactions in the bacterial two-hybrid system, so as to screen interactions between these five proteins and Mam proteins. Preliminary screening results showed that three proteins related to Fe also had close link with MamA, MamS and MamT, thereby providing certain clues for further revealing the functions of MamA.