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MAGNETOSOME FORMATION

Introduction

Motivation

Studies of synthetic biology produced various E. coli which functions are multiply expanded. To utilixze such E. coli in real world, it is necessary to place them proper spot and have them stay there. However, we have very limited technique to realize it. Solving this problem and utilizing transformed E. coli, we tried to create E. coli which have magnet inside their cells. Therefore, we focused on magnetotactic bacteria and introduce its gene to E. coli.

About magnetotactic bacteria

In 1975, microbiologist Richard P. Blakemore first discovered a kind of magnetotactic bacteria, which now known as Magnetospirillum magnetotacticum (MS-1). He observed this bacterium under his microscope and realized that they move along the magnetic field lines of Earth's magnetic field and named this microorganism as magnetotactic bacteria (or MTB)[1]. After that, many other species of MTB were discovered, such as Magnetospirillum magneticum (AMB-1), Magnetospirillum gryphiswaldense (MSR-1). And most of these MTB are only able to thrive in an oxic-anoxic transition zone[2].

The most impressive feature of them is the magnetosomes (Fig. 2). The magnetosomes are intracellular structures that consist of magnetic, iron-mineral crystals enveloped by a membrane vesicle known as the magnetosome membrane. The magnetosome crystals typically size from 35 nm to 120 nm. Inside the MTB, the magnetosomes are organized in one or more straight chains parallel to the long axis of the cell, which function as the compass aid MTB to reach regions of optimal oxygen concentration[3].

In M. magneticum or M. gryphiswaldense, the hypothetical formation process of magnetosome can be roughly divided into three steps (Fig. 3). The first step is vesicle formation. The inner membrane of MTB swells out and makes a vesicle. The second step is iron uptake. Transporters in the magnetosome membrane pump in Fe²⁺/Fe³⁺ creating high iron ion concentration in the vesicle. The third step is biomineralization. Providing high iron ion concentration, magnetosome proteins crystalize iron ion making magnetite crystal (Fe₃O₄)[4].

Gene cluster involved in magnetosome formation

A gene cluster called Magnetosome Island (MAI) is a group of genes related to magnetosome formation[5].

MAI is highly conserved among MTB and contains 4 operons – mamAB operon, mamGFDC operon, mamXY operon, mms6 operon. In a previous research published early in this year, Kolinko et al. introduced these 4 operons from M. gryphiswaldense into photosynthetic bacteria Rhodospirillum rubrum, which is phylogenetically close to Magnetospirillum sp. yet does not make magnetosomes. They observed small vesicles formed in R. rubrum after these genes were introduced [6] (Fig. 4). They also demonstrated that the R. rubrum strain carrying these vesicles can be collected by a permanent magnet, showing that the small vesicles indeed function as magnetosomes.

To unravel the function of genes in MAI, researches had been conducted by Dirk Schüler et al. through knocking out each one of them. In MTB, magnetosomes membrane cannot be formed if either one of mamL, mamQ or mamB, which belongs to mamAB operon, is knocked out. Proteins of the three genes, though their function mechanisms are not so clear, are considered to be integrated with the inner membrane and triggers vesicle formation [4] (Fig. 5).

Magnetosome vesicle formation in E. coli

OUC-China 2013 also tried to magnetosome membrane formation in E. coli. They introduced mamL, mamQ, mamB, mamI and mamK to E. coli and indicated that introducing these genes affected localization of MamC: GFP fusion protein in the cells. However, their constructs lack RBS in proper position, that is, they did not insert RBS 6 bases upstream of start codon. In addition, they did not have a mean to detect vesicles at high resolution. These difficulties hinder their experiments from the observation of the vesicle formation. Moreover, in 2014, new paper was published and mamL, mamQ and mamB were considered to be essential for the first step of magnetosome membrane formation.

Generally, E. coli do not have any internal structures enclosed by lipid membranes. Therefore, it is impossible for E. coli to make magnetosomes without forming lipid vesicles first. As we described above, in magnetosome formation of MTB, three proteins — MamL, MamQ and MamB, are considered to be integrated with the inner membrane and triggers vesicle formation. Here we started from magnetosome vesicle formation in E. coli by expressing these three proteins. In the result below, we show successful reconstitution of the magnetosome-like vesicles in E. coli. The characterization of the induced vesicles and roles for MamL, MamQ and MamB are also discussed.

Experiments & Results

Why we choose AMB-1 genome

In our experiment, we decided to choose AMB-1 genome rather than MSR-1 genome as gene source. There are mainly two reasons. The first one is that in a past iGEM project, conducted by OUC-China in 2013, genes from AMB-1 genome were used to make the inner membrane. However, the result was not so clear. We can make improvement on the iGEM parts and the observation method of their project. The second one is availability. Almost all previous work about MTB conducted in Japan was using AMB-1 strain; we can get its genome easily. And considering the rigorous biosafety Investigation in Japanese universities, the obstacles to get a genome of a rare organism, i. e. MSR-1, is daunting.

To check if mamL, mamQ and mamB can function in vesicle formation also in E. coli, we constructed plasmid pLQB (Fig. 6). Observing negative control (E. coli that is transformed pSB1C3 inserted only T7promoter and RBS) and the transformant by transmission electron microscope (TEM), the transformants formed magnetosome-like lipid vesicles with a radius of about 100 nm. (Fig. 7)

Since the pLQB transformant could make vesicles, it is unclear whether the three proteins are all necessary for vesicle formation in E. coli or not. And it is interesting that E. coli has some proteins homologous to mamB, like Chain A zinc transporter (Fig. 6-1). So we constructed plasmid pLQ (Fig. 6) and introduced it to E. coli. The pLQ transformant also made lipid vesicles (Fig. 8). Additionally, we observed the magnetosome-like lipid vesicle formation in the pLQ transformant. The inner membrane is first curved. Then gradually the space between the inner membrane and the outer membrane become larger. Finally the vesicle was formed (Fig. 9).

Also, we counted the number of lipid vesicles per sections of E. coli. Finely we detect a significant difference between negative control and pLQB or pLQ (Fig. 10). These results suggest pLQ as well as pLQB would induce vesicle formation in E. coli.

Moreover, to confirm the translation of mamL, mamQ and mamB, we conducted Western blotting. ウェスタンの結果は後日(Fig. 11)

Then, we evaluated our parts because not all sections we observed had vesicles. (Fig. 12-1) Firstly, we estimated the efficiency of our parts. We determined the percentage of E. coli that formed vesicles as our parts efficiency. To estimate the percentage of vesicle formed E. coli, we used possibility that we can detect magnetosome vesicle in the sections of E. coli and we determined this possibility as vesicle-observation-rate (VOR), that is, we compared theoretical VOR and measured VOR. We used the pictures of TEM to estimated measured VOR. We must made model case because we should have calculate theoretical VOR and compared theoretical and measured VOR. Thus, we made model case and estimated theoretical VOR (Fig. 12-2). When we observe E. coli by TEM, we made some slice of samples that is E. coli fixed by resin. In the slice, E. coli is cut by various degrees so we deal with only the sections that is considered to be cut by the side vertical to x-axis because E. coli that are cut vertical can be identified easily. The thickness of the slice is about 100nm. Therefore, we assume that when a part of a vesicle is in the 100 nm, we can detect the vesicle, that is, when the shortest distance of the center of modeled vesicle (a spherical shape) and the face of the slice is lower than the vesicle's radius (The value of "a" is lower than "b"/2). We measured height and radius of negative control E. coli, pLQ transformants and pLQB transformants (Fig. 12-2). There is no significant difference between pLQ and pLQB transformants' radius, so we can assumed E. coli shape as column whose height is "c" and calculate the probability by following formula.

(b+200)/c

Then, magnetosome have various radius so we use maximum value we observed (b = 250 nm). In the case of pLQ, c = 2875 and in the case of pLQB, c = 49008. Using these value, theoretical VORs are 22.8 % (pLQB) and 13.8 % (pLQB).

To adapt this model on our transformants, we needed to count only the sections of pLQ and pLQB cut by vertical side and vesicles in them, so we used long axis and short axis of the sections. When the ratio of long axis and short axis is lower than 2.0(Fig. 12-3), we determined the section as vertical one because the gap of height and radius is more than 4 times (Fig. 12-4). Using this criteria, measured VORs were 9.1 % (pLQ) and 10.0 % (pLQB). Thus, calculating parts efficiencies by means of dividing measured VOR by that theoretical one, the efficiencies of the parts were 40.0 % (pLQ) and 72.4 % (pLQB). This model used maximum measured value, so we next used more rough criteria, that is, we assume the vesicle size is 50 (magnetosome of magnetotactic bacteria is 50 - 100 nm and that is small compared to the thickness of the slices), the probabilities of the model are 8.1 % (pLQ) and 4.9 % (pLQB) so the efficiencies are 112 % and 204 %.

When we observed E. coli to make model, we found some pLQ and pLQB transformants have 1.44(pLQ) and 2.46(pLQB) times larger cells than negative control. (Fig-12)

We thought that E. coli manipulation was inhibited due to the transformation. So we investigated growth curve of negative control, pL, pLQ, and pLQB transformants. (Fig. 13) The growth rate of pL transformant was same as the control, but that of pLQ and pLQB transformants were lower than control.

Reference

• [1] Blakemore, Richard. "Magnetotactic bacteria." Science 190.4212 (1975): 377-379.
• [2] Bazylinski, Dennis A. "Controlled biomineralization of magnetic minerals by magnetotactic bacteria." Chemical Geology 132.1 (1996): 191-198.
• [3] Richard B. Frankel and Dennis A. Bazylinski Magnetosome Mysteries, ASM news (2004)
• [4] Lohße A, Borg S, Raschdorf O, et al. Genetic dissection of the mamAB and mms6 operons reveals a gene set essential for magnetosome biogenesis in Magnetospirillum gryphiswaldense[J]. Journal of bacteriology, 2014: JB. 01716-14.
• [5] Grünberg K, Wawer C, Tebo B M, et al. A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria[J]. Applied and environmental microbiology, 2001, 67(10): 4573-4582.
• [6] Müller R, Zhang Y, Schüler D. Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters[J]. 2014.
• [7] iGEM OUC-China 2013
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