Team:Hong Kong-CUHK/Description

Azotobacter

Magnetosome - Nanostructure with Great Application Potentials

Figure 1: Magnetosome

Magnetosome is a kind of rare intracellular membrane-bound structure in a specific type of prokaryotes, of nano-size ranging about 35 - 120 nm. They comprise of a magnetic mineral crystal encapsulated by a lipid bilayer about 3 – 4 nm thick (Figure 1) [1], which might be utilized in various applications involving magnetic field.

The magnetosome membrane is highly significant for its biogenesis as it creates an isolated environment within the cell crucial for mineral crystal nucleation and growth [2]. These inorganic crystals are magnetic in nature (hence its name), which compose of either magnetite (Fe3O4) or greigite (Fe3S4). The magnetosomes usually arrange in one or multiple chains along the cell axis. Different varieties of crystal morphologies such as cubo-octahedral, elongated hexagonal prismatic, and bullet-shaped morphologies were discovered in different magnetotactic bacteria [1].

Magnetotactic Bacteria - The Magnetosome Producer

Figure 2: Micrograph of a Magnetotactic Bacteria, Magnetospirillum gryphiswaldense (image from Departamento de Inmunología, Microbiología y Parasitología, University of the Basque Country)

Magnetosomes are organelles synthesized by magnetotactic bacteria for its movement along magnetic field. First discovered in 1975 by Richard Blakemore, these magnetotactic bacteria are mobile, aquatic, gram-negative prokaryotes [3] with an array of cellular morphologies, including coccoid, rod-shaped, vibrioid, helical or even multi-cellular. Some of them are more extensively studied, including Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense. They are found optimally grown at the oxic-anoxic interface in aquatic habitats, and in fact grow less happily under atmospheric oxygen concentration.

Magnetosomes form a chain and are aligned along the axis within the bacteria. With these magnetosomes inside them, they are able to align passively to the earth’s magnetic field so as to swim along geomagnetic field lines. This behaviour is called magnetotaxis and is beneficial to their survival by aiding them to reach regions of optimal oxygen concentrations at minimal energy cost [4].

Azotobacter vinelandii - What and Why?

Although magnetotactic bacteria produces magnetosomes, these bacteria are notorious for the difficulty to cultivate owing to their micro-aerophilic nature. Elaborate growth techniques are required, and they are difficult to grow on the surface of agar plates, introducing problems in mutant screening [5].

The lack of effective methods of DNA transfer in these microorganisms is a challenge too. Luckily, the situation is improving due to better technologies recently and some of the genes from M. magnetotacticum were confirmed functionally expressed in Escherichia coli, a common lab bacteria strain. This shows that the transcriptional and translational elements of the two microorganisms are compatible. With such good news, a number of previous iGEM teams including Kyoto-2014, OCU-China-2013, Washington-2011 and UNIK_Copenhagen-2013 have been working with transferring the magnetosome-related genes to E. coli. Some exciting results about the formation of magnetosome membrane in E. coli (by the Kyoto-2014 team) has been reported, however, never the whole magnetosome.

Figure 3: Azotobacter vinelandii

We wonder why magnetosomes seem so hard to be formed in E. coli. And then, we come up with a hypothesis - the formation of magnetosome requires a micro-aerobic or anaerobic environment as the magnetotatic bacteria are all living micro-aerobically.

Therefore, we chose a new bacteria to work on our magnetosome project - Azotobacter vinelandii.

A. vinelandii is gram-negative diazotroph (nitrogen-fixing microorganism). It is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while having enzymatic mechanisms protecting its oxygen-sensitive nitrogenase from oxidative damage. This finding shows that A. vinelandii could be an excellent host for the production and characterization of oxygen-sensitive proteins or organelles in our case [6].

With the biggest advantage of using Azotobacter that it being an aerobe providing an intracellular anaerobic environment, we can grow it easily in normal lab conditions without expensive equipments, while fulfilling the formation criteria for magnetosome. Besides, most parts in registry are compatible in Azotobacter and it is of safety level group 1. One more important thing is that it can conduct homologous recombination for stable genome integration, which is a critical process we need in our project.

The Magnetosome island and the genes involved

For the synthesis of magnetosome, it is strictly controlled by a group of genes clustered in the magnetosome island (MAI). The magnetosome island comprise of four operons, namely the mms6 operon, the mamGFDC operon, mamAB and mamXY [5]. The actual size and organisation of the MAI might differ between species, but the operons seems to be highly conserved within the MAI.

Through gene knockdown and other comprehensive experiments, researches has shown with the deletion of the mamAB operon would lead to non-magnetic phenotype. All in all, it shows the importance of the mamAB operon as it is the most responsible for magnetosome formation and have important functions such as membrane invagination, iron transport, and magnetite biomineralization [7].

>In the bacteria Magnetospirillum gryphiswaldense, the mamAB operon consists of 17 genes

(mamH, -I, -E, -J, -K, -L, -M, -N, -O, -P, -A, -Q, -R, -B, -S, -T, and -U) .

How is magnetosome formed

We can look at the biosynthesis of magnetosomes as a multistep complex process.

Figure 4: The overview of magnetosome formation

First, the inner membrane of the magnetotatic bacteria swells outwards facilitating invagination of vesicles. The following step is to sort magnetosome proteins to the magnetotactic membrane. This enables the magnetosome membrane to perform specific functions in the transport and accumulation of iron. Apart from that, the magnetosome membrane also plays a crucial role in nucleation of crystallization and pH control.

After the sorting of essential magnetosome proteins to the MM, the next step is iron uptake. The need of importation of iron to make magnetite makes MTB differ from other bio-mineralizers. Iron transporters in the MM, would pump Fe2+/Fe3+into the vesicle.

Additionally, magnetosome proteins MamM, MamB, and MamH have also been suggested as additional iron transporters for magnetite biomineralization. As the concentration of iron ions increases inside the vesicle, bio-mineralization occurs[8].

The process of biomineralization of magnetite is tightly regulated through specific conditions such as: pH and the concentration of iron within the vesicle. Furthermore, from research, it shows that the formation of magnetite only occurs below a threshold value of 10 millibar of atmospheric pressure. Magnetite formation is inhibited at higher oxygen concentrations. In other words, the size of particles can be limited by atmospheric pressure and oxygen concentration. It is found that at 0.25 mbar magnetite bio-mineralization can produce particles up to 42nm. As the condition rise to 10 mbar, the particle size can drop to about 20 nm. [9] As the biomineralization of the magnetosome is reported to be highly affected by oxygen, we propose an educated guess that magnetosome will have a higher probability to be successfully formed in our bacteria Azotobacter rather than E.coli due to its intracellular microaerobic characteristic.

References

1. BAZYLINSKI, Dennis A.; FRANKEL, Richard B. Magnetosome formation in prokaryotes. Nature Reviews Microbiology, 2004, 2.3: 217-230.

2. NUDELMAN, Hila; ZARIVACH, Raz. Structure prediction of magnetosome-associated proteins. Frontiers in microbiology, 2014, 5.

3. BLAKEMORE, Richard. Magnetotactic bacteria. Science, 1975, 190.4212: 377-379.

4. FRANKEL, Richard B.; BAZYLINSKI, Dennis A. Magnetosomes and magneto-aerotaxis. 2009.

5. YAN, Lei, et al. Magnetotactic bacteria, magnetosomes and their application. Microbiological research, 2012, 167.9: 507-519.

6. MAIER, R. J.; MOSHIRI, F. Role of the Azotobacter vinelandiinitrogenase-protective shethna protein in preventing oxygen-mediated cell death. Journal of bacteriology, 2000, 182.13: 3854-3857.

7. LOHßE, Anna, et al. Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization. PLoS One, 2011, 6.10: e25561.

8. LOHßE, Anna, et al. Genetic dissection of the mamAB and mms6 operons reveals a gene set essential for magnetosome biogenesis in Magnetospirillum gryphiswaldense. Journal of bacteriology, 2014, 196.14: 2658-2669.

9. ODENBACH, Stefan (ed.). Colloidal Magnetic Fluids: Basics, Development and Application of Ferrofluids. Springer, 2009.