Team:OUC-China/Design

Design

As for the design of intracellular compartment, it is divided into two parts, artificial gene cluster and natural gene cluster mamAB from Magnetospirillum Magneticum AMB-1 strain.

1. Why magnetosome?

It is generally considered that intracellular membrane structures are unique to eukaryotes, but rare in bacteria. The magnetosome structure of M.magneticum has obvious similarities to eukaryote membranous organelles. Similar to organelles of prokaryotes, magnetosome is packaged by 3 ~ 4 nm thick biofilms, which resemble plasma membrane. Its inner diameter is 10 to 120nm, arranged in chains in wild-type strain. Due to the barrier effect of the MMB, magnetosome membrane cavity is a nanoscale reactor, for the formation of Fe3O4 (iron oxide) requires strict pH environment.

So, we plan to construct the magnetosome membrane compartment in E.coli as a reactor for future work.


Figure 1.Three-dimensional organization of magnetosomes. An ECT reconstruction of Magnetospirillum magneticum sp.AMB-1. Image courtesy of Zhuo Li & Grant Jensen[3]

2. What is mamAB?

The construction of magnetosome is guided by a 100Kb long gene sequence which is formed by 14 regions, called magnetosome island (MAI). According to gene knock-out experiments, we know that natural R6, R7, R8, R9 dominant negative the artificial R1, R2, R3, R4, R6, R9, R10, R11, R12, R13, R14 dominant negative contain normal magentosome compartment chain, while magnetism of the R2, R3,R3, R6, R9, R11, R12, R13, R14 dominant negative reduce varying degrees, and only when R5 region (also called mamAB) is knocked out, the magnetosome can’t exist anymore.

Above all, R5 is required for the construction of magnetosome. That’s why we have hopes in constructing the mamAB gene cluster in E.coli according to homologous recombination. This is what we wish to achieve in the future.


Figure 2.Genomic organization of the MAI and the mamAB gene cluster in Magnetospirillum magneticum AMB-1.[13]

3. How did we design the artificial gene cluster?

The genes related to the formation of magnetosome are mainly concentrated in MAI, which contains a large number of repeats and antisense gene fragments. But, as we know, because it contains too many regulate sequences; it can mutate easily when oxygen concentration fluctuates. What’s more, it will also be much more difficult to transform such a long sequence into cell. So we decided to transform a plasmid that contains only necessary genes into E.coli to explore the necessary and sufficient condition of MMB formation.

Magnetosomes are formed in three steps. First, mamK lead the membrane invagination derived from the inner membrane, and magnetosome proteins sorted away from cell membrane proteins. Second, individual invaginations assembly into a chain with the help of MamJ and MamK proteins. Third, iron is transformed into highly ordered magnetite crystals within the magnetosome membrane. In this step, there are many special magnetosome protein such as mamI, mamL, mamB and mamQ assembled onto the membrane while playing an important role on the construction of MMB.

Two separate models can be envisioned for the role of MamK in organizing the chain.

It is assumed that MamK interacts to establish the chain by bring newly formed magnetosomes into the magnetosome chain. Alternatively, MamK maintains the magnetosome chain perhaps by preventing the insertion of newly synthesized membrane lipids in the magnetosome chain.

This model can be envisioned for the role of MamK in organizing the chain. Which is assumed that MamK interacts to establish the chain by bring newly formed magnetosomes into themagnetosome chain. Alternatively, MamK maintains the magnetosome chain perhaps bypreventing the insertion of newly synthesized membrane lipids in the magnetosome chain.


Figure 3.Model for magnetosome formation. Magnetosomes are formed in three steps.[14]


Above all, we designed an artificial gene cluster in order to construct the compartment we need, which includes mamI, mamL, mamB, mamQ and mamK.


Figure 4.The design of artificial magnetosome gene cluster.

4. What is the point of applying MamC::GFP fusion protein?

MamC is the mostly expressed protein among all the MMB associated proteins, and its structure fits to be an anchor protein. In our project we combine MamC and GFP as a fusion protein and express it on high efficiency expression vector to show the existence of our intracellular compartment under the fluorescent microscope.(The fusion protein was designed by Prof.Longfei Wu.)


Figure 5.The MamC::GFP expression vector (provided by Prof.Longfei Wu) we use to shows the existence of our artificial organelle under the fluorescent microscope.

Reference

[1]Richard Blakemore. Magnetotactic Bacteria. Science, New Seris,Vol.190, No.4212:377-379.
[2]Christian Jogler, Gerhard Wanner, Sebastian Kolinko. Conservation of proteobacterial magnetosome genes and structures in an uncultivated member of the deep-branching Nitrospiraphylum. PNAS 2011,108:1134-1139.
[3]Arash Komeili. Molecular Mechanisms of Magnetosome Formation. Annu. Rev. Biochem. 2007,76:351–366
[4]Anna Loh?e, Susanne Ullrich, Emanuel Katzmann.Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization. PloS one, 2011,6(10).
[5]Dorothée Murat, Anna Quinlan, Hojatollah Vali, and Arash Komeili. Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. PNAS 2010, 107(12): 5593–5598.
[6]Shoji Ohuchi, Dirk Schuler. In Vivo Display of a Multisubunit Enzyme Complex on Biogenic Magnetic Nanoparticles. Applied and Environmental Microbiology Dec. 2009: 7734–7738.
[7] Anna Quinlan, Dorothée Murat, Hojatollah Vali. The HtrA/DegP family protease MamE is a biofunctional protein with roles in magnetosome protein localization and magnetite biomineralization. Molecular Microbiology 2011, 80(4), 1075–1087.
[8]Arash Komeili, Zhuo Li, Dianne K. Newman. Magnetosomes are Cell Membrane Invaginations Organized by the Actin-Like Protein MamK. Science 311:242-245.
[9]Masayoshi Tanaka, Eri Mazuyama, Atsushi Arakaki. MMS6 Protein Regulates Crystal Morphology during Nano-sized Magnetite Biomineralizationin Vivo. Joural of Biological Chemistry 2011, 286(8):6386-6392.
[10]Jean-Baptiste Rioux, Nadege Philippe, Sandrine Pereira. A Second Actin-Like MamK Protein in Magnetospirillum magneticumAMB-1 Encoded Outside the Genomic Magnetosome Island. PLoS ONE, 2010, 5(2).
[11]Nicolas Ginet, Romain Pardoux, Geraldine Adryanczyk Single-Step Production of a Recyclable Nanobiocatalyst for Organophosphate Pesticides Biodegradation Using Functionalized Bacterial Magnetosomes. PLoS ONE,2011,6(6).
[12]Christian Jogler, Gerhard Wanner, Sebastian Kolinko. Conservation of proteobacterial magnetosome genes and structures in an uncultivated member of the deep-branching Nitrospiraphylum. PNAS 2011, 108:1134-1139.
[13]徐争辉陈奇韩秀英趋磁细菌磁小体合成相关蛋白的研究进展安徽农业科学 2012,40（1）：32-35
[14]杨靖张同伟黄修良趋磁细菌磁小体合成机制研究进展微生物学通报 2011,38（8）：1262-1269