Interaction of Proteins Associated with the Magnetosome Assembly in Magnetotactic Bacteria As Revealed by Two-Hybrid Two-Photon Excitation Fluorescence Lifetime Imaging Microscopy Förster Resonance Energy Transfer

Abstract

Bacteria have recently revealed an unexpectedly complex level of intracellular organization. Magnetotactic bacteria represent a unique class of such organization through the presence of their magnetosome organelles, which are organized along the magnetosome filament. Although the role of individual magnetosomes-associated proteins has started to be unraveled, their interaction has not been addressed with current state-of-the-art optical microscopy techniques, effectively leaving models of the magnetotactic bacteria protein assembly arguable. Here we report on the use of FLIM-FRET to assess the interaction of MamK (actin-like protein) and MamJ, two magnetosome membrane associated proteins essential to the assembly of magnetosomes in a chain. We used a host organism (E. coli) to express eGFP_MamJ and MamK_mCherry, the latest expectedly forming a filament. We found that in the presence of MamK the fluorescence of eGFP_MamJ is distributed along the MamK filament. FRET analysis using the fluorescence lifetime of the donor, eGFP, revealed a spatial proximity of MamK_mCherry and eGFP_MamJ typical of a stable physical interaction between two proteins. Our study effectively led to the reconstruction of part of the magnetotactic apparatus in vivo.

Bacterial protein networks: properties and functions

“…Such shortening of the donor lifetime provides an alternative readout that can be used to detect FRET and can be measured using fluorescence lifetime imaging microscopy (FLIM) 137 . Fluorescence anisotropy also declines as a result of energy transfer, and this can be useful in the detection of interactions between two proteins labelled with the same fluorophore (for example, YFP) 67 .…”

Genetically Engineered Organization: Protein Template, Biological Recognition Sites, and Nanoparticles

“…Furthermore, the fluorescence of our constructs is useful for imaging and provides an additional functionality to the template that could, in conjunction with the appropriate reporting fluorescent probe, be used to probe protein interaction [15] or to quantify local microenvironment such as pH, [28,29] temperature, [30] and viscosity. The attachment of these different NPs to the mCherry fusion partner or to the hexahistidine tag indicates the availability of these recognition sites and highlights the possibility of attaching other NPs that are able to interact with the anti-mCherry or anti-HisTag antibodies.…”

“…In particular, iron oxides and gold particles coated with different DNA fragments were assembled in a controlled manner. [13] MamK is also able to form filaments in host organisms such as E. coli [14,15] as well as when recombinantly produced. [6] Here, we show that the functionality of engineered and recombinantly expressed fusion proteins can be used for the directed assembly of different types of nanoparticles (NPs).…”

Magnetoreception and Magnetotaxis

“…The mechanical stability of the intracellular mineral chain is an important characteristic in order to provide sufficient magnetic torque and a reliable navigation bias to the cells. In MSR-1, the roles of two proteins, MamJ and MamK, are important for the mechanical stability of the chain and have been subject to numerous studies [55][56][57][58][59][60][61][62]. MamK is a bacterial homolog of the eukaryotic protein actin.…”

“…As reported by Scheffel et al [61] and Komeili et al [60], deletion of either MamJ or MamK leads to the partial or total loss of the chain-like mineral structure. MamJ forms a stable interaction with MamK [56] and plays the role of an anchor between the magnetosomes and the filament bundle (Figure 22.6b).…”