Abstract
Living organisms produce finely tuned biomineral architectures with the aid of biomineral-associated proteins. The functional amino acid residues in these proteins have been previously identified using in vitro and in silico experimentation in different biomineralization systems. However, the investigation in living organisms is limited owing to the difficulty in establishing appropriate genetic techniques. Mms6 protein, isolated from the surface of magnetite crystals synthesized in magnetotactic bacteria, was shown to play a key role in the regulation of crystal morphology. In this study, we have demonstrated a defect in the specific region or substituted acidic amino acid residues in the Mms6 protein for observing their effect on magnetite biomineralization in vivo. Analysis of the gene deletion mutants and transformants of Magnetospirillum magneticum AMB-1 expressing partially truncated Mms6 protein revealed that deletions in the N-terminal or C-terminal regions disrupted proper protein localization to the magnetite surface, resulting in a change in the crystal morphology. Moreover, single amino acid substitutions at Asp123, Glu124, or Glu125 in the C-terminal region of Mms6 clearly indicated that these amino acid residues had a direct impact on magnetite crystal morphology. Thus, these consecutive acidic amino acid residues were found to be core residues regulating magnetite crystal morphology.
Highlights
We identified a series of proteins: Mms[5], Mms[6], Mms[7], and Mms[13], localized onto magnetite crystals in the Magnetospirillum magneticum strain AMB-120
The mms[6] gene deletion mutant strain (Δmms[6] strain) was found to synthesize elongated magnetite crystals with a smaller size and lower shape factor than that of the cubo-octahedral crystals synthesized by the wild-type strain[29]
Western blot analysis revealed that all the variants of single amino acid substituted Mms[6] (Mms6D116A, Mms6I117A, Mms6E118A, Mms6S122A, Mms6D123A, Mms6E124A, Mms6E125A, Mms6V126A, Mms6E127A) were expressed and localized in the magnetosome membrane (Fig. 3C). These results clearly indicated that the single amino acid substitutions at D123, E124, and E125 were responsible for the morphological change of the magnetite crystals
Summary
We identified a series of proteins: Mms[5], Mms[6], Mms[7], and Mms[13], localized onto magnetite crystals in the Magnetospirillum magneticum strain AMB-120. Their amino acid sequences contain a C-terminal hydrophilic region comprising of acidic amino acids, and an N-terminal hydrophobic region, with a Gly and Leu (GL) repetitive region. Iron binding[20,34,35] and iron oxide nucleation at the C-terminal acidic region were confirmed[36] According to these in vitro studies, the acidic amino acids in the C-terminal region are most likely to be responsible for controlling the crystal morphology. A single amino acid substitution in the C-terminal region of Mms[6] was investigated in order to identify the amino acid residues essential for the function of Mms[6]
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