RESUMEN

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

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RESUMEN

Individual magnetosome-containing magnetic mineral particles (MMP) from magnetotactic bacteria grow rapidly such that only a small fraction (5%) of all magnetosomes contain dwarf (< or =20 nm) MMP. Studies of the developmental stages in the growth of MMP are difficult due to the absence of techniques to separate dwarf from mature particles and because the former are sensitive to extraction procedures. Here, O2 stress was used to inhibit MMP expression in Magnetospirillum magneticum strain AMB-1. In addition, defined growth conditions not requiring chemical monitoring or manipulation of the gas composition during growth resulted in the production of cells containing high numbers of dwarf MMP. Cells exposed to different incubation treatments and cells with dwarf MMP were compared to cells with normal MMP with respect to growth, respiration, iron content, and relative magnetite load (RML). The cells were examined by electron microscopy, low temperature magnetometry, X-ray diffraction (XRD), and Mössbauer spectroscopy. In the 0-110 microM O2(aq) range, growth was positively correlated with [O2] and negatively correlated with RML. Most MMP formed during exponential growth of the cells. At 50-100 microM O2(aq) with stirring (150 rpm) and 30% O2 loss during incubation, MMP expression was strongly inhibited whereas MMP nucleation was not. Cells highly enriched (~95%) in dwarf MMP were obtained at the end of the exponential phase in stirred (150 rpm) cultures containing 45 microM O2(aq). Only one dwarf MMP formed in each MMP vesicle and the chain arrangement was largely preserved. O2-stress-induced dwarf MMP consisted of non-euhedral spheroids (~25 nm) that were similar in shape and size to immature MMP from normal cells. They consisted solely of magnetite, with a single domain signature, no superparamagnetic behavior, and magnetic signatures, Fe(II)/Fe(III) ratios, and XRD patterns very similar to those of mature MMP. These results show that O2 stress in liquid cultures amended with an inorganic redox buffer (S2O3 2-/S0) can be used to produce abundant dwarf MMP that are good proxies for studying MMP development (AU)

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