Isolation and cultivation of a novel sulfate-reducing magnetotactic bacterium belonging to the genus Desulfovibrio

Abstract

Magnetotactic bacteria (MTB) synthesize magnetosomes composed of membrane-enveloped magnetite (Fe3O4) and/or greigite (Fe3S4) nanoparticles in the cells. It is known that the magnetotactic Deltaproteobacteria are ubiquitous and inhabit worldwide in the sediments of freshwater and marine environments. Mostly known MTB belonging to the Deltaproteobacteria are dissimilatory sulfate-reducing bacteria that biomineralize bullet-shaped magnetite nanoparticles, but only a few axenic cultures have been obtained so far. Here, we report the isolation, cultivation and characterization of a dissimilatory sulfate-reducing magnetotactic bacterium, which we designate "strain FSS-1". We found that the strain FSS-1 is a strict anaerobe and uses casamino acids as electron donors and sulfate as an electron acceptor to reduce sulfate to hydrogen sulfide. The strain FSS-1 produced bullet-shaped magnetite nanoparticles in the cells and responded to external magnetic fields. On the basis of 16S rRNA gene sequence analysis, the strain FSS-1 is a member of the genus Desulfovibrio, showing a 96.7% sequence similarity to Desulfovibrio putealis strain B7-43T. Futhermore, the magnetosome gene cluster of strain FSS-1 was different from that of Desulfovibrio magneticus strain RS-1. Thus, the strain FSS-1 is considered to be a novel sulfate-reducing magnetotactic bacterium belonging to the genus Desulfovibrio.

Fig 1. Morphological features of strain FSS-1.

(A) TEM image of a vibroid cell of strain FSS-1 incubated in the MD medium under anaerobic conditions. (B) TEM image of a magnetosome synthesized in the cell corresponding to the dashed-line box indicated in panel (A). (C) TEM image of the same cell as shown in panel (A). A single polar flagellum is indicated by a black arrow. (D) TEM image of a spiral cell of strain FSS-1.

Fig 2. Phylogenetic tree based on the 16S rRNA gene sequences of strain FSS-1 and some other related Desulfovibrio.

The tree was constructed by the neighbor-joining method and rooted using Escherichia coli as an outgroup. Bootstrap values per 1,000 replicates are indicated. The GenBank accession numbers are shown in the parentheses. Bar, 0.01 changes per nucleotide position.

Fig 3. Growth of strain FSS-1 in the MD medium containing casamino acids and sodium sulfate.

(A) MD medium containing 0.025, 0.05, 0.1, 0.2 and 0.4 g/liter of casamino acids after 13 day incubation. Cells grew only when 0.1 g/liter of casamino acids was present in the medium as indicated by the production of ferrous sulfide. (B) MD containing 0.1, 0.2, 0.4 and 0.8 g/liter of sodium sulfate in the presence of 0.1 g/liter casamino acids after 13 day incubation. Cells grew irrespective of the difference in the concentration of sodium sulfate in the medium. (C) Growth curve of strain FSS-1 in the liquid medium containing 0.1 g/liter of casamino acids in the absence of sodium sulfate. The average values were calculated from three independent experiments. The error bars represent the standard deviations.

Fig 4. Response of strain FSS-1 cells to an external magnetic field, the number of magnetic nanoparticles in each cell and the size distribution of magnetic nanoparticles.

(A) Differential interface contrast (DIC) optical microscopic image of strain FSS-1 cells at the edge of a ‘hanging drop’ in an external magnetic field. (B) Distribution of the number of magnetic nanoparticles in each cell. (C) Distribution of the length of magnetic nanoparticles. (D) Distribution of the width of magnetic nanoparticles.

Fig 5. STEM-EDS analysis, and TEM and HRTEM images of magnetic nanoparticles in each cell.

(A) STEM image of a magnetosome. (B), (C) STEM-EDS elemental maps corresponding to iron (Fe) and oxygen (O). (D) TEM image of a magnetosome in a cell corresponding to the dashed-line box indicated in the inset. (E) HRTEM image of a magnetic nanoparticle indicated by the dashed-line box in panel (D). (F) HRTEM image of the same nanoparticle as shown in panel (E). (G) Fast Fourier transform (FFT) pattern of the same nanoparticle as shown in panel (F).

Fig 6. Comparison of Magnetosome Gene Clusters (MGCs) between strain FSS-1 and Desulfovibrio magneticus strain RS-1^T.

13 mam genes, 16 mad genes and 6 non-homologous genes are present in the MGC of strain FSS-1. Dashed-lines between two MGCs show homologous genes.