Toward cloning of the magnetotactic metagenome: identification of magnetosome island gene clusters in uncultivated magnetotactic bacteria from different aquatic sediments

Abstract

In this report, we describe the selective cloning of large DNA fragments from magnetotactic metagenomes from various aquatic habitats. This was achieved by a two-step magnetic enrichment which allowed the mass collection of environmental magnetotactic bacteria (MTB) virtually free of nonmagnetic contaminants. Four fosmid libraries were constructed and screened by end sequencing and hybridization analysis using heterologous magnetosome gene probes. A total of 14 fosmids were fully sequenced. We identified and characterized two fosmids, most likely originating from two different alphaproteobacterial strains of MTB that contain several putative operons with homology to the magnetosome island (MAI) of cultivated MTB. This is the first evidence that uncultivated MTB exhibit similar yet differing organizations of the MAI, which may account for the diversity in biomineralization and magnetotaxis observed in MTB from various environments.

FIG. 1.

(A) Direct magnetic collection at a microcosm as previously described (15). (B) “MTB trap” that allows the simultaneous mass collection of north- and south-seeking bacteria (for details, see text). (C) After a collection period of approximately 1 h, a visible cell pellet was found to have resulted from accumulation at the bottom of the collection tube.

FIG. 2.

Examples of different morphotypes present in the sampled habitats. TEM revealed the virtual absence of nonmagnetic contaminants among the highly enriched MTB. The diverse morphotypes included large rod-like “Ca. Magnetobacterium bavaricum” cells (C and E). Two different cocci (A, B, C, G, and I) and spirillum-like MTB (D) were observed. The diversity of magnetosome crystals ranged from arrow- or bullet-shaped magnetic particles (F) to cubic and cubo-octahedral morphologies (H). Crystals were arranged either as irregular clusters in cocci of the Bilophococcus type (A, G, and H) or in single or multiple chains (B, C, D, F, and I). Scale bars, 500 (E) and 200 (I) mm.

FIG. 3.

Molecular organization of two clones harboring magnetosome operons in comparison to the homologous regions from the MAIs of cultivated MTB. In addition to those of magnetosome genes, the entire annotated regions of both metagenomic clones are shown. “Conserved magnetosome proteins” are defined as proteins that occur universally in all magnetospirilla, strain MC-1, and strain MV-1. Magnetosome genes are indicated by capital letters, (e.g., in the case of mamH, as “H”), while italic notation indicates homologues with a lower degree of conservation (e value < 10E5); e.g., “R” for strain MV-1 stands for “mamR like” (exception: “F” should be read as mmsF). MSR-1, M. gryphiswaldense; AMB-1, “M. magneticum”; MS-1, M. magnetotacticum; MV-1, magnetic vibrio strain MV-1; MC-1, magnetic coccus strain MC-1.

FIG. 4.

Similarity trees illustrating the phylogenetic relationships of selected genes. The upper panel shows the phylogenetic affiliations of MamB proteins from all MTB sequenced thus far as an example. Other magnetosome proteins form congruent trees (with some exceptions, as mentioned in the text). The lower panel shows the similarity tree of the Fur (ferric uptake regulator) protein identified in studies of Fos002 that serves as an alternative phylogenetic marker. Shaded boxes indicate Fur proteins from MTB, Fos002, and Rhodospirillum rubrum.