Frequent mutations within the genomic magnetosome island of Magnetospirillum gryphiswaldense are mediated by RecA

Abstract

Genes for magnetosome formation in magnetotactic bacteria are clustered in large genomic magnetosome islands (MAI). Spontaneous deletions and rearrangements were frequently observed within these regions upon metabolic stress. This instability was speculated to be due to RecA-dependent homologous recombination between the numerous sequence repeats present within the MAI. Here we show that a RecA-deficient strain of Magnetospirillum gryphiswaldense (IK-1) no longer exhibits genetic instability of magnetosome formation. Strain IK-1 displayed higher sensitivity to oxygen and UV irradiation. Furthermore, the lack of RecA abolished allelic exchange in the mutant. Cells of strain IK-1 displayed a slightly altered (i.e., more elongated) morphology, whereas the absence of RecA did not affect the ability to synthesize wild-type-like magnetosomes. Our data provide evidence that the observed genetic instability of magnetosome formation in the wild type is due predominantly to RecA-mediated recombination. In addition, increased genetic stability could make strain IK-1 a useful tool for the expression of genes and further genetic engineering, as well as for biotechnological production of bacterial magnetosomes.

Fig. 1.

Growth (OD₅₆₅) and C_mag of M. gryphiswaldense IK-1 and the WT (●, WT C_mag; ○, IK-1 C_mag; ▴, WT OD₅₆₅; Δ, IK-1 OD₅₆₅) during incubation for 30 h under microaerobic (A), aerobic (B), and anaerobic (C) conditions.

Fig. 2.

(A) Magnetosome size distributions under anaerobic conditions (Mann-Whitney P value, ≥0.05) in IK-1 and the WT. (B) Distributions of different cell sizes of strain IK-1 and the WT were estimated. Morphotypes were investigated under aerobic, microaerobic, and anaerobic conditions. (C) DIC micrographs showing representative short, medium, and long cells. (D) TEM of the WT and strain IK-1. For TEM pictures, cells were incubated at 30°C under microaerobic conditions.

Fig. 3.

(A) Recombination assay results. A suicide vector carrying a 2-kb insert homologous to a partial region of the MAI was transferred into the WT and the recA mutant. The frequency of cells with genomic insertions was estimated by the number of Km-resistant clones after 7 days of microaerobic incubation (4 × 10⁸ recipient cells per conjugation). (B) UV irradiation assay results. Cells were irradiated with 15 mJ/cm² UV light. Both assays were performed in triplicates. Survival rates (CFU/ml) after irradiation were estimated after 7 days of microaerobic incubation.