Abstract
BackgroundMagnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the ‘magnetosome island’ (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain.ResultsHere we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content.ConclusionsOur results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.
Highlights
Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the ‘magnetosome island’ (MAI), which harbors numerous mobile genetic elements, repeats, and genetic junk
Besides their function as magnetic sensors and importance as models for prokaryotic organelle biosynthesis, magnetosomes formed by magnetotactic bacteria represent magnetic nanoparticles that are highly attractive
Mutagenesis by several large, overlapping deletions of up to 61 kb has already demonstrated that a total of 115 kb of the MAI can be eliminated without any detectable effects on growth and magnetosome formation [28, 29]
Summary
Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the ‘magnetosome island’ (MAI), which harbors numerous mobile genetic elements, repeats, and genetic junk. The exquisite properties of magnetosomes, such as high chemical purity and crystallinity, strong magnetization, uniform shapes and sizes [25] are due to the strict control over their biomineralization This is orchestrated by more than 30 biosynthetic genes, which were mostly found to be clustered in a single chromosomal region, the genomic magnetosome island (MAI) [26,27,28,29]. The five key operons are separated by stretches containing genes of yet unknown, but irrelevant function for magnetosome biosynthesis [29] These intervening MAI regions harbor numerous mobile genetic elements, repeats and genetic “junk” (e.g., several incomplete and pseudogenes as well as non-coding genetic content), which are thought to be responsible for genetic instability, i.e., frequent rearrangements, deletions and the spontaneous loss of the magnetic phenotype during subcultivation of M. gryphiswaldense [26, 27, 30]. The role of distal and MAI-adjacent regions remains unclear
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