Dataset Information

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

ABSTRACT:

Background

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 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.

Results

Here 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.

Conclusions

Our 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.

SUBMITTER: Zwiener T

PROVIDER: S-EPMC7908775 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

Zwiener Theresa T Mickoleit Frank F Dziuba Marina M Rückert Christian C Busche Tobias T Kalinowski Jörn J Faivre Damien D Uebe René R Schüler Dirk D

BMC microbiology 20210225 1

<h4>Background</h4>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 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 ...[more]

PMID: 33632118

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Project description:One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth’s magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under suboxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of >30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear. Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the >4300 genes was significantly enhanced during magnetosome formation. The about 40 top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. mam and mms gene clusters specifically controlling magnetosome biosynthesis were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria. Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource.

Project description:Magnetosomes are complex membrane organelles synthesized by magnetotactic bacteria (MTB) for navigation in the Earth's magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense, all steps of magnetosome formation are tightly controlled by >30 specific genes arranged in several gene clusters. However, the transcriptional organization of the magnetosome gene clusters has remained poorly understood. Here, by applying Cappable-seq and whole-transcriptome shotgun RNA sequencing, we show that mamGFDCop and feoAB1op are transcribed as single transcriptional units, whereas multiple transcription start sites (TSS) are present in mms6op, mamXYop, and the long (>16 kb) mamABop. Using a bioluminescence reporter assay and promoter knockouts, we demonstrate that most of the identified TSS originate from biologically meaningful promoters which mediate production of multiple transcripts and are functionally relevant for proper magnetosome biosynthesis. In addition, we identified a strong promoter in a large intergenic region within mamXYop, which likely drives transcription of a noncoding RNA important for gene expression in this operon. In summary, our data suggest a more complex transcriptional architecture of the magnetosome operons than previously recognized, which is largely conserved in other magnetotactic Magnetospirillum species and, thus, is likely fundamental for magnetosome biosynthesis in these organisms. IMPORTANCE Magnetosomes have emerged as a model system to study prokaryotic organelles and a source of biocompatible magnetic nanoparticles for various biomedical applications. However, the lack of knowledge about the transcriptional organization of magnetosome gene clusters has severely impeded the engineering, manipulation, and transfer of this highly complex biosynthetic pathway into other organisms. Here, we provide a high-resolution image of the previously unappreciated transcriptional landscape of the magnetosome operons. Our findings are important for further unraveling the complex genetic framework of magnetosome biosynthesis. In addition, they will facilitate the rational reengineering of magnetic bacteria for improved bioproduction of tunable magnetic nanoparticles, as well as transplantation of magnetosome biosynthesis into foreign hosts by synthetic biology approaches. Overall, our study exemplifies how a genetically complex pathway is orchestrated at the transcriptional level to ensure the balanced expression of the numerous constituents required for the proper assembly of one of the most intricate prokaryotic organelles.

Dataset Information

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

Background

Results

Conclusions

Publications

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

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