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Transcriptional organization and regulation of magnetosome operons in Magnetospirillum gryphiswaldense.

ABSTRACT: Genes involved in magnetite biomineralization are clustered within the genomic magnetosome island of Magnetospirillum gryphiswaldense. Their transcriptional organization and regulation were studied by several approaches. Cotranscription of genes within the mamAB, mamDC, and mms clusters was demonstrated by reverse transcription-PCR (RT-PCR) of intergenic regions, indicating the presence of long polycistronic transcripts extending over more than 16 kb. The transcription start points of the mamAB, mamDC, and mms operons were mapped at 22 bp, 52 bp, and 58 bp upstream of the first genes of the operons, respectively. Identified -10 and -35 boxes of the P(mamAB), P(mamDC), and P(mms) promoters showed high similarity to the canonical sigma(70) recognition sequence. The transcription of magnetosome genes was further studied in response to iron and oxygen. Transcripts of magnetosome genes were detected by RT-PCR both in magnetic cells grown microaerobically under iron-sufficient conditions and in nonmagnetic cells grown either aerobically or with iron limitation. The presence of transcripts was found to be independent of the growth phase. Further results from partial RNA microarrays targeting the putative magnetosome transcriptome of M. gryphiswaldense and real-time RT-PCR experiments indicated differences in expression levels depending on growth conditions. The expression of the mam and mms genes was down-regulated in nonmagnetic cells under iron limitation and, to a lesser extent, during aerobic growth compared to that in magnetite-forming cells grown microaerobically under iron-sufficient conditions.

SUBMITTER: Schubbe S

PROVIDER: S-EPMC1563640 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Transcriptional organization and regulation of magnetosome operons in Magnetospirillum gryphiswaldense.

Schübbe Sabrina S Würdemann Chris C Peplies Jörg J Heyen Udo U Wawer Cathrin C Glöckner Frank Oliver FO Schüler Dirk D

Applied and environmental microbiology 20060901 9

Genes involved in magnetite biomineralization are clustered within the genomic magnetosome island of Magnetospirillum gryphiswaldense. Their transcriptional organization and regulation were studied by several approaches. Cotranscription of genes within the mamAB, mamDC, and mms clusters was demonstrated by reverse transcription-PCR (RT-PCR) of intergenic regions, indicating the presence of long polycistronic transcripts extending over more than 16 kb. The transcription start points of the mamAB, ...[more]

PMID: 16957191

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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.

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:BackgroundOne 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 microoxic to anoxic 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.ResultsHere, 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. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The 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.ConclusionsOur 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 (GEO Series accession number GSE197098).

Project description:Magnetospirillum gryphiswaldense employs iron-rich nanoparticles for magnetic navigation within environmental redox gradients. This behavior termed magneto-aerotaxis was previously shown to rely on the sensory pathway CheOp1, but the precise localization of CheOp1-related chemoreceptor arrays during the cell cycle and its possible interconnection with three other chemotaxis pathways have remained unstudied. Here, we analyzed the localization of chemoreceptor-associated adaptor protein CheW1 and histidine kinase CheA1 by superresolution microscopy in a spatiotemporal manner. CheW1 localized in dynamic clusters that undergo occasional segregation and fusion events at lateral sites of both cell poles. Newly formed smaller clusters originating at midcell before completion of cytokinesis were found to grow in size during the cell cycle. Bipolar CheA1 localization and formation of aerotactic swim halos were affected depending on the fluorescent protein tag, indicating that CheA1 localization is important for aerotaxis. Furthermore, polar CheW1 localization was independent of cheOp2 to cheOp4 but lost in the absence of cheOp1 or cheA1 Results were corroborated by the detection of a direct protein interaction between CheA1 and CheW1 and by the observation that cheOp2- and cheOp3-encoded CheW paralogs localized in spatially distinct smaller clusters at the cell boundary. Although the findings of a minor aerotaxis-related CheOp4 phenotype and weak protein interactions between CheOp1 and CheOp4 by two-hybrid analysis implied that CheW1 and CheW4 might be part of the same chemoreceptor array, CheW4 was localized in spatially distinct polar-lateral arrays independent of CheOp1, suggesting that CheOp1 and CheOp4 are also not connected at the molecular level.IMPORTANCE Magnetotactic bacteria (MTB) use the geomagnetic field for navigation in aquatic redox gradients. However, the highly complex signal transduction networks in these environmental microbes are poorly understood. Here, we analyzed the localization of selected chemotaxis proteins to spatially and temporally resolve chemotaxis array localization in Magnetospirillum gryphiswaldense Our findings suggest that bipolar localization of chemotaxis arrays related to the key signaling pathway CheOp1 is important for aerotaxis and that CheOp1 signaling units assemble independent of the three other chemotaxis pathways present in M. gryphiswaldense Overall, our results provide deeper insights into the complex organization of signaling pathways in MTB and add to the general understanding of environmental bacteria possessing multiple chemotaxis pathways.

Project description:Magnetosomes of magnetotactic bacteria (MTB) consist of structurally perfect, nano-sized magnetic crystals enclosed within vesicles of a proteo-lipid membrane. In species of Magnetospirillum, biosynthesis of their cubo-octahedral-shaped magnetosomes was recently demonstrated to be a complex process, governed by about 30 specific genes that are comprised within compact magnetosome gene clusters (MGCs). Similar, yet distinct gene clusters were also identified in diverse MTB that biomineralize magnetosome crystals with different, genetically encoded morphologies. However, since most representatives of these groups are inaccessible by genetic and biochemical approaches, their analysis will require the functional expression of magnetosome genes in foreign hosts. Here, we studied whether conserved essential magnetosome genes from closely and remotely related MTB can be functionally expressed by rescue of their respective mutants in the tractable model Magnetospirillum gryphiswaldense of the Alphaproteobacteria. Upon chromosomal integration, single orthologues from other magnetotactic Alphaproteobacteria restored magnetosome biosynthesis to different degrees, while orthologues from distantly related Magnetococcia and Deltaproteobacteria were found to be expressed but failed to re-induce magnetosome biosynthesis, possibly due to poor interaction with their cognate partners within multiprotein magnetosome organelle of the host. Indeed, co-expression of the known interactors MamB and MamM from the alphaproteobacterium Magnetovibrio blakemorei increased functional complementation. Furthermore, a compact and portable version of the entire MGCs of M. magneticum was assembled by transformation-associated recombination cloning, and it restored the ability to biomineralize magnetite both in deletion mutants of the native donor and M. gryphiswaldense, while co-expression of gene clusters from both M. gryphiswaldense and M. magneticum resulted in overproduction of magnetosomes. IMPORTANCE We provide proof of principle that Magnetospirillum gryphiswaldense is a suitable surrogate host for the functional expression of foreign magnetosome genes and extended the transformation-associated recombination cloning platform for the assembly of entire large magnetosome gene cluster, which could then be transplanted to different magnetotactic bacteria. The reconstruction, transfer, and analysis of gene sets or entire magnetosome clusters will be also promising for engineering the biomineralization of magnetite crystals with different morphologies that would be valuable for biotechnical applications.

Dataset Information

Transcriptional organization and regulation of magnetosome operons in Magnetospirillum gryphiswaldense.

Publications

Transcriptional organization and regulation of magnetosome operons in Magnetospirillum gryphiswaldense.

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