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A novel role for Crp in controlling magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1.

ABSTRACT: Magnetotactic bacteria (MTB) are specialized microorganisms that synthesize intracellular magnetite particles called magnetosomes. Although many studies have focused on the mechanism of magnetosome synthesis, it remains unclear how these structures are formed. Recent reports have suggested that magnetosome formation is energy dependent. To investigate the relationship between magnetosome formation and energy metabolism, a global regulator, named Crp, which mainly controls energy and carbon metabolism in most microorganisms, was genetically disrupted in Magnetospirillum gryphiswaldense MSR-1. Compared with the wild-type or complemented strains, the growth, ferromagnetism and intracellular iron content of crp-deficient mutant cells were dramatically decreased. Transmission electron microscopy (TEM) showed that magnetosome synthesis was strongly impaired by the disruption of crp. Further gene expression profile analysis showed that the disruption of crp not only influenced genes related to energy and carbon metabolism, but a series of crucial magnetosome island (MAI) genes were also down regulated. These results indicate that Crp is essential for magnetosome formation in MSR-1. This is the first time to demonstrate that Crp plays an important role in controlling magnetosome biomineralization and provides reliable expression profile data that elucidate the mechanism of Crp regulation of magnetosome formation in MSR-1.

SUBMITTER: Wen T

PROVIDER: S-EPMC4754748 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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A novel role for Crp in controlling magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1.

Wen Tong T Guo Fangfang F Zhang Yunpeng Y Tian Jiesheng J Li Ying Y Li Jilun J Jiang Wei W

Scientific reports 20160216

Magnetotactic bacteria (MTB) are specialized microorganisms that synthesize intracellular magnetite particles called magnetosomes. Although many studies have focused on the mechanism of magnetosome synthesis, it remains unclear how these structures are formed. Recent reports have suggested that magnetosome formation is energy dependent. To investigate the relationship between magnetosome formation and energy metabolism, a global regulator, named Crp, which mainly controls energy and carbon metab ...[more]

PMID: 26879571

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Project description:The bacterium Magnetospirillum gryphiswaldense MSR-1 forms nanosized membrane-enclosed organelles termed magnetosomes. The mamXY operon, part of the magnetosome island (MAI), includes the mamY, mamX, mamZ, and ftsZ-like genes, which initiate gene transcription via the same promoter. We used a combination of molecular biological techniques (targeting of cross-linking reagents) and high-resolution mass spectrometry to investigate the coordinated activity of the four MamXY proteins in magnetite biomineralization. The FtsZ-like protein was shown by confocal laser scanning microscopy to be dispersed in the cytoplasm in the early stage of cell growth and then gradually polymerized along the magnetosome chain. Interactions of various pairs of MamXY proteins were observed using a bacterial two-hybrid system. We constructed a recombinant FtsZ-like-overexpressing strain, examined its growth patterns, and extracted magnetosome membrane proteins using a modified SDS/boiling method with BS2G-d0/d4 reagent, which helped stabilize interactions among MamXY proteins. In liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, MamY expression was detected first and remained highest among the four proteins throughout all stages of cell growth. MamX and MamZ expression was detected subsequently. The four proteins displayed coordinated expression patterns during the magnetosome maturation process. Unique peptides discovered in the MamXY protein sequences appeared to constitute "hidden" interaction sites involved in the formation of MamXY complex that helped control magnetosome shape and size.IMPORTANCE mamXY operon genes play an essential role in magnetite biomineralization, participate in redox reactions, and control magnetosome shape and size. However, mechanisms whereby the four MamXY proteins function together in iron oxidoreduction and transport are poorly understood. We used a combination of targeted cross-linking techniques and high-resolution mass spectrometry to elucidate the coordinated activity patterns of the MamXY proteins during magnetite biomineralization. Our findings indicate that the FtsZ-like protein undergoes polymerization and then recruits MamY, MamX, and MamZ in turn, and that these interactions depend on unique peptides present in the protein sequences. A hypothetical model of the functionalities of these proteins is proposed that accounts for the findings and provides a basis for further studies of coordination among magnetosome island (MAI) gene clusters during the process of magnetosome formation.

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:Magnetotactic bacteria (MTB) stand out by their ability to manufacture membrane-enclosed magnetic organelles, so-called magnetosomes. Previously, it has been assumed that a genomic region of approximately 100 kbp, the magnetosome island (MAI), harbors all genetic determinants required for this intricate biosynthesis process. Recent evidence, however, argues for the involvement of additional auxiliary genes that have not been identified yet. In the present study, we set out to delineate the full gene complement required for magnetosome production in the alphaproteobacterium Magnetospirillum gryphiswaldense using a systematic genome-wide transposon mutagenesis approach. By an optimized procedure, a Tn5 insertion library of 80,000 clones was generated and screened, yielding close to 200 insertants with mild to severe impairment of magnetosome biosynthesis. Approximately 50% of all Tn5 insertion sites mapped within the MAI, mostly leading to a nonmagnetic phenotype. In contrast, in the majority of weakly magnetic Tn5 insertion mutants, genes outside the MAI were affected, which typically caused lower numbers of magnetite crystals with partly aberrant morphology, occasionally combined with deviant intracellular localization. While some of the Tn5-struck genes outside the MAI belong to pathways that have been linked to magnetosome formation before (e.g., aerobic and anaerobic respiration), the majority of affected genes are involved in so far unsuspected cellular processes, such as sulfate assimilation, oxidative protein folding, and cytochrome c maturation, or are altogether of unknown function. We also found that signal transduction and redox functions are enriched in the set of Tn5 hits outside the MAI, suggesting that such processes are particularly important in support of magnetosome biosynthesis.IMPORTANCEMagnetospirillum gryphiswaldense is one of the few tractable model magnetotactic bacteria (MTB) for studying magnetosome biomineralization. So far, knowledge on the genetic determinants of this complex process has been mainly gathered using reverse genetics and candidate approaches. In contrast, nontargeted forward genetics studies are lacking, since application of such techniques in MTB has been complicated for a number of technical reasons. Here, we report on the first comprehensive transposon mutagenesis study in MTB, aiming at systematic identification of auxiliary genes necessary to support magnetosome formation in addition to key genes harbored in the magnetosome island (MAI). Our work considerably extends the candidate set of novel subsidiary determinants and shows that the full gene complement underlying magnetosome biosynthesis is larger than assumed. In particular, we were able to define certain cellular pathways as specifically important for magnetosome formation that have not been implicated in this process so far.

Dataset Information

A novel role for Crp in controlling magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1.

Publications

A novel role for Crp in controlling magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1.

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