Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.

Background Magnetosomes produced by magnetotactic bacteria represent magnetic nanoparticles with unprecedented characteristics. However, their use in many biotechnological applications has so far been hampered by their challenging bioproduction at larger scales. Results Here, we developed an oxystat batch fermentation regime for microoxic cultivation of Magnetospirillum gryphiswaldense in a 3 L bioreactor. An automated cascade regulation enabled highly reproducible growth over a wide range of precisely controlled oxygen concentrations (1–95% of air saturation). In addition, consumption of lactate as the carbon source and nitrate as alternative electron acceptor were monitored during cultivation. While nitrate became growth limiting during anaerobic growth, lactate was the growth limiting factor during microoxic cultivation. Analysis of microoxic magnetosome biomineralization by cellular iron content, magnetic response, transmission electron microscopy and small-angle X-ray scattering revealed magnetosomal magnetite crystals were highly uniform in size and shape. Conclusion The fermentation regime established in this study facilitates stable oxygen control during culturing of Magnetospirillum gryphiswaldense. Further scale-up seems feasible by combining the stable oxygen control with feeding strategies employed in previous studies. Results of this study will facilitate the highly reproducible laboratory-scale bioproduction of magnetosomes for a diverse range of future applications in the fields of biotechnology and biomedicine.

This study explores lab-based small-angle X-ray scattering (SAXS) as a novel quantitative stand-alone technique to monitor the size, shape, and arrangement of magnetosomes during different stages of particle biogenesis in the model organism Magnetospirillum gryphiswaldense. The SAXS data sets contain volume-averaged, statistically accurate information on both the diameter of the inorganic nanocrystal and the enveloping protein-rich magnetosome membrane. As a robust and nondestructive in situ technique, SAXS can provide new insights into the physicochemical steps involved in the biosynthesis of magnetosome nanoparticles as well as their assembly into well-ordered chains. The proposed fit model can easily be adapted to account for different particle shapes and arrangements produced by other strains of magnetotactic bacteria, thus rendering SAXS a highly versatile method.

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.

Background 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 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. Results 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. 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. Conclusions 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 (GEO Series accession number GSE197098).

Genomic information from various magnetotactic bacteria suggested that besides their common ability to form magnetosomes, they potentially also represent a source of bioactive natural products. By using targeted deletion and transcriptional activation, we connected a large biosynthetic gene cluster (BGC) of the trans-acyltransferase polyketide synthase (trans-AT PKS) type to the biosynthesis of a novel polyketide in the alphaproteobacterium Magnetospirillum gryphiswaldense. Structure elucidation by mass spectrometry and nuclear magnetic resonance spectroscopy (NMR) revealed that this secondary metabolite resembles sesbanimides, which were very recently reported from other taxa. However, sesbanimide R exhibits an additional arginine moiety the presence of which reconciles inconsistencies in the previously proposed sesbanimide biosynthesis pathway observed when comparing the chemical structure and the potential biochemistry encoded in the BGC. In contrast to the case with sesbanimides D, E, and F, we were able to assign the stereocenter of the arginine moiety experimentally and two of the remaining three stereocenters by predictive biosynthetic tools. Sesbanimide R displayed strong cytotoxic activity against several carcinoma cell lines. IMPORTANCE The findings of this study contribute a new secondary metabolite member to the glutarimide-containing polyketides. The determined structure of sesbanimide R correlates with its cytotoxic bioactivity, characteristic for members of this family. Sesbanimide R represents the first natural product isolated from magnetotactic bacteria and identifies this highly diverse group as a so-far-untapped source for the future discovery of novel secondary metabolites.

Genomic information from various magnetotactic bacteria suggested that besides their common ability to form magnetosomes they potentially also represent a source of bioactive natural products. By using targeted deletion and transcriptional activation, we connected a large biosynthetic gene cluster (BGC) of the trans-AT PKS type to the biosynthesis of a novel polyketide in the alphaproteobacterium Magnetospirillum gryphiswaldense. Structure elucidation by mass spectrometry and NMR revealed that this secondary metabolite resembles sesbanimides which were very recently reported from other taxa. However, sesbanimide R exhibits an additional arginine moiety the presence of which reconciles inconsistencies in the previously proposed sesbanimide biosynthesis pathway when comparing the chemical structure and the potential biochemistry encoded in the BGC. In contrast to sesbanimides D, E and F, we were able to assign the stereocenter of the arginine moiety experimentally and two of the remaining three stereocenters by predictive biosynthetic tools. Sesbanimide R displayed strong cytotoxic activity against several carcinoma cell lines. ImportanceThe finding of this study contributes a new secondary metabolite member to the glutarimide-containing polyketides. The determined structure of sesbanimide R correlates with its cytotoxic bioactivity characteristic for members of this family. Sesbanimide R represents the first natural product isolated from magnetotactic bacteria and identifies this highly diverse group as a so far untapped source for the future discovery of novel secondary metabolites.