Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins

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

The transcriptomic landscape of Magnetospirillum gryphiswaldense during magnetosome biomineralization

Riese CN, Wittchen M, Jerome V, et al. The transcriptomic landscape of Magnetospirillum gryphiswaldense during magnetosome biomineralization. BMC Genomics . 2022;23(1): 699.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 sigma(70) 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)

The Complex Transcriptional Landscape of Magnetosome Gene Clusters in Magnetospirillum gryphiswaldense

Dziuba M, Riese CN, Borgert L, et al. The Complex Transcriptional Landscape of Magnetosome Gene Clusters in Magnetospirillum gryphiswaldense. mSystems. 2021;6(5): e00893-21.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 magneto some 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

Determination of subunit stoichiometry using single molecule microscopy.

<p>A) and B) 20 consecutive images of a streams acquisition of one cell expressing FloT-YFP, 20 ms time intervals. The triangles indicate a relatively stationary focus that bleaches during the stream acquisition. White bar 2 μm. C to E) Graphs showing bleaching events. One individual bleaching step is indicated in panel C), an event of addition of a molecule (positive increase in fluorescence) in panel D), panel E) shows all clear bleaching steps in an acquisition.</p

Localization of flotillins and associated protein in exponentially growing <i>B</i>. <i>subtilis</i> cells expressing two protein fusions.

<p>A) FloA-CFP and KinC-YFP, Nomarski-DIC, overlay of Nomarski-DIC, FloA-CFP (green) and KinC-YFP. B) FloT-CFP, FloA-YFP, overlay of Nomarski-DIC and FloT-CFP (red) and FloA-YFP (green). C) KinC-YFP, FloT-CFP, overlay of Nomarski-DIC and KinC-YFP (red) and FloT-CFP (green). D) NfeD2-YFP and FloT-CFP, overlay of Nomarski-DIC and NfeD2-YFP (red) and FloT-CFP (green). E) SecA-YFP and FloT-CFP, overlay of Nomarski-DIC and SecA-YFP (red) and FloT-CFP (green), F) overlay of FtsH-YFP (green) and FloA-CFP (red), images of the two panels were taken with an interval of 200 ms. White triangles indicate cases of colocalization, white bars 2 μm.</p

The soluble part of FloA (FloAsp) forms multimers.

<p>A) SDS-PAGE showing purification by Ni-NTA chromatography of FloAsp carrying a 6xHis tag at the N-terminus, lane 1 flow through, lane 2 wash step, lane 3 peak elution. B) Circular dichroism analysis of FloAsp (25 μM). C) Size-exclusion chromatography (SEC) of FloAsp; elution of marker proteins and of the void peak are indicated by arrows, numbered letters indicate fractions, which are shown in D) SDS PAGE showing corresponding fractions from SEC. E-F) SDS PAGE of fractions from 5 to 20% (w/v) sucrose gradient centrifugation, position of marker proteins is indicated by arrows, size of denatured proteins is indicated by the marker lane. E) Loading of fraction B5 (dimeric FloAsp) from SEC, F) loading of fraction A5 (multimeric FloAsp); the last lane of the SDS PAGE shows the 35 and 25 kDa proteins from the marker lane. G) Electron microscopy (EM, uranyl acetate negative stain) of the LMW fraction of Strep-FloAsp, H) EM of the HMW fraction of Strep-FloAsp, scale bars 100 nm.</p

Dynamics of <i>Bacillus</i> flotillin structures.

<p>A) overlay of a bright field image of a single <i>Bacillus</i> cells with the first frame of fluorescence (green signals) and of 9 trajectories, each having a different colour, of YFP tagged protein FloA, displayed over a time course of 8 seconds. B) individual mean squared displacement curves of the 9 particles observed (in corresponding colours) C) Example of about 50 tracks in 2 <i>Bacillus</i> cells, which are analysed in D) MSD curves for FloA; E) MSD curves of FloT for an experiment analogous to that shown in A) and C). F) weighted mean over all tracked MSD curves of FloA (3152 tracks, red curve) and of FloT (N = 1024, black curve). Error bars indicate the confidence of the mean values, (given by the weighted standard deviation, divided by the number of degrees of freedom). Dotted lines show the fits to the diffusion constants. G) Plot like in panel F), but differentiated into movement in the y-direction or in the x-direction for FloA-YFP; H) distribution of instantaneous velocities of FloA and along the perpendicular (v_x, blue bars) and longitudinal (v_y, red bars) cell axis, No = number. I) same as H) for FloT. For FloA, the tails of the distribution are visibly broader than for FloT, indicating a higher variance and therefore a higher diffusion constant of FloA.</p