Thomas Heimerl

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.

A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.

Evolutionary Remodeling of Bacterial Motility Checkpoint Control

Summary Regulatory networks play a central role in the relationship between genotype and phenotype in all organisms. However, the mechanisms that underpin the evolutionary plasticity of these networks remain poorly understood. Here, we used experimental selection for enhanced bacterial motility in a porous environment to explore the adaptability of one of the most complex networks known in bacteria. We found that the resulting phenotypic changes are mediated by adaptive mutations in several functionally different proteins, including multiple components of the flagellar motor. Nevertheless, this evolutionary adaptation could be explained by a single mechanism, namely remodeling of the checkpoint regulating flagellar gene expression. Supported by computer simulations, our findings suggest that the specific “bow-tie” topology of the checkpoint facilitates evolutionary tuning of the cost-benefit trade-off between motility and growth. We propose that bow-tie regulatory motifs, which are widespread in cellular networks, play a general role in evolutionary adaptation.

The Mitotic Exit Network Regulates Spindle Pole Body Selection During Sporulation of Saccharomyces cerevisiae

Age-based inheritance of centrosomes in eukaryotic cells is associated with faithful chromosome distribution in asymmetric cell divisions. During Saccharomyces cerevisiae ascospore formation, such an inheritance mechanism targets the yeast centrosome equivalents, the spindle pole bodies (SPBs) at meiosis II onset. Decreased nutrient availability causes initiation of spore formation at only the younger SPBs and their associated genomes. This mechanism ensures encapsulation of nonsister genomes, which preserves genetic diversity and provides a fitness advantage at the population level. Here, by usage of an enhanced system for sporulation-induced protein depletion, we demonstrate that the core mitotic exit network (MEN) is involved in age-based SPB selection. Moreover, efficient genome inheritance requires Dbf2/20-Mob1 during a late step in spore maturation. We provide evidence that the meiotic functions of the MEN are more complex than previously thought. In contrast to mitosis, completion of the meiotic divisions does not strictly rely on the MEN whereas its activity is required at different time points during spore development. This is reminiscent of vegetative MEN functions in spindle polarity establishment, mitotic exit, and cytokinesis. In summary, our investigation contributes to the understanding of age-based SPB inheritance during sporulation of S. cerevisiae and provides general insights on network plasticity in the context of a specialized developmental program. Moreover, the improved system for a developmental-specific tool to induce protein depletion will be useful in other biological contexts.

Molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion.

Significance Lacking a canonical signal peptide for translocation into the endoplasmic reticulum, galectin-3 (Gal3) is exported by unconventional secretion to play key roles in various cellular processes of fundamental importance. However, molecular details about the sorting components, the signals involved, and the underlying mechanism have remained elusive. Here, we identify a highly conserved tetrapeptide motif P(S/T)AP in the amino-terminal domain of Gal3 that directly interacts with the endosomal sorting complex required for transport (ESCRT) component Tsg101, resulting in exosomal release. This study thus defines a unique molecular mechanism based on a late domain-like motif known from many viruses by which an endogenous non-ESCRT protein is secreted in exosomes. The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.

The Central Vacuole of the Diatom Phaeodactylum tricornutum: Identification of New Vacuolar Membrane Proteins and of a Functional Di-leucine-based Targeting Motif

Diatoms are unicellular organisms evolved by secondary endosymbiosis. Although studied in many aspects, the functions of vacuolar-like structures of these organisms are rarely investigated. One of these structures is a dominant central vacuole-like compartment with a marbled phenotype, which is supposed to represent a chrysolaminarin-storing and carbohydrate mobilization compartment. However, other functions as well as targeting of proteins to this compartment are not shown experimentally. In order to study trafficking of membrane proteins to the vacuolar membrane, we scanned the genome for intrinsic vacuolar membrane proteins and used one representative for targeting studies. Our work led to the identification of several proteins located in the vacuolar membrane as well as the sub-compartmentalized localization of one protein. In addition, we show that a di-leucine-based motif is an important signal for correct targeting to the central vacuole of diatoms, like it is in plants.

Seven-transmembrane receptor protein RgsP and cell wall-binding protein RgsM promote unipolar growth in Rhizobiales

Members of the Rhizobiales (class of α-proteobacteria) display zonal peptidoglycan cell wall growth at one cell pole, contrasting with the dispersed mode of cell wall growth along the sidewalls of many other rod-shaped bacteria. Here we show that the seven-transmembrane receptor (7TMR) protein RgsP (SMc00074), together with the putative membrane-anchored peptidoglycan metallopeptidase RgsM (SMc02432), have key roles in unipolar peptidoglycan formation during growth and at mid-cell during cell division in Sinorhizobium meliloti. RgsP is composed of a periplasmic globular 7TMR-DISMED2 domain, a membrane-spanning region, and cytoplasmic PAS, GGDEF and EAL domains. The EAL domain confers phosphodiesterase activity towards the second messenger cyclic di-GMP, a key regulatory player in the transition between bacterial lifestyles. RgsP and RgsM localize to sites of zonal cell wall synthesis at the new cell pole and cell divison site, suggesting a role in cell wall biogenesis. The two proteins are essential for cell wall biogenesis and cell growth. Cells depleted of RgsP or RgsM had an altered muropeptide composition and RgsM binds to peptidoglycan. RgsP and RgsM orthologs are functional when interchanged between α-rhizobial species pointing to a conserved mechanism for cell wall biogenesis/remodeling within the Rhizobiales. Overall, our findings suggest that RgsP and RgsM contribute to the regulation of unipolar cell wall biogenesis in α-rhizobia.

Identification and localization of peroxisomal biogenesis proteins indicates the presence of peroxisomes in the cryptophyte Guillardia theta and other ‘chromalveolates’

Abstract Peroxisomes are single-membrane-bound organelles with a huge metabolic versatility, including the degradation of fatty acids (β-oxidation) and the detoxification of reactive oxygen species as most conserved functions. Although peroxisomes seem to be present in the majority of investigated eukaryotes, where they are responsible for many eclectic and important spatially separated metabolic reactions, knowledge about their existence in the plethora of protists (eukaryotic microorganisms) is scarce. Here, we investigated genomic data of organisms containing complex plastids with red algal ancestry (so-called “chromalveolates”) for the presence of genes encoding peroxins—factors specific for the biogenesis, maintenance, and division of peroxisomes in eukaryotic cells. Our focus was on the cryptophyte Guillardia theta, a marine microalga, which possesses two phylogenetically different nuclei of host and endosymbiont origin, respectively, thus being of enormous evolutionary significance. Besides the identification of a complete set of peroxins in G. theta, we heterologously localized selected factors as GFP fusion proteins via confocal and electron microscopy in the model diatom Phaeodactylum tricornutum. Furthermore, we show that peroxins, and thus most likely peroxisomes, are present in haptophytes as well as eustigmatophytes, brown algae, and alveolates including dinoflagellates, chromerids, and noncoccidian apicomplexans. Our results indicate that diatoms are not the only “chromalveolate” group devoid of the PTS2 receptor Pex7, and thus a PTS2-dependent peroxisomal import pathway, which seems to be absent in haptophytes (Emiliania huxleyi) as well. Moreover, important aspects of peroxisomal biosynthesis and protein import in “chromalveolates”are highlighted.

Ereboglobus luteus gen. nov. sp. nov. from cockroach guts, and new insights into the oxygen relationship of the genera Opitutus and Didymococcus (Verrucomicrobia: Opitutaceae)

We isolated a novel member of the phylum Verrucomicrobia from the hindgut of the cockroach Shelfordella lateralis. Strain Ho45 is a yellow-pigmented, motile coccus that represents a new genus-level lineage with less than 93% sequence similarity to the 16S rRNA genes of other species in the family Opitutaceae. Ultrastructural analysis revealed a Gram-negative cell envelope with an outer membrane and a periplasmic space. In its ability to ferment sugars to propionate and acetate as major products, strain Ho45 resembles its closest relative, Opitutus terrae. However, the strains differed in their relationship to oxygen. Although strain Ho45 grew and consumed oxygen at sub-atmospheric concentrations (1-4%), both growth rate and cell yield decreased strongly with increasing oxygen concentration in the headspace. By contrast, O. terrae, previously described as an obligate anaerobe, proved to be facultatively aerobic, with highest growth rates and cell yields at 2% and 16% oxygen, respectively. Also the closely related Didymococcus (Diplosphaera) colitermitum, previously described as an obligately aerobic microaerophile, showed a fermentative metabolism under anoxic conditions, forming the same products from glucose as strain Ho45 and O. terrae. Based on phenotypic and phylogenetic evidence, we propose strain Ho45 as the type strain of a novel genus, Ereboglobus luteus gen. nov. sp. nov., and provide an emended description of the family Opitutaceae and the genera Opitutus and Didymococcus.