Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

Protein adsorption onto nanoparticles induces conformational changes: Particle size dependency, kinetics, and mechanisms.

The use of nanomaterials in bioapplications demands a detailed understanding of protein-nanoparticle interactions. Proteins can undergo conformational changes while adsorbing onto nanoparticles, but studies on the impact of particle size on conformational changes are scarce. We have shown that conformational changes happening upon adsorption of myoglobin and BSA are dependent on the size of the nanoparticle they are adsorbing to. Out of eight initially investigated model proteins, two (BSA and myoglobin) showed conformational changes, and in both cases this conformational change was dependent on the size of the nanoparticle. Nanoparticle sizes ranged from 30 to 1000 nm and, in contrast to previous studies, we attempted to use a continuous progression of sizes in the range found in live viruses, which is an interesting size of nanoparticles for the potential use as drug delivery vehicles. Conformational changes were only visible for particles of 200 nm and bigger. Using an optimized circular dichroism protocol allowed us to follow this conformational change with regard to the nanoparticle size and, thanks to the excellent temporal resolution also in time. We uncovered significant differences between the unfolding kinetics of myoglobin and BSA. In this study, we also evaluated the plausibility of commonly used explanations for the phenomenon of nanoparticle size-dependent conformational change. Currently proposed mechanisms are mostly based on studies done with relatively small particles, and fall short in explaining the behavior seen in our studies.

The 3D pore structure and fluid dynamics simulation of macroporous monoliths: High permeability due to alternating channel width.

Polymethacrylate-based monoliths have excellent flow properties. Flow in the wide channel interconnected with narrow channels is theoretically assumed to account for favorable permeability. Monoliths were cut into 898 slices in 50nm distances and visualized by serial block face scanning electron microscopy (SBEM). A 3D structure was reconstructed and used for the calculation of flow profiles within the monolith and for calculation of pressure drop and permeability by computational fluid dynamics (CFD). The calculated and measured permeabilities showed good agreement. Small channels clearly flowed into wide and wide into small channels in a repetitive manner which supported the hypothesis describing the favorable flow properties of these materials. This alternating property is also reflected in the streamline velocity which fluctuated. These findings were corroborated by artificial monoliths which were composed of regular (interconnected) cells where narrow cells followed wide cells. In the real monolith and the artificial monoliths with interconnected flow channels similar velocity fluctuations could be observed. A two phase flow simulation showed a lateral velocity component, which may contribute to the transport of molecules to the monolith wall. Our study showed that the interconnection of small and wide pores is responsible for the excellent pressure flow properties. This study is also a guide for further design of continuous porous materials to achieve good flow properties.

A microscale method of protein extraction from bacteria: Interaction of Escherichia coli with cationic microparticles.

We developed a simple, highly selective, efficient method for extracting recombinant proteins from Escherichia coli. Our recombinant protein yield was equivalent to those obtained with high pressure homogenization, and did not require exposure to harsh thermal, chemical, or other potentially denaturing factors. We first ground conventional resin, designed for the exchange of small anions, into microparticles about 1µm in size. Then, these cationic microparticles were brought convectively into close contact with bacteria, and cell membranes were rapidly perforated, but solid cell structures were not disrupted. The released soluble components were adsorbed onto the cell wall associated microparticles or diffused directly into the supernatant. Consequently, the selective adsorption and desorption of acidic molecules is built into our extraction method, and replaces the equally effective subsequent capture on anion exchange media. Simultaneously to cell perforation flocculation was induced by the microparticles facilitating separation of cells yet after desorption of proteins with NaCl. Relative to high pressure homogenization, endogenous component release was reduced by up to three orders of magnitude, including DNA, endotoxins, and host cell proteins, particularly outer membrane protein, which indicates the presence of cell debris.

Protein O-glucosylation in Lactobacillus buchneri.

Based on the previous demonstration of surface (S-) layer protein glycosylation in Lactobacillus buchneri 41021/251 and because of general advantages of lactic acid bacteria for applied research, protein glycosylation in this bacterial species was investigated in detail. The cell surface of L. buchneri CD034 is completely covered with an oblique 2D crystalline array (lattice parameters, a = 5.9 nm; b = 6.2 nm; γ ~ 77°) formed by self-assembly of the S-layer protein SlpB. Biochemical and mass spectrometric analyses revealed that SlpB is the most abundant protein and that it is O-glycosylated at four serine residues within the sequence S(152)-A-S(154)-S(155)-A-S(157) with, on average, seven Glc(α1-6) residues, each. Subcellular fractionation of strain CD034 indicated a sequential order of SlpB export and glucosylation as evidenced by lack of glucosylation of cytosolic SlpB. Protein glycosylation analysis was extended to strain L. buchneri NRRL B-30929 where an analogous glucosylation scenario could be detected, with the S-layer glycoprotein SlpN containing an O-glycosylation motif identical to that of SlpB. This corroborates previous data on S-layer protein glucosylation of strain 41021/251 and let us propose a species-wide S-layer protein O-glucosylation in L. buchneri targeted at the sequence motif S-A-S-S-A-S. Search of the L. buchneri genomes for the said glucosylation motif revealed one further ORF, encoding the putative glycosyl-hydrolase LbGH25B and LbGH25N in L. buchneri CD034 and NRRL B-30929, respectively, for which we have indications of a glycosylation comparable to that of the S-layer proteins. These findings demonstrate the presence of a distinct protein O-glucosylation system in Gram-positive and beneficial microbes.

Prediction of inclusion body solubilization from shaken to stirred reactors.

Inclusion bodies (IBs) were solubilized in a µ-scale system using shaking microtiter plates or a stirred tank reactor in a laboratory setting. Characteristic dimensionless numbers for mixing, the Phase number Ph and Reynolds number Re did not correlate with the kinetics and equilibrium of protein solubilization. The solubilization kinetics was independent of the mixing system, stirring or shaking rate, shaking diameter, and energy input. Good agreement was observed between the solubilization kinetics and yield on the µ-scale and laboratory setting. We show that the IB solubilization process is controlled predominantly by pore diffusion. Thus, for the process it is sufficient to keep the IBs homogeneously suspended, and additional power input will not improve the process. The high-throughput system developed on the µ-scale can predict solubilization in stirred reactors up to a factor of 500 and can therefore be used to determine optimal solubilization conditions on laboratory and industrial scale.

Characterizing the S-layer structure and anti-S-layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X-ray scattering.

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22-nm-thick, 2D crystalline surface (S-) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium-host cross-talk. The S-layer is composed of two intercalating glycoproteins (TfsA-GP, TfsB-GP) that are aligned into a periodic lattice. To characterize this unique S-layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and single-molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild-type and S-layer-deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high-resolution imaging of the outer surface of T. forsythia wild-type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti-TfsA antibody coupled to the AFM tip and the S-layer as present on T. forsythia wild-type and mutant cells, displaying TfsA-GP alone. Unbinding forces between the antibody and wild-type cells were greater than with mutant cells. This indicated that the TfsA-GP is not so strongly attached to the mutant cell surface when the co-assembling TfsB-GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S-layer architecture with two intercalating S-layer glycoproteins and TfsA-GP being mainly outwardly oriented.

Intracellular targeting of ascomycetous catalase-peroxidases (KatG1s).

Bifunctional catalase-peroxidases (KatGs) are heme oxidoreductases widely spread among bacteria, archaea and among lower eukaryotes. In fungi, two KatG groups with different localization have evolved, intracellular (KatG1) and extracellular (KatG2) proteins. Here, the cloning, expression analysis and subcellular localization of two novel katG1 genes from the soil fungi Chaetomium globosum and Chaetomium cochliodes are reported. Whereas, the metalloenzyme from Ch. globosum is expressed constitutively, Ch. cochliodes KatG1 reveals a slight increase in expression after induction of oxidative stress by cadmium ions and hydrogen peroxide. The intronless open reading frames of both Sordariomycetes katG1 genes as well as of almost all fungal katG1s possess two peroxisomal targeting signals (PTS1 and PTS2). Peroxisomal localization of intracellular eukaryotic catalase-peroxidases was verified by organelle separation and immunofluorescence microscopy. Co-localization with the peroxisomal enzyme 3-ketoacyl-CoA-thiolase was demonstrated for KatGs from Magnaporthe grisea, Chaetomium globosum and Chaetomium cochliodes. The physiological role of fungal catalase-peroxidases is discussed.

Small-angle X-ray scattering for imaging of surface layers on intact bacteria in the native environment.

Crystalline cell surface layers (S-layers) represent a natural two-dimensional (2D) protein self-assembly system with nanometer-scale periodicity that decorate many prokaryotic cells. Here, we analyze the S-layer on intact bacterial cells of the Gram-positive organism Geobacillus stearothermophilus ATCC 12980 and the Gram-negative organism Aquaspirillum serpens MW5 by small-angle X-ray scattering (SAXS) and relate it to the structure obtained by transmission electron microscopy (TEM) after platinum/carbon shadowing. By measuring the scattering pattern of X rays obtained from a suspension of bacterial cells, integral information on structural elements such as the thickness and lattice parameters of the S-layers on intact, hydrated cells can be obtained nondestructively. In contrast, TEM of whole mounts is used to analyze the S-layer lattice type and parameters as well as the physical structure in a nonaqueous environment and local information on the structure is delivered. Application of SAXS to S-layer research on intact bacteria is a challenging task, as the scattering volume of the generally thin (3- to 30-nm) bacterial S-layers is low in comparison to the scattering volume of the bacterium itself. For enhancement of the scattering contrast of the S-layer in SAXS measurement, either silicification (treatment with tetraethyl orthosilicate) is used, or the difference between SAXS signals from an S-layer-deficient mutant and the corresponding S-layer-carrying bacterium is used for determination of the scattering signal. The good agreement of the SAXS and TEM data shows that S-layers on the bacterial cell surface are remarkably stable.

Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees.

The gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis.

Analysis of the cell surface layer ultrastructure of the oral pathogen Tannerella forsythia.

The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium.

Glycobiology Aspects of the Periodontal Pathogen Tannerella forsythia.

Glycobiology is important for the periodontal pathogen Tannerella forsythia, affecting the bacterium's cellular integrity, its life-style, and virulence potential. The bacterium possesses a unique Gram-negative cell envelope with a glycosylated surface (S-) layer as outermost decoration that is proposed to be anchored via a rough lipopolysaccharide. The S-layer glycan has the structure 4­MeO-b-ManpNAcCONH2-(1→3)-[Pse5Am7Gc-(2→4)-]-b-ManpNAcA-(1→4)-[4-MeO-a-Galp-(1→2)-]-a-Fucp-(1→4)-[-a-Xylp-(1→3)-]-b-GlcpA-(1→3)-[-b-Digp-(1→2)-]-a-Galp and is linked to distinct serine and threonine residues within the D(S/T)(A/I/L/M/T/V) amino acid motif. Also several other Tannerella proteins are modified with the S­layer oligosaccharide, indicating the presence of a general O­glycosylation system. Protein O­glycosylation impacts the life-style of T. forsythia since truncated S-layer glycans present in a defined mutant favor biofilm formation. While the S­layer has also been shown to be a virulence factor and to delay the bacterium's recognition by the innate immune system of the host, the contribution of glycosylation to modulating host immunity is currently unraveling. Recently, it was shown that Tannerella surface glycosylation has a role in restraining the Th17-mediated neutrophil infiltration in the gingival tissues. Related to its asaccharolytic physiology, T. forsythia expresses a robust enzymatic repertoire, including several glycosidases, such as sialidases, which are linked to specific growth requirements and are involved in triggering host tissue destruction. This review compiles the current knowledge on the glycobiology of T. forsythia.