N-Glycosylation Is Important for Halobacterium salinarum Archaellin Expression, Archaellum Assembly and Cell Motility (original) (raw)
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A way to identify archaellins in Halobacterium
In the current study, haloarchaea Halobacterium salinarum cells were transformed individually with each of the modified archaellin genes (flaA1, flaA2 and flaB2) containing an oligonucleotide insert encoding the FLAG peptide (DYKDDDDK). The insertion site was selected to expose the FLAG peptide on the archaella filament surface. Three types of transformed cells synthesizing archaella, containing A1, A2, or B2 archaellin modified with FLAG peptide were obtained. Electron microscopy of archaella has demonstrated that in each case the FLAG peptide is available for the specific antibody binding. It was shown for the first time that the B2 archaellin, like archaellins A1 and A2, is found along the whole filament length. © Versita Sp. z o.o.
A way to identify archaellins in Halobacterium salinarum archaella by FLAG-tagging
Central European Journal of Biology, 2013
In the current study, haloarchaea Halobacterium salinarum cells were transformed individually with each of the modified archaellin genes (flaA1, flaA2 and flaB2) containing an oligonucleotide insert encoding the FLAG peptide (DYKDDDDK). The insertion site was selected to expose the FLAG peptide on the archaella filament surface. Three types of transformed cells synthesizing archaella, containing A1, A2, or B2 archaellin modified with FLAG peptide were obtained. Electron microscopy of archaella has demonstrated that in each case the FLAG peptide is available for the specific antibody binding. It was shown for the first time that the B2 archaellin, like archaellins A1 and A2, is found along the whole filament length. © Versita Sp. z o.o.
Identifying Components of a Halobacterium salinarum N-Glycosylation Pathway
Frontiers in Microbiology, 2021
Whereas N-glycosylation is a seemingly universal process in Archaea, pathways of N-glycosylation have only been experimentally verified in a mere handful of species. Toward expanding the number of delineated archaeal N-glycosylation pathways, the involvement of the putative Halobacterium salinarum glycosyltransferases VNG1067G, VNG1066C, and VNG1062G in the assembly of an N-linked tetrasaccharide decorating glycoproteins in this species was addressed. Following deletion of each encoding gene, the impact on N-glycosylation of the S-layer glycoprotein and archaellins, major glycoproteins in this organism, was assessed by mass spectrometry. Likewise, the pool of dolichol phosphate, the lipid upon which this glycan is assembled, was also considered in each deletion strain. Finally, the impacts of such deletions were characterized in a series of biochemical, structural and physiological assays. The results revealed that VNG1067G, VNG1066C, and VNG1062G, renamed Agl25, Agl26, and Agl27 according to the nomenclature used for archaeal N-glycosylation pathway components, are responsible for adding the second, third and fourth sugars of the N-linked tetrasaccharide decorating Hbt. salinarum glycoproteins. Moreover, this study demonstrated how compromised N-glycosylation affects various facets of Hbt. salinarum cell behavior, including the transcription of archaellin-encoding genes.
Microorganisms
Halobacterium salinarum NRC-1 is an extremophile that grows optimally at 4.3 M NaCl concentration. In spite of being an established model microorganism for the archaea domain, direct comparisons between its proteome and transcriptome during osmotic stress are still not available. Through RNA-seq-based transcriptomics, we compared a low salt (2.6 M NaCl) stress condition with 4.3 M of NaCl and found 283 differentially expressed loci. The more commonly found classes of genes were: ABC-type transporters and transcription factors. Similarities, and most importantly, differences between our findings and previously published datasets in similar experimental conditions are discussed. We validated three important biological processes differentially expressed: gas vesicles production (due to down-regulation of gvpA1b, gvpC1b, gvpN1b, and gvpO1b); archaellum formation (due to down-regulation of arlI, arlB1, arlB2, and arlB3); and glycerol metabolism (due to up-regulation of glpA1, glpB, and g...
Identification of the new protein participating in the archaea motility regulation
Biochemistry (moscow) Supplement Series A: Membrane and Cell Biology, 2010
A new family of archaeal proteins, CheM, having no homologues among bacteria and eukaryotes, was identified. Genes cheM are represented only in archaea possessing the chemotaxis and generally located close to che and fla loci. There is only one copy of the cheM gene in thermophilic and methanogenic archaea. Halophilic archaea have an additional paralog of the cheM gene. Mutant strains of Halobacterium salinarum R1 with deletions of the cheM1 (OE2402F) and cheM2 (OE2404R) genes were obtained. Mutant strains were not differ from the wild type strain by speed of movement in liquid medium but had appreciable differences in the diameter of a swarm on semi-liquid agar, indicative of reduced chemotaxis. It was demonstrated that the CheM2 protein from H. salinarum R1 co-isolates with protein CheY, the chemotaxis regulator in the conditions of its activation. The specific interaction between proteins CheM and CheY from hyperthermophilic archaea Pyrococcus horikoshii OT3 was also found. We suppose that CheM proteins provide adaptation of the chemotaxis system universal for bacteria and archaea to the specific archaeal flagellar motor apparatus.
Journal of Biological Chemistry, 2019
Edited by Wolfgang Peti Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. Forkhead-associated (FHA) and von Willebrand type A (vWA) domains are involved in several phosphorylation-dependent processes of multiprotein complex assemblies. Although well-studied in eukaryotes and bacteria, the structural and functional contexts of these domains are not yet understood in Archaea. Here, we report the structural base for such an interacting pair of FHA and vWA domain-containing proteins, ArnA and ArnB, in the thermoacidophilic archaeon Sulfolobus acidocaldarius, where they act synergistically and negatively modulate motility. The structure of the FHA domain of ArnA at 1.75 Å resolution revealed that it belongs to the subclass of FHA domains, which recognizes double-pSer/pThr motifs. We also solved the 1.5 Å resolution crystal structure of the ArnB paralog vWA2, disclosing a complex topology comprising the vWA domain, a -sandwich fold, and a C-terminal helix bundle. We further show that ArnA binds to the C terminus of ArnB, which harbors all the phosphorylation sites identified to date and is important for the function of ArnB in archaellum regulation. We also observed that expression levels of the archaellum components in response to changes in nutrient conditions are independent of changes in ArnA and ArnB levels and that a strong interaction between ArnA and ArnB observed during growth on rich medium sequentially diminishes after nutrient limitation. In summary, our findings unravel the structural features in ArnA and ArnB important for their interaction and functional archaellum expression and reveal how nutrient conditions affect this interaction. Stress response that yields adaptation to changing environmental conditions is one of the most important prerequisites to ensure survival in all living organisms. A vast amount of modules has evolved to receive, process, and transfer these signals within the cell. A well-known key element of cellular signal transduction is phosphorylation, which at the same time is one of the most important posttranslational modifications in all three domains of life (1). In prokaryotes sensor kinases receive environmental signals and transmit them to receivers within the cell, which mostly regulate gene expression. A variety of protein families exists, which specifically recognize and bind phosphorylated side chains in target proteins. One representative is the family of proteins containing forkhead-associated (FHA) 4 domains, which have been intensely studied since their identification in 1995 (2). FHA domains are ubiquitously found and involved in a variety of cellular processes. For example, FHA domains are part of the eukaryotic DNA damage-response, DNA-repair, and DNA-replication systems (3). This domain type is often part of larger kinases, e.g. in Rad53 of Saccharomyces cerevisiae, where it is involved in the signaling cascade initiated after DNA damage by interaction with other proteins (4). In bacteria, FHA domain-containing proteins are involved in a variety of processes such as amino acid production, sporulation, or resistance to antimicrobial substances. In the latter, either they are part of a protein that directly interacts
Nature Microbiology, 2016
Motile archaea swim using a rotary filament, the archaellum, a surface appendage that resembles bacterial flagella structurally, but is homologous to bacterial type IV pili. Little is known about the mechanism by which archaella produce motility. To gain insights into this mechanism, we characterized archaellar function in the model organism Halobacterium salinarum. Three-dimensional tracking of quantum dots enabled visualization of the left-handed corkscrewing of archaea in detail. An advanced analysis method combined with total internal reflection fluorescence microscopy, termed cross-kymography, was developed and revealed a right-handed helical structure of archaella with a rotation speed of 23 ± 5 Hz. Using these structural and kinetic parameters, we computationally reproduced the swimming and precession motion with a hydrodynamic model and estimated the archaellar motor torque to be 50 pN nm. Finally, in a tethered-cell assay, we observed intermittent pauses during rotation with ∼36°or 60°intervals, which we speculate may be a unitary step consuming a single adenosine triphosphate molecule, which supplies chemical energy of 80 pN nm when hydrolysed. From an estimate of the energy input as ten or six adenosine triphosphates per revolution, the efficiency of the motor is calculated to be ∼6-10%.
Molecular microbiology, 2013
Cell surfaces are decorated by a variety of proteins that facilitate interactions with their environments and support cell stability. These secreted proteins are anchored to the cell by mechanisms that are diverse, and, in archaea, poorly understood. Recently published in silico data suggest that in some species a subset of secreted euryarchaeal proteins, which includes the S-layer glycoprotein, is processed and covalently linked to the cell membrane by enzymes referred to as archaeosortases. In silico work led to the proposal that an independent, sortase-like system for proteolysis-coupled, carboxy-terminal lipid modification exists in bacteria (exosortase) and archaea (archaeosortase). Here, we provide the first in vivo characterization of an archaeosortase in the haloarchaeal model organism Haloferax volcanii. Deletion of the artA gene (HVO_0915) resulted in multiple biological phenotypes: (a) poor growth, especially under low-salt conditions, (b) alterations in cell shape and th...
The archaellum: how Archaea swim
Frontiers in microbiology, 2015
Recent studies on archaeal motility have shown that the archaeal motility structure is unique in several aspects. Although it fulfills the same swimming function as the bacterial flagellum, it is evolutionarily and structurally related to the type IV pilus. This was the basis for the recent proposal to term the archaeal motility structure the "archaellum." This review illustrates the key findings that led to the realization that the archaellum was a novel motility structure and presents the current knowledge about the structural composition, mechanism of assembly and regulation, and the posttranslational modifications of archaella.
2007
Background: Differential expression of genes can be regulated on many different levels. Most global studies of gene regulation concentrate on transcript level regulation, and very few global analyses of differential translational efficiencies exist. The studies have revealed that in Saccharomyces cerevisiae, Arabidopsis thaliana, and human cell lines translational regulation plays a significant role. Additional species have not been investigated yet. Particularly, until now no global study of translational control with any prokaryotic species was available. Results: A global analysis of translational control was performed with two haloarchaeal model species, Halobacterium salinarum and Haloferax volcanii. To identify differentially regulated genes, exponentially growing and stationary phase cells were compared. More than 20% of H. salinarum transcripts are translated with non-average efficiencies. By far the largest group is comprised of genes that are translated with above-average efficiency specifically in exponential phase, including genes for many ribosomal proteins, RNA polymerase subunits, enzymes, and chemotaxis proteins. Translation of 1% of all genes is specifically repressed in either of the two growth phases. For comparison, DNA microarrays were also used to identify differential transcriptional regulation in H. salinarum, and 17% of all genes were found to have non-average transcript levels in exponential versus stationary phase. In H. volcanii, 12% of all genes are translated with non-average efficiencies. The overlap with H. salinarum is negligible. In contrast to H. salinarum, 4.6% of genes have non-average translational efficiency in both growth phases, and thus they might be regulated by other stimuli than growth phase.