RESUMEN
Archaea play indispensable roles in global biogeochemical cycles, yet many crucial cellular processes, including cell-shape determination, are poorly understood. Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. Through phenotypic characterization of deletion strains, we established that rod-determining factor A (RdfA) and disk-determining factor A (DdfA) are required for the formation of rods and disks, respectively. We also identified structural proteins, including an actin homolog that plays a role in disk-shape morphogenesis, which we named volactin. Using live-cell imaging, we determined volactin's cellular localization and showed its dynamic polymerization and depolymerization. Our results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains.
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Proteínas Arqueales , Haloferax volcanii , Forma de la Célula , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismoRESUMEN
Despite the ecological, evolutionary and economical significance of archaea, key aspects of their cell biology, metabolic pathways, and adaptations to a wide spectrum of environmental conditions, remain to be elucidated. Proteomics allows for the system-wide analysis of proteins, their changes in abundance between different conditions, as well as their post-translational modifications, providing detailed insights into the function of proteins and archaeal cell biology. In this chapter, we describe a sample preparation and mass spectrometric analysis workflow that has been designed for Haloferax volcanii but can be applied to a broad range of archaeal species. Furthermore, proteomics experiments provide a wealth of data that is invaluable to various disciplines. Therefore, we previously initiated the Archaeal Proteome Project (ArcPP), a community project that combines the analysis of multiple datasets with expert knowledge in various fields of archaeal research. The corresponding bioinformatic analysis, allowing for the integration of new proteomics data into the ArcPP, as well as the interactive exploration of ArcPP results is also presented here. In combination, these protocols facilitate an optimized, detailed and collaborative approach to archaeal proteomics.
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Haloferax volcanii , Proteoma , Análisis de Datos , Espectrometría de Masas/métodos , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Biofilms are cellular aggregates encased in extracellular polymeric substances and are commonly formed by single-celled eukaryotes, bacteria, and archaea. In addition to attaching to solid surfaces, these cellular aggregates can also be observed floating on or immersed within liquid cultures. While biofilms on surfaces have been studied in some archaea, little is known about liquid biofilms. Surprisingly, immersed liquid biofilms of the model archaeon Haloferax volcanii do not require the same set of machinery needed to form surface-attached biofilms. In fact, to date not a single gene has been identified that is involved in forming immersed liquid biofilms. Interestingly, after an immersed liquid biofilm forms, removal of the Petri dish lid induces rapid, transient, and reproducible honeycomb patterns within the immersed liquid biofilm itself, triggered by a reduction in humidity. In this chapter, we outline a protocol for both immersed liquid biofilm and honeycomb pattern formations. This protocol will be essential for determining the novel components required for the formation of immersed liquid biofilms and honeycomb patterns.
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Haloferax volcanii , Bacterias , Biopelículas , Haloferax volcanii/genéticaRESUMEN
Biofilms are aggregates of cells surrounded by an extracellular matrix providing protection from external stresses. While biofilms are commonly studied in bacteria, archaea also form such cell aggregates both in liquid cultures and on solid surfaces. Biofilm architectures vary when in liquid cultures versus on surfaces as well as when incubated under static conditions versus under shear forces of flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe surface adhesion assays employing a cost-effective, 3D-printed coverslip holder that can be used under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows the same culture to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analysis of the dynamics of biofilm formation. Additionally, a traditional surface adhesion assay in a 12-well plate under standing conditions is outlined as well. We anticipate the combination of these protocols to be useful for analyzing a wide array of biofilms and answering a multitude of biological questions.
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Archaea , Biopelículas , Análisis Costo-BeneficioRESUMEN
Early exposure to science is critical to incite interest in scientific careers, promote equity and retention in STEM fields, and increase the general understanding of the scientific method. For many educators, however, the myriad resources that many scientific experiments require are not readily available. Microbiology experiments in particular can often be inaccessible for a lot of classrooms. In addition, microbiological studies often involve eukaryotic microbes and bacteria while excluding an entire domain of life: archaea. Archaea are more closely related to eukaryotes than are bacteria, and although all prokaryotic cells lack a nucleus, various key aspects of the cell biology of archaea and bacteria are fundamentally different. In addition to being useful for teaching about the diversity and evolution of living organisms, these differences between archaea and bacteria can also be harnessed to teach and emphasize other important biological topics. Haloferax volcanii is a non-pathogenic model haloarchaeon that allows for safe, affordable, and accessible microbiological experiments, as the requirement of high-salt media to grow H. volcanii presents a low risk of contamination. Here, we describe how H. volcanii can be used in the classroom and outline a protocol demonstrating their resistance to a broad spectrum of antibiotics, underscoring the distinct cell biology of bacteria and archaea. Finally, we introduce strategies and protocols to perform this and other H. volcanii experiments such that they can be performed based on the resources available in a high school or undergraduate classroom.
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Haloferax volcanii , Antibacterianos , Bacterias , EucariontesRESUMEN
Archaea, once thought to only live in extreme environments, are present in many ecosystems, including the human microbiome, and they play important roles ranging from nutrient cycling to bioremediation. Yet this domain is often overlooked in microbiology classes and rarely included in laboratory exercises. Excluding archaea from high school and undergraduate curricula prevents students from learning the uniqueness and importance of this domain. Here, we have modified a familiar and popular microbiology experiment-the Kirby-Bauer disk diffusion antibiotic susceptibility test-to include, together with the model bacterium Escherichia coli, the model archaeon Haloferax volcanii. Students will learn the differences and similarities between archaea and bacteria by using antibiotics that target, for example, the bacterial peptidoglycan cell wall or the ribosome. Furthermore, the experiment provides a platform to reiterate basic cellular biology concepts that students may have previously discussed. We have developed two versions of this experiment, one designed for an undergraduate laboratory curriculum and the second, limited to H. volcanii, that high school students can perform in their classrooms. This nonpathogenic halophile can be cultured aerobically at ambient temperature in high-salt media, preventing contamination, making the experiment low-cost and safe for use in the high school setting.
RESUMEN
Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multipanel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δphz) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that an H. volcanii mutant that lacks archaella is impaired in early stages of biofilm formation under shaking conditions. IMPORTANCE Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.
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Biopelículas , Técnicas Microbiológicas , Células Procariotas , Análisis Costo-Beneficio , Haloferax volcanii , Técnicas Microbiológicas/métodos , Células Procariotas/fisiología , Pseudomonas aeruginosaRESUMEN
The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets.
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Análisis de Datos , Bases de Datos de Proteínas , Metadatos , Proteómica , Macrodatos , Humanos , Reproducibilidad de los Resultados , Programas Informáticos , TranscriptomaRESUMEN
Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.
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Proteínas Arqueales/metabolismo , Glicopéptidos/metabolismo , Glicoproteínas/metabolismo , Haloferax volcanii/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , Bioensayo , Forma de la Célula/efectos de los fármacos , Bases de Datos de Proteínas , Glicopéptidos/química , Glicoproteínas/química , Glicosilación/efectos de los fármacos , Haloferax volcanii/efectos de los fármacos , Mutación/genética , Fenotipo , Filogenia , Proteómica , Cloruro de Sodio/farmacologíaRESUMEN
The identification of peptide sequences and their post-translational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs). Whereas the benefits of combining results from multiple protein database search engines have been previously established, similar approaches for OMS results have been missing so far. Here we compare and combine results from three different OMS engines, demonstrating an increase in peptide spectrum matches of 8-18%. The unification of search results furthermore allows for the combined downstream processing of search results, including the mapping to potential PTMs. Finally, we test for the ability of OMS engines to identify glycosylated peptides. The implementation of these engines in the Python framework Ursgal facilitates the straightforward application of the OMS with unified parameters and results files, thereby enabling yet unmatched high-throughput, large-scale data analysis.
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Algoritmos , Programas Informáticos , Bases de Datos de Proteínas , Procesamiento Proteico-Postraduccional , Proteómica , Motor de BúsquedaRESUMEN
MOTIVATION: Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. RESULTS: Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae. AVAILABILITY AND IMPLEMENTATION: The source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
RESUMEN
The ability to form biofilms is shared by many microorganisms, including archaea. Cells in a biofilm are encased in extracellular polymeric substances that typically include polysaccharides, proteins, and extracellular DNA, conferring protection while providing a structure that allows for optimal nutrient flow. In many bacteria, flagella and evolutionarily conserved type IV pili are required for the formation of biofilms on solid surfaces or floating at the air-liquid interface of liquid media. Similarly, in many archaea it has been demonstrated that type IV pili and, in a subset of these species, archaella are required for biofilm formation on solid surfaces. Additionally, in the model archaeon Haloferax volcanii, chemotaxis and AglB-dependent glycosylation play important roles in this process. H. volcanii also forms immersed biofilms in liquid cultures poured into petri dishes. This study reveals that mutants of this haloarchaeon that interfere with the biosynthesis of type IV pili or archaella, as well as a chemotaxis-targeting transposon and aglB deletion mutants, lack obvious defects in biofilms formed in liquid cultures. Strikingly, we have observed that these liquid-based biofilms are capable of rearrangement into honeycomb-like patterns that rapidly form upon removal of the petri dish lid, a phenomenon that is not dependent on changes in light or oxygen concentration but can be induced by controlled reduction of humidity. Taken together, this study demonstrates that H. volcanii requires novel, unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements.IMPORTANCE This first molecular biological study of archaeal immersed liquid biofilms advances our basic biological understanding of the model archaeon Haloferax volcanii Data gleaned from this study also provide an invaluable foundation for future studies to uncover components required for immersed liquid biofilms in this haloarchaeon and also potentially for liquid biofilm formation in general, which is poorly understood compared to the formation of biofilms on surfaces. Moreover, this first description of rapid honeycomb pattern formation is likely to yield novel insights into the underlying structural architecture of extracellular polymeric substances and cells within immersed liquid biofilms.
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Biopelículas , Proteínas Fimbrias/metabolismo , Haloferax volcanii/metabolismo , Proteínas Fimbrias/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Glicosilación , Haloferax volcanii/citología , Haloferax volcanii/genética , Polisacáridos/metabolismoRESUMEN
While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as N-terminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics.
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Proteínas Arqueales/metabolismo , Haloferax volcanii/fisiología , Proteoma/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Conjuntos de Datos como Asunto , Glicosilación , Espectrometría de MasasRESUMEN
The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination.IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.
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Proteínas Arqueales/metabolismo , Haloferax volcanii/enzimología , Haloferax volcanii/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Fosfolípidos/metabolismo , Proteínas Arqueales/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Carboxiliasas/genética , Carboxiliasas/metabolismo , Haloferax volcanii/genética , Proteínas de la Membrana/genética , Péptido Hidrolasas/genéticaRESUMEN
In this infographic we present the main tools available for the halophilic archaeon Haloferax volcanii, which have enabled successful research on its biology, including its genetics, proteostasis, cell surface structures, metabolic pathways, and adaptation to high salt environments. Isolated from the Dead Sea in 1975, Haloferax volcanii thrives in high salt environments and has emerged as an important archaeal model system. An extensive repertoire of genetic, molecular biological, and biochemical tools has been developed for this fast-growing, easily cultivated haloarchaeon, including expression vectors and gene-deletion strategies, including CRISPR. Its low mutation rate and ability to grow on defined media allow straightforward application of methods such as metabolic labeling, and the sequenced genome laid the foundation for transcriptomics and proteomics studies. These tools have allowed examination of key pathways such as transcription, noncoding RNAs, protein synthesis and degradation, protein glycosylation, motility, and biofilm formation. With the collaborative spirit of the H. volcanii community, this model system has become invaluable not only for enhancing our understanding of archaea but also for improving the development of biotech applications.
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Genética Microbiana/métodos , Haloferax volcanii/genética , Haloferax volcanii/fisiología , Biología Molecular/métodos , Genómica/métodos , Haloferax volcanii/clasificación , Haloferax volcanii/aislamiento & purificación , Redes y Vías Metabólicas/genética , Proteómica/métodosRESUMEN
Chlamydomonas reinhardtii (C. reinhardtii) N-glycans carry plant typical ß1,2-core xylose, α1,3-fucose residues, as well as plant atypical terminal ß1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N-Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii. To further elucidate enzymes important for N-glycosylation, novel single knockdown mutants of candidate genes involved in the N-glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known N-glycosylation pathway genes were generated. By characterizing N-glycan compositions of intact N-glycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N-glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N-glycan core epitopes modulate protein function in C. reinhardtii.
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Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.
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Chlamydomonas reinhardtii/metabolismo , Complejo de Citocromo b6f/química , Electrones , Complejos de Proteína Captadores de Luz/química , Complejos Multiproteicos/química , Fotosíntesis , Complejo de Proteína del Fotosistema I/química , Anaerobiosis , Chlamydomonas reinhardtii/crecimiento & desarrollo , Complejo de Citocromo b6f/metabolismo , Transporte de Electrón , Complejos de Proteína Captadores de Luz/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/metabolismo , Conformación ProteicaRESUMEN
Cell surfaces are critical for diverse functions across all domains of life, from cell-cell communication and nutrient uptake to cell stability and surface attachment. While certain aspects of the mechanisms supporting the biosynthesis of the archaeal cell surface are unique, likely due to important differences in cell surface compositions between domains, others are shared with bacteria or eukaryotes or both. Based on recent studies completed on a phylogenetically diverse array of archaea, from a wide variety of habitats, here we discuss advances in the characterization of mechanisms underpinning archaeal cell surface biogenesis. These include those facilitating co- and post-translational protein targeting to the cell surface, transport into and across the archaeal lipid membrane, and protein anchoring strategies. We also discuss, in some detail, the assembly of specific cell surface structures, such as the archaeal S-layer and the type IV pili. We will highlight the importance of post-translational protein modifications, such as lipid attachment and glycosylation, in the biosynthesis as well as the regulation of the functions of these cell surface structures and present the differences and similarities in the biogenesis of type IV pili across prokaryotic domains.
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Archaea/metabolismo , Proteínas Arqueales/biosíntesis , Proteínas de la Membrana/biosíntesis , Procesamiento Proteico-Postraduccional , Proteínas Arqueales/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
The female sex in honeybees (Apis spp.) comprises a reproductive queen and a sterile worker caste. Nurse bees feed all larvae progressively with a caste-specific food jelly until the prepupal stage. Only those larvae that are exclusively fed a large amount of royal jelly (RJ) develop into queens [1]. RJ is a composite secretion of two specialized head glands: the mandibular glands, which produce mainly fatty acids [2], and the hypopharyngeal glands, which contribute proteins, primarily belonging to the major royal jelly protein (MRJP) family [3]. Past research on RJ has focused on its nutritional function and overlooked its central role with regard to the orientation of the larva in the royal brood cell. Whereas workers are reared in the regular horizontal cells of the comb, the queen cells are specifically built outside of the normal comb area to accommodate for the larger queen [4, 5]. These cells hang freely along the bottom of the comb and are vertically oriented, opening downward [6]. Queen larvae are attached by their RJ diet to the cell ceiling. Thus, the physical properties of RJ are central to successful retention of larvae in the cell. Here, we show that the main protein of RJ (MRJP1) polymerizes in complex with another protein, apisimin, into long fibrous structures that build the basis for the high viscosity of RJ to hold queen larvae on the RJ surface.
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Abejas/crecimiento & desarrollo , Ácidos Grasos/metabolismo , Gravitación , Proteínas de Insectos/metabolismo , Reproducción , Conducta Social , Animales , Abejas/fisiología , Ácidos Grasos/química , Femenino , Larva , ViscosidadRESUMEN
At present, only little is known about the enzymatic machinery required for N-glycosylation in Chlamydomonas reinhardtii, leading to the formation of N-glycans harboring Xyl and methylated Man. This machinery possesses new enzymatic features, as C. reinhardtii N-glycans are independent of ß1,2-N-acetylglucosaminyltransferase I. Here we have performed comparative N-glycoproteomic analyses of insertional mutants of mannosidase 1A (IM Man1A ) and xylosyltransferase 1A (IM XylT1A ). The disruption of man1A affected methylation of Man and the addition of terminal Xyl. The absence of XylT1A led to shorter N-glycans compared to the wild type. The use of a IM Man1A xIM XylT1A double mutant revealed that the absence of Man1A suppressed the IM XylT1A phenotype, indicating that the increased N-glycan trimming is regulated by core ß1,2-Xyl and is dependent on Man1A activity. These data point toward an enzymatic cascade in the N-glycosylation pathway of C. reinhardtii with interlinked roles of Man1A and XylT1A. The results described herein represent the first step toward a functional characterization of the enzymatic N-glycosylation machinery in C. reinhardtii.