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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., While archaeal proteomics advanced rapidly, a comprehensive proteome database for archaea is lacking. Therefore, the authors here launch the Archaeal Proteome Project, a community-effort providing insights into archaeal cell biology via the combined reanalysis of Haloferax volcanii proteomics data.

Fil: Ferrari, Maria Celeste. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones Biologicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biologicas; Argentina

[Aims]: To examine the antioxidant activity of Bacterioruberin (Bctr)‐rich extracts isolated from a hyperpigmented, genetically modified Haloferax volcanii strain (HVLON 3) and to investigate the effect on cold‐sensitive ram sperm cells., [Methods and Results]: The strain HVLON 3 produces higher Bctr amounts than most haloarchaea (220 ± 13 mg g−1 DW ). HVLON 3‐Bctr extract has higher antioxidant activity than β‐carotene (threefold) as evaluated using 2,2 diphenyl‐1‐picrylhydrazyl combined with Electron Paramagnetic Resonance analysis (EC 50 4·5 × 10−5 mol l−1 vs 13·9 × 10−5 mol l−1 respectively). Different concentrations of HVLON 3‐Bctr extracts were assayed on ram sperm after freezing/thawing and physiologically relevant parameters were examined. Extracts containing 7 and 20 μ mol l−1 Bctr significantly improved cell viability (P < 0·0001), total and progressive motility (P < 0·0001) and sperm velocities (P = 0·0172 for curvilinear velocity VCL , P = 0·0268 for average path velocity VAP and P = 0·0181 for straight line velocity VSL ) and did not affect other parameters evaluated., [Conclusions]: HVLON 3 is an excellent source of natural microbial C50 carotenoids with applicability in Biotechnology, Biomedical and Veterinary fields. HVLON 3 Bctr extract improves the quality of cryopreserved ram sperm cells and could be applied to increase insemination yields., [Significance and Impact of the Study]: This study provides an insight on the bioactive properties of a bioproduct derived from haloarchaea (carotenoids) which are so far underexploited., This research was supported by the National Agency for Scientific and Technological Promotion (ANPCyT) grants PICT‐2015‐3682 and PICT‐2014‐1477 awarded to A.C. and R.D.C., respectively; UNMDP grants 15/E818, EXA819/17 and EXA823/17 awarded to M.S.C., A.C. and R.D.C, respectively.

The dynamic changes that take place along the phases of microbial growth (lag, exponential, stationary, and death) have been widely studied in bacteria at the molecular and cellular levels, but little is known for archaea. In this study, a high-throughput approach was used to analyze and compare the proteomes of two haloarchaea during exponential and stationary growth: the neutrophilic Haloferax volcanii and the alkaliphilic Natrialba magadii. Almost 2000 proteins were identified in each species (≈50% of the predicted proteome). Among them, 532 and 432 were found to be differential between growth phases in H. volcanii and N. magadii, respectively. Changes upon entrance into stationary phase included an overall increase in proteins involved in the transport of small molecules and ions, stress response, and fatty acid catabolism. Proteins related to genetic processes and cell division showed a notorious decrease in amount. The data reported in this study not only contributes to our understanding of the exponential–stationary growth phase transition in extremophilic archaea but also provides the first comprehensive analysis of the proteome composition of N. magadii. The MS proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier JPST000395. Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Gimenez, Maria Ines. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Tröetschel, Christian. Ruhr Universität Bochum; Alemania Fil: D'alessandro, Celeste Paola. Universidade de Sao Paulo; Brasil Fil: Poetsch, Ansgar. University Of Plymouth; . Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Paggi, Roberto Alejandro. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

The membrane protease LonB is an essential protein in the archaeon Haloferax volcanii and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a H. volcanii LonB mutant under reduced and physiological protease levels. LC-MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis. Fil: Cerletti, Micaela. Universidad Nacional de Mar del Plata; Argentina Fil: Paggi, Roberto Alejandro. Universidad Nacional de Mar del Plata; Argentina Fil: Troetschel, Christian. Ruhr Universität Bochum; Alemania Fil: Ferrari, María Celeste. Universidad Nacional de Mar del Plata; Argentina Fil: Guevara, Carina Ramallo. Ruhr Universität Bochum; Alemania Fil: Albaum, Stefan. Universitat Bielefeld; Alemania Fil: Poetsch, Ansgar. Plant Biochemistry, Ruhr University Bochum; Alemania Fil: de Castro, Rosana Esther. Universidad Nacional de Mar del Plata; Argentina

Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO-1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism. Fil: Costa, Mariana Inés. Universidad Nacional de Mar del Plata; Argentina Fil: Cerletti, Micaela. Universidad Nacional de Mar del Plata; Argentina Fil: Paggi, Roberto Alejandro. Universidad Nacional de Mar del Plata; Argentina Fil: Trötschel, Christian. Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Poetsch, Ansgar. Ruhr Universität Bochum; Alemania Fil: Gimenez, Maria Ines. Universidad Nacional de Mar del Plata; Argentina

This data article provides information in support of the research article "Global role of the membrane protease LonB in Archaea: Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii" [1]. The proteome composition of a wt and a LonB protease mutant strain (suboptimal expression) in the archaeon Haloferax volcanii was assessed by a quantitative shotgun proteomic approach. Membrane and cytosol fractions of H. volcanii strains were examined at two different growth stages (exponential and stationary phase). Data is supplied in the present article. This study represents the first proteome examination of a Lon-deficient cell of the Archaea Domain. Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Paggi, Roberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Ramallo Guevara, Carina. Ruhr Universität Bochum; Alemania Fil: Poetsch, Ansgar. Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Mar del Plata. Instituto de Investigaciones Biologicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina

The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii. Fil: Gimenez, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina

The membrane-associated LonB protease is essential for viability in Haloferax volcanii, however, the cellular processes affected by this protease in archaea are unknown. In this study, the impact of a lon conditional mutation (down-regulation) on H. volcanii physiology was examined by comparing proteomes of parental and mutant cells using shotgun proteomics. A total of 1778 proteins were identified (44% of H. volcanii predicted proteome) and 142 changed significantly in amount (≥. 2 fold). Of these, 66 were augmented in response to Lon deficiency suggesting they could be Lon substrates. The "Lon subproteome" included soluble and predicted membrane proteins expected to participate in diverse cellular processes. The dramatic stabilization of phytoene synthase (57 fold) in concert with overpigmentation of lon mutant cells suggests that Lon controls carotenogenesis in H. volcanii. Several hypothetical proteins, which may reveal novel functions and/or be involved in adaptation to extreme environments, were notably increased (300 fold). This study, which represents the first proteome examination of a Lon deficient archaeal cell, shows that Lon has a strong impact on H. volcanii physiology evidencing the cellular processes controlled by this protease in Archaea. Additionally, this work provides a platform for the discovery of novel targets of Lon proteases. Biological Significance: The proteome of a Lon-deficient archaeal cell was examined for the first time showing that Lon has a strong impact on H. volcanii physiology and evidencing the proteins and cellular processes controlled by this protease in Archaea. This work will facilitate future investigations aiming to address Lon function in archaea and provides a platform for the discovery of endogenous targets of the archaeal-type Lon as well as novel targets/processes regulated by Lon proteases. This knowledge will advance the understanding on archaeal physiology and the biological function of membrane proteases in microorganisms. Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Paggi, Roberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Ramallo Guevara, Carina. Ruhr Universität Bochum; Alemania Fil: Poetsch, Ansgar. Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

The transition from balanced growth to the stationary phase induces changes in the morphology and physiology of most bacteria with the aim of preparing the cells to withstand unfavorable conditions (Nystrom, 2004; Siegele and Kolter, 1992). In Archaea, the third domain of life, many species are extremophiles, meaning that they have adapted their physiology to optimally live in environments with conditions that are lethal for most life forms. The effect of the growth stage in archaeal physiology has been investigated to a limited extent (Lange et al., 2007) and may reveal conserved as well as novel strategies of adaptation. The accurate regulation of gene expression is essential in all living cells for the adaptation to different environmental conditions (Soppa, 2006). In the haloarchaea (optimum growth at ＞2 M NaCl) growth phase-dependent gene expression has been investigated through genomewide analysis of transcription and/or translation in the neutrophilic species Haloferax volcanii and Halobacterium salinarum (Facciotti et al., 2010; Lange et al., 2007). These studies showed that a signifi cant fraction of genes were subjected to differential transcriptional and/or translational control; however, neither the fractions nor the identity of the regulated genes were conserved between these archaea. Fil: Madrid, Enrique A. . Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Paggi, Roberto A. . Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Sastre, Diego Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

Aims: The alkaliphilic haloarchaeon Natrialba magadii secretes a halolysin-like protease (Nep) that is active and stable in high salt and in organic solvents, which represents a potential resource for biocatalysis in low water activity conditions. In this study, the effect of the growth stage on Nep biosynthesis was examined. Methods and Results: Nep mRNA and extracellular protease activity were measured by RT-PCR and azocaseinolytic activity determination, respectively. Increased abundance in Nep mRNA was observed in Nab. magadii cells with culture age, which correlated with accumulation of extracellular protease activity. Moreover, a ‘stationary phase behavior’ on synthesis of Nep was evidenced in low-density cultures incubated with stationary phase medium. Conclusions: nep gene expression is up-regulated during the transition to the stationary phase in response to ‘factors’ (metabolite and/or regulatory molecule) occurring in high-density cultures of Nab. magadii. Although the identity of these molecules remains to be determined, preliminary evidence suggests that they are hydrophobic and stable in high salt and high pH values (3·5 mol l−1 NaCl, pH 10). Significance and impact of study: This study contributes to gain insight into the regulation of haloarchaeal protease biosynthesis, facilitating the large-scale production of this extremozyme for basic studies or potential applications.