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Phytoene synthase (PSY) converts two molecules of geranyl-geranyl diphosphate to phytoene, the key regulatory step in carotenogenesis. However, post-translational mechanisms that control PSY expression are scarcely understood. Carotenoid biosynthesis (mainly bacterioruberin) is a distinctive feature of haloarchaea thriving in hypersaline environments. Carotenogenesis is negatively regulated by the AAA+ LonB protease in the haloarchaeon Haloferax volcanii as it controls PSY degradation. We investigated the relevance of the C-terminal portion of HvPSY as a regulatory element for carotenoid biosynthesis. H. volcanii mutants were constructed to express full-length HvPSY protein (strain HVPSYwt) and truncated HvPSY lacking 10 (HVPSY10), 20 (HVPSY20) or 34 amino acids (HVPSY34) at the C-terminus. Cells of HVPSY20 and HVPSY34 showed hyperpigmentation (bacterioruberin content 3-fold higher than HVPSYwt) which correlated with increased PSY protein abundance (2-fold in HVPSY34) while they contained less psy transcript level compared with HVPSYwt. In vivo degradation assays showed that HvPSY34 was more stable than HvPSYwt. Collectively, these results show that the C-terminal region of HvPSY contains a ‘recognition determinant’ for proteolysis in H. volcanii. Preliminary evidence suggests that LonB is involved in the recognition mechanism. This study provides the first identification of a regulatory sequence in an archaeal PSY for the post-translational control of carotenogenesis.

Sperm capacitation in mammals is a fundamental requirement to acquire their fertilizing capacity. Little is known about the action mechanism of the molecules that prevent capacitation from occurring prematurely. These molecules are known as decapacitation factors (DFs) and they must be removed from the sperm surface for capacitation to occur successfully. Serine protease inhibitor Kazal type 3 (SPINK3) has been proposed as one of these DFs. Here, we evaluate how this protein binds to mouse sperm and its removal kinetics. We describe that SPINK3 is capable of binding to the membrane of mature epididymal sperm through protein–lipid interactions, specifically to lipid rafts subcellular fraction. Moreover, cholera toxin subunit b (CTB) avoids SPINK3 binding. We observe that SPINK3 is removed from the sperm under in vitro capacitating conditions and by the uterine fluid from estrus females. Our ex vivo studies show the removal kinetics of this protein within the female tract, losing SPINK3 formerly from the apical region of the sperm in the uterus and later from the flagellar region within the oviduct. The presence of acrosome-reacted sperm in the female duct concurs with the absence of SPINK3 over its surface.

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.

[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

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 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