Algoriphagus machipongonensis sp. nov., co-isolated with a colonial choanoflagellate

A Gram-negative, non-motile, non-spore-forming bacterial strain, PR1[superscript T], was isolated from a mud core sample containing colonial choanoflagellates near Hog Island, Virginia, USA. Strain PR1[superscript T] grew optimally at 30 °C and with 3 % (w/v) NaCl. Strain PR1[superscript T] contained MK-7 as the major menaquinone as well as carotenoids but lacked pigments of the flexirubin-type. The predominant fatty acids were iso-C15 : 0 (29.4 %), iso-C17 : 1ω9c (18.5 %) and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 11.3 %). The major polar lipids detected in strain PR1[superscript T] were phosphatidylethanolamine, an unknown phospholipid, an aminophospholipid, an aminolipid and two lipids of unknown character. The DNA G+C content was 38.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain PR1[superscript T] fell within the cluster comprising the genus Algoriphagus and was most closely related to Algoriphagus halophilus JC 2051[superscript T] (95.4 % sequence similarity) and Algoriphagus lutimaris S1-3[superscript T] (95.3 % sequence similarity). The 16S rRNA gene sequence similarity between strain PR1[superscript T] and the type strains of other species of the genus Algoriphagus were in the range 91–95 %. Differential phenotypic properties and phylogenetic and genetic distinctiveness of strain PR1[superscript T] demonstrated that this strain was distinct from other members of the genus Algoriphagus, including its closest relative, A. halophilus. Based on phenotypic, chemotaxonomic, phylogenetic and genomic data, strain PR1[superscript T] should be placed in the genus Algoriphagus as a representative of a novel species, for which the name Algoriphagus machipongonensis sp. nov. is proposed. The type strain is PR1[superscript T] ( = ATCC BAA-2233[superscript T]  = DSM 24695[superscript T]).Gordon and Betty Moore Foundation (Investigator Award (581))National Institutes of Health (U.S.) (NIH National Research Service Award and Fellowship grant (5F32GM086054))United States. National Aeronautics and Space Administration (NASA Astrobiology Institute (NNA08CN84A

Cooperativity and communication in archaeal Cdc48·20S, an ancient proteolytic machine

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences, 2015."September 2015." Cataloged from PDF version of thesis.Includes bibliographical references.ATP dependent proteolysis is a process essential for life and is carried out by AAA+ proteases. AAA+ unfoldases use the energy of ATP hydrolysis to power the unfolding and translocation of protein substrates into compartmentalized peptidases for regulated proteolysis. Cdc48 is a highly conserved AAA+ homohexameric unfoldase which is made up of two AAA+ rings. Each ring can, in principle, bind and hydrolyzing ATP, but it is unclear what roles are played by each ring and how they coordinate their activities. A regulatory N domain functions to control the activity of the enzyme and binding to its partner peptidase, the 20S proteasome. In this thesis I present experiments which investigate the role of inter-ring communication in ATP hydrolysis, protein unfolding, and allosteric interactions with the 20S and show how these features affect enzyme function. Experiments also show how the N domain controls D1-D2 interactions that govern ATP hydrolysis and substrate unfolding. Finally, I present experiments that take steps toward developing a system for screening protein substrates of Cdc48-20S and identify several substrates from E. coli lysates.by Jonathan Dean Grabenstatter.Ph. D

Architecture and assembly of the archaeal Cdc48*20S proteasome

ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48⋅20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.National Institutes of Health (U.S.) (Grant AI-16892)National Institutes of Health (U.S.) (Grant GM-49224)German Science Foundation (Grant BA 4890/1

Sterol and genomic analyses validate the sponge biomarker hypothesis

Molecular fossils (or biomarkers) are key to unraveling the deep history of eukaryotes, especially in the absence of traditional fossils. In this regard, the sterane 24-isopropylcholestane has been proposed as a molecular fossil for sponges, and could represent the oldest evidence for animal life. The sterane is found in rocks ∼650–540 million y old, and its sterol precursor (24-isopropylcholesterol, or 24-ipc) is synthesized today by certain sea sponges. However, 24-ipc is also produced in trace amounts by distantly related pelagophyte algae, whereas only a few close relatives of sponges have been assayed for sterols. In this study, we analyzed the sterol and gene repertoires of four taxa (Salpingoeca rosetta, Capsaspora owczarzaki, Sphaeroforma arctica, and Creolimax fragrantissima), which collectively represent the major living animal outgroups. We discovered that all four taxa lack C30 sterols, including 24-ipc. By building phylogenetic trees for key enzymes in 24-ipc biosynthesis, we identified a candidate gene (carbon-24/28 sterol methyltransferase, or SMT) responsible for 24-ipc production. Our results suggest that pelagophytes and sponges independently evolved C30 sterol biosynthesis through clade-specific SMT duplications. Using a molecular clock approach, we demonstrate that the relevant sponge SMT duplication event overlapped with the appearance of 24-isopropylcholestanes in the Neoproterozoic, but that the algal SMT duplication event occurred later in the Phanerozoic. Subsequently, pelagophyte algae and their relatives are an unlikely alternative to sponges as a source of Neoproterozoic 24-isopropylcholestanes, consistent with growing evidence that sponges evolved long before the Cambrian explosion ∼542 million y ago.Agouron Institute (Geobiology Fellowship)NASA Astrobiology Institute (Contract NNA13AA90A