Archaea of the phylum Thaumarchaeota are among the most abundant prokaryotes on Earth and are widely distributed in marine, terrestrial, and geothermal environments. All studied Thaumarchaeota couple the oxidation of ammonia at extremely low concentrations with carbon fixation. As the predominant nitrifiers in the ocean and in various soils, ammonia-oxidizing archaea contribute significantly to the global nitrogen and carbon cycles. Here we provide biochemical evidence that thaumarchaeal ammonia oxidizers assimilate inorganic carbon via a modified version of the autotrophic hydroxypropionate/hydroxybutyrate cycle of Crenarchaeota that is far more energy efficient than any other aerobic autotrophic pathway. The identified genes of this cycle were found in the genomes of all sequenced representatives of the phylum Thaumarchaeota, indicating the environmental significance of this efficient CO 2 -fixation pathway. Comparative phylogenetic analysis of proteins of this pathway suggests that the hydroxypropionate/hydroxybutyrate cycle emerged independently in Crenarchaeota and Thaumarchaeota, thus supporting the hypothesis of an early evolutionary separation of both archaeal phyla. We conclude that high efficiency of anabolism exemplified by this autotrophic cycle perfectly suits the lifestyle of ammonia-oxidizing archaea, which thrive at a constantly low energy supply, thus offering a biochemical explanation for their ecological success in nutrient-limited environments.Nitrosopumilus maritimus | autotrophy

Clostridium difficile toxin (CDT) is a binary actin-ADP-ribosylating toxin that causes depolymerization of the actin cytoskeleton and formation of microtubule-based membrane protrusions, which are suggested to be involved in enhanced bacterial adhesion and colonization of hypervirulent C. difficile strains. Here, we studied the involvement of membrane lipid components of human colon adenocarcinoma (Caco-2) cells in formation of membrane protrusions. Depletion of cholesterol by methyl-␤-cyclodextrin inhibited protrusion formation in a concentration-dependent manner but had no major effect on the toxin-catalyzed modification of actin in target cells. Repletion of cholesterol reconstituted formation of protrusions and increased velocity and total amount of protrusion formation. Methyl-␤-cyclodextrin had no effect on the CDT-induced changes in the dynamics of microtubules. Formation of membrane protrusions was also inhibited by the cholesterol-binding polyene antibiotic nystatin. Degradation or inhibition of synthesis of sphingolipids by sphingomyelinase and myriocin, respectively, blocked CDT-induced protrusion formation. Benzyl alcohol, which increases membrane fluidity, prevented protrusion formation. CDT-induced membrane protrusions were stained by flotillin-2 and by the fluorescent-labeled lipid raft marker cholera toxin subunit B, which selectively interacts with GM1 ganglioside mainly located in lipid microdomains. The data suggest that formation and especially the initiation of CDTinduced microtubule-based membrane protrusions depend on cholesterol-and sphingolipid-rich lipid microdomains.Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis (1). Both diseases depend on the production of toxins. Major virulence factors are the glycosylating C. difficile toxins A and B, which inactivate Rho GTPases (2-5). Especially hypervirulent strains additionally produce C. difficile transferase (CDT) 3 (6). CDT belongs to the family of actin-ADP-ribosylating toxins like Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin (7-9). These toxins are binary in structure and consist of an enzymatic component, which possesses ADP-ribosyltransferase activity, and a separated binding/translocation component. The binding component is proteolytically activated and forms heptamers, which interact with the enzymatic component (8, 10). After binding of the toxin to a cell surface receptor, the toxin complex is endocytosed (11,12). At low pH of endosomal compartments, the binding component inserts into the membrane of endosomes and forms pores, which allow the translocation of the enzymatic component into the cytosol (10, 13). Here, the toxin ADP-ribosylates actin at arginine 177 (14). Modification at this site inhibits actin polymerization and causes destruction of the actin cytoskeleton.Recently, we reported that following ADP-ribosylation of actin, CDT induces the formation of microtubule-based protrusions in epithelial cells (15). These protrusions are in general Ͻ1 m in diameter ...

The recently described ammonia-oxidizing archaea of the phylum Thaumarchaeota are highly abundant in marine, geothermal, and terrestrial environments. All characterized representatives of this phylum are aerobic chemolithoautotrophic ammonia oxidizers assimilating inorganic carbon via a recently described thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. Although some genes coding for the enzymes of this cycle have been identified in the genomes of Thaumarchaeota, many other genes of the cycle are not homologous to the characterized enzymes from other species and can therefore not be identified bioinformatically. Here we report the identification and characterization of malonic semialdehyde reductase Nmar_1110 in the cultured marine thaumarchaeon Nitrosopumilus maritimus. This enzyme, which catalyzes the reduction of malonic semialdehyde with NAD(P)H to 3-hydroxypropionate, belongs to the family of iron-containing alcohol dehydrogenases and is not homologous to malonic semialdehyde reductases from Chloroflexus aurantiacus and Metallosphaera sedula. It is highly specific to malonic semialdehyde (K m , 0.11 mM; V max , 86.9 mol min ؊1 mg ؊1 of protein) and exhibits only low activity with succinic semialdehyde (K m , 4.26 mM; V max , 18.5 mol min ؊1 mg ؊1 of protein). Homologues of N. maritimus malonic semialdehyde reductase can be found in the genomes of all Thaumarchaeota sequenced so far and form a well-defined cluster in the phylogenetic tree of iron-containing alcohol dehydrogenases. We conclude that malonic semialdehyde reductase can be regarded as a characteristic enzyme for the thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle.A ll cultured members of a recently described archaeal phylum, Thaumarchaeota (1), are chemolithoautotrophs which aerobically oxidize ammonia to nitrite (2). These ammonia-oxidizing archaea are highly abundant in nature and contribute significantly to nitrification as well as to primary production in marine, terrestrial, and geothermal environments (2-11). Nitrosopumilus maritimus, the first cultured thaumarchaeon, may be regarded as a model organism since it has served to unravel characteristic cellular, genomic, and physiological features of this group (12)(13)(14)(15)(16). N. maritimus couples ammonia oxidation at extremely low ammonia concentrations (of low nanomolar range) to autotrophic CO 2 fixation (14). Chemolithotrophic life in extremely nutrient-limited environments results in a permanently low energy supply and requires special metabolic adaptation to enable growth. Indeed, CO 2 fixation, the central anabolic process in autotrophs, proceeds in N. maritimus via a novel variant of the 3-hydroxypropionate/ 4-hydroxybutyrate (HP/HB) cycle, which represents the most energy-efficient aerobic autotrophic pathway (13). Genes of this HP/HB cycle were found in all thaumarchaeal genomes, indicating the potential operation in all members of the phylum Thaumarchaeota ( Fig. 1) and its ecological significance in various habitats (13).The HP...

Ammonia-oxidizing archaea of the phylum Thaumarchaeota are among the most abundant organisms that exert primary control of oceanic and soil nitrification and are responsible for a large part of dark ocean primary production. They assimilate inorganic carbon via an energetically efficient version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. In this cycle, acetyl-CoA is carboxylated to succinyl-CoA, which is then converted to two acetyl-CoA molecules with 4-hydroxybutyrate as the key intermediate. This conversion includes the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Here, we heterologously produced the protein Nmar_1028 catalyzing this reaction in thaumarchaeon Nitrosopumilus maritimus, characterized it biochemically and performed its phylogenetic analysis. This NAD-dependent dehydrogenase is highly active with its substrate, (S)-3-hydroxybutyryl-CoA, and its low Km value suggests that the protein is adapted to the functioning in the 3-hydroxypropionate/4-hydroxybutyrate cycle. Nmar_1028 is homologous to the dehydrogenase domain of crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase that is present in many Archaea. Apparently, the loss of the dehydratase domain of the fusion protein in the course of evolution was accompanied by lateral gene transfer of 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase from Bacteria. Although (S)-3-hydroxybutyryl-CoA dehydrogenase studied here is neither unique nor characteristic for the HP/HB cycle, Nmar_1028 appears to be the only (S)-3-hydroxybutyryl-CoA dehydrogenase in N. maritimus and is thus essential for the functioning of the 3-hydroxypropionate/4-hydroxybutyrate cycle and for the biology of this important marine archaeon.