Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.</p

Adaptations to Submarine Hydrothermal Environments Exemplified by the Genome of Nautilia profundicola

Submarine hydrothermal vents are model systems for the Archaean Earth environment, and some sites maintain conditions that may have favored the formation and evolution of cellular life. Vents are typified by rapid fluctuations in temperature and redox potential that impose a strong selective pressure on resident microbial communities. Nautilia profundicola strain Am-H is a moderately thermophilic, deeply-branching Epsilonproteobacterium found free-living at hydrothermal vents and is a member of the microbial mass on the dorsal surface of vent polychaete, Alvinella pompejana. Analysis of the 1.7-Mbp genome of N. profundicola uncovered adaptations to the vent environment—some unique and some shared with other Epsilonproteobacterial genomes. The major findings included: (1) a diverse suite of hydrogenases coupled to a relatively simple electron transport chain, (2) numerous stress response systems, (3) a novel predicted nitrate assimilation pathway with hydroxylamine as a key intermediate, and (4) a gene (rgy) encoding the hallmark protein for hyperthermophilic growth, reverse gyrase. Additional experiments indicated that expression of rgy in strain Am-H was induced over 100-fold with a 20°C increase above the optimal growth temperature of this bacterium and that closely related rgy genes are present and expressed in bacterial communities residing in geographically distinct thermophilic environments. N. profundicola, therefore, is a model Epsilonproteobacterium that contains all the genes necessary for life in the extreme conditions widely believed to reflect those in the Archaean biosphere—anaerobic, sulfur, H2- and CO2-rich, with fluctuating redox potentials and temperatures. In addition, reverse gyrase appears to be an important and common adaptation for mesophiles and moderate thermophiles that inhabit ecological niches characterized by rapid and frequent temperature fluctuations and, as such, can no longer be considered a unique feature of hyperthermophiles

A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens.

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a DeltafccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The DeltafccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor

The single cysteine residue of the Sud protein is required for its function as a polysulfide-sulfur transferase in Wolinella succinogenes.

The periplasmic Sud protein which is induced in Wolinella succinogenes growing by polysulfide respiration, has been previously proposed to serve as a polysulfide binding protein and to transfer polysulfide-sulfur to the active site of polysulfide reductase [Klimmek, O, Kreis, V., Klein, C., Simon, J., Wittershagen, A. & Kröger, A. (1998) Eur. J. Biochem. 253, 263-269.]. The results presented in this communication suggest that polysulfide-sulfur is covalently bound to the single cysteine residue (Cys109) of the Sud monomer, and that Cys109 is required for tight binding of polysulfide-sulfur and for sulfur transfer. A modified Sud protein [(C109S)Sud-His6] in which the cysteine residue was replaced by serine, did not catalyze sulfur transfer from polysulfide to cyanide and did not stimulate electron transport to polysulfide, in contrast to Sud-His6. The polysulfide-sulfur bound to (C109S)Sud-His6 was fully removed upon dialysis against sulfide. After this treatment, Sud-His6 retained one sulfur atom per monomer; thiocyanate was formed upon addition of cyanide to the preparation. After incubation of Sud-His6 with polysulfide, a proportion of the Sud-His6 monomers carried one or two sulfur atoms, as shown by matrix-assisted laser desorption ionization mass spectrometry. The sulfur atoms were absent from monomers derived from Sud-His6 treated with cyanide and from (C109S)Sud-His6 incubated with polysulfide

Acta Crystallogr. Sect. D-Biol. Crystallogr.

Crystals of the complex between the enzyme cytochrome c nitrite reductase (NrfA) and the membrane-bound quinol oxidase and electron carrier NrfH were grown by vapour diffusion using ammonium sulfate as a precipitant. In the epsilon- proteobacterium Wolinella succinogenes, NrfA and NrfH form a functional membrane-bound complex which catalyzes the last step in the metabolic pathway of nitrate dissimilation. NrfH represents a prototype of a large family of putative bacterial quinol oxidases, the NapC/NirT family, which have been proposed to serve as electron donors for a variety of reductases. Crystal growth of the NrfHA complex was strongly dependent on the presence of detergent; the crystals grown belonged to space group I422