Functional compartmentalization and metabolic separation in a prokaryotic cell.

The prokaryotic cell is traditionally seen as a "bag of enzymes," yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the "cytoplasm" or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

A novel interdomain consortium from a Costa Rican oil well composed of Methanobacterium cahuitense sp. nov. and Desulfomicrobium aggregans sp. nov.

A novel interdomain consortium composed of a methanogenic Archaeon and a sulfate-reducing bacterium was isolated from a microbial biofilm in an oil well in Cahuita National Park, Costa Rica. Both organisms can be grown in pure culture or as stable co-culture. The methanogenic cells were non-motile rods producing CH4 exclusively from H2/CO2. Cells of the sulfate-reducing partner were motile rods forming cell aggregates. They utilized hydrogen, lactate, formate, and pyruvate as electron donors. Electron acceptors were sulfate, thiosulfate, and sulfite. 16S rRNA sequencing revealed 99% gene sequence similarity of strain CaP3V-M-L2AT to Methanobacterium subterraneum and 98.5% of strain CaP3V-S-L1AT to Desulfomicrobium baculatum. Both strains grew from 20 to 42 °C, pH 5.0-7.5, and 0-4% NaCl. Based on our data, type strains CaP3V-M-L2AT (= DSM 113354 T = JCM 39174 T) and CaP3V-S-L1AT (= DSM 113299 T = JCM 39179 T) represent novel species which we name Methanobacterium cahuitense sp. nov. and Desulfomicrobium aggregans sp. nov.

Photodynamic inactivation of different pathogenic bacteria on human skin using a novel photosensitizer hydrogel.

BACKGROUND: The colonization of skin with pathogenic, partially antibiotic-resistant bacteria is frequently a severe problem in dermatological therapies. For instance, skin colonization with Staphylococcus aureus is even a disease-promoting factor in atopic dermatitis. The photodynamic inactivation (PDI) of bacteria could be a new antibacterial procedure. Upon irradiation with visible light, a special photosensitizer exclusively generates singlet oxygen. This reactive oxygen species kills bacteria via oxidation independent of species or strain and their antibiotic resistance profile causing no bacterial resistance on its part. OBJECTIVE: To investigate the antibacterial potential of a photosensitizer, formulated in a new hydrogel, on human skin ex vivo. METHODS: The photochemical stability of the photosensitizer and its ability to generate singlet oxygen in the hydrogel was studied. Antimicrobial efficacy of this hydrogel was tested step by step, firstly on inanimate surfaces and then on human skin ex vivo against S. aureus and Pseudomonas aeruginosa using standard colony counting. NBTC staining and TUNEL assays were performed on skin biopsies to investigate potential necrosis and apoptosis effects in skin cells possibly caused by PDI. RESULTS: None of the hydrogel components affected the photochemical stability and the life time of singlet oxygen. On inanimate surfaces as well as on the human skin, the number of viable bacteria was reduced by up to 4.8 log10 being more effective than most other antibacterial topical agents. Histology and assays showed that PDI against bacteria on the skin surface caused no harmful effects on the underlying skin cells. CONCLUSION: Photodynamic inactivation hydrogel proved to be effective for decolonization of human skin including the potential to act against superficial skin infections. Being a water-based formulation, the hydrogel should be also suitable for the mucosa. The results of the present ex vivo study form a good basis for conducting clinical studies in vivo.

Methanofollis propanolicus sp. nov., a novel archaeal isolate from a Costa Rican oil well that uses propanol for methane production.

A novel methanogenic strain, CaP3V-MF-L2AT, was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8-1.8 µm in diameter, stained Gram-negative and were motile. The strain utilized H2/CO2, formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25-40 °C, pH 6.0-7.5 and 0-2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis. It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus. The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2AT (= DSM 113321T = JCM 39176T) represents a novel species, Methanofollis propanolicus sp. nov.

The hyperthermophilic partners Nanoarchaeum and Ignicoccus stabilize their tRNA T-loops via different but structurally equivalent modifications.

The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m5U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m5U54 is subsequently thiolated to form m5s2U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m1s4Ψ. The methyl and thiol groups in m1s4Ψ and m5s2U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m1A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.

Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC.

Radiation of ionizing or non-ionizing nature has harmful effects on cellular components like DNA as radiation can compromise its proper integrity. To cope with damages caused by external stimuli including radiation, within living cells, several fast and efficient repair mechanisms have evolved. Previous studies addressing organismic radiation tolerance have shown that radiotolerance is a predominant property among extremophilic microorganisms including (hyper-) thermophilic archaea. The analysis of the ionizing radiation tolerance of the chemolithoautotrophic, obligate anaerobic, hyperthermophilic Crenarchaeon Ignicoccus hospitalis showed a D10-value of 4.7 kGy, fourfold exceeding the doses previously determined for other extremophilic archaea. The genome integrity of I. hospitalis after γ-ray exposure in relation to its survival was visualized by RAPD and qPCR. Furthermore, the discrimination between reproduction, and ongoing metabolic activity was possible for the first time indicating that a potential viable but non-culturable (VBNC) state may also account for I. hospitalis.

Recognition of the unsuitability of DSM 12173 as the deposited type strain of Thermocrinis ruber Huber et al. 1999, recognition of DSM 23557 as an authentic sub-culture of strain OC 1/4, the nomenclatural type of Thermocrinis ruber Huber et al. 1999 and an emended description of Thermocrinis ruber Huber et al. 1999.

During the course of growing cell material for the extraction of genomic DNA for the Genomic Encyclopedia of Bacteria and Archaea, strain OC 1/4, the designated type strain of Thermocrinis ruber was cultivated at the Institute for Microbiology and Archaea Center of the University of Regensburg, Regensburg, Germany. Partial sequencing of the 16S rRNA gene indicated that the cell material initially cultivated and the strain held in the DSMZ as DSM 12173 did not correspond with that deposited as AJ005640 and was probably a strain of Thermocrinis albus. A subsequent search of the strain collection of the Institute for Microbiology and Archaea Center of the University of Regensburg held in liquid nitrogen indicated that a strain could be recovered from the liquid nitrogen stocks that corresponded with the properties originally given for strain OC 1/4. We report here on the characterization of this strain that has subsequently been deposited in the DSMZ as DSM 23557.

Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics of Ignicoccus Species.

In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized, and structurally compared to the respective enzymes of the other Ignicoccus species, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genus Ignicoccus comprises three described species, i.e., I. hospitalis and Ignicoccus islandicus from hot marine sediments near Iceland and Ignicoccus pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeon Nanoarchaeum equitansI. hospitalis grows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A1AO complex seemed to be at pH 9. Interestingly, the soluble A1 subcomplexes of the different Ignicoccus species exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCE The Crenarchaeota represent one of the major phyla within the Archaea domain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among crenarchaeotes, since it is the only known host of N. equitans The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

Bypassing rRNA methylation by RsmA/Dim1during ribosome maturation in the hyperthermophilic archaeon Nanoarchaeum equitans.

In all free-living organisms a late-stage checkpoint in the biogenesis of the small ribosomal subunit involves rRNA modification by an RsmA/Dim1 methyltransferase. The hyperthermophilic archaeon Nanoarchaeum equitans, whose existence is confined to the surface of a second archaeon, Ignicoccus hospitalis, lacks an RsmA/Dim1 homolog. We demonstrate here that the I. hospitalis host possesses the homolog Igni_1059, which dimethylates the N6-positions of two invariant adenosines within helix 45 of 16S rRNA in a manner identical to other RsmA/Dim1 enzymes. However, Igni_1059 is not transferred from I. hospitalis to N. equitans across their fused cell membrane structures and the corresponding nucleotides in N. equitans 16S rRNA remain unmethylated. An alternative mechanism for ribosomal subunit maturation in N. equitans is suggested by sRNA interactions that span the redundant RsmA/Dim1 site to introduce 2΄-O-ribose methylations within helices 44 and 45 of the rRNA.

Influence of osmotic stress on desiccation and irradiation tolerance of (hyper)-thermophilic microorganisms.

This study examined the influence of prior salt adaptation on the survival rate of (hyper)-thermophilic bacteria and archaea after desiccation and UV or ionizing irradiation treatment. Survival rates after desiccation of Hydrogenothermus marinus and Archaeoglobus fulgidus increased considerably when the cells were cultivated at higher salt concentrations before drying. By doubling the concentration of NaCl, a 30 times higher survival rate of H. marinus after desiccation was observed. Under salt stress, the compatible solute diglycerol phosphate in A. fulgidus and glucosylglycerate in H. marinus accumulated in the cytoplasm. Several different compatible solutes were added as protectants to A. fulgidus and H. marinus before desiccation treatment. Some of these had similar effects as intracellularly produced compatible solutes. The survival rates of H. marinus and A. fulgidus after exposure to UV-C (254 nm) or ionizing X-ray/gamma radiation were irrespective of the salt-induced synthesis or the addition of compatible solutes.

Analysis of the complete genome sequence of the archaeon Pyrococcus chitonophagus DSM 10152 (formerly Thermococcus chitonophagus).

Here we analyze the first complete genome sequence of Pyrococcus chitonophagus. The archaeon was previously suggested to belong to the Thermococcus rather than the Pyrococcus genus. Whole genome phylogeny as well as whole proteome comparisons using all available complete genomes in Thermococcales clearly showed that the species belongs to the Pyrococcus genus. P. chitonophagus was originally isolated from a hydrothermal vent site and it has been described to effectively degrade chitin debris, and therefore is considered to play a major role in the sea water ecology and metabolic activity of microbial consortia within hot sea water ecosystems. Indeed, an obvious feature of the P. chitonophagus genome is that it carries proteins showing complementary activities for chitin degradation, i.e. endo- and exo-chitinase, diacetylchitobiose deacetylase and exo-ß-D glucosaminidase activities. This finding supports the hypothesis that compared to other Thermococcales species P. chitonophagus is adapted to chitin degradation.

Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue.

Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the -EKHVP- motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (-E23T24HVP-), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild-type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

Three multihaem cytochromes c from the hyperthermophilic archaeon Ignicoccus hospitalis: purification, properties and localization.

Three different multihaem cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetrahaem cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron-sulfur protein Igni_0529. Two octahaem cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambiguous for Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight haems per subunit and so both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase (HAO) family (29 % mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol min(-1) (mg hydrogenase)(-1) while activity was non-existent for Igni_0530 and low for Igni_1359. Immuno-electron microscopy of ultra-thin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases, whereas Igni_0530 might be part of the sulfur-reducing mechanism.

Novel cultivation-based approach to understanding the miscellaneous crenarchaeotic group (MCG) archaea from sedimentary ecosystems.

The uncultured miscellaneous crenarchaeotic group (MCG) archaea comprise one of the most abundant microbial groups in the Earth's subsurface environment. However, very little information is available regarding the lifestyle, physiology, and factors controlling the distribution of members of this group. We established a novel method using both cultivation and molecular techniques, including a pre-PCR propidium monoazide treatment, to investigate viable members of the MCG in vitro. Enrichment cultures prepared from estuarine sediment were provided with one of a variety of carbon substrates or cultivation conditions and incubated for 3 weeks. Compared with the samples from time zero, there was an order-of-magnitude increase in the number of MCG 16S rRNA genes in almost all cultures, indicating that MCG archaea are amenable to in vitro cultivation. None of the tested substrates or conditions significantly stimulated growth of MCG archaea more than the basal medium alone; however, glycerol (0.02%) had a significantly inhibitory effect (P < 0.05). Diversity analysis of populations resulting from four culture treatments (basal medium, addition of amino acids, H2-CO2 as the gas phase, or initial aerobic conditions) revealed that the majority of viable MCG archaea were affiliated with the MCG-8 and MCG-4 clusters. There were no significant differences in MCG diversity between these treatments, also indicating that some members of MCG-4 and MCG-8 are tolerant of initially oxic conditions. The methods outlined here will be useful for further investigation of MCG archaea and comparison of substrates and cultivation conditions that influence their growth in vitro.

Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis.

ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and H(2):sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane.

Photodynamic Inactivation of Bacteria in Ionic Environments Using the Photosensitizer SAPYR and the Chelator Citrate†.

Many studies show that photodynamic inactivation (PDI) is a powerful tool for the fight against pathogenic, multiresistant bacteria and the closing of hygiene gaps. However, PDI studies have been frequently performed under standardized in vitro conditions comprising artificial laboratory settings. Under real-life conditions, however, PDI encounters substances like ions, proteins, amino acids and fatty acids, potentially hampering the efficacy of PDI to an unpredictable extent. Thus, we investigated PDI with the phenalene-1-one-based photosensitizer SAPYR against Escherichia coli and Staphylococcus aureus in the presence of calcium or magnesium ions, which are ubiquitous in potential fields of PDI applications like in tap water or on tissue surfaces. The addition of citrate should elucidate the potential as a chelator. The results indicate that PDI is clearly affected by such ubiquitous ions depending on its concentration and the type of bacteria. The application of citrate enhanced PDI, especially for Gram-negative bacteria at certain ionic concentrations (e.g. CaCl2 or MgCl2 : 7.5 to 75 mmol L-1 ). Citrate also improved PDI efficacy in tap water (especially for Gram-negative bacteria) and synthetic sweat solution (especially for Gram-positive bacteria). In conclusion, the use of chelating agents like citrate may facilitate the application of PDI under real-life conditions.

AMP-forming acetyl coenzyme A synthetase in the outermost membrane of the hyperthermophilic crenarchaeon Ignicoccus hospitalis.

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.

Coupled TLC and MALDI-TOF/MS analyses of the lipid extract of the hyperthermophilic archaeon Pyrococcus furiosus.

The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80-90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

What is an autotroph?

The concept of autotrophy depends on the growth media for pure cultures supplying a single one carbon source for anabolism. Secondary carbon compounds added to the medium as chelators and/or vitamins confuse the meaning. This note suggests a clarification of definition suitable for contemporary biochemical studies of true autotrophs.