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.

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.

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.

Combining a robust thermophilic methanogen and packing material with high liquid hold-up to optimize biological methanation in trickle-bed reactors.

The hydrogen gas-to-liquid mass transfer is the limiting factor in biological methanation. In trickle-bed reactors, mass transfer can be increased by high flow velocities in the liquid phase, by adding a packing material with high liquid hold-up or by using methanogenic archaea with a high methane productivity. This study developed a polyphasic approach to address all methods at once. Various methanogenic strains and packings were investigated from a microbial and hydrodynamic perspective. Analyzing the ability to produce high-quality methane and to form biofilms, pure cultures of Methanothermobacter performed better than those of the genus Methanothermococcus. Liquid and static hold-up of a packing material and its capability to facilitate attachment was not attributable to a single property. Consequently, it is recommended to carefully match organism and packing for optimized performance of trickle-bed reactors. The ideal combination for the ORBIT-system was identified as Methanothermobacter thermoautotrophicus IM5 and DuraTop®.

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.

Interplay of phosphate and carbonate ions with flavin photosensitizers in photodynamic inactivation of bacteria.

Photodynamic inactivation (PDI) of pathogenic bacteria is a promising technology in different applications. Thereby, a photosensitizer (PS) absorbs visible light and transfers the energy to oxygen yielding reactive oxygen species (ROS). The produced ROS are then capable of killing microorganisms via oxidative damage of cellular constituents. Among other PS, some flavins are capable of producing ROS and cationic flavins are already successfully applied in PDI. When PDI is used for example on tap water, PS like flavins will encounter various ions and other small organic molecules which might hamper the efficacy of PDI. Thus, the impact of carbonate and phosphate ions on PDI using two different cationic flavins (FLASH-02a, FLASH-06a) was investigated using Staphylococcus aureus and Pseudomonas aeruginosa as model organisms. Both were inactivated in vitro at a low light exposure of 0.72 J cm-2. Upon irradiation, FLASH-02a reacts to single substances in the presence of carbonate or phosphate, whereas the photochemical reaction for FLASH-06a was more unspecific. DPBF-assays indicated that carbonate and phosphate ions decreased the generation of singlet oxygen of both flavins. Both microorganisms could be easily inactivated by at least one PS with up to 6 log10 steps of cell counts in low ion concentrations. Using the constant radiation exposure of 0.72 J cm-2, the inactivation efficacy decreased somewhat at medium ion concentrations but reached almost zero for high ion concentrations. Depending on the application of PDI, the presence of carbonate and phosphate ions is unavoidable. Only upon light irradiation such ions may attack the PS molecule and reduce the efficacy of PDI. Our results indicate concentrations for carbonate and phosphate, in which PDI can still lead to efficient reduction of bacterial cells when using flavin based PS.

Optimized biological CO2-methanation with a pure culture of thermophilic methanogenic archaea in a trickle-bed reactor.

In this study, a fully automated process converting hydrogen and carbon dioxide to methane in a high temperature trickle-bed reactor was developed from lab scale to field test level. The reactor design and system performance was optimized to yield high methane content in the product gas for direct feed-in to the gas grid. The reaction was catalyzed by a pure culture of Methanothermobacter thermoautotrophicus IM5, which formed a biofilm on ceramic packing elements. During 600 h in continuous and semi-continuous operation in countercurrent flow, the 0.05 m3 reactor produced up to95.3 % of methane at a methane production rate of 0.35 [Formula: see text] . Adding nitrogen as carrier gas during startup, foam control and dosing of ammonium and sodium sulfide as nitrogen and sulfur source were important factors for process automation.

Convergent Evolution of a Promiscuous 3-Hydroxypropionyl-CoA Dehydratase/Crotonyl-CoA Hydratase in Crenarchaeota and Thaumarchaeota.

The autotrophic 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle functions in thermoacidophilic, (micro)aerobic, hydrogen-oxidizing Crenarchaeota of the order Sulfolobales as well as in mesophilic, aerobic, ammonia-oxidizing Thaumarchaeota. Notably, the HP/HB cycle evolved independently in these two archaeal lineages, and crenarchaeal and thaumarchaeal versions differ regarding their enzyme properties and phylogeny. These differences result in altered energetic efficiencies between the variants. Compared to the crenarchaeal HP/HB cycle, the thaumarchaeal variant saves two ATP equivalents per turn, rendering it the most energy-efficient aerobic pathway for carbon fixation. Characteristically, the HP/HB cycle includes two enoyl coenzyme A (CoA) hydratase reactions: the 3-hydroxypropionyl-CoA dehydratase reaction and the crotonyl-CoA hydratase reaction. In this study, we show that both reactions are catalyzed in the aforementioned archaeal groups by a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase (Msed_2001 in crenarchaeon Metallosphaera sedula and Nmar_1308 in thaumarchaeon Nitrosopumilus maritimus). Although these two enzymes are homologous, they are closely related to bacterial enoyl-CoA hydratases and were retrieved independently from the same enzyme pool by the ancestors of Crenarchaeota and Thaumarchaeota, despite the existence of multiple alternatives. This striking similarity in the emergence of enzymes involved in inorganic carbon fixation from two independently evolved pathways highlights that convergent evolution of autotrophy could be much more widespread than anticipated.IMPORTANCE Inorganic carbon fixation is the most important biosynthetic process on Earth and the oldest type of metabolism. The autotrophic HP/HB cycle functions in Crenarchaeota of the order Sulfolobales and in ammonia-oxidizing Archaea of the phylum Thaumarchaeota that are highly abundant in marine, terrestrial, and geothermal environments. Bioinformatic prediction of the autotrophic potential of microorganisms or microbial communities requires identification of enzymes involved in autotrophy. However, many microorganisms possess several isoenzymes that may potentially catalyze the reactions of the cycle. Here, we studied the enzymes catalyzing 3-hydroxypropionyl-CoA dehydration and crotonyl-CoA hydration in Nitrosopumilus maritimus (Thaumarchaeota) as well as in Metallosphaera sedula (Crenarchaeota). We showed that both reactions were catalyzed by homologous promiscuous enzymes, which evolved independently from each other from their bacterial homologs. Furthermore, the HP/HB cycle is of applied value, and knowledge of its enzymes is necessary to transfer them to a heterologous host for synthesis of various value-added products.

A promiscuous ancestral enzyme´s structure unveils protein variable regions of the highly diverse metallo-ß-lactamase family.

The metallo-ß-lactamase fold is an ancient protein structure present in numerous enzyme families responsible for diverse biological processes. The crystal structure of the hyperthermostable crenarchaeal enzyme Igni18 from Ignicoccus hospitalis was solved at 2.3 Å and could resemble a possible first archetype of a multifunctional metallo-ß-lactamase. Ancestral enzymes at the evolutionary origin are believed to be promiscuous all-rounders. Consistently, Igni18´s activity can be cofactor-dependently directed from ß-lactamase to lactonase, lipase, phosphodiesterase, phosphotriesterase or phospholipase. Its core-domain is highly conserved within metallo-ß-lactamases from Bacteria, Archaea and Eukarya and gives insights into evolution and function of enzymes from this superfamily. Structural alignments with diverse metallo-ß-lactamase-fold-containing enzymes allowed the identification of Protein Variable Regions accounting for modulation of activity, specificity and oligomerization patterns. Docking of different substrates within the active sites revealed the basis for the crucial cofactor dependency of this enzyme superfamily.

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.

The Rnf complex is a Na+ coupled respiratory enzyme in a fermenting bacterium, Thermotoga maritima.

rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.

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.

Enzymes Catalyzing Crotonyl-CoA Conversion to Acetoacetyl-CoA During the Autotrophic CO2 Fixation in Metallosphaera sedula.

Autotrophic Crenarchaeota use two different cycles for carbon dioxide fixation. Members of the Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle, whereas Desulfurococcales and Thermoproteales use the dicarboxylate/4-hydroxybutyrate cycle. While these two cycles differ in the carboxylation reactions resulting in the conversion of acetyl-CoA + 2 CO2 to succinyl-CoA, they have a common regeneration part in which succinyl-CoA is reconverted to two acetyl-CoA molecules. This common part includes crotonyl-CoA conversion to acetoacetyl-CoA, which has unequivocally been shown in Ignicoccus hospitalis (Desulfurococcales) and Pyrobaculum neutrophilus (Thermoproteales) to be catalyzed by a bifunctional crotonase/3-hydroxybutyryl-CoA dehydrogenase. It is a fusion protein consisting of an enoyl-CoA hydratase and a dehydrogenase domain. As the homologous bifunctional protein is present in Sulfolobales as well, its common functioning in the conversion of crotonyl-CoA to acetoacetyl-CoA was proposed. Here we show that a model autotrophic member of Sulfolobales, Metallosphaera sedula, possesses in addition to the bifunctional protein (Msed_0399) several separate genes coding for crotonyl-CoA hydratase and (S)-3-hydroxybutyryl-CoA dehydrogenase. Their genes were previously shown to be transcribed under autotrophic and mixotrophic conditions. The dehydrogenase Msed_1423 (and not the bifunctional protein Msed_0399) appears to be the main enzyme catalyzing the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Homologs of this dehydrogenase are the only (S)-3-hydroxybutyryl-CoA dehydrogenases present in all autotrophic Sulfolobales, strengthening this conclusion. Two uncharacterized crotonase homologs present in M. sedula genome (Msed_0336 and Msed_0384) were heterologously produced and characterized. Both proteins were highly efficient crotonyl-CoA hydratases and may contribute (or be responsible) for the corresponding reaction in the HP/HB cycle in vivo.

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.

First Report on Photodynamic Inactivation of Archaea Including a Novel Method for High-Throughput Reduction Measurement.

Archaea are considered third, independent domain of living organisms besides eukaryotic and bacterial cells. To date, no report is available of photodynamic inactivation (PDI) of any archaeal cells. Two commercially available photosensitizers (SAPYR and TMPyP) were used to investigate photodynamic inactivation of Halobacterium salinarum. In addition, a novel high-throughput method was tested to evaluate microbial reduction in vitro. Due to the high salt content of the culture medium, the physical and chemical properties of photosensitizers were analyzed via spectroscopy and fluorescence-based DPBF assays. Attachment or uptake of photosensitizers to or in archaeal cells was investigated. The photodynamic inactivation of Halobacterium salinarum was evaluated via growth curve method allowing a high throughput of samples. The presented results indicate that the photodynamic mechanisms are working even in high salt environments. Either photosensitizer inactivated the archaeal cells with a reduction of 99.9% at least. The growth curves provided a fast and precise measurement of cell viability. The results show for the first time that PDI can kill not only bacterial cells but also robust archaea. The novel method for generating high-throughput growth curves provides benefits for future research regarding antimicrobial substances in general.

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.

A Closer Look at Dark Toxicity of the Photosensitizer TMPyP in Bacteria.

Photodynamic inactivation of bacteria (PIB) is based on photosensitizers which absorb light and generate reactive oxygen species (ROS), killing cells via oxidation. PIB is evaluated by comparing viability with and without irradiation, where reduction of viability in the presence of the photosensitizer without irradiation is considered as dark toxicity. This effect is controversially discussed for photosensitizers like TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluensulfonate). TMPyP shows a high absorption coefficient for blue light and a high yield of ROS production, especially singlet oxygen. Escherichia coli and Bacillus atrophaeus were incubated with TMPyP and irradiated with different light sources at low radiant exposures (µW per cm²), reflecting laboratory conditions of dark toxicity evaluation. Inactivation of E. coli occurs for blue light, while no effect was detectable for wavelengths >450 nm. Being more susceptible toward PIB, growth of B. atrophaeus is even reduced for light with emission >450 nm. Decreasing the light intensities to nW per cm² for B. atrophaeus, application of TMPyP still caused bacterial killing. Toxic effects of TMPyP disappeared after addition of histidine, quenching residual ROS. Our experiments demonstrate that the evaluation of dark toxicity of a powerful photosensitizer like TMPyP requires low light intensities and if necessary additional application of substances quenching any residual ROS.

High Tolerance of Hydrogenothermus marinus to Sodium Perchlorate.

On Mars, significant amounts (0.4-0.6%) of perchlorate ions were detected in dry soil by the Phoenix Wet Chemistry Laboratory and later confirmed with the Mars Science Laboratory. Therefore, the ability of Hydrogenothermus marinus, a desiccation tolerant bacterium, to survive and grow in the presence of perchlorates was determined. Results indicated that H. marinus was able to tolerate concentrations of sodium perchlorate up to 200 mM ( 1.6%) during cultivation without any changes in its growth pattern. After the addition of up to 440 mM ( 3.7%) sodium perchlorate, H. marinus showed significant changes in cell morphology; from single motile short rods to long cell chains up to 80 cells. Furthermore, it was shown that the known desiccation tolerance of H. marinus is highly influenced by a pre-treatment with different perchlorates; additive effects of desiccation and perchlorate treatments are visible in a reduced survival rate. These data demonstrate that thermophiles, especially H. marinus, have so far, unknown high tolerances against cell damaging treatments and may serve as model organisms for future space experiments.