Microbial Metabolism of Amino Acids—Biologically Induced Removal of Glycine and the Resulting Fingerprint as a Potential Biosignature

The identification of reliable biomarkers, such as amino acids, is key for the search of extraterrestrial life. A large number of microorganisms metabolize, synthesize, take up and excrete amino acids as part of the amino acid metabolism during aerobic and/or anaerobic respiration or in fermentation. In this work, we investigated whether the anaerobic microbial metabolism of amino acids could leave a secondary biosignature indicating biological activity in the environment around the cells. The observed fingerprints would reflect the physiological capabilities of the specific microbial community under investigation. The metabolic processing of an amino acid mixture by two distinct anaerobic microbial communities collected from Islinger Mühlbach (ISM) and Sippenauer Moor (SM), Germany was examined. The amino acid mixture contained L-alanine, β-alanine, L-aspartic acid, DL-proline, L-leucine, L-valine, glycine, L-phenylalanine and L-isoleucine. In parallel, an amino acid spiked medium without microorganisms was used as a control to determine abiotic changes over time. Liquid chromatography mass spectrometry (LC-MS) was used to track amino acid changes over time. When comparing to the control samples that did not show significant changes of amino acids concentrations over time, we found that glycine was almost completely depleted from both microbial samples to less than 3% after the first two weeks- This results indicates a preferential use of this simple amino acid by these microbial communities. Although glycine degradation can be caused by abiotic processes, these results show that its preferential depletion in an environment would be consistent with the presence of life. We found changes in most other amino acids that varied between amino acids and communities, suggesting complex dynamics with no clear universal pattern that might be used as a signature of life. However, marked increases in amino acids, caused by cellular synthesis and release into the extracellular environment (e.g., alanine), were observed and could be considered a signature of metabolic activity. We conclude, that substantial anomalous enhancements of some amino acids against the expected abiotic background concentration may be an agnostic signature of the presence of biological processes

BIOMARKERS DETECTION IN MARS ANALOGUE SITES WITHIN MASE PROJECT

Life is a physico-chemical process by which tell-tale signals or traces are left on the environment. These signals are indicators of life and are known as biomarkers. Besides, the traces of some kinds of microorganisms can be well preserved, provided that they are rapidly mineralized and that the sediments in which they occur are rapidly cemented [1]. The search for these traces of life is one of the main objectives of Mars exploration [1] and to improve and optimize the search and detection of them forms part of MASE project targets. In MASE project (Mars Analogues for Space Exploration) we work to improve approaches and methods for biomarker detection in samples with low biomass from Mars analogue sites. A developed antibody multiarray competitive immunoassay (MACIA) for the simultaneous detection of compounds of a wide range of molecular sizes or whole spores and cells [2] [3] has revealed as suitable option to achieve this purpose

Mars Analogues for space exploration - from anaerobic field site to culture collection

Astrobiology seeks to understand the limits of life and to determine the physiology of organisms in order to be able to better assess the potential habitability of other worlds and improve our ability to assay them for the presence of life. To successfully achieve this we require representative microorganisms from environments on Earth that in physical and/or chemical conditions approximate to extraterrestrial environments. The most challenging of these environments with respect to the sample collection and follow on isolation and cultivation of microorganisms are anaerobic environments. Here we describe a systematic approach to this challenge and aim to provide a guideline for future fieldwork and sampling campaigns. We selected a number of anaerobic environments based on characteristics that make them analogous to past and present locations on Mars (Icelandic lakes, sulfidic springs, deep hypersaline environments, acidic iron-rich environments, and permafrost). We implemented a culturing approach to enrich organisms from these environments under anaerobic conditions using a defined medium that would allow for all organisms to be grown under identical culturing conditions m future physiological comparisons. We then isolated anaerobic microorganisms, carried out a study of their basic physiology and deposited these organisms in the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) culture collection to make them available to astrobiologists and microbiologists. This project represents the first attempt to implement a coordinated effort from the selection of extraterrestrial analog sites through to the isolation and the characterisation of organisms and their deposition within a culture collection

The responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today

Cumulative exposure to biological therapy and risk of cancer in patients with psoriasis: a meta-analysis of Psonet studies from Israel, Italy, Spain, the U.K. and Republic of Ireland.

Cancer risk following long-term exposure to systemic immunomodulatory therapies in patients with psoriasis is possible. To assess a dose-response relationship between cumulative length of exposure to biological therapy and risk of cancer. Four national studies (a healthcare database from Israel, and prospective cohorts form Italy, Spain and the U.K. and Republic of Ireland) collaborating through Psonet (European Registry of Psoriasis) participated in these nested case-control studies, including nearly 60 000 person-years of observation. 'Cases' were patients who developed an incident cancer. Patients with previous cancers and benign or in situ tumours were excluded. Four cancer-free controls were matched to each case on year of birth, sex, geographic area and registration year. Follow-up for controls was censored at the date of cancer diagnosis for the matched case. Conditional logistic regression was performed by each registry. Results were pooled using random-effects meta-analysis. A total of 728 cases and 2671 controls were identified. After matching, differences between cases and controls were present for the Charlson Comorbidity Index in all three registries, and in the prevalence of previous exposure to psoralen-ultraviolet A and smoking (the British Association of Dermatologists Biologic Interventions Register only). The risk of first cancers was not significantly associated with cumulative exposure to biologics (adjusted odds ratio per year of exposure 1·02, 95% confidence interval 0·92-1·13). Results were similar if squamous and basal cell carcinomas were included in the outcome. Cumulative length of exposure to biological therapies in patients with psoriasis in real-world clinical practice does not appear to be linked to a higher risk of cancer after several years of use

Assessing the habitability of the MASE Mars analogue sites

The MASE (Mars Analogues for Space Exploration) [1] project is a four-year collaborative research project supported by the European Commission Seventh Framework Contract. The aim of the project is to understand how combined environmental stresses influence the habitability of a number of Mars analogue environments in Europe and beyond. Field sites sampled for MASE include deep subsurface salts at Boulby Mine in the UK, sulfidic springs In Germany and an acidic cold lake in lceland. Samples and isolates have also been provided to the project from acidic deep subsurface environments at the Rio Tinto in Spain as well as permafrost sites in Russian Siberia and northern Canada. Crucial to assessing the habitability of any environmental system to be used as an astrobiological analogue • whether for life in general or, as in this case, for anaerobic microorganisms • is a detailed understanding of their geological and physiochemical context [2, 3]. One of the key outcomes of the MASE project is a comparison and synthesis of just such a set of context data from a varied set of analogue sites, the core of which is presented here, and complemented by an analysis of field samples to detect and quantify amino acids, other organics as well as biologically relevant molecules. We show that anaerobic environments provide some of the best fidelity environments for examining the potential habitability of environments on Mars, which are also anaerobic, but that even these analogue environments show the signatures of Earth's very different geological history, such as high carbon abundance in some environments (from 0.1% in lcelandic lake sediments to 22. 7% in deep .permafrost environments)

Survival of MASE strains under different aspects of simulated Martian conditions

During the project MASE (Mars Analogues for Space Exploration) several microbial isolates were obtained and some of these strains were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions; e.g. radiation, low water activity, high salt concentrations, oxidizing compounds. All sampling, isolation, and cultivation steps, as well as the stress tests were performed under anoxic conditions. So far, five only distantly related microorganisms are under detailed investigation: \textit{Buttiauxella} sp. MASE-IM-7, \textit{Clostridium} sp. MASE-IM-4, \textit{Halanaerobium} sp. MASE-BB-1, \textit{Trichococcus} sp. MASE-IM-5, and \textit{Yersinia} sp. MASE-LG-1. It was shown that tolerance to desiccation and to ionizing radiation, applied separately was not correlated. If desiccation and exposure to radiation was applied together typical additive effects could be observed in each species. The survival after addition of oxidizing compounds (hydrogen peroxide; perchlorates) for a dedicated time (15 minutes; 24 hours) was very divers. If the five microorganisms were compared with each other, \textit{Trichococcus} sp. MASE-IM-5 was the most sensitive strain and survived only 10 mM hydrogen peroxide for 15 minutes and 24 hours, respectively. The most tolerant organism was \textit{Halanaerobium} sp. MASE-BB-1 which was able to survive 100 mM hydrogen peroxide for both tested time points. In between, the other Bacteria were arranged with different tolerances against hydrogen peroxide. Comparable, species specific results were obtained after the addition of different perchlorates. In general, the strategy of the MASE project has proven to be useful to gain new model microorganisms. The isolated and characterized MASE strains have so far unknown high tolerances against cell damaging treatments and may serve as model organisms for future space exposure experiments

Anaerobic microorganisms in astrobiological analogue environments:from field site to culture collection

Astrobiology seeks to understand the limits of life and to determine the physiology of organisms in order to better assess the habitability of other worlds. To successfully achieve these goals we require microorganisms from environments on Earth that approximate to extraterrestrial environments in terms of physical and/or chemical conditions. The most challenging of these environments with respect to sample collection, isolation and cultivation of microorganisms are anoxic environments. In this paper, an approach to this challenge was implemented within the European Union’s MASE (Mars Analogues for Space Exploration) project. In this review paper, we aim to provide a set of methods for future field work and sampling campaigns. A number of anoxic environment based on characteristics that make them analogous to past and present locations on Mars were selected. They included anoxic sulphur-rich springs (Germany), the salt-rich Boulby Mine (UK), a lake in a basaltic context (Iceland), acidic sediments in the Rio Tinto (Spain), glacier samples (Austria) and permafrost samples (Russia and Canada). Samples were collected under strict anoxic conditions to be used for cultivation and genomic community analysis. Using the samples, a culturing approach was implemented to enrich anaerobic organisms using a defined medium that would allow for organisms to be grown under identical conditions in future physiological comparisons. Anaerobic microorganisms were isolated and deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) culture collection to make them available to other scientists. In MASE, the selected organisms are studied with respect to survival and growth under Mars relevant stresses. They are artificially fossilized and the resulting biosignatures studied and used to investigate the efficacy of life detection instrumentation for planetary missions. Some of the organisms belong to genera with medical and environmental importance such as Yersinia spp., illustrating how astrobiology field research can be used to increase the availability of microbial isolates for applied terrestrial purposes