Abstract 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.

Our understanding of the habitability of Mars is hampered by a lack of knowledge of the stress respons-es of anaerobic organisms. In the project MASE (Mars Analogues for Space Exploration) representative (fac-ultative) anaerobic microorganisms are isolated from Mars analogue environments on Earth and exposed to stresses typical for the Martian environment. The aim is to gain fundamental insights into the limits of anaer-obic microbial life on Earth and to use these data to assess the habitability of Mars.

A recent study on members of the genus Actinobaculum revealed that cultures of the species Actinobaculum massiliense CCUG 47753(T) (=DSM 19118(T)) currently being distributed do not conform to the properties of the type strain of A. massiliense CIP 107404(T) given by Greub & Raoult [Greub, G. & Raoult, D. (2002). J Clin Microbiol 40, 3938-3941]. The original strain, CIP 107404(T) is no longer available from the Biological Resource Center of Institut Pasteur, Paris. Based on data currently available, the organism currently deposited as CCUG 47753(T) and DSM 19118(T) is a member of the species Actinobaculum schaalii. Clearly, the organism deposited as CCUG 47753(T) and DSM 19118(T) as the type strain of the species Actinobaculum massiliense does not have the properties given by Greub & Raoult. Based on the absence of an authentic type strain, the Judicial Commission is requested to examine the status of the name Actinobaculum massiliense Greub and Raoult 2006 and to issue an Opinion.

16S rRNA gene sequences deposited for the type strains of Paraoerskovia marina (CTT-37(T); GenBank accession no. AB445007) and Koreibacter algae (DSW-2(T); FM995611) show a similarity of 100 %. Consequently, the type strains were subjected to a polyphasic recharacterization under direct comparison in order to clarify their taxonomic position. PvuII RiboPrint patterns and quantitative ratios of cellular fatty acids revealed strain-specific differences between P. marina DSM 21750(T) ( = CTT-37(T)) and K. algae DSM 22126(T) ( = DSW-2(T)). The percentage of DNA-DNA binding of 94 % indicated that the two type strains belong to the same genomospecies. Agreement in the peptidoglycan structure and polar lipid pattern, highly similar fatty acid profiles and MALDI-TOF mass spectra, the ability to produce acid from the same carbon sources, corresponding enzymic activities and DNA G+C contents of 70.8 ± 0.3 mol%, in addition to the consistent characteristics reported in the original descriptions, support the view that the two strains should be affiliated to the same species. According to Rules 38 and 42 of the Bacteriological Code, Koreibacter algae should be reclassified as later heterotypic synonym of Paraoerskovia marina, and the descriptions of the genus Paraoerskovia Khan et al. 2009 and of Paraoerskovia marina Khan et al. 2009 are emended accordingly.

The taxonomic status of three Bacillus species, Bacillus insolitus, B. psychrodurans and B. psychrotolerans was reexamined using a polyphasic approach. In our analysis, these three Bacillus species formed a cluster separate from other members of Bacillus rRNA group 2 [5] and from Bacillus sensu stricto. These three species shared high 16S rRNA gene sequence similarities between them (97.8–99.7%) and showed closest sequence similarity (95.3–96.3%) to Paenisporosarcina quisquiliarum gen. nov., sp. nov. [18]. Sequence similarities with other related genera ranged between 90.9% and 94.5%. Phylogenetic coherence of the three species was supported by phenotypic characteristics, such as growth at low temperatures, negative oxidation and assimilation of many carbohydrates, MK8 as the major isoprenoid quinine and broadly similar polar lipid profiles. All three species had a similar peptidoglycan type of the variation A4β and similar genomic G + C contents (35.7–36.6 mol% [1]). Genomic relatedness among them was shown to be less than 70% and justified their separate species status [1]. These three species could be differentiated from each other and from related taxa on the basis of phenotypic, including chemotaxonomic, characteristics and ribotype patterns. On the basis of our analysis, we propose a new genus Psychrobacillus gen. nov. and to transfer B. insolitus, B. psychrodurans and B. psychrotolerans to the new genus as Psychrobacillus insolitus comb. nov. (type species of the genus; type strain W16BT = DSM 5T), P. psychrodurans comb. nov. (type strain 68E3T = DSM 11713T) and P. psychrotolerans comb. nov. (type strain 3H1T = DSM 11706T).

A novel phenanthrene-degrading bacterium, designated strain Sphe3, was isolated from a creosote-contaminated soil in Greece. Cells were non-motile, Gram-positive, aerobic, and rod- to coccus-shaped. The strain was isolated on the basis of formation of a clear zone on agar plates sprayed with phenanthrene. Optimal growth occurred at 30 degrees C. The G+C content of the DNA was 65.7 mol%. The polar lipid pattern of strain Sphe3 (T) consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant fatty acids were iso-C-15:0, anteiso-C-15:0, iso-C-16:0, C-16:0 and anteiso-C-17:0, representing > 86% of the total fatty acids. The predominant isoprenoid quinone of strain Sphe3(T) was menaquinone-8 (MK-8). Based on 16S rRNA gene sequence analysis, strain Sphe3(T) showed 99 and 98.9% similarity to the type strains of Arthrobacter oxydans and Arthrobacter polychromogenes, respectively. Strain Sphe3(T) showed 91% similarity to homologues of A. oxydans and A. polychromogenes based on recA gene sequence analysis. Based on 16S rRNA and recA gene sequence analysis and DNA-DNA hybridization analysis, as well as physiological and chemotaxonomic characteristics, it is concluded that strain Sphe3(T) represents a novel species of the genus Arthrobacter, for which the name Arthrobacter phenanthrenivorans sp. nov. is proposed. The type strain is Sphe3(T) (=DSM 18606(T)=LMG 23796(T)).

A polyphasic taxonomic study was performed on the type strain of Bacillus thermosphaericus DSM 10633T and three related soil isolates. On the basis of phenotypic characteristics, chemotaxonomic profiles and phylogenetic data a new genus, Ureibacillus gen. nov., is proposed for the strains in the Bacillus thermosphaericus cluster. Strains of this cluster fall into two DNA-DNA similarity groups: while one group contains the type strain of Ureibacillus thermosphaericus comb. nov. and a single soil isolate, the other contains two soil isolates. The two groups differed in the composition of isoprenoid quinones and some phenotypic properties. These data support the description of a novel species of Ureibacillus for which the name Ureibacillus terrenus is proposed. The type strain of this new species is TH9AT (= DSM 12654T = LMG 19470T).

an opportunistic pathogen causing valve endocarditis, hemorrhagic pneumonia, and pitted keratolysis. It is strictly aerobic and can only grow when several amino acids are provided in the medium. The strain described in this report is a free-living, nonmotile, Gram-positive bacterium, origi-nally isolated from a marine environment. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Dermacoccaceae and the 2,785,024 bp long sin-gle replicon genome with its 2639 protein-coding and 64 RNA genes is part of the G enomic E ncyclopedia of B acteria and A rchaea project.

Based on phenotypic properties, 16S rRNA gene sequence and MALDI-TOF analysis, strains DSM 15084T and CIP 107628T, deposited as the type strain of Bacillus aeolius, do not represent the original type strain, strain 4-1T. It is therefore proposed that the Judicial Commission of the International Committee on Systematics of Prokaryotes places the name Bacillus aeolius on the list of rejected specific and subspecific epithets in names of species and subspecies of bacteria if a suitable replacement for the type strain or a neotype cannot be found within 2 years of publication of this Request.

Within a collection of marine strains that were shown to contain the photosynthesis reaction-centre genes pufL and pufM, a novel group of alphaproteobacteria was found and was characterized phenotypically. The 16S rRNA gene sequence data suggested that the strains belonged to the order Rhizobiales and were closest (98.5% sequence similarity) to the recently described species Hoeflea marina. The cells contained bacteriochlorophyll a and a carotenoid, presumably spheroidenone, in small to medium amounts. Cells of the novel strains were small rods and were motile by means of single polarly inserted flagella. Good growth occurred in complex media with 0.5-7.0% sea salts, at 25-33 degrees C (optimum, 31 degrees C) and at pH values in the range 6-9. With the exception of acetate and malate, organic carbon sources tested supported poor growth or no growth at all. Growth factors were required; these were provided by small amounts of yeast extract, but not by standard vitamin solutions. Growth occurred under aerobic to microaerobic conditions, but not under anaerobic conditions, either in the dark or light. Nitrate was not reduced. Photosynthetic pigments were formed at low to medium salt concentrations, but not at the salt concentration of sea water (3.5%). On the basis of smaller cell size, different substrate utilization profile and photosynthetic pigment content, the novel strains can be classified as representatives of a second species of Hoeflea, for which the name Hoeflea phototrophica sp. nov. is proposed. The type strain of Hoeflea phototrophica sp. nov. is DFL-43(T) (= DSM 17068(T) = NCIMB 14078(T)).

A Gram-positive, rod-shaped, non-spore-forming bacterium, strain CSCT, was isolated from diseased, surface-sterilized specimens of the predatory mite Phytoseiulus persimilis Athias-Henriot and subjected to polyphasic taxonomic analysis. Comparative analysis of the 16S rRNA gene sequence revealed that the strain was a new member of the family Micrococcaceae. Nearest phylogenetic neighbours were determined as Renibacterium salmoninarum (94.0%), Arthrobacter globiformis (94.8%) and Arthrobacter russicus (94.6%). Although the predominant fatty acids (anteiso C15:0), cell-wall sugars (galactose, glucose) and polar lipids (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol) are in accordance with those of members of the genus Arthrobacter, strain CSCT can be distinguished from members of the genus Arthrobacter by biochemical tests, the absence of a rod-coccus life cycle and the occurrence of the partially saturated menaquinone MK-10(H2) as the predominant menaquinone. The DNA G+C content is 57.7 mol%. On the basis of morphological, chemotaxonomic and phylogenetic differences from other species of the Micrococcaceae, a novel genus and species are proposed, Acaricomes phytoseiuli gen. nov., sp. nov. The type strain is CSCT (=DSM 14247T=CCUG 49701T).

A novel group of aerobic anoxygenic phototrophic bacteria was isolated from marine dinoflagellates, and two strains were characterized in detail. Cells were Gram-negative cocci or ovoid rods and were motile by means of a single, polarly inserted flagellum. They were obligate aerobes requiring 1-7 % salinity. The optimal pH range for growth was 6.5-9.0 and the temperature optimum was 33 degrees C. The bacteria contained bacteriochlorophyll a and spheroidenone as the only carotenoid. The in vivo absorption spectrum displayed two maxima in the infrared region at 804 and 868 nm. The distinct 804 nm band indicates the presence of light-harvesting system 2. Various organic carbon sources were assimilated, including many carboxylic acids, glucose and glycerol, but not butyrate, ethanol or methanol. Dissimilatory nitrate reduction was found for both strains. The physiological characteristics of the new strains resembled those of Roseobacter denitrificans, but there were differences in the lipid composition. Based on 16S rRNA gene sequence analysis the new strains are relatively distant from other recognized species, with the closest relatives Jannaschia helgolandensis, Ruegeria atlantica and Rhodobacter veldkampii showing 94.1-93.4 % similarity. Similarity to Roseobacter denitrificans was only 92.2 %, in line with numerous other species of the Roseobacter group. Therefore, it is proposed to classify the strains in a new genus and species within the Roseobacter clade, Dinoroseobacter shibae gen. nov., sp. nov. The type strain is DFL 12(T) (=DSM 16493(T)=NCIMB 14021(T)).

More than 100 bacterial isolates from various marine habitats were screened for AHL production by using gfp reporter constructs based on the lasR system of Pseudomonas aeruginosa and the luxR system of Vibrio fischeri. Of the 67 Alphaproteobacteria tested, most of which belonged into the so-called Roseobacter clade, 39 induced fluorescence in either one or both sensor strains up to 103-fold compared to controls. Acylated homoserine lactones were identified by GC-MS analysis and shown to have chain lengths of C8, C10, C13-C16, and C18. One or two double bonds were often present, while a keto or hydroxyl group occurred only rarely in the side chain. Most strains produced several different AHLs. C18-en-HSL and C18-dien-HSL were produced by Dinoroseobacter shibae, an aerobic anoxygenic phototrophic bacterium isolated from dinoflagellates, and are among the longest AHLs found to date. Z7-C14-en-HSL, which has previously been detected in Rhodobacter sphaeroides, was produced by Roseovarius tolerans and Jannaschia helgolandensis. This signal molecule was synthesised and shown to induce a similar response to the culture supernatant in the respective sensor strain. The widespread occurrence of quorum-sensing compounds in marine Alphaproteobacteria, both free-living strains and those associated to eukaryotic algae, points to a great importance of this signalling mechanism for the adaptation of the organisms to their widely different ecological niches.

ABSTRACT A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis , B. mojavensis , B. pumilus , or B. fusiformis , by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis ) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42°C, and no reduction in activity was observed even after 24 h of growth at 42°C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22°C. None of the cytotoxic strains produced B. cereus -like enterotoxins as tested by PCR or by immunological methods.

Among a group of marine isolates that were found to be positive for genes of the bacterial photosynthetic reaction centre, a strain was selected for characterization that was phylogenetically close to the genus Roseovarius. The strain, designated DFL-24T, originated from a culture of Alexandrium ostenfeldii (dinoflagellate) and contained small amounts of bacteriochlorophyll a, corresponding to about 1 % of the amount found in intensely pigmented aerobic phototrophs such as Roseobacter litoralis. Cells were rods of 0.5-0.7x1.3-3.0 microm often with uneven ends, suggesting a budding mode of division. True motility was not observed. Electron micrographs of ultrathin sections revealed a Gram-negative cell-wall structure. Cultures did not grow without addition of sea salts and tolerated up to 10 % (w/v) sea-water salts. Acetate, butyrate, tricarboxylic acid cycle intermediates, glutamate and glycerol were used as growth substrates, but not glucose or fructose. Biotin, thiamine and nicotinic acid were required as growth factors. The DNA G+C content was 63 mol%. 16S rRNA gene sequence analysis placed the strain within the Roseobacter lineage of the 'Alphaproteobacteria'. Its closest phylogenetic neighbour was Roseovarius tolerans showing 96.4 % 16S rRNA gene sequence similarity. Based also on its physiological and biochemical characteristics, the strain is considered to represent a novel species of the genus Roseovarius, Roseovarius mucosus sp. nov. Strain DFL-24T (=DSM 17069T=NCIMB 14077T) is the type strain. The 16S rRNA gene sequence of DFL-24T was found to have a similarity of 99.7 % with an unidentified strain, 253-13 (=DSM 17070), which was likewise characterized and shown to constitute another strain of the species.

The 'Lactobacillus plantarum group' encompasses the taxa Lactobacillus plantarum subsp. plantarum, Lactobacillus plantarum subsp. argentoratensis, Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus arizonensis. In this study, the phylogenetic position of L. arizonensis was examined using 16S rRNA gene-specific methodologies (16S rRNA sequencing and ribotyping) and genomic DNA-based investigations [repetitive extragenic palindromic DNA (rep)-PCR and DNA-DNA hybridization]. Our results show that the L. arizonensis type strain could not be distinguished from the type strain of L. plantarum or from various L. plantarum reference strains. Therefore, it is proposed that the species L. arizonensis should be reclassified as a heterotypic synonym of L. plantarum.

Two Gram-positive strains isolated from cysts of the brine shrimp Artemia franciscana were subjected to a polyphasic taxonomic analysis. Based on 16S rRNA gene sequence comparison and composition of isoprenoid quinones, peptidoglycan and fatty acids, these organisms are members of the genus Exiguobacterium. Both strains showed 95.9% 16S rRNA gene sequence similarity to one another. The 16S rRNA gene sequences of strain 8NT and 9ANT were 97.5% and 98.9% similar to those of Exiguobacterium aurantiacum DSM 6208T and Exiguobacterium undae DSM 14481T, respectively. Based on differences in chemotaxonomic and physiological characteristics, results of DNA–DNA hybridization and automated riboprinting, two novel species of the genus Exiguobacterium are proposed, Exiguobacterium mexicanum sp. nov. (type strain 8NT=DSM 16483T=CIP 108859T) and Exiguobacterium artemiae sp. nov. (type strain 9ANT=DSM 16484T=CIP 108858T).

Using a lake sediment mat sample from Lake Fryxell, Antarctica, different DNA extraction and purification methods were compared by denaturing gradient gel electrophoresis (DGGE). Based on the analyses of cloned 16S rRNA gene sequences a high degree of as yet uncultured prokaryotes have been reported in this sample. Although the vast majority of these as yet uncultured organisms seem to be classified as representatives of Firmicutes, Proteobacteria and Bacteriodetes, many of these taxa should be regarded novel species as judged from the distance of their gene sequences to those of their nearest cultured phylogenetic neighbours. The physiological properties of cultured strains from Lake Fryxell and of those of described species that are phylogenetically affiliated to the as yet uncultured species from this environment, suggest the presence of a well developed food web of primary producers, anaerobic degraders and fermenters, and aerobes. The few novel species described from this sample add to the increasing number of species characterized from various Antarctic habitats. Determination of the phylogenetic relatedness of the mat clone sequences of Clostridia with recent entries into public databases revealed that many of the putative species are closely related to other putative species detected in a broad range of environments, ranging from rumen and gut, anaerobic and polluted soil to sediment and groundwater samples.

Strain XLG9A10.2T was isolated from an alkaline salt marsh soil in western China. 16S rRNA gene sequence analysis indicated that strain XLG9A10.2T constitutes a distinct lineage within the family Promicromonosporaceae, sharing 94.8-95.1% gene similarity with members of the genus Promicromonospora and 94.4-95.7% similarity with those of Xylanimonas and related genera. The general colony and cell morphology of strain XLG9A10.2T is similar to that of members of Promicromonospora, but differs from members of the genus Xylanimonas in forming a well-developed branching mycelium and production of coccoid spores. Strain XLG9A10.2T shows the peptidoglycan type A4alpha (L-lys<--L-thr<--D-Glu), contains glucose, mannose and galactose as whole cell sugars and has MK-9(H4) and MK-9(H6) as major menaquinones, while phospholipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, three unidentified phospholipids and one unidentified glycolipid. The DNA base composition is 71.9 mol% G+C. On the basis of morphological, chemotaxonomic, metabolic and phylogenetic differences from other species of Promicromonosporaceae, a new genus and species, Myceligenerans xiligouense gen. nov., sp. nov., is proposed. The type strain is XLG9A10.2T (=DSM 15700T=CGMCC 1.3458T.)

A water sample from the North Sea was used to isolate the abundant heterotrophic bacteria that are able to grow on complex marine media. Isolation was by serial dilution and spread plating. Phylogenetic analysis of nearly complete 16S rRNA gene sequences revealed that one of the strains, HEL-45T, had 97.4% sequence similarity to Sulfitobacter mediterraneus and 96.5 % sequence similarity to Staleya guttiformis. Strain HEL-45T is a Gram-negative, non-motile rod and obligate aerobe and requires sodium and 1-7% sea salts for growth. It contains storage granules and does not produce bacteriochlorophyll. Optimal growth temperatures are 25-30 degrees C. The DNA base composition (G+C content) is 60.1 mol%. Strain HEL-45T has Q10 as the dominant respiratory quinone. The major polar lipids are phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine and an aminolipid. The fatty acids comprise 18 : 1omega7c, 18 : 0, 16 : 1omega7c, 16 : 0, 3-OH 10 : 0, 3-OH 12 : 1 (or 3-oxo 12 : 0) and traces of an 18 : 2 fatty acid. Among the hydroxylated fatty acids only 3-OH 12 : 1 (or 3-oxo 12 : 0) appears to be amide linked, whereas 3-OH 10 : 0 appears to be ester linked. The minor fatty acid components (between 1 and 7%) allow three subgroups to be distinguished in the Sulfitobacter/Staleya clade, placing HEL-45T into a separate lineage characterized by the presence of 3-OH 12 : 1 (or 3-oxo 12 : 0) and both ester- and amide-linked 16 : 1omega7c phospholipids. HEL-45T produces indole and derivatives thereof, several cyclic dipeptides and thryptanthrin. Phylogenetic analysis of 16S rRNA gene sequences and chemotaxonomic data support the description of a new genus and species, to include Oceanibulbus indolifex gen. nov., sp. nov., with the type strain HEL-45T (=DSM 14862T=NCIMB 13983T).