Response of Methanogenic Archaea from Siberian Permafrost and Non-permafrost Environments to Simulated Mars-like Desiccation and the Presence of Perchlorate.

Numerous preflight investigations were necessary prior to the exposure experiment BIOMEX on the International Space Station to test the basic potential of selected microorganisms to resist or even to be active under Mars-like conditions. In this study, methanogenic archaea, which are anaerobic chemolithotrophic microorganisms whose lifestyle would allow metabolism under the conditions on early and recent Mars, were analyzed. Some strains from Siberian permafrost environments have shown a particular resistance. In this investigation, we analyzed the response of three permafrost strains (Methanosarcina soligelidi SMA-21, Candidatus Methanosarcina SMA-17, Candidatus Methanobacterium SMA-27) and two related strains from non-permafrost environments (Methanosarcina mazei, Methanosarcina barkeri) to desiccation conditions (-80°C for 315 days, martian regolith analog simulants S-MRS and P-MRS, a 128-day period of simulated Mars-like atmosphere). Exposure of the different methanogenic strains to increasing concentrations of magnesium perchlorate allowed for the study of their metabolic shutdown in a Mars-relevant perchlorate environment. Survival and metabolic recovery were analyzed by quantitative PCR, gas chromatography, and a new DNA-extraction method from viable cells embedded in S-MRS and P-MRS. All strains survived the two Mars-like desiccating scenarios and recovered to different extents. The permafrost strain SMA-27 showed an increased methanogenic activity by at least 10-fold after deep-freezing conditions. The methanogenic rates of all strains did not decrease significantly after 128 days S-MRS exposure, except for SMA-27, which decreased 10-fold. The activity of strains SMA-17 and SMA-27 decreased after 16 and 60 days P-MRS exposure. Non-permafrost strains showed constant survival and methane production when exposed to both desiccating scenarios. All strains showed unaltered methane production when exposed to the perchlorate concentration reported at the Phoenix landing site (2.4 mM) or even higher concentrations. We conclude that methanogens from (non-)permafrost environments are suitable candidates for potential life in the martian subsurface and therefore are worthy of study after space exposure experiments that approach Mars-like surface conditions.

Mechanical and cell-to-cell adhesive properties of aggregated Methanosarcina.

The mechanical and adhesive properties as well as the turgor pressure of microbes play an important role in cell growth and aggregation. By applying AFM together with finite element modelling, one can determine the cell wall structural homogeneity, mechanical and cell-to-cell adhesive properties for aggregated Methanosarcina barkeri cells. This also allows a novel approach to determine in-aggregate turgor pressure determination. Analyzing the AFM force-indentation response of the aggregates under loads less than 10 nN, our study reveals structural inhomogeneity of the polymeric part of the cell wall material and suggests that the cell wall consists of two layers of methanochondroitin (external: with a thickness of 3 ± 1 nm and internal: with a thickness of 169 ± 30 nm). On average, the hyperelastic finite element model showed that the internal layer is more rigid (µ = 14 ± 4 MPa) than the external layer (µ = 2.8 ± 0.9 MPa). To determine the turgor pressure and adhesiveness of the cells, a specific mode of indentation (under a load of 45 nN), aimed towards the centre of the individual aggregate, was performed. By modelling the AFM induced decohesion of the aggregate, the turgor pressure and the cell-to-cell adhesive interface properties could be determined. On average, the turgor pressure is estimated to be 59 ± 22 kPa, the interface strength is 78 ± 12 kPa and the polymer network extensibility is 2.8 ± 0.9 nm. We predict that internal cell wall comprised highly compressed methanochondroitin chains and we are able to identify a conceptual model for stress dependent inner cell wall growth.

Towards a computational model of a methane producing archaeum.

Progress towards a complete model of the methanogenic archaeum Methanosarcina acetivorans is reported. We characterized size distribution of the cells using differential interference contrast microscopy, finding them to be ellipsoidal with mean length and width of 2.9 µ m and 2.3 µ m, respectively, when grown on methanol and 30% smaller when grown on acetate. We used the single molecule pull down (SiMPull) technique to measure average copy number of the Mcr complex and ribosomes. A kinetic model for the methanogenesis pathways based on biochemical studies and recent metabolic reconstructions for several related methanogens is presented. In this model, 26 reactions in the methanogenesis pathways are coupled to a cell mass production reaction that updates enzyme concentrations. RNA expression data (RNA-seq) measured for cell cultures grown on acetate and methanol is used to estimate relative protein production per mole of ATP consumed. The model captures the experimentally observed methane production rates for cells growing on methanol and is most sensitive to the number of methyl-coenzyme-M reductase (Mcr) and methyl-tetrahydromethanopterin:coenzyme-M methyltransferase (Mtr) proteins. A draft transcriptional regulation network based on known interactions is proposed which we intend to integrate with the kinetic model to allow dynamic regulation.

Methanosarcina semesiae sp. nov., a dimethylsulfide-utilizing methanogen from mangrove sediment.

Methanosarcina semesiae MD1T (T = type strain), a novel obligately methylotrophic methanogenic archaeon is described. Strain MD1T was isolated from an enrichment on dimethylsulfide inoculated with mangrove sediment. The cells were irregularly coccoid, non-motile, 1.4+/-0.2 microm in diameter and stained Gram-positive. The catabolic substrates used included dimethylsulfide, methanethiol, methanol and methylated amines, but not acetate, formate, H2/CO2 or a combination of these substrates. When cells grown on dimethylsulfide were transferred to trimethylamine or methanol and vice versa, a lag phase was observed. The same lag phase occurred when cells grown on trimethylamine were transferred to methanol and vice versa, indicating that for each substrate different enzymes were induced. Fastest growth occurred within a temperature range of 30-35 degrees C and a pH of 6.5-7.5. Both Na+ and Mg2+ were required for growth, with maximum growth rates at 200-600 mM Na+ and 20-100 mM Mg2+. The cells exhibited specific growth rates (h-1) of 0.07+/-0.02, 0.15+/-0.04 and 0.18-/+0.05 on dimethylsulfide, methanol and trimethylamine, respectively. Analysis of the 16S rRNA gene sequence showed that strain MD1T was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (94-97% sequence similarity).

Characterization of Methanosarcina mazei N2M9705 isolated from an aquaculture fishpond.

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H2-CO2, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6-0.8 micrometer in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32 degrees -37 degrees C with 1% NaCl. The pH range of growth was 6.2-7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond.

Isolation of a methanogenic bacterium, Methanosarcina sp. strain FR, for its ability to degrade high concentration of perchloroethylene.

Tetrachloroethylene (PCE) is a toxic compound essentially used as a degreasing and dry-cleaning solvent. A methanogenic and sulfate-reducing consortium that dechlorinates and mineralizes high concentrations of PCE was derived from anaerobically digested sludge obtained from a waste water treatment plant (Bourg-en-Bresse, France). A methanogenic bacterium, strain FR, was isolated from this acclimated consortium. On the basis of morphological and physiological characteristics, strain FR was classified in the genus of Methanosarcina. Phylogeny analysis with the 16S rRNA gene sequence revealed that strain FR is highly related to Methanosarcina mazei and Methanosarcina frisia (99.6 and 99.5% identity, respectively). High concentrations (50-87 microM) of PCE were completely dechlorinated by strain FR cultures at the rate of 76 nM-mg protein(-1).day(-1). PCE dechlorination produced a nonidentified compound. The tracer experiments with [13C]PCE revealed that the product was nonchlorinated. Dechlorination of PCE to trichloroethylene was still active in the presence of boiled cell extract of the strain FR. However, no further dechlorination was observed. This result suggests that a cofactor rather than an enzymatic system is responsible for the first dechlorination of PCE. Dechlorination-active fractions purified from cell extracts on a XAD-4 column revealed the presence of F(420), F(430), and cobamides cofactors. This is the first report of the isolation of a methanogenic bacterium with the ability to dechlorinate high concentrations of PCE to a nonchlorinated product.

Heat-shock response in Methanosarcina mazei S-6.

The dnaK locus of Methanosarcina mazei S-6, a mesophilic organism of the phylogenetic domain Archaea, contains the heat-shock genes 5'-grpE-dnaK-dnaJ-3'. Parameters known to affect the response of these genes in organisms of the other two domains, Bacteria and Eucarya, were tested to determine their effects on the archaeal homologs. The mRNA from the three genes increased after heat shock more in lamina than in single cells (these S-6 morphologic stages can be grown in the same substrate). Single cells in early stationary phase showed the highest levels of dnaK mRNA after heat shock, as compared with cells in exponential, or in late stationary, phase. The dnaK mRNA always had the size of a monocistronic transcript. dnaK was also found in the thermophileMethanosarcina thermophila TM-1, and its response to heat shock showed distinctive characteristics. However, dnaK was not revealed in other archaea: three hyperthermophiles (Methanothermus fervidus,Methanococcus jannaschii, and Sulfolobus sp.), and one mesophilic methanogen (Methanospirillum hungateii).

Immunochemical differences among Methanosarcina mazei S-6 morphologic forms.

Methanosarcinae are the only archaeobacteria known to undergo major morphologic changes during growth involving unicellular and multicellular forms, and Methanosarcina mazei S-6 is the only strain for which three distinct forms, packets, single cells, and lamina, have so far been observed. It is reported that two pairs of these forms, either packets and single cells or single cells and lamina, grew and interconverted in medium with the same composition, Ca2+ and Mg2+ concentrations, and growth substrate, and that the two forms in each pair displayed distinctive differences revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, the same growth medium-substrate notwithstanding.

Lamina, a novel multicellular form of Methanosarcina mazei S-6.

A novel multicellular form of Methanosarcina mazei S-6 is described. It was termed lamina, and it formed during the exponential growth phase when packets or single cells were grown in 40 mM trimethylamine and a total concentration of 8.3 to 15.6 mM Ca2+ and/or Mg2+, in cultures that were not shaken. A distinct molecular event represented by the increment in expression and a spatial redistribution of an antigen during lamina formation is documented.