Corien Bakermans

Subfreezing activity of microorganisms and the potential habitability of Mars' polar regions.

The availability of water-ice at the surface in the Mars polar cap and within the top meter of the high-latitude regolith raises the question of whether liquid water can exist there under some circumstances and possibly support the existence of biota. We examine the minimum temperatures at which liquid water can exist at ice grain-dust grain and ice grain-ice grain contacts, the minimum subfreezing temperatures at which terrestrial organisms can grow or multiply, and the maximum temperatures that can occur in martian high-latitude and polar regions, to see if there is overlap. Liquid water can exist at grain contacts above about -20 degrees C. Measurements of growth in organisms isolated from Siberian permafrost indicate growth at -10 degrees C and metabolism at -20 degrees C. Mars polar and high-latitude temperatures rise above -20 degrees C at obliquities greater than ~40 degrees, and under some conditions rise above 0 degrees C. Thus, the environment in the Mars polar regions has overlapped habitable conditions within relatively recent epochs, and Mars appears to be on the edge of being habitable at present. The easy accessibility of the polar surface layer relative to the deep subsurface make these viable locations to search for evidence of life.

The Genome Sequence of Psychrobacter arcticus 273-4, a Psychroactive Siberian Permafrost Bacterium, Reveals Mechanisms for Adaptation to Low-Temperature Growth

ABSTRACT Psychrobacter arcticus strain 273-4, which grows at temperatures as low as −10°C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (−10°C to −12°C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.

Diversity of 16S rDNA and Naphthalene Dioxygenase Genes from Coal-Tar-Waste-Contaminated Aquifer Waters

Microbial diversity in four wells along a groundwater flowpath in a coal-tar-waste-contaminated aquifer was examined using RFLP analysis of both 16S rDNA and naphthalene dioxygenase (NDO) genes. Amplified ribosomal DNA restriction analysis (ARDRA) relied upon eubacteria-specific primers to generate four clone libraries. From each library, 100 clones were randomly picked for analysis. Sixty percent of 400 clones contained unique ARDRA patterns. Diversity indices calculated for each community were high (Shannon–Weaver, H? = 3.53 to 3.69). Clones representing ARDRA patterns found in the highest abundance were sequenced (31 total). Sequences related to aerobic bacteria (e.g., Nitrospira, Methylomonas, and Gallionella) predominated among those retrieved from the uncontaminated area of the site, whereas sequences related to facultatively aerobic and anaerobic bacteria (e.g. Azoarcus, Syntrophus, and Desulfotomaculum) predominated among those retrieved from contaminated areas of the site. Using NDO-specific primers and low-stringency PCR conditions, variability in RFLP patterns was only detected in community-derived DNA (3 of 4 wells) and not in 5 newly isolated naphthalene-degrading pure cultures. The ARDRA patterns of the pure culture isolates were not found in the clone libraries. Polymorphisms in community 16S rDNA and NDO genes found in well-water microorganisms reflected distinctive geochemical conditions across the site. Sequences related to sulfate-reducing bacteria were found in groundwater that contained sulfide, while sequences related to Gallionella, Syntrophus, and nitrate-reducing aromatic hydrocarbon-degrading bacteria were found in groundwater that contained ferrous iron, methane, and naphthalene, respectively.

Psychrobacter arcticus 273-4 Uses Resource Efficiency and Molecular Motion Adaptations for Subzero Temperature Growth

Permafrost soils are extreme environments that exert low-temperature, desiccation, and starvation stress on bacteria over thousands to millions of years. To understand how Psychrobacter arcticus 273-4 survived for >20,000 years in permafrost, transcriptome analysis was performed during growth at 22 degrees C, 17 degrees C, 0 degrees C, and -6 degrees C using a mixed-effects analysis of variance model. Genes for transcription, translation, energy production, and most biosynthetic pathways were downregulated at low temperatures. Evidence of isozyme exchange was detected over temperature for D-alanyl-D-alanine carboxypeptidases (dac1 and dac2), DEAD-box RNA helicases (csdA and Psyc_0943), and energy-efficient substrate incorporation pathways for ammonium and acetate. Specific functions were compensated by upregulation of genes at low temperature, including genes for the biosynthesis of proline, tryptophan, and methionine. RNases and peptidases were generally upregulated at low temperatures. Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of a resource efficiency response. In contrast to results observed for other psychrophiles and mesophiles, only clpB and hsp33 were upregulated at low temperature, and there was no upregulation of other chaperones and peptidyl-prolyl isomerases. relA, csdA, and dac2 knockout mutants grew more slowly at low temperature, but a dac1 mutant grew more slowly at 17 degrees C. The combined data suggest that the basal biological machinery, including translation, transcription, and energy metabolism, is well adapted to function across the growth range of P. arcticus from -6 degrees C to 22 degrees C, and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth.

Geochemical and Physiological Evidence for Mixed Aerobic and Anaerobic Field Biodegradation of Coal Tar Waste by Subsurface Microbial Communities

We used geochemical analyses of groundwater and laboratory-incubated microcosms to investigate the physiological responses of naturally occurring microorganisms to coal-tar-waste constituents in a contaminated aquifer. Waters were sampled from wells along a natural hydrologic gradient extending from uncontaminated (1 well) into contaminated (3 wells) zones. Groundwater analyses determined the concentrations of carbon and energy sources (pollutants or total organic carbon), final electron acceptors (oxygen, nitrate, sulfate), and metabolic byproducts (dissolved inorganic carbon [DIC], alkalinity, methane, ferrous iron, sulfide, Mn2+). In the contaminated zone of the study site, concentrations of methane, hydrogen, alkalinity, and DIC were enhanced, while dissolved oxygen and nitrate were depleted. Field-initiated biodegradation assays using headspace-free serum bottle microcosms filled with groundwater examined metabolism of the ambient organic contaminants (naphthalene, 2-methylnaphthalene, benzothiophene, and indene) by the native microbial communities. Unamended microcosms from the contaminated zone demonstrated the simultaneous degradation of several coal-tar-waste constituents at the in situ temperature (10°C). Lag phases prior to the onset of biodegradation indicated the prevalence of both aerobic and anaerobic conditions in situ. Electron acceptor-amended microcosms from the most contaminated well waters demonstrated only aerobic naphthalene degradation. Collectively, the geochemical and microbial evidence show that biodegradation of coal-tar-waste constituents occurs via both aerobic and anaerobic terminal electron accepting processes at this site.

Methanogenesis in Subglacial Sediments

Methanogenic archaea have a unique role in Earths global carbon cycle as producers of the greenhouse gas methane (CH4 ). However, despite the fact that ice covers 11% of Earths continental landmass, evidence for methanogenic activity in subglacial environments has yet to be clearly demonstrated. Here we present genetic, biochemical and geochemical evidence indicative of an active population of methanogens associated with subglacial sediments from Robertson Glacier (RG), Canadian Rockies. Porewater CH4 was quantified in two subglacial sediment cores at concentrations of 16 and 29 ppmv. Coenzyme M (CoM), a metabolic biomarker for methanogens, was detected at a concentration of 1.3 nmol g sediment(-1) corresponding to ∼3 × 10(3) active cells g sediment(-1) . Genetic characterization of communities associated with subglacial sediments indicated the presence of several archaeal 16S rRNA and methyl CoM reductase subunit A (mcrA) gene phylotypes, all of which were affiliated with the euryarchaeal order Methanosarcinales. Further, CH4 was produced at 9-51 fmol g dry weight sediment(-1)  h(-1) in enrichment cultures of RG sediments incubated at 4°C. Collectively, these findings have important implications for the global carbon cycle in light of recent estimates indicating that the Earths subglacial biome ranges from 10(4) to 10(6)  km(3) sediment.

Relationship of Critical Temperature to Macromolecular Synthesis and Growth Yield in Psychrobacter cryopegella

Most microorganisms isolated from low-temperature environments (below 4 degrees C) are eury-, not steno-, psychrophiles. While psychrophiles maximize or maintain growth yield at low temperatures to compensate for low growth rate, the mechanisms involved remain unknown, as does the strategy used by eurypsychrophiles to survive wide ranges of temperatures that include subzero temperatures. Our studies involve the eurypsychrophilic bacterium Psychrobacter cryopegella, which was isolated from a briny water lens within Siberian permafrost, where the temperature is -12 degrees C. P. cryopegella is capable of reproducing from -10 to 28 degrees C, with its maximum growth rate at 22 degrees C. We examined the temperature dependence of growth rate, growth yield, and macromolecular (DNA, RNA, and protein) synthesis rates for P. cryopegella. Below 22 degrees C, the growth of P. cryopegella was separated into two domains at the critical temperature (T(critical) = 4 degrees C). RNA, protein, and DNA synthesis rates decreased exponentially with decreasing temperatures. Only the temperature dependence of the DNA synthesis rate changed at T(critical). When normalized to growth rate, RNA and protein synthesis reached a minimum at T(critical), while DNA synthesis remained constant over the entire temperature range. Growth yield peaked at about T(critical) and declined rapidly as temperature decreased further. Similar to some stenopsychrophiles, P. cryopegella maximized growth yield at low temperatures and did so by streamlining growth processes at T(critical). Identifying the specific processes which result in T(critical) will be vital to understanding both low-temperature growth and growth over a wide range of temperatures.

Proteomic analysis of Psychrobacter cryohalolentis K5 during growth at subzero temperatures.

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and −4°C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (∼31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at −4°C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at −4°C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at −4°C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to −4°C.

Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification

The ideal ecological metabolic activity assay would be applied to naturally occurring microbial populations immediately fixed in the field, and the assay would focus upon intracellular parameters indicative of a dynamic biogeochemical process. In this study, fluorescent in situ hybridization (FISH) with tyramide signal amplification (TSA) detected intracellular mRNA in bacteria. Detection sensitivity was enhanced by using a Hamamatsu color chilled CCD camera and extended exposure times. Pseudomonas putida NCIB 9816-4, a model naphthalene degrading bacterium, was used to refine the protocol. Probe Ac627BR was developed for detecting naphthalene dioxygenase (nahAc) mRNA transcripts. Only induced cells showed positive hybridization to probe Ac627BR. Results were verified by RNase A or DNase I digestion of samples prior to hybridization. When applied to field-fixed groundwater samples, the naphthalene dioxygenase mRNA probe conferred fluorescence on a subset (approximately 1%) of the cells present in the contaminated groundwater. This methodology represents progress towards achieving one of the longstanding goals of environmental microbiology: to simultaneously ascertain the identity, activity, and biogeochemical impact of individual microorganisms in situ-in soil, water, or sediment where they dwell.

Microbial metabolism in ice and brine at -5°C.

Metabolic activity, but not growth, has been observed in ice at temperatures from -5°C to -32°C. To improve understanding of metabolism in ice, we simultaneously examined various aspects of metabolism ((14) C-acetate utilization, macromolecule syntheses and viability via reduction of CTC) of the glacial isolates Sporosarcina sp. B5 and Chryseobacterium sp. V3519-10 during incubation in nutrient-rich ice and brine at -5°C for 50 days. Measured rates of acetate utilization and macromolecule syntheses were high in the first 20 days suggesting adjustment to the lower temperatures and higher salt concentrations of both the liquid vein network in the ice and the brine. Following this adjustment, reproductive growth of both organisms was evident in brine, and suggested for Sporosarcina sp. B5 in ice by increases in cell numbers and biomass. Chryseobacterium sp. V3519-10 cells incubated in ice remained active. These data indicate that neither low temperature nor high salt concentrations prohibit growth in ice, but some other aspect of living within ice slows growth to within the detection limits of current methodologies. These results imply that microbial growth is plausible in natural ice systems with comparable temperatures and sufficient nutrients, such as debris-rich basal ices of glaciers and ice masses.