Daniel H. Shain

Four kingdoms on glacier ice: convergent energetic processes boost energy levels as temperatures fall

A diverse group of glacially obligate organisms coexist on temperate glaciers between Washington State and Alaska. A fundamental challenge for these and other cold–adapted species is the necessity to maintain an energy flux capable of sustaining life at low physiological temperatures. We show here that ice–adapted psychrophiles from four kingdoms (Animalia, Eubacteria, Fungi, Protista) respond to temperature fluctuations in a similar manner; namely, ATP levels and the total adenylate pool increase as temperatures fall (within their viable temperature limits, respectively), yet growth rate increases with temperature. By contrast, mesophilic representatives of each kingdom respond in an opposite manner (i.e. adenylates increase with temperature). These observations suggest that elevated adenylate levels in psychrophiles may offset inherent reductions in molecular diffusion at low physiological temperatures.

Distinctions in adenylate metabolism among organisms inhabiting temperature extremes

Microbiota from multiple kingdoms (e.g., Eubacteria, Fungi, Protista) thrive at temperature optima ranging from 0–20°C (psychrophiles) to 40–85°C (thermophiles). In this study, we have monitored changes in adenylate levels and growth rate as a function of temperature in disparate thermally adapted organisms. Our data indicate that growth rate and adenylate levels increase with temperature in mesophilic and thermophilic species, but rapid losses of adenosine 5′-triphosphate (ATP) occur upon cold or heat shock. By contrast, psychrophilic species decrease adenylate levels but increase growth rate as temperatures rise within their viable range. Moreover, psychrophilic ATP levels fell rapidly upon heat shock, but dramatic gains in ATP (~20–50%) were observed upon cold shock, even at sub-zero temperatures. These results suggest that energy metabolism in thermophiles resembles that in mesophiles, but that elevated adenylate nucleotides in psychrophiles may constitute a compensatory strategy for maintaining biochemical processes at low temperature.

Morphologic characterization of the ice worm Mesenchytraeus solifugus.

Ice worms occupy a unique position in metazoan phylogeny in that they are the only known annelid that completes its life cycle in ice. The mechanism(s) associated with this adaptation are likely to occur at different levels, ranging from modification of their metabolism to changes in morphology. In this study, we examined specimens of Mesenchytraeus solifugus by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in an effort to identify morphologic structures that may aid in its glacial habitation. We report that M. solifugus contains an elongated head pore at the tip of its prostomium, numerous sensory structures, and differentially oriented setae that curve abruptly at their distal end. J. Morphol. 246:192–197, 2000.

An AMP nucleosidase gene knockout in Escherichia coli elevates intracellular ATP levels and increases cold tolerance.

Disparate psychrophiles (e.g. glacier ice worms, bacteria, algae and fungi) elevate steady-state intracellular ATP levels as temperatures decline, which has been interpreted as a compensatory mechanism to offset reductions in molecular motion and Gibbs free energy of ATP hydrolysis. In this study, we sought to manipulate steady-state ATP levels in the mesophilic bacterium, Escherichia coli, to investigate the relationship between cold temperature survivability and elevated intracellular ATP. Based on known energetic pathways and feedback loops, we targeted the AMP nucleotidase (amn) gene, which is thought to encode the primary AMP degradative enzyme in prokaryotes. By knocking out amn in wild-type E. coli DY330 cells using recombineering methodology, we generated a mutant (AMNk) that elevated intracellular ATP levels by more than 30% across its viable temperature range. As temperature was lowered, the relative ATP disparity between AMNk and DY330 cells increased to approximately 66% at 10°C, and was approximately 100% after storage at 0°C for 5–7 days. AMNk cells stored at 0°C for 7 days displayed approximately fivefold higher cell viability than wild-type DY330 cells treated in the same manner.

A cysteine-rich protein in the Theromyzon (Annelida: Hirudinea) cocoon membrane.

The aquatic leech, Theromyzon rude, secretes a flexible, proteinaceous cocoon that is resistant to a broad range of denaturing conditions (e.g. heat, denaturing chemicals). We have partially solubilized the Theromyzon cocoon membrane in 10% acetic acid and identified two major protein fragments. Microsequencing of both Theromyzon cocoon protein (Tcp) fragments generated an identical stretch of the amino‐terminal sequence that was used to clone the corresponding gene. The predicted linear amino acid sequence of the resulting cDNA contained an unusually high cysteine content (17.8%). Sequence analysis identified six internal repeats, each comprising 12 ordered Cys residues in a ∼62 amino acid repeating unit. Sequence comparisons identified homology with undescribed, Cys‐rich repeats across animal phyla (i.e. Arthropod, Nematoda).

Fungal toxins and multiple sclerosis: A compelling connection

Multiple sclerosis occurs as a consequence of central nervous system neuronal demyelination. Decades of research suggest that the primary suspects (e.g., viruses, genes, immune system) are associative rather than causative agents, but a surprisingly coherent relationship can be made between multiple sclerosis and fungal toxins. Specifically, certain pathogenic fungi sequester in non-neuronal tissue and release toxins that target and destroy CNS astrocytes and oligodendrocytes. Without these glial support cells, myelin degrades triggering the onset of multiple sclerosis and its associated symptoms. We propose here that fungal toxins are the underlying cause of multiple sclerosis and thus may offer an avenue towards an effective cure.

Cocoon Deposition and Hatching in the Aquatic Leech, Theromyzon tessulatum (Annelida, Hirudinea, Glossiphoniidae)

Abstract Cocoons of the leech, Theromyzon tessulatum, are secreted underwater and sealed by two opercula (plugs) that are asymmetrically positioned on the upper aspect of the cocoon membrane. The opercula are protein-based, but their amino acid composition appears to differ from the surrounding cocoon membrane. Fluid deposited inside the cocoon by the parent leech contains several proteins ranging in size from ∼40–100 kDa, most of which have pIs below 6.8. While inside the cocoon, late stage embryos display peristaltic contractions that may contribute to weakening of the opercula/membrane boundary. Juveniles break through the opercula after 2–3 wk and exit the cocoon over the course of several hours. The asymmetrical position of the opercula on the upper surface of the cocoon membrane may facilitate contact between the brooding parent leech and the emerging young, which attach to the venter of the parent and are brought to their first blood meal.

Molecular Adaptation in the Ice Worm, Mesenchytraeus solifugus: Divergence of Energetic-Associated Genes

The ice worm, Mesenchytraeus solifugus, is the largest glacially obligate metazoan and among only a few metazoan species that complete their life cycle at 0°C. We conducted a large-scale sequencing analysis of cDNAs isolated from ice worm anterior segments. Sequence comparisons among an available group of ice worm, arthropod, chordate, and nematode homologues suggest that ice worms encode proteins that are less bulky, are less polar, and contain fewer charged residues. Also, subunits of the catalytic F1 ATP synthase complex appear to have diverged more rapidly than other ice worm genes examined, suggesting a role in cold-temperature adaptation. Modeling of F1 ATP synthase β and γ subunits identified nonconservative, ice worm-specific amino acid substitutions at subunit contact sites and at sites proximal to the catalytic site.

Is intracellular pH a clock for mitosis

Experiments have shown that the intracellular pH of many cells rises to a maximum at the onset of mitosis, subsequently decreasing 0.3 to 0.5 pH units by the end of mitosis. This result, and observations that tubulin net charge depends strongly on pH, may be critical for microtubule (MT) dynamics during mitosis. In vivo studies demonstrate that MT dynamics is sensitive to pH, with MT growth favored by higher pH values. Therefore it seems likely that the shift from the dominance of microtubule growth during prophase, and to a lesser extent during prometaphase, to a parity between MT polymerization and depolymerization during metaphase chromosome oscillations is a consequence of gradually decreasing intracellular pH during mitosis. Thus the timing and sequencing of prophase, prometaphase, and metaphase chromosome motions may be understood as an increase in the MT disassembly to assembly probability ratio resulting from a continuously declining intracellular pH.

Metagenome Sequencing of Prokaryotic Microbiota Collected from Byron Glacier, Alaska

ABSTRACT Cold environments, such as glaciers, are large reservoirs of microbial life. The present study employed 16S rRNA gene amplicon metagenomic sequencing to survey the prokaryotic microbiota on Alaskan glacial ice, revealing a rich and diverse microbial community of some 2,500 species of bacteria and archaea.