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

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.Temperature is one of the most important parameters that determine the distribution and extent of life on earth, and it does this by affecting cell structure and function. High temperatures break covalent bonds and ionic interactions between molecules, inactivating proteins and disrupting cell structures. Low temperatures reduce biochemical reaction rates and substrate transport and induce the formation of ice that damages cell structures. Not surprisingly, an organism''s compatibility with the temperature of its habitat is ultimately determined by its underlying genetic architecture.The strong emphasis in research on mesophile biology (temperatures in the 20°C to 37°C range) has given us a misimpression of the importance of cold on earth. However, 70% of the Earth''s surface is covered by oceans with average temperatures between 1°C and 5°C (11), 20% of the Earth''s terrestrial surface is permafrost (47), and a larger portion of the surface undergoes seasonal freezing, making our planet a predominantly cold environment. Hence, cold adaptation in the microbial world should be expected (55).Permafrost is defined as soils or sediments that are continuously exposed to a temperature of 0°C or less for at least 2 years (44). Permafrost temperatures range from −10°C to −20°C in the Arctic and from −10°C to −65°C in the Antarctic, and permafrost has low water activity, often contains small amounts of carbon (0.85 to 1%), and is subjected to prolonged exposure to damaging gamma radiation from ⁴⁰K in soil minerals (49). Liquid water occurs as a very thin, salty layer surrounding the soil particles in the frozen layer. Despite the challenges of the permafrost, a variety of microorganisms successfully colonize this environment, and many microorganisms have been isolated from it (54, 70). The bacterial taxa most frequently isolated from the Kolyma permafrost of northeast Siberia include Arthrobacter, Exiguobacterium, Flavobacterium, Sphingomonas, and Psychrobacter (71). Rhode and Price (56) proposed that microorganisms can survive in frozen ice for very long periods due to the very thin film of water surrounding each cell that serves as a reserve of substrates. Permafrost is a more favorable environment than ice as a result of its heterogeneous soil particles and larger reservoirs of nutrients.The genus Psychrobacter comprises a group of Gram-negative, rod-shaped, heterotrophic bacteria, and many Psychrobacter species are capable of growth at low temperatures. Members of this genus can grow at temperatures between −10°C and 42°C, and they have frequently been isolated from various cold environments, including Antarctic sea ice, ornithogenic soil and sediments, the stomach contents of Antarctic krill (Euphausia), deep seawater, and permafrost (9, 36, 57, 70, 71, 76; http://www.bacterio.cict.fr/p/psychrobacter.html). Psychrobacter arcticus 273-4 is a recently described species (4) that was isolated from a 20,000- to 30,000-year-old continuously frozen permafrost horizon in the Kolyma region in Siberia that was not exposed to temperatures higher than 4°C during isolation (70). This strain, the type strain of the species, grows at temperatures ranging from −10°C to 28°C, has a generation time of 3.5 days at −2.5°C, exhibits excellent long-term survival under freezing conditions, and has temperature-dependent physiological modifications in membrane composition and carbon source utilization (50). The fact that Psychrobacter has been found to be an indicator genus for permafrost and other polar environments (66) suggests that many of its members are adapted to low temperatures and increased levels of osmotica and have evolved molecular-level changes that aid survival at low temperatures.Early studies on cold adaptation in microorganisms revealed physiological strategies to deal with low temperatures, such as changes in membrane saturation, accumulation of compatible solutes, and the presence of cold shock proteins (CSPs) and many other proteins with general functions (62). However, many of the studies were conducted with mesophilic microorganisms, which limits the generality of the conclusions. We addressed the question of cold adaptation by studying microorganisms isolated from subzero environments using physiologic and genomic methods. We chose P. arcticus as our model because of its growth at subzero temperatures and widespread prevalence in permafrost. This paper focuses on the more novel potential adaptations.     

Cathepsins F and S block HDL3-induced cholesterol efflux from macrophage foam cells

In atherosclerosis, accumulation of cholesterol in macrophages may partially depend on its defective removal by high-density lipoproteins (HDL). We studied the proteolytic effect of cathepsins F, S, and K on HDL(3) and on lipid-free apoA-I, and its consequence on their function as inductors of cholesterol efflux from cholesterol-filled mouse peritoneal macrophages in vitro. Incubation of HDL(3) with cathepsin F or S, but not with cathepsin K, led to rapid loss of prebeta-HDL, and reduced cholesterol efflux by 50% in only 1min. Cathepsins F or K partially degraded lipid-free apoA-I and reduced its ability to induce cholesterol efflux, whereas cathepsin S totally degraded apoA-I, leading to complete loss of apoA-I cholesterol acceptor function. These results suggest that cathepsin-secreting cells induce rapid depletion of lipid-poor (prebeta-HDL) and lipid-free apoA-I and inhibit cellular cholesterol efflux, so tending to promote the formation and maintenance of foam cells in atherosclerotic lesions.     

Experimental prediction of the natural evolution of antibiotic resistance

The TEM family of beta-lactamases has evolved to confer resistance to most of the beta-lactam antibiotics, but not to cefepime. To determine whether the TEM beta-lactamases have the potential to evolve cefepime resistance, we evolved the ancestral TEM allele, TEM-1, in vitro and selected for cefepime resistance. After four rounds of mutagenesis and selection for increased cefepime resistance each of eight independent populations reached a level equivalent to clinical resistance. All eight evolved alleles increased the level of cefepime resistance by a factor of at least 32, and the best allele improved by a factor of 512. Sequencing showed that alleles contained from two to six amino acid substitutions, many of which were shared among alleles, and that the best allele contained only three substitutions.     

Transients in acetylcholine receptor site density and degradation during reinnervation of mouse sternomastoid muscle

The degradation rates of acetylcholine receptors (AchRs) were evaluated at the neuromuscular junction during and just after reinnervation of denervated muscles. When mouse sternomastoid muscles are denervated by multiple nerve crush, reinnervation begins 2-4 days later and is complete by day 7-9 after the last crush. In fully innervated muscles, the AChR degradation rate is stable and slow (t1/2 approximately 10 days), whereas after denervation the newly inserted receptors degrade rapidly (t1/2 approximately 1.2 days). The composite profile of degradation, which a mixture of the stable and the rapid receptors would give, is not observed during reinnervation. Instead, the receptors inserted between 2.5 and 7.5 days after the last crush all have an intermediate degradation rate of t1/2 approximately 3.7 days with standard error +/- 0.3 days. The total receptor site density at the endplate was evaluated during denervation and during reinnervation. As predicted theoretically, the site density increased substantially, but temporarily, after denervation. An analogous deleterious substantial decrease in density would be expected during reinnervation, without the intermediate receptor. This decrease is not observed, however, because of a large insertion rate at intermediate times (3000 +/- 700 receptor complexes per micro m2 per day). The endplate density of receptors thus remains relatively constant.     

Nesprin-1alpha self-associates and binds directly to emerin and lamin A in vitro

The mitogen-stimulated protein kinase p70(s6k)/p85(s6k) (S6K) plays an essential role in cell proliferation and growth, with inhibitors of the S6K signalling pathway showing promise as anti-tumour therapeutics. Here, we report that the bisindolylmaleimide derivative Ro 31-6045, previously reported to be inactive as a kinase inhibitor, inhibited S6K activity in vivo with an IC50=8 microM. Structure/function analysis using mutant forms of S6K indicates that Ro 31-6045 inhibition is independent of the upstream activator mTOR. Ro 31-6045 will prove useful in elucidating the complex activation mechanism of S6K and its independence from mTOR will allow confirmation of functional data obtained using the mTOR inhibitor rapamycin.     

Current perspectives on the mechanism of action of artemisinins

Artemisinin derivatives are the most recent single drugs approved and introduced for public antimalarial treatment. Although their recommended use is for treatment of Plasmodium falciparum infection, these drugs also act against other parasites, as well as against tumor cells. The mechanisms of action attributed to artemisinin include interference with parasite transport proteins, disruption of parasite mitochondrial function, modulation of host immune function and inhibition of angiogenesis. Artemisinin combination therapies are currently the preferred treatment for malaria. These combinations may prevent the induction of parasite drug resistance. However, in view of the multiple mechanisms involved, especially when additional drugs are used, the combined therapy should be carefully examined for antagonistic effects. It is now a general theory that the crucial mechanism is interference with plasmodial SERCA. Therefore, future development of resistance may be associated with overproduction or mutations of this transporter. However, a general mechanism, such as alterations in general drug transport pathways, is feasible. In this article, we review the evidence for each mechanism of action suggested.