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.     

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.     

Methylenetetrahydrofolate reductase gene C677T polymorphism,homocysteine, vitamin B12, and DNA damage in coronary artery disease

Elevated levels of plasma homocysteine (Hcy), a risk factor for coronary artery disease (CAD), can result from genetic errors, e.g., the methylenetetrahydrofolate reductase (MTHFR) polymorphism, or nutritional deficiencies, e.g., in vitamin B12 and folate. The mechanism by which Hcy induces atherosclerosis is not fully understood. Recently, Hcy has also been observed to induce DNA damage. In this study, we have investigated whether DNA damage is related to the C677T variant in the MTHFR gene and to plasma levels of Hcy, B12, and folate in patients with CAD. Patients ( n=46) with angiographically proven CAD were studied by using the micronucleus (MN) test, an accepted method for evaluating genetic instability. TT patients had plasma Hcy levels higher than those with the CT or CC genotypes (27.8+/-5.2 vs 13.7+/-2.2 and 12.9+/-1.9 micro mol/l, respectively; P=0.02). Patients with multi-vessel disease had higher plasma Hcy levels (11.6+/-1.2, 22.0+/-4.7, 19.3+/-3.9 micromol/l for one-, two- and three-vessel disease, respectively; P=0.05). The MN index increased with the number of affected vessels (8.4+/-0.7, 11.1+/-2.0, 14.2+/-1.7 for one-, two-, and three-vessels disease, respectively; P=0.02) and was significantly higher in subjects with the TT genotype compared with the CC or CT genotypes (15.7+/-2.4 vs 8.9+/-1.7 and 9.9+/-0.8; P=0.02). The MN index was also correlated negatively with plasma B12 concentration ( r=-0.343; P=0.019) and positively with plasma Hcy ( r=0.429, P=0.005). These data indicate that the MN index is associated with the severity of CAD and is related to the MTHFR polymorphism, suggesting an interesting link between coronary atherosclerosis and genetic instability in humans.     

Endogenous retroviral insertion in Cryge in the mouse No3 cataract mutant

No3 (nuclear opacity 3) is a novel congenital nuclear cataract in mice. Microsatellite mapping placed the No3 locus on chromosome 1 between D1Mit480 (32cM) and D1Mit7 (41cM), a region containing seven crystallin genes; Cryba2 and the Cryga-Crygf cluster. Although polymorphic variants were observed, no candidate mutations were found for six of the genes. However, DNA walking identified a murine endogenous retrovirus (IAPLTR1: ERVK) insertion in exon 3 of Cryge, disrupting the coding sequence for gammaE-crystallin. Recombinant protein for the mutant gammaE was completely insoluble. The No3 cataract is mild compared with the effects of similar mutations of gammaE. Quantitative RT-PCR showed that gammaE/F mRNA levels are reduced in No3, suggesting that the relatively mild phenotype results from suppression of gammaE levels due to ERVK insertion. However, the severity of cataract is also strain dependent suggesting that genetic background modifiers also play a role in the development of opacity.     

PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans embryos

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Highlights? PP2A phosphatase is essential for centriole formation in C. elegans ? PP2A subunits genetically and physically interact with the SAS-5/SAS-6 complex ? PP2A-mediated dephosphorylation of SAS-5 is required for SAS-5/SAS-6 centriolar targeting ? Human PP2A is required for HsSAS-6 centriolar targeting and centriole formation     

Sexually dimorphic expression of secreted frizzled-related (SFRP) genes in the developing mouse Müllerian duct

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.