At which temperature did life originate and where is the root of the organismal tree?

Nicolas Glansdorff

Department of Microbiology, Free University of Brussels, Belgium

The current "textbook" view of the universal organismal tree points to a procaryotic-like and extreme thermophilic last common ancestor ( LCA ) for the three Domains of life: Archaea, Bacteria and Eucarya. We will argue , on the contrary, that the LCA probably was a non-thermophilic protoeucaryote from which the two procaryotic domains emerged by reductive evolution.

Molecular adaptations of enzymes to extreme temperatures

Charles Gerday

Laboratory of Biochemistry – Institute of Chemistry B6, University of Liège – Sart-Tilman, B4000 Liège – Belgium

Microorganisms adapted to extreme temperature environments are of major importance since the environments of low and high temperatures are particularly abundant at the surface of our planet. These organisms successfully grow and breed within a temperature range of about –2°C to 115°C and are called psychrophiles and thermophiles respectively.
· How can macromolecular structure such as proteins be stable at temperatures higher than 100°C ?
· How is it possible to display appropriate metabolic fluxes at temperatures as low as –2°C ?
In thermophiles, the selective pressure is exerted towards stability factors. In order to acquire a good equilibrium between flexibility and stability, the stabilisation energy of thermophilic enzymes must increase by about 40 kJ/mole. This is achieved through multiple ways leading to a reinforcement of the intramolecular interactions network. Due to their high stability thermophilic enzymes have usually a low specific activity.
At the opposite, psychrophilic enzymes have to cope with the exponential decrease of reaction rates with temperature. The strategy is to improve the flexibility of the proteins reducing in this way the energy cost of induced fit mechanisms.
All the cold-adapted enzymes investigated display two general properties :
· a high specific activity at low and moderate temperature
· a high thermosensitivity
The case of an alpha-amylase originating from an Antarctic bacteria will be discussed.
The adaptation strategy depends on the nature of the chemical reaction to be catalyzed and in particular, on the size of the substrate. Commonly however, the adaptation leads to a decrease of the activation enthalpy partially compensated by a decrease in the activation entropy.

Transport systems for transition metals, heavy metals and metalloids.

Barry P. Rosen

Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201, USA.

Life may have first arisen in deep oceanic hydrothermal vents that were rich in metals such as arsenic, lead, copper and zinc. Maintaining suitable intracellular concentrations of essential metals such as copper and zinc while excluding toxic metals such as arsenic, lead and cadmium was one of the earliest challenges of the first cells. This ancient environmental challenge has been the driving force for the evolution of mechanisms for metal ion homeostasis and detoxification. Even today toxic metals such as arsenic enter the ecosphere from geochemical sources (http://co.water.usgs.gov/trace/arsenic/). It is little wonder that in every organism examined there are transport systems that detoxify metal ions by catalyzing extrusion from the cytosol (3, 5).
This presentation will focus on transporters that provide homeostasis for required metals copper and zinc and/or confer resistance to ions of the soft metals cadmium, lead, silver, arsenic and antimony in Saccharomyces cerevisiae and Escherichia coli. In yeast there are two transporters confer resistance to As(III) and other heavy metals (1). Acr3p is a plasma membrane efflux system that is specific for As(III). Ycf1p, a member of the ABC superfamily of transport ATPases, confers resistance to As(III), Sb(III), Cd(II) and Hg(II) by sequestering the GSH conjugates of the metals into the vacuole. In E. coli CopA is a P-type ATPase that extrudes Cu(I) from cells (2). ZntA is a second P-type ATPase that catalyzes Zn(II), Pb(II) and Cd(II) efflux (4). ArsAB is a novel ATPase that pumps As(III) and Sb(III) out of the cells (6). The ars operon of E. coli plasmid R773 encodes both the ArsAB ATPase and the ArsC arsenate reductase that reduces As(V) to As(III). Aspects o! f the crystal structures and mechanisms of ArsA (7) and ArsC will be discussed.
Supported by US Public Health Service Grants GM55425 and GM52216

1. Ghosh, M., J. Shen, and B. P. Rosen. 1999. Pathways of As(III) detoxification in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 96:5001-5006.
2. Rensing, C., B. Fan, R. Sharma, B. Mitra, and B. P. Rosen. 2000. CopA: an Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci USA 97:652-656.
3. Rensing, C., M. Ghosh, and B. P. Rosen. 1999. Families of soft metal ion transporting ATPases. J Bacteriol 181(19):5891-5897.
4. Rensing, C., B. Mitra, and B. P. Rosen. 1997. The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase. Proc Natl Acad Sci U S A 94(26):14326-31.
5. Rosen, B. P. 1999. Families of arsenic transporters. Trends Microbiol 7:207-212.
6. Rosen, B. P., H. Bhattacharjee, T. Zhou, and A. R. Walmsley. 1999. Mechanism of the ArsA ATPase. Biochim Biophys Acta 1461(2):207-15.
7. Zhou, T., S. Radaev, B. P. Rosen, and D. L. Gatti. 2000. Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump. Embo J 19(17):1-8.

Biomarkers in fish gonads to detect pollution in freshwater fish

Barnhoorn, IEJ, Van Vuren, JHJ, Pieterse, GM & GJ Steyn

Department of Zoology, Rand Arfikaans University, PO Box 524, Auckland Park, 2006, South Africa

The survival of any species in a specific environment depends on successful reproduction. Little information is available on the effect of metal pollutants on gonad structure and function in freshwater fish. Biomarkers to assess the effects of metal pollution on gametogenesis can be useful in the determination of critical levels of aquatic pollution.
In recent studies different biomarkers in the gonads of Oreochromis mossambicus were investigated. Sub-lethal experiments were conducted to assess the effects of iron on selected enzyme levels in the gonads. Gonads of fish sampled from polluted environments were also analysed. The selected enzymes could not in all instances be positively identified as biomarkers. Techniques employed make it now possible to determine the changes in testis structure during sperm cell development. This information on abnormalities in the testis can be used as a biomarker to determine damage to the reproductive ability of O. mossambicus. Experiments were carried out to determine if HSP 70 expression could be a sensitive indicator of stress caused by metal pollution in the gonads of male and female O. mossambicus, and limited success has been achieved.

Common loons and methyl mercury: characterizing exposure and quantifying effects

William H. Karasov¹, François Fournier^1,4, Kevin Kenow², and Michael W. Meyer³

¹ Department of Wildlife Ecology, 226 Russell Labs, University of Wisconsin-Madison, Madison, WI, 53706
² Upper Midwest Environmental Sciences Center, USGS-Biological Resources Division, 2630 Fanta Reed Road, La Crosse, WI 54603
³ Wisconsin Department of Natural Resources, 107 Sutliff Avenue, Rhinelander, WI 54501
⁴ Present address: Département de biologie, Université Laval, Ste-Foy, Québec, Canada G1K 7P4

Common loons nesting on low-pH lakes in northern Wisconsin in North America received greater Hg exposure than loons nesting on neutral-pH lakes, and production of loon chicks was significantly lower at lakes where chicks had elevated blood Hg levels. The putative cause-effect linkages suggested by these correlations were tested in two kinds of controlled dosing studies with loon chicks.
In toxicokinetic studies we determined that the bioavailability of methyl Hg in fish prey was 85%, similar to values reported in laboratory animals. The half-time for elimination of methyl Hg from the plasma compartment was much shorter in younger loons aged 35-80 days than older loons aged >84 days, probably because feather growth in the younger birds effectively added an extra pathway of Hg elimination. Steady state plasma methyl Hg concentrations were modeled as a function of fish Hg concentration using a 2-compartment model and then compared with measurements in captive loons fed fish with Hg concentrations ranging 0.1 - 0.5 µg/g.
Using the same captives in toxicodynamic studies, we tested for behavioral and physiological impacts of methyl Hg. The results of these experiments, along with additional observations of wild loons, will help us evaluate the likely ecological consequences of exposure at different levels and test whether the correlations in the field between Hg exposure and loon performance reflect a cause-effect relationship. It is conceivable to develop models of environmental transport and fate of Hg linked to models of exposure and toxicity in fish and then piscivores such as loons. This approach will lead to improved, scientifically defensible regulatory goals and a detailed understanding of how, and at what cost, various regulatory scenarios will achieve those goals.

The use of multivariate statistical analyses to elucidate biomarker responses in Mytilus edilus along a pollution gradient in the Scheldt Estuary

V. Wepener¹, R. Blust² and W. De Coen²

¹Department of Zoology, Rand Afrikaans University, P.O. Box 524, Aucklanpark, 2006, South Africa
²Dept. of Biology, University of Antwerp (RUCA), 171 Groenenborgerlaan Antwerp, 2020, Belgium.
(vw@na.rau.ac.za)

The use of biomarkers are currently being advocated as a possible bioindicator of environmental conditions. However, the potential can only be realised once specific biomarkers can be linked to higher levels of effects and shown to provide early warning of adverse effects, and not just exposure. It is recognized that a single biomarker should not be used in isolation, as no single biomarker is sensitive to all pollutants. It is therefore necessary to develop and/or adapt tools to interpret biomarker data. One such tool that is currently being applied with success involves using multi dimensional scaling statistical techniques. The application of this technique was used to assess field-based biomarker data from resident mussel populations along a pollution gradient in the Scheldt Estuary. The biomarkers used in this study included condition index, scope for growth, survival in air, cell membrane stability, DNA damage and metallothionein concentrations. Similarity matrices and multi dimensionally scaling were used to highlight differences in responses. Although the chemical analyses of the water samples indicated a gradient in metal pollution from the upper reaches of the Scheldt to the mouth, but the biomarker responses did not display the same pattern. Possible reasons for the discrepancy between biomarker responses and physico-chemical conditions are discussed.

Effects of copper on the freshwater crab, Potamonautes warreni Calman - an assesment for future research

André Vosloo¹, Willie van Aardt¹ & Japie Mienie²

¹School of Environmental Science and Development and ²School of Chemistry and Biochemistry, Potchefstroom University for CHE, Potchefstroom, 2520, South Africa

The sublethal effects of copper has been studied in the freshwater crab for a number of years. At this stage we are attempting to integrate the various effects in order to determine the impacts of heavy metal pollution on animal health. This presentation will focus only on effects in animals exposed to 1 mg Cu l^-1 at 25°C in laboratory studies, and assess the prospects for future research.

From our experiments the following mode of action of copper is proposed:
1. Lipid peroxidation is initiated within 3 hours after start of exposure and is maintained for (at least) 21 days. This is an uncontrolled breakdown with hepatopancreas free fatty acid levels (quantified and identified by GC-MS) five times above control levels. The long-term impact on survival and reproductive success needs to be assessed
2. Glycolytic action is reversed for the duration af a 7 day experiment, and ¹⁴C-glucose and ¹⁴C-glycogen is anabolized from injected ¹⁴C-lactate in an animal that is, under normal conditions, predominantly reliant on carbohydrates for energy. As the importance of carbohydrate metabolism fluctuates during the moulting cycle, impacts on different moulting stages needs to be assessed.
3. Chloride concentrations and osmotic pressure in haemolymph and urine are unchanged over a 7 day exposure period. The simplistic deduction would be that copper causes no perturbation of osmoregulatory mechanisms, but more detailed investigation gives us an idea of the strain placed on osmoregulatory mechanisms to maintain salt balance. In copper-exposed animals the kidney's salt-concentrating capability, as measured by ⁵¹Cr-EDTA experiments, is impaired, leading to an increase in urine filtration rate to prevent excess salt loss. It is unclear how ion uptake mechanisms on the gills or gill membrane permeabilities are impaired.

Mortality as a measurable end-point is not feasible in monitoring pollution impacts. We argue that integrated measures of animal health and long-term effects on reproductive success are required in order to better quantify the long-term survival of species.

Metabolic rate in freshwater fish as biomarker for metal toxicity studies

van Aardt, WJ; Hough, M; Booysen, A; Venter LCR

School of Environmental Sciences and Development, Potchefstroom University,Potchefstroom, South Africa.
(drkwjva@puknet.puk.ac.za)

Since the development of the Clark polarographic oxygen electrode in 1953 and its later use to measure the amount of oxygen consumed by aquatic organisms the method has been applied to a broad field in biology. This research reports the application and results of closed system respirometry using a Radiometer electrode (Copenhagen) to measure toxic effects of heavy metals after 96 hours exposure to fish.
Oxygen consumption rates (MO₂) from large and small specimens were calculated from regression lines and expressed per standard 30 gram fish at 20 °C.
Handling stress (using 5 or 1.8 litre capacity respirometers) on the chiclid Tilapia sparrmanii increase the oxygen consumption rate from 4.5 to 6.8 mmol O2 Kg^-1 hr^-1 but returns to resting levels after 6 hours. The same values were also found for the large mouth bass Micropteris salmoides (3.2 to 6.1) and the mud fish, Labeo capenis. (3.5 to 6.7). Crowding stress: When ten individuals from any of the three species were simultaneously subjected to measurements in a 23 litre capacity respirometer, resting MO₂ increased more than 200%.compared to individual resting MO₂ measurements.
Ten milligram copper per litre water decreased both the MO₂ and gill frequency for M. salmoides and L. capensis by about 20% in hard water from dolomitic origin. No effect on MO₂ was found when 20 or 50 milligram per litre cadmium or lead was exposed to the three fish species in dolomitic water. These two metals precipitate out completely, 96 hours after they were placed in the experimental water in a dissolved state. In very soft water the MO₂, following 10 and 20 milligram per litre cadmium exposed to T. sparrmanii decreased significantly from 4.5 mmol O₂ Kg^-1 h^-1 to less than 1 mmol O₂ Kg^-1 h^-1 indicating the toxicity of cadmium. This was not found for the same concentrations of lead dissolved in soft water at pH 8.3.
It is concluded that by careful selection of respirometer size, appropriately designed low level oxygen consumption Clark electrodes, elimination of handling stress, using large keep tanks to hold acclimatised fish in open respirometers and short MO₂ measuring times, MO₂ can be confidently used as an indicator of metal toxicity on fish for short term (96 hours) experiments.

Radioactive environment contamination and human genome damage

Valery N. Soyfer, V.V. Vlassov, I.V. Morozov and N.I. Soyfer

George Mason University, Fairfax, VA 22030, USA.
(vnsoyfer@gmu.edu)

A series of radioactive catastrophes (from 1948 to 1961) in the Southern Urals in the USSR has led to intensive environmental contamination for more than 40 years. In all, 76 million m³ of liquid radioactive wastes of general activity of beta particles 2.75 million Curies was released into the Techa River. The present work goal was to study the DNA damage at the nucleotide resolution level of the genome of the inhabitants of the river region, who presumably received chronic doses of irradiation. Studies were conducted through the direct sequencing of genes after their PCR-amplification and preselection of allegedly mutated DNA molecules. In the presented study we have sequenced the gene of dopamine receptor D1 (gene D1 subfamily of the G-protein coupled receptor L-DOPA) and the intron 12 of the gene for phenylalanine hydroxylase (PAH) responsible for phenylketonuria or hyperphenylalaninemia . Five polymorphic loci have been revealed, 2 of them being wide spread and 3 are unique. One of the 2 wide spread mutations is a deletion and the other 4 are substitutions. The distribution pattern of deletion appeared to be very unusual: wide spread heterozygotes but a complete absence of mutant homozygotes.