Selecting representative model micro-organisms

Background  

Micro-biological research relies on the use of model organisms that act as representatives of their species or subspecies, these are frequently well-characterized laboratory strains. However, it has often become apparent that the model strain initially chosen does not represent important features of the species. For micro-organisms, the diversity of their genomes is such that even the best possible choice of initial strain for sequencing may not assure that the genome obtained adequately represents the species. To acquire information about a species' genome as efficiently as possible, we require a method to choose strains for analysis on the basis of how well they represent the species.     

Influence of salt concentration and temperature on the fatty acid compositions of Ectothiorhodospira and other halophilic phototrophic purple bacteria

Influences of the salt concentration on the fatty acid composition of Ectothiorhodospira species and other phototrophic purple bacteria have been analysed. Major fatty acids in bacteria of the genera Rhodobacter, Rhodopseudomonas, Chromatium, and Ectothiorhodospira were straight chain saturated and monounsaturated C-16 and C-18 fatty acids. Salt-dependent responses of all investigated bacteria revealed relations to their salt optima. Minimum values of C-16 and saturated fatty acids and maximum values of C-18 and unsaturated fatty acids were found at or close to the salt optima. Responses of Ectothiorhodospira mobilis upon changes in salinity were nearly identical, whether cells were grown in batch culture or in continuous culture with identical dilution rates at all salt concentrations. With increasing temperature, the fatty acid composition of Ectothiorhodospira mobilis and Ectothiorhodospira halophila strains showed decreasing portions of C-18 and of unsaturated fatty acids, while the contents of C-16 and saturated fatty acids increased. The results are discussed with respect to bilayer stabilisation and membrane fluidity.Abbreviations PC phosphatidylcholine - PG phosphatidylglycerol - CL cardiolipin - PE phosphatidylethanolamine     

Correlation of viable cell counts,metabolic activity of sulphur-oxidizing bacteria and chemical parameters of marine sediments

Viable counts of aerobic and anaerobic chemotrophic sulphur-oxidizers as well as phototrophic sulphur bacteria were determined in sediment samples taken from two different areas along the Baltic Sea shore which were known to regularly develop sulphidic conditions. Depth profiles of bacterial cell counts were correlated with concentration profiles of chloride, sulphate, sulphide, nitrate and phosphate in the pore water of these sediments and with potential activities of nitrate reduction, thiosulphate transformation and sulphate formation. The data revealed a complex multilayered structure within the sediments. Sulphide was released into the water from sediments of both sampling areas, but it was found that light and the availability of oxygen significantly reduced this amount. In the highly reduced sediment at Hiddensee, the highest numbers of phototrophic and chemotrophic sulphur-oxidizers were found near the sediment surface. Therefore, it was concluded that the combined action of both groups of bacteria most efficiently oxidizes reduced sulphur compounds in the top layers of the sediments. Nitrate may replace oxygen as final electron acceptor and will support oxidation of sulphide, in particular when oxygen and light are limiting.     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.     

The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.     

Ectothiorhodospira vacuolata sp. nov., a new phototrophic bacterium from soda lakes

A new phototrophic bacterium was isolated from Jordanian and Kenyan alkaline salt lakes. Cells are rod shaped, 1.5 m wide and 2–4 m long, and motile by polar flagella. They divide by binary fission, and possess photosynthetic membranes as lamellar stacks similar to those in the other species of the genus Ectothiorhodospira and the brown colored Rhodospirillum species. The presence of bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series is indicated by the absorption spectra of living cells. Under certain growth conditions the cells form gas vacuoles, may become immotile and float to the top of the culture medium. Sulfide and thiosulfate are used as photosynthetic electron donors. During the oxidation of sulfide to sulfate, elemental sulfur is formed, which is accumulated outside the cells. The organisms are strictly anaerobic, do not require vitamins, are moderately halophilic and need alkaline pH-values for growth. The new species Ectothiorhodospira vacuolata is proposed.     

Sulfate assimilation in Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to grow on sulfate as sole source of sulfur, but only at concentrations below 1 mM. Good growth was observed with thiosulfate, cysteine or methionine as sulfur sources. Tetrathionate supported slow growth. Sulfide and sulfite were growth inhibitory. Growth inhibition by higher sulfate concentrations was overcome by the addition of O-acetylserine, which is known as derepressor of sulfate-assimilating enzymes, and by reduced glutathione. All enzymes of the sulfate assimilation pathway. ATP-sulfurylase, adenylylphosphate-sulfotransferase, thiosulfonate reductase and O-acetylserine sulfhydrylase are present in R. globiformis. Sulfate was taken up by the cells and the sulfur incorporated into the amino acids cysteine, methionine and homocysteine. It is concluded, that the failure of R. globiformis to grow on higher concentrations of sulfate is caused by disregulation of the sulfate assimilation pathway. Some preliminary evidence for this view is given in comparing the activities of some of the involved enzymes after growth on different sulfur sources and by examining the effect of O-acetylserine on these activities.Abbreviations DTE dl-dithioerythritol - APS adenosine 5-phosphosulfate, adenylyl sulfate - PAPS 3-phosphoadenosine 5-phosphosulfate, 3-phosphoadenylylsulfate     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

The Wadi Natrun: Chemical composition and microbial mass developments in alkaline brines of Eutrophic Desert Lakes

Six lakes of the Wadi Natrun, Egypt, were studied with respect to the chemical composition of their brines and the occurrence of microbial mass developments. All investigated lakes showed pH values of approximately 11 and a total salt content of generally more than 30%. The main components were sulfate, carbonate, chloride, sodium, and minor amounts of potassium. Only traces of magnesium and calcium were present, but unusually high concentrations of organic carbon compounds, nitrogen compounds, and phosphate were found. Mass developments of phototrophic sulfur bacteria, halobacteria, cyanobacteria, and green algae were observed. The functions of complete nitrogen and sulfur cycles in the alkaline brines are discussed. The properties of the lakes and their ecology are compared with data on the Dead Sea and Great Salt Lake, Utah.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.