Use of marker genes in competition studies of Rhizobium

Use of marker genes has several advantages in studying rhizobial competition compared to traditional approaches. Reporter genes such as the ß-glucuronidase gene (gusA) or a thermostable ß-glucosidase gene (celB) allow detection of rhizobial strains in nodules when they are still attached to the root system. Analysis is extremely simple, fast and permits a high data throughput. This detection technique is therefore highly suitable for the study of rhizobial competition and studies using gusA-marked strains of Rhizobium are presented. By making use of gusA and celB, differentially marked strains can be produced and distinguished easily on roots. The availability of two marker genes permits competition studies of two or more than two strains and analysis of dual nodule occupancy. As this methodology does not require sophisticated equipment, a GUS Gene Marking Kit was developed.     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

Arctic charr in Lake Myvatn: The centennial catch record in the light of recent stock estimates

In Lake Myvatn, Arctic charr (Salvelinus alpinus) is economically the most important fish species. It is fast growing fish and its size at maturity is 35–50 cm at the age of 4 to 5 years. Catch statistics that are available for the whole 20th century show considerable long-term variation with the highest catch in the 1920s. The catches after 1970 are about 40% lower than the average from 1930–1969 and the annual catches for the last decade are the lowest. Stock size during the winter fishing season fluctuated considerably between years, with average annual fishing mortality of 83.9%. The Arctic charr population has been monitored annually since 1986, using standard series of gill nets of different mesh sizes. In 1988 one to three-year-old fish were heavily reduced in numbers during the summer months. There are indications of a similar event in 1997. In both years the charr changed its main diet from Cladocera and chironomid midges to three-spined sticklebacks Gasterosteus aculeatus and snails. At the same time its condition deteriorated. The catch in the monitoring fishery in Lake Myvatn correlates with the stock size in the beginning of the following winter fishing season deduced from Leslie's method and can give prospects for the fishery in the successive fishing season. An index of abundance of young charr also correlates with the number of chironomids and cladocerans and also with ducklings that feed on the same food as the charr. Large fluctuations in the Arctic charr population in Lake Myvatn seem to be related to changes in the main food species. The catch records available from Lake Myvatn can to a large extent be used as a measure of changes of the Arctic charr population in the lake for the past century.     

The ecology of Lake Myvatn and the River Laxá: Variation in space and time

Ecological features of Lake Myvatn and the outflowing River Laxá show a wide range of spatial and temporal variations. The physical division of the lake into three main basins and the variation in chemical composition and temperature of the artesian springs feeding this shallow productive lake have large spatial effects. Variation in groundwater characteristics depends on percolation time and proximity to geothermal sources. Variation in precipitation is evened out by the porous volcanic soil and bedrock and the spring-water discharge is therefore very stable. A pulse of volcanic activity in 1975–1984 (the Krafla Fires) heated the groundwater entering the North Basin of the lake and changed its chemistry. Although much reduced, these effects have not disappeared yet, but overall the impact of the volcanic activity on the biota seemed minimal. Recycling of nutrients through internal loading is important and occurs on various time scales. In winter, when the lake is ice-covered, the topmost 5-cm layer of sediment pore water has a hundredfold concentration of nutrients relative to the overlying lake water. The nutrients are released during the ice-free period by sediment resuspension, diffusion, bioturbation and recycling. In spring, resuspension events sometimes lead to spikes in dissolved phosphorus and nitrogen, but there is little evidence of any major desorption of nutrients from suspended particles during such events later in the summer. In contrast to the stable groundwater, the biota show more or less regular fluctuations with no straightforward correlation with external signals. The most prominent fluctuations, those of the chironomid Tanytarsus gracilentus seem to be driven by interactions between the species and its sediment resources. Fluctuations in other invertebrates could be a consequence of the Tanytarsus cycles due to the large impact this species has on the benthic environment of this detritus-driven ecosystem. Temporal variation in epibenthic chironomids and Cladocera translates into variable production of vertebrate predators (Arctic charr, Salvelinus alpinus, and ducks), body condition and mortality of fish and sometimes into return rates of migrating adult ducks. The waterfowl show large temporal variation on a centennial scale, e.g., the invasion of the tufted duck (Aythya fuligula) which arrived by the end of the 19th century and has by now outnumbered other species. Fluctuations of Cyanobacteria (Anabaena) and the fish Gasterosteus aculeatus (three-spined stickleback) harmonize with the cycles in the benthic community. Palaeolimnological studies indicate that primary production in the South Basin became increasingly benthic as the lake depth was reduced by sedimentation (around 2 mm year^–1). Other trends include a decrease in Tanytarsus and Daphnia and an exponential increase in green algae (Cladophorales, Pediastrum) and associated organisms.     

Isolation of thermophilic obligately autotrophic hydrogen-oxidizing bacteria,similar to Hydrogenobacter thermophilus,from Icelandic hot springs

Thermophilic obligately autotrophic hydrogen-oxidizing bacteria were isolated from several alkaline hot springs in Iceland. The bacteria were Gram negative rods, 0.4–0.5 m in diameter and 3–4 m long but 6–7 m long cells without septa were often seen. Long and short laments are formed. Spores, flagella or lipid granules were not observed. Strains H1 and H12 grew optimally at 70° C and pH 6.5 under mixture of air plus 0.6 atm H₂ and 0.1 atm CO₂. The cells contained cytochromes and carotenoid-like pigments. They would not grow on agar or silicia gel plates. The cells would not grow heterotrophically on organic substrates and were inhibited by most of these same organic compounds and agar in low concentrations. They were very sensitive to common antibiotics. The role of these bacteria in the hot spring ecosystem is discussed.     

Populations of ducks and trout of the River Laxá, Iceland, in relation to variation in food resources

We examined annual variation in production, recruitment and density of the three most abundant vertebrate species of the River Laxá at Lake Myvatn, Iceland: Barrow’s goldeneye, Bucephala islandica, harlequin duck, Histrionicus histrionicus, and brown trout, Salmo trutta, in relation to food resources and other environmental variables. The study is largely based on correlations from long-term monitoring series in the period 1975–2002. Production of young in the harlequin duck was significantly correlated with food resources (the blackfly, Simulium vittatum) of the river, as was the recruitment of brown trout to the angling stock. In Barrow’s goldeneye, which uses both the lake and the river, dispersion of adults in spring and young in August was influenced by the availability of aquatic insects in each habitat. The dispersion of Barrow’s goldeneye tracks the availability of aquatic insects in each of these two main habitats. Introduced Amercian mink, Mustela vison, may have affected spring numbers and dispersion of harlequin ducks, but the evidence was not conclusive. Numbers of both duck species and the trout (as CPUE) were relatively stable, although a sharp drop in numbers followed by slow recovery was observed in Barrow’s goldeneye, and an increase was observed in harlequin ducks in the first year of study.     

Characterization of alanine and malate dehydrogenases from a marine psychrophile strain PA-43

Alanine dehydrogenase (AlaDH: EC 1.4.1.1), malate dehydrogenase (MDH: EC 1.1.1.37), and glutamate dehydrogenase (EC 1.4.1.2), all NAD+ dependent, were detected in extracts from a psychrophilic bacterium, strain PA-43, isolated from a sea urchin off the Icelandic coast. Characterization tests suggested that the strain had a close relationship to Vibrio, but sequencing of part of the 16S rDNA gene placed the bacterium among Shewanella species in a constructed phylogenetic tree. The bacterium had an optimum growth temperature of 16.5 degrees C, and maximum dehydrogenase expression was obtained in a rich medium supplemented with NaCl. Both AlaDH and MDH were purified to homogeneity. AlaDH is a hexamer, with an approximate relative molecular mass of 260,000, whereas MDH is dimeric, with an apparent relative molecular mass of approximately 70,000. Both enzymes were thermolabile, and the optimum temperatures for activity were shifted toward lower temperatures than those found in the same enzymes from mesophiles, 37 degrees C for MDH and approximately 47 degrees C for AlaDH. The pH optima for AlaDH in the forward and reverse reactions were 10.5 and 9, respectively, whereas those for MDH were 10-10.2 and 8.8, respectively. Partial amino acid sequences, comprising approximately 30% of the total sequences from each enzyme, were determined for N-terminal, tryptic, and chymotryptic fragments of the enzymes. The AlaDH showed the highest similarity to AlaDHs from the psychrotroph Shewanella Ac10 and the mesophile Vibrio proteolyticus, whereas MDH was most similar to the MDHs from the mesophiles Escherichia coli and Haemophilus influenzae, with lower identity to the psychrophilic malate dehydrogenases from Vibrio 5710 and Photobacterium SS9.     

Alanine dehydrogenase from the psychrophilic bacterium strain PA-43: overexpression,molecular characterization,and sequence analysis

The gene encoding alanine dehydrogenase (AlaDH; EC 1.4.1.1) from the marine psychrophilic bacterium strain PA-43 was cloned, sequenced, and overexpressed in Escherichia coli. The primary structure was deduced on the basis of the nucleotide sequence. The enzyme subunit contains 371 amino acid residues, and the sequence is 90% and 77% identical, respectively, to AlaDHs from Shewanella Ac10 and Vibrio proteolyticus. The half-life of PA-43 AlaDH at 52 degrees C is 9 min, and it is thus more thermolabile than the AlaDH from Shewanella Ac10 or V. proteolyticus. The enzyme showed strong specificity for NAD(+) and l-alanine as substrates. The apparent K(m) for NAD(+) was temperature dependent (0.04 mM-0.23 mM from 15 degrees C to 55 degrees C). A comparison of the PA-43 deduced amino acid sequence to the solved three-dimensional structure of Phormidium lapideum AlaDH showed that there were likely to be fewer salt bridges in the PA-43 enzyme, which would increase enzyme flexibility and decrease thermostability. The hydrophobic surface character of the PA-43 enzyme was greater than that of P. lapideum AlaDH, by six residues. However, no particular modification or suite of modifications emerged as being clearly responsible for the psychrophilic character of PA-43 AlaDH.     

Distribution of Thermus spp. in Icelandic Hot Springs and a Thermal Gradient

The growth range in nature of bacteria belonging to the genus Thermus was investigated by sampling 55 different hot springs in Iceland. The springs ranged in temperature from 32 to 99°C, and in pH from 2.1 to 10.1. Viable counts of Thermus spp. ranging from 10 to 10⁴ CFU/100 ml of spring water were found in 27 of the springs sampled. The temperature range for these bacteria was found to be 55 to 85°C, and the pH range was from about 6.5 to above 10. Thermus spp. were found in springs containing up to 1 mM dissolved sulfide and having conductivity up to 2,000 μS/cm. The distribution of Thermus spp. in a hot spring thermal gradient was also investigated and found to agree well with the overall distribution in individual springs.     

Estimating crop N uptake from organic residues using a new approach to the 15N isotope dilution technique

Experiments were conducted to test a new approach to the ¹⁵N isotope dilution technique for estimating crop N uptake from organic inputs. Soils were pre-labelled with ¹⁵N fertiliser and a carbon source. These were then incubated until there was stabilisation of the ¹⁵N abundance of the inorganic N pool and resumption of inorganic N concentrations. Residues were then applied to the soils and planted with ryegrass (Lolium perenneL.) to determine the nitrogen derived from the residue (Ndfr) using the isotope dilution equations. This method was compared with the direct method, i.e. where ¹⁵N-labelled residues were added to the soil and Ndfr in the ryegrass calculated directly. Estimates of percentage nitrogen derived from the residue (%Ndfr) alfalfa (Medicago sativaL.) in the ryegrass, were similar, 22 and 23% for the direct and soil pre-labelling methods, respectively, in the Wechsel sandy loam. Also, estimates of the %Ndfr from soybean (Glycine max (L.) Merr) residues in the Krumbach sandy loam were similar 34% (direct) and 36% (soil pre-labelling approach). However, in the Seibersdorf clay loam, the %Ndfr from soybean was 49% using the direct method and 61% using the soil pre-labelling method; yet Ndfr from common bean residue was 46% using the direct approach and 40% using the pre-labelling, not significantly different (P > 0.05). The soil pre-labelling approach appears to give realistic values for Ndfr. It was not possible to obtain an estimate of Ndfr using the soil pre-labelling method from the maize residues (Zea mays L.) in two of the soils, as there was no increase in the total N of the ryegrass over the growing period. This was probably due to microbial immobilisation of inorganic N, as a result of the wide C:N ratio of the residue added. The results suggest that the new soil pre-labelling method is feasible and that it is a potentially useful technique for measuring N release from a wide range or organic residues, but it requires further field-testing. This revised version was published online in June 2006 with corrections to the Cover Date.