Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: Complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin

Summary The ¹H, ¹⁵N and ¹³C backbone and ¹H and ¹³C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, ³J_Hn_H coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel -sheet and five -helices. One of the outer regions of the -sheet shows no regular extended conformation, whereas the outer strand 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five -helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the -helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn¹¹, Ser⁶⁸ and Asn⁷² are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.Abbreviations SQ semiquinone - FMN riboflavin 5-monophosphate; nif, nitrogen fixation - TSP 3-(trimethylsilyl)propionate sodium salt - DSS 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt Supplementary Material is available on request, comprising a Materials and Methods section for the expression and purification of the A. chroococcum flavodoxin, a Table S1 containing the parameters of the titration of A. chroococcum flavodoxin with the Fe protein, and a Table S2 containing the ¹⁵N, H^N, ¹³C, ¹H, ¹³C, ¹H and ¹³CO chemical shifts.To whom correspondence should be addressed.     

The Influence of Genotype and Environment on the Physiological and Metabolic Diversity ofFusarium compactum

Talbot, N. J., Vincent, P., and Wildman, H. G. 1996. The influence of genotype and environment on the physiological and metabolic diversity ofFusarium compactum. Fungal Genetics and Biology20,254–267. Fungal species produce a large variety of secondary metabolites which are of considerable interest to the pharmaceutical industry. It is clear that the secondary metabolite production of a species varies significantly in strains from different geographic locations and from different habitats. The influence of genotype and environment on metabolite production is, however, poorly understood. In this study we examined the influence of genotypic variability, physiological variability, environmental location, and habitat on metabolite production byFusarium compactum.Isolates of the fungus from two geographic locations and two distinct habitat types were examined for growth on 95 different carbon sources, and genotypic variability was determined using RAPDs and rDNA–RFLP analysis. In a blind test secondary metabolite production was assessed using HPLC profiles of methanolic cell extracts. A number of correlations were observed between genotypic groupings, as determined using parsimony, and specific metabolite production. Similar correlations were also observed with physiological groups although genotypic analysis proved to be a more sensitive predictor of metabolite variability. The data suggest a complex relationship between environment, genotype, and metabolite production but highlight the use of genetic screening as a means of optimizing the chances of identifying a wide range of metabolites from a given species.     

The impact of mycorrhizal colonization upon nitrogen source utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook

Analysis of soil solution from forest sites dominated by Eucalyptus grandis and Eucalyptus maculata indicates that soluble forms of organic nitrogen (amino acids and protein) are present in concentrations similar to those of mineral nitrogen (nitrate and ammonium). Experiments were conducted to determine the extent to which mycorrhizal associations might broaden nitrogen source utilization in Eucalyptus seedlings to include organic nitrogen. In isolation, species of ectomycorrhizal fungi from northern Australia show varying abilities to utilize mineral and organic forms of nitrogen as sole sources. Pisolithus sp. displayed strongest growth on NH₄⁺, glutamine and asparagine, but grew poorly on protein, while Amanita sp. grew well both on mineral sources and on a range of organic sources (e.g. arginine, asparagine, glutamine and protein). In sterile culture, non-mycorrhizal seedlings of Eucalyptus grandis and Eucalyptus maculata grew well on mineral sources of nitrogen, but showed no ability to grow on sources of organic nitrogen other than glutamine. In contrast, mycorrhizal seedlings grew well on a range of organic nitrogen sources. These observations indicate that mycorrhizal associations confer on species of Eucalyptus the ability to broaden their resource base substantially with respect to nitrogen. This ability to utilize organic nitrogen was not directly related to that of the fungal symbiont in isolation. Seedlings mycorrhizal with Pisolithus sp. were able to assimilate sources of nitrogen (in particular histidine and protein) on which the fungus in pure culture appeared to grow weakly. Experiments in which plants were fed ¹⁵N-labelled ammonium were undertaken in order to investigate the influence of mycorrhizal colonization on the pathway of nitrogen metabolism. In roots and shoots of all seedlings, ¹⁵N was incorporated into the amide group of glutamine, and label was also found in the amino groups of glutamine, glutamic acid, γ-aminobutyric acid and alanine. Mycorrhizal colonization appeared to have no effect on the assimilation pathway and metabolism of [¹⁵N]H₄⁺; labelling data were consistent with the operation of the glutamate synthase cycle in plants infected with either Pisolithus sp. (which in isolation assimilates via the glutamate synthase cycle) or Elaphomyces sp. (which assimilates via glutamate dehydrogenase). It is likely that the control of carbon supply to the mycorrhizal fungus from the host may have a profound effect on both the assimilatory pathway and the range of nitrogen sources that can be utilized by the association.     

Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460     

Colonization of artificial substrates by benthos in a second-order high altitude river in Kenya

An investigation on the colonization of artificial substrate baskets by benthos was carried out in the Naro Moru River, a second-order high altitude river in Kenya, from November, 1986 to October, 1987. Simuliidae dominated the colonizing benthos. The mean (±95% CL) maximum colonization time was achieved after 9.63±1.57 days. The highest mean diversity (H) occurred after 6 days of exposure whilst the highest mean H_maximum, evenness (J) and species richness (s) were achieved after 10, 8 and 10 days respectively.The benthos arrival rate (AR) declined exponentially with time. The changes in benthos departure rate (DR) with time was insignificant. Equilibrial state (AR:DR=1.0) was not achieved in the present study.     

Gas phase composition effects on suspension cultures of Taxus cuspidata

The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. (c) 1995 John Wiley & Sons, Inc.     

THE FOUNDING OF A NEW POPULATION OF DARWIN'S FINCHES

We report the natural colonization of the small Galápagos island Daphne Major by the large ground finch (Geospiza magnirostris). Immigrants of this species were present in every year of a 22-yr study, 1973–1994. Typically they arrived after a breeding season and left at the beginning of the next one. Geospiza magnirostris bred on the island for the first time in the exceptionally wet El Niño year of 1982–1983, and bred in all subsequent years except drought years. In agreement with theoretical expectations the frequency of inbreeding was unusually high. Pronounced fluctuating asymmetry in tarsus length, together with slightly reduced breeding success of inbreeding pairs, suggests a low level of inbreeding depression. Despite this, the population increased from 5 breeding individuals in 1983 to 20 breeding individuals in 1992, and probably more than twice that number in 1993, largely through recruitment of locally born birds.     

Distribution and production ofChironomus salinarius (Diptera: Chironomidae) in a shallow coastal lagoon in the Bay of Cádiz

The abundance, generation time and production ofChironomus salinarius larvae in a lagoon fish-pond system in the Bay of Cádiz were studied by taking monthly samples at 3 sites during 1991 and 1992. Numerical abundance and biomass of larvae showed considerable spatial, seasonal and interannual variation (ANCOVAs,P<0.001). The maximum mean annual density was 7048 larvae m^–2, and corresponded to a biomass of 3.08 g dry weight (DW) m^–2. It was recorded at the site with the lowest rate of water renewal. Seasonal patterns were similar at all sites, with main annual peaks of abundance and biomass in autumn-early winter. Chironomid density was positively related to the biomass of benthic macroalgae (P<0.001). The population studied was multivoltine with a probable average of five generations per year, with overlapping cohorts and a predominance of third- and fourth-instar larvae. Estimates of annual production ranged between 72.2 g DW m^–2 yr^–1 at the site with the lowest rate of water renewal in 1991 and 0.1 g DW m^–2 yr^–1 at the site with the highest rate of water renewal in 1992. Mean annual production and the production/biomass ratio for the system was estimated to be 16.8 g DW m^–2 yr^–1 and 12.7, respectively. Possible factors leading to the observed density fluctuations are discussed, as well as possible sources of error in production estimates.     

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form.

Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling. The combination of three-dimensional 15N-edited TOCSY, 15N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain 1H and 13C assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104. The secondary structure also shows four beta-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet almost identical to that found in E. coli glutaredoxin-1. Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.