Eutrophication in Australian Rivers, Reservoirs and Estuaries – A Southern Hemisphere Perspective on the Science and its Implications

Australian science has made rapid advances in the last decade in understanding eutrophication processes in inland waters and estuaries. The freshwater research on which these advances are based was triggered by well-publicised blooms of cyanobacteria during the 1980s and early 1990s, particularly a 1000 km long bloom on the Darling River. In estuaries the study which greatly enhanced our understanding but simultaneously served to stimulate further research into estuarine eutrophication, the Port Phillip Bay Study, was initially designed to address perceived problems of toxicants in the Bay but provided profound insights into drivers for, and ecosystem responses to, eutrophication. Subsequent estuarine research has largely been stimulated by management questions arising from Australia’s increasing coastal development for residential purposes. The research has shown that some of the beliefs extant at the time of the blooms were incorrect. For example, it is now clear that stratification and light penetration, not nutrient availability, are the triggers for blooms in the impounded rivers of southeastern Australia, although nutrient exhaustion limits the biomass of blooms. Again, nitrogen seems to play as important a role as phosphorus does in controlling the biomass of these freshwater blooms. The research has also shown that aspects of eutrophication, such as nutrient transport, are dominated by different processes in different parts of Australia. Many of the biophysical processes involved in eutrophication have now been quantified sufficiently for models to be developed of such processes as sediment-nutrient release, stratification, turbidity and algal growth in both freshwater and estuarine systems. In some cases the models are reliable enough for the knowledge gained in particular waterbodies to be applied elsewhere. Thus, there is now a firm scientific foundation for managers to rely upon when managing algal blooms. Whilst these findings have already been presented to managers and communities throughout Australia, there is still a considerable way to go before they are absorbed into their modus operandi.     

Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana

De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.     

High dynamic range characterization of the trauma patient plasma proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.     

Phylogenomic analysis supports the monophyly of cryptophytes and haptophytes and the association of rhizaria with chromalveolates

Here we use phylogenomics with expressed sequence tag (EST) data from the ecologically important coccolithophore-forming alga Emiliania huxleyi and the plastid-lacking cryptophyte Goniomonas cf. pacifica to establish their phylogenetic positions in the eukaryotic tree. Haptophytes and cryptophytes are members of the putative eukaryotic supergroup Chromalveolata (chromists [cryptophytes, haptophytes, stramenopiles] and alveolates [apicomplexans, ciliates, and dinoflagellates]). The chromalveolates are postulated to be monophyletic on the basis of plastid pigmentation in photosynthetic members, plastid gene and genome relationships, nuclear "host" phylogenies of some chromalveolate lineages, unique gene duplication and replacements shared by these taxa, and the evolutionary history of components of the plastid import and translocation systems. However the phylogenetic position of cryptophytes and haptophytes and the monophyly of chromalveolates as a whole remain to be substantiated. Here we assess chromalveolate monophyly using a multigene dataset of nuclear genes that includes members of all 6 eukaryotic supergroups. An automated phylogenomics pipeline followed by targeted database searches was used to assemble a 16-protein dataset (6,735 aa) from 46 taxa for tree inference. Maximum likelihood and Bayesian analyses of these data support the monophyly of haptophytes and cryptophytes. This relationship is consistent with a gene replacement via horizontal gene transfer of plastid-encoded rpl36 that is uniquely shared by these taxa. The haptophytes + cryptophytes are sister to a clade that includes all other chromalveolates and, surprisingly, two members of the Rhizaria, Reticulomyxa filosa and Bigelowiella natans. The association of the two Rhizaria with chromalveolates is supported by the approximately unbiased (AU)-test and when the fastest evolving amino acid sites are removed from the 16-protein alignment.     

Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates

MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (L-Ala/L-Ser) and second (L-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402-6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-L-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is L-Ala-L-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.     

Adsorption of Binary Mixtures in a Zeolite Micropore

Abstract

We investigate the selective adsorption of xenon, argon, and methane in zeolite NaA by applying the grand canonical ensemble Monte Carlo simulation technique to an adsorbed binary mixture and to two reference systems: i) an adsorbed single component system and ii) a bulk mixture. We define and calculate selectivities and excess densities due to i) mixing and ii) adsorption in terms of differences between the binary adsorbed system and these reference systems. We observe that xenon selectively adsorbs in both xenon-argon and xenon-methane mixtures at low chemical potential (low pressure) due to its greater energetic interaction with the zeolite. However, a reversal in selectivity occurs at higher chemical potential in both of these mixtures. This is due in large part to the greater efficiency in which the smaller component “packs” in the pore as compared to the bulk. We show that the crossover in selectivity occurs at a lower chemical potential for a mixture where one component can occupy regions of the porespace inaccessible to the other. We suggest that this crossover in selectivity may be a general feature of microporous adsorption.