Quorum sensing influences growth and photosynthetic membrane production in high-cell-density cultivations of Rhodospirillum rubrum

BACKGROUND: The facultative anoxygenic photosynthetic bacterium Rhodospirillum rubrum exhibits versatile metabolic activity allowing the adaptation to rapidly changing growth conditions in its natural habitat, the microaerobic and anoxic zones of stagnant waters. The microaerobic growth mode is of special interest as it allows the high-level expression of photosynthetic membranes when grown on succinate and fructose in the dark, which could significantly simplify the industrial production of compounds associated with PM formation. However, recently we showed that PM synthesis is no longer inducible when R. rubrum cultures are grown to high cell densities under aerobic conditions. In addition a reduction of the growth rate and the continued accumulation of precursor molecules for bacteriochlorophyll synthesis were observed under high cell densities conditions. RESULTS: In the present work, we demonstrate that the cell density-dependent effects are reversible if the culture supernatant is replaced by fresh medium. We identified six N-acylhomoserine lactones and show that four of them are produced in varying amounts according to the growth phase and the applied growth conditions. Further, we demonstrate that N-acylhomoserine lactones and tetrapyrrole compounds released into the growth medium affect the growth rate and PM expression in high cell density cultures. CONCLUSIONS: In summary, we provide evidence that R. rubrum possesses a Lux-type quorum sensing system which influences the biosynthesis of PM and the growth rate and is thus likely to be involved in the phenotypes of high cell density cultures and the rapid adaptation to changing environmental conditions

Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.EU Joint Programme JPND/0003/2011, FCT grant: Pest-OE/EQB/LA0004/2011, FCT PhD fellowship

Use of approximations of Hamilton-Jacobi-Bellman inequality for solving periodic optimization problems

We show that necessary and sufficient conditions of optimality in periodic optimization problems can be stated in terms of a solution of the corresponding HJB inequality, the latter being equivalent to a max-min type variational problem considered on the space of continuously differentiable functions. We approximate the latter with a maximin problem on a finite dimensional subspace of the space of continuously differentiable functions and show that a solution of this problem (existing under natural controllability conditions) can be used for construction of near optimal controls. We illustrate the construction with a numerical example.Comment: 29 pages, 2 figure

Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated

Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering

Sensitive and specific visualization of small biomolecules in living systems is highly challenging. We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors

The Photosynthetic Apparatus and Its Regulation in the Aerobic Gammaproteobacterium Congregibacter litoralis gen. nov., sp. nov

BACKGROUND: There is accumulating evidence that in some marine environments aerobic bacteriochlorophyll a-producing bacteria represent a significant part of the microbial population. The interaction of photosynthesis and carbon metabolism in these interesting bacteria is still largely unknown and requires further investigation in order to estimate their contribution to the marine carbon cycle. METHODOLOGY/PRINCIPAL FINDINGS: Here, we analyzed the structure, composition and regulation of the photosynthetic apparatus in the obligately aerobic marine gammaproteobacterium KT71(T). Photoheterotrophically grown cells were characterized by a poorly developed lamellar intracytoplasmic membrane system, a type 1 light-harvesting antenna complex and a photosynthetic reaction center associated with a tetraheme cytochrome c. The only photosynthetic pigments produced were bacteriochlorophyll a and spirilloxanthin. Under semiaerobic conditions KT71(T) cells expressing a photosynthetic apparatus showed a light-dependent increase of growth yield in the range of 1.3-2.5 fold. The expression level of the photosynthetic apparatus depended largely on the utilized substrate, the intermediary carbon metabolism and oxygen tension. In addition, pigment synthesis was strongly influenced by light, with blue light exerting the most significant effect, implicating that proteins containing a BLUF domain may be involved in regulation of the photosynthetic apparatus. Several phenotypic traits in KT71(T) could be identified that correlated with the assumed redox state of growing cells and thus could be used to monitor the cellular redox state under various incubation conditions. CONCLUSIONS/SIGNIFICANCE: In a hypothetical model that explains the regulation of the photosynthetic apparatus in strain KT71(T) we propose that the expression of photosynthesis genes depends on the cellular redox state and is maximal under conditions that allow a balanced membrane redox state. So far, bacteria capable of an obligately aerobic, photosynthetic metabolism constitute a unique phenotype within the class Gammaproteobacteria, so that it is justified to propose a new genus and species, Congregibacter litoralis gen. nov, sp. nov., represented by the type strain KT71(T) ( = DSM 17192(T) = NBRC 104960(T))

Global Analysis of Protein N-Myristoylation and Exploration of N-Myristoyltransferase as a Drug Target in the Neglected Human Pathogen Leishmania donovani

N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen

Evaluating Process Quality Based on Change Request Data – An Empirical Study of the Eclipse Project

Abstract. The information routinely collected in change request management systems contains valuable information for monitoring of the process quality. However this data is currently utilized in a very limited way. This paper presents an empirical study of the process quality in the product portfolio of the Eclipse project. It is based on a systematic approach for the evaluation of process quality characteristics using change request data. Results of the study offer insights into the development process of Eclipse. Moreover the study allows assessing applicability and limitations of the proposed approach for the evaluation of process quality