Alginate biosynthesis in mucoid recombinants of Pseudomonas aeruginosa overproducing GDP-mannose dehydrogenase.

The Pseudomonas aeruginosa algD gene, encoding GDP-mannose dehydrogenase (GMD) and cloned at Chakrabarty's Laboratory in the expression vector pMMB24 (plasmid pVD211), was mobilized into P.aeruginosa strains 8821 and 8821M. Strain 8821M was a high-alginate-producing variant, spontaneously obtained from mucoid strain 8821, with derepressed levels of GMD, a key enzyme in the regulation of alginate biosynthesis, leading to the irreversible oxidation of GDP-mannose to GDP-mannuronic acid. A slight increase in the level of GMD, in both strains harboring the plasmid pVD211 and batch-grown at 37 degrees C without IPTG induction, led to the increase of production rate and the final concentration of alginate produced by control strains harboring the cloning vector. However, the viscosity of the aqueous solutions prepared with the alginate (3 g l-1) produced by mucoid strains harboring pVD211 was lower than those with the alginate produced by the controls (shear rates in the range 0.6-12 s-1). The specific activity of GMD assayed in crude extracts from cells harboring pVD211 and subjected to IPTG induction (0.5 and 3 mM) presented the highest values. However, either the rate of biosynthesis and final concentration of alginate or the viscosity of solutions prepared with the alginate produced by recombinants grown with IPTG were lower than that possible without overproduction. Therefore, the stimulation of the alginate pathway only by manipulating the rate of the step catalysed by GMD, although possible within certain levels, was at the expense of the final exopolysaccharide quality.     

Enzymes involved in the biosynthesis of alginate byPseudomonas aeruginosa

Summary Analysis of the enzymes involved in the biosynthesis of alginic acid by mucoidPseudomonas aeruginosa PAO strain's determined the presence of enzymes required to synthesise GDP-mannuronic acid. Addition of polymannuronic acid to an ammonium sulphate precipitate of a cell free alginate suspension indicated the presence of an enzyme which catalysed the epimerisation of mannuronic acid to guluronic acidafter the polymer had been synthesised. The epimerase was shown to be calcium dependant.Various non-mucoid mutants were also studied. The non-mucoid parental strain PAO 381 also contained the enzymes required for alginate synthesis but they were not expressed. Synthesis of alginic acid led to an increase in the level of these enzymes. In the non-mucoid mutants derived from mucoid parents GDP-mannose dehydrogenase was absent in all strains studied. In some of these strains GDP-mannose pyrophosphorylase was also absent, while in other strains, phosphomannase isomerase was absent or greatly reduced.     

Allosterism and cooperativity in Pseudomonas aeruginosa GDP-mannose dehydrogenase

GDP-Mannose dehydrogenase catalyzes the formation of GDP-mannuronic acid, which is the monomeric unit from which the polysaccharide alginate is formed. Alginate is secreted by the pathogenic bacterium Pseudomonas aeruginosa and is believed to play an important role in the bacteria's resistance to antibiotics and the host immune response. We have characterized the kinetic behavior of GDP-mannose dehydrogenase in detail. The enzyme displays cooperative behavior with respect to NAD(+) binding, and phosphate and GMP act as allosteric effectors. Binding of the allosteric effectors causes the Hill coefficient for NAD(+) binding to decrease from 6 to 1, decreases K(1/2) for NAD(+) by a factor of 10, and decreases V(max) by a factor of 2. The cooperative binding of NAD(+) is also sensitive to pH; deprotonation of two residues with identical pK's of 8.0 is required for maximally cooperative behavior. The kinetic behavior of GDP-mannose dehydrogenase suggests that it must be at least hexameric under turnover conditions; however, dynamic light-scattering measurements do not provide a clear determination of the size of the active enzyme complex.     

Crystal structure of GDP-mannose dehydrogenase: a key enzyme of alginate biosynthesis in P. aeruginosa

The enzyme GMD from Pseudomonas aeruginosa catalyzes the committed step in the synthesis of the exopolysaccharide alginate. Alginate is a major component of P. aeruginosa biofilms that protect the bacteria from the host immune response and antibiotic therapy. The 1.55 A crystal structure of GMD in ternary complex with its cofactor NAD(H) and product GDP-mannuronic acid reveals that the enzyme forms a domain-swapped dimer with two polypeptide chains contributing to each active site. The extensive dimer interface provides multiple opportunities for intersubunit communication. Comparison of the GMD structure with that of UDP-glucose dehydrogenase reveals the structural basis of sugar binding specificity that distinguishes these two related enzyme families. The high-resolution structure of GMD provides detailed information on the active site of the enzyme and a template for structure-based inhibitor design.     

Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose

Background: GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII). Results: The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis. Conclusions: The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.     

褐藻77 K荧光特异性研究

测定了裙带菜、叉开网地藻、海带、囊藻、海蒿子、鼠尾藻、萱藻和水云等8种褐藻的77K荧光光谱并同菠菜和红藻条斑紫菜作了比较。结果表明与红藻和高等植物明显不同,褐藻没有作为PSⅠ特征的730 nm荧光峰。按荧光主峰的波长,可以分为二种类型：裙带菜、叉开网地藻、海带和囊藻的荧光主峰位于690 nm,海蒿子、萱藻、水云和鼠尾藻的荧光主峰在705-720 nm。这种77K荧光特异性预示褐藻同高等植物之间在PSⅠ结构上的差异。     

Lipoxygenase pathway in brown algae: The biosynthesis of novel oxylipins ‘ectocarpins’ by hydroperoxide bicyclase CYP5164A3 of Ectocarpus siliculosus

The sequence encoding the CYP5164A3 of the brown alga Ectocarpus siliculosus (Stramenopiles, SAR) was heterologously expressed in E. coli cells. The resulting recombinant CYP74 clan-related protein CYP5164A3 possessed a selective activity towards the α-linolenic acid 13(S)-hydroperoxide (13-HPOTE) and eicosapentaenoic acid 15(S)-hydroperoxide (15-HPEPE). The major products were the heterobicyclic oxylipins. For instance, the 13-HPOTE was converted into plasmodiophorols A, B, and C formed at about 14:3:2 ratio. Plasmodiophorols A-C have been recently described as the products of enzyme hydroperoxide bicyclase CYP50918A1 of cercozoan Plasmodiophora brassicae (Rhizaria, SAR). Furthermore, an unknown compound 1 was detected. Purified product 1 (Me) was identified as a novel substituted 3-propenyl-6-oxabicyclo[3.1.0]hexane based on its MS and NMR spectral data. Conversion of 15-HPEPE by CYP5164A3 resulted in products 7 and 8, analogous to plasmodiophorols A and B. This work uncovered the CYP5164A3 as the first hydroperoxide bicyclase in brown algae. Apparently, this enzyme plays a crucial role in the biosynthesis of heterobicyclic oxylipins like hybridalactone, ecklonilactones, and related natural products, widespread in brown algae.     

Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research

Brown algae share several important features with land plants, such as their photoautotrophic nature and their cellulose-containing wall, but the two groups are distantly related from an evolutionary point of view. The heterokont phylum, to which the brown algae belong, is a eukaryotic crown group that is phylogenetically distinct not only from the green lineage, but also from the red algae and the opisthokont phylum (fungi and animals). As a result of this independent evolutionary history, the brown algae exhibit many novel features and, moreover, have evolved complex multicellular development independently of the other major groups already mentioned. In 2004, a consortium of laboratories, including the Station Biologique in Roscoff and Genoscope, initiated a project to sequence the genome of Ectocarpus siliculosus, a small filamentous brown alga that is found in temperate, coastal environments throughout the globe. The E. siliculosus genome, which is currently being annotated, is expected to be the first completely characterized genome of a multicellular alga. In this review we look back over two centuries of work on this brown alga and highlight the advances that have led to the choice of E. siliculosus as a genomic and genetic model organism for the brown algae.     

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae

Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [¹⁸O₂]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.     

Development and characteristics of an adhesion bioassay for ectocarpoid algae

Species of filamentous brown algae in the family Ectocarpaceae are significant members of fouling communities. However, there are few systematic studies on the influence of surface physico-chemical properties on their adhesion. In the present paper the development of a novel, laboratory-based adhesion bioassay for ectocarpoid algae, at an appropriate scale for the screening of sets of experimental samples in well-replicated and controlled experiments is described. The assays are based on the colonization of surfaces from a starting inoculum consisting of multicellular filaments obtained by blending the cultured alga Ectocarpus crouaniorum. The adhesion strength of the biomass after 14 days growth was assessed by applying a hydrodynamic shear stress. Results from adhesion tests on a set of standard surfaces showed that E. crouaniorum adhered more weakly to the amphiphilic Intersleek? 900 than to the more hydrophobic Intersleek? 700 and Silastic? T2 coatings. Adhesion to hydrophilic glass was also weak. Similar results were obtained for other cultivated species of Ectocarpus but differed from those obtained with the related ectocarpoid species Hincksia secunda. The response of the ectocarpoid algae to the surfaces was also compared to that for the green alga, Ulva.     

PASSAGE OF A MARINE BROWN ALGAL DNA VIRUS FROM ECTOCARPUS FASCICULATUS (ECTOCARPALES,PHAEOPHYCEAE) TO MYRIOTRICHIA CLAVAEFORMIS (DICTYOSIPHONALES,PHAEOPHYCEAE): INFECTION SYMPTOMS AND RECOVERY1

A dsDNA virus (EfasV-1) isolated from Ectocarpus fasciculatus Harvey infected Myriotrichia clavaeformis Harvey, a species belonging to a different brown algal order. The virus did not complete its infection cycle in the foreign host but caused infertility due to malformed reproductive structures. After some time in culture, the host's reproductive capacity was sometimes restored with concomitant loss of at least part of the viral genome. This incidence of interordinal virus transfer is discussed in relation to possibilities for virus-mediated horizontal gene transfer in brown algae.     

Rapid,Blue-Light-Induced Acidifications at the Surface of Ectocarpus and Other Marine Macroalgae

In most brown algae, photosynthesis saturated with red light can be stimulated by continuous blue light. Pulses of blue light lead to transient increases in photosynthetic rate. When a CO2-sensitive electrode was used, occasionally blue light was observed to cause an apparent increase of CO2 instead of the expected decrease. This was changed by buffering the seawater medium and, under these conditions, blue light caused stimulation of CO2 consumption. These results led to investigations of blue-light-dependent pH changes at the outer surface of the plants. Shifts of the pH were recorded in the presence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In all brown algae tested and in the green algae Ulva and Enteromorpha, blue-light pulses caused transient acidification of 0.03 to 0.18 pH units, depending on the species. The kinetics showed lag phases of a few seconds and the minimum was reached after 5 to 9 min. Fluence response relationships indicated that the sensitivity (threshold) to blue light was very similar in all species. The responses in Ectocarpus changed with time, and about 5 h after the beginning of red light or darkness, a second component became evident, which peaked 20 min after the blue-light pulse. The refractory period of the whole system was about 3 h in Ectocarpus. The blue-light-dependent pH changes show striking similarities to those of higher plant guard cells, and it is possible that similar responses may occur in other tissues of higher plants. In red algae, however, no blue-light-dependent acidifications could be detected. The possible role of the observed pH shifts in a mechanism of CO2 acquisition is discussed.     

Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa.

Alginate (Alg), a random polymer of mannuronic acid and glucuronic acid residues, is synthesized and secreted by Pseudomonas aeruginosa primarily during its infection of the lungs of cystic fibrosis patients. The molecular biology and biochemistry of the enzymatic steps leading to the production of the Alg precursor GDP-mannuronic acid have been elucidated, but the mechanism of polymer formation and export of Alg are not understood. We report the nucleotide sequence of a 2.4-kb DNA fragment containing the algE gene, previously designated alg76, encoding the AlgE protein (Mr 54,361) that is believed to be involved in these late steps of Alg biosynthesis. Expression of algE appears to occur from its own promoter. The promoter region contains several direct and inverted repeat sequences and shares structural similarity with promoters of several other alg genes from P. aeruginosa. In addition, the AlgE protein was overproduced from the tac promoter in P. aeruginosa. N-terminal amino acid sequence analysis showed that the polypeptide contains a signal peptide which is cleaved to form the mature protein during AlgE export from the cell cytoplasm.     

Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina

Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k_m values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi.     

The Demand for Iodine and Bromine of three Marine Brown Algae grown in Bacteria-free Cultures

Three marine brown algae have been cultivated with different additions of iodine and bromine in bacteria-free cultures. Ectocarpus jasciculatus appeared to have an absolute demand for iodine and was inhibited by a concentration of 64 μmol of KJ per 1. Lithosiphon pusillus had the best growth in the highest concentration tested (64 μmol/1) but there was always some growth in the series without iodine. Additions could be made either as inorganic iodine or as organically bound iodine. Additions of KJ to a culture medium consisting of vitamin-free Asp 6 F with B₁₂ (1 μg/1) and kinetin (20 μmol/1) remarkably increased the growth of the zoospores of Pylaiella litoralis. Lithosiphon pusilius proved to be indifferent to bromide additions in media containing KJ. In media lacking KJ addition of 1 μmol of KBr per 1 is stimulating but higher concentrations of KBr are inhibiting. The inhibiting effect is overcome by iodide addition.     

Toward systems biology in brown algae to explore acclimation and adaptation to the shore environment

Brown algae belong to a phylogenetic lineage distantly related to land plants and animals. They are almost exclusively found in the intertidal zone, a harsh and frequently changing environment where organisms are submitted to marine and terrestrial constraints. In relation with their unique evolutionary history and their habitat, they feature several peculiarities, including at the level of their primary and secondary metabolism. The establishment of Ectocarpus siliculosus as a model organism for brown algae has represented a framework in which several omics techniques have been developed, in particular, to study the response of these organisms to abiotic stresses. With the recent publication of medium to high throughput profiling data, it is now possible to envision integrating observations at the cellular scale to apply systems biology approaches. As a first step, we propose a protocol focusing on integrating heterogeneous knowledge gained on brown algal metabolism. The resulting abstraction of the system will then help understanding how brown algae cope with changes in abiotic parameters within their unique habitat, and to decipher some of the mechanisms underlying their (1) acclimation and (2) adaptation, respectively consequences of (1) the behavior or (2) the topology of the system resulting from the integrative approach.     

Monoclonal Antibodies Directed to Fucoidan Preparations from Brown Algae

Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.