Mannuronan C-5 epimerase activity in protoplasts of Laminaria digitata

Biosynthesis of alginate in algae may be studied by following the cell wall regeneration of brown seaweed protoplasts in culture. The enzyme mannuronan C-5 epimerase will control the composition of the alginate being synthetized.Freshly isolated protoplasts from the thallus of young Laminaria digitata plants showed only low expression of this enzyme. However, after prolonged periods in culture, this activity increased 15-fold. The synthesis of C-5 epimerase by the protoplasts is probably essential for the formation of a new cell wall.After cellular disruption by osmotic shock and centrifugation, most of the epimerase activity resided in the pellet fraction. This may indicate that the enzyme is membrane associated.     

REPRODUCTIVE ISOLATION BY SEX PHEROMONES IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

The Closterium peracerosum–strigosum–littorale (C. psl.) complex consists of unicellular algae and is known to be composed of several reproductively isolated mating groups of heterothallic strains. Group I‐E is completely isolated from mating groups II‐A and II‐B, groups II‐A and II‐B are partially isolated from each other, and only mating‐type plus (mt⁺) cells of group II‐A and mating‐type minus (mt^?) cells of group II‐B form zygotes. Based on the alignment of 1506 group I introns, significant phylogenetic relationships were observed among mating groups II‐A and II‐B, while mating group I‐E was distant from groups II‐A and II‐B. Sexual cell division in both mating‐type cells of group II‐A was stimulated in conditioned media in which cells of group II‐B had been cultured. When mt^? cells of group II‐B were stimulated in conditioned medium derived from group II‐A, mt⁺ cells of group II‐B did not respond to the conditioned medium. Conditioned media derived from group I‐E did not exhibit sexual cell division (SCD)–inducing activity against any strain except those within its own group. From the alignment of deduced amino acid sequences from orthologous protoplast‐release‐inducing protein (PR‐IP) Inducer genes, we detected a significant similarity among groups II‐A and II‐B, and mating group I‐E had low similarity to other mating groups. The existing degree of reproductive isolation can be partially explained by differences in molecular structures and physiological activities of sex pheromones of these heterothallic mating groups.     

Prevailing sea surface temperatures inhibit summer reproduction of the kelp Laminaria digitata at Helgoland (North Sea)

The impact of abiotic factors on kelp sporophyte reproduction has rarely been investigated. Laminaria digitata (Hudson) J.V. Lamouroux is one of the few summer fertile Laminaria species worldwide and reproduction is subjected to relatively high water temperatures. We investigated the impact of prevailing summer temperatures (~18°C in August) on the induction of sporangia, meiospore release, and germination at the island of Helgoland (North Sea). At Helgoland, fertile sporophytes are found between April and December with a maximum in late summer. While released meiospore numbers were constant between June and October, germination rates decreased significantly in summer. Short‐term exposure of mature sori to 17°C–22°C induced a significantly higher meiospore release indicating enhancement of sporulation by elevated temperatures. Induction of sporangia on vegetative blade disks was not possible at 20°C, and fertility was only 20% at 18°C–19°C, but it was 100% in cool temperatures of 1°C–10°C. It was shown for the first time in a kelp species that “sporogenesis” is the life‐cycle process with the narrowest temperature window compared to growth or survival of the sporophyte or reproduction, growth, and survival of the gametophyte. We incorporated several parameters (induction time, fertile area, and relative fertility) into a “Reproductive efficiency index.” This indicates that sporogenesis of L. digitata is a cold‐adapted process with an optimum at (5)–10°C. The results show that the population at Helgoland is at its reproduction limit despite the existence of other geographically more southerly located populations.     

Crystal structures of the archaeal UDP‐GlcNAc 2‐epimerase from Methanocaldococcus jannaschii reveal a conformational change induced by UDP‐GlcNAc

Uridine diphosphate N ‐ acetylglucosamine (UDP‐GlcNAc) 2‐epimerase catalyzes the interconversion of UDP‐GlcNAc to UDP‐N‐acetylmannosamine (UDP‐ManNAc), which is used in the biosynthesis of cell surface polysaccharides in bacteria. Biochemical experiments have demonstrated that mutation of this enzyme causes changes in cell morphology and the thermoresistance of the cell wall. Here, we present the crystal structures of Methanocaldococcus jannaschii UDP‐GlcNAc 2‐epimerase in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDP‐GlcNAc binding, the enzyme undergoes conformational changes involving a rigid‐body movement of the C‐terminal domain. We also present the crystal structure of Bacillus subtilis UDP‐GlcNAc 2‐epimerase in the closed conformation in the presence of UDP and UDP‐GlcNAc. Although a structural overlay of these two closed‐form structures reveals that the substrate‐binding site is evolutionarily conserved, some areas of the allosteric site are distinct between the archaeal and bacterial UDP‐GlcNAc 2‐epimerases. This is the first report on the crystal structure of archaeal UDP‐GlcNAc 2‐epimerase, and our results clearly demonstrate the changes between the open and closed conformations of this enzyme. Proteins 2014; 82:1519–1526. © 2014 Wiley Periodicals, Inc.     

Baculoviral p94 homologs encoded in Cotesia plutellae bracovirus suppress both immunity and development of the diamondback moth,Plutellae xylostella

Polydnaviruses (PDVs) are a group of insect DNA viruses, which exhibit a mutual symbiotic relationship with their specific host wasps. Moreover, most encapsidated genes identified so far in PDVs share homologies with insect‐originated genes, but not with virus‐originated genes. In the meantime, PDVs associated with 2 wasp genera Cotesia and Glytapanteles encode some genes presumably originated from other viruses. Cotesia plutellae bracovirus (CpBV) encodes 4 genes homologous to baculoviral p94: CpBV‐E94k1, CpBV‐E94k2, CpBV‐E94k3, and CpBV‐E94k4. This study was conducted to predict the origin of CpBV‐E94ks by comparing their sequences with those of baculoviral orthologs and to determine the physiological functions by their transient expressions in nonparasitized larvae and subsequent specific RNA interference. Our phylogenetic analysis indicated that CpBV‐E94ks were clustered with other E94ks originated from different PDVs and shared high similarity with betabaculoviral p94s. These 4 CpBV genes were expressed during most developmental stages of the larvae of Plutella xylostella parasitized by C. plutellae. Expression of these 4 E94ks was mainly detected in hemocytes and fat body. Subsequent functional analysis by in vivo transient expression showed that all 4 viral genes significantly inhibited both host immune and developmental processes. These results suggest that CpBV‐E94ks share an origin with betabaculoviral p94s and play parasitic roles in suppressing host immune and developmental processes.     

Zur Biochemie und Regulation des heteromorphen Generationswechsels der Grünalge Derbesia-Halicystis

Summary During the life cycle of the green marine alga Derbesia marina-Halicystix ovalis, a diploid filamentous sporophyte called Derbesia alternates with a haploid spherical gametophyte called Halicystis. Beside this wild type a so-called mutant and a parthenogenetic enogenetic form stand at our disposal. The mutant is a haploid form with a Derbesia-like morphology. The morphology of the parthenogenetic form is also very similar to that of Derbesia. Biochemically sporophyte and gametophyte are characterized by a different cell-wall composition and enzyme pattern. The main polysaccharide of the cell wall of the sporophyte (Derbesia) is a mannan, whereas the cell wall of the gametophyte is built up mainly of glucose- and xylose-containing polysaccharides. The cell wall composition of the mutant is nearly identical with that of the Derbesia wild type. On the other hand the parthogenetic form lies to a certain degree between Derbesia and Halicystis with respect to its cell wall composition. Differences in enzyme pattern exist especially in the enzymes of GDPM- and mannan synthesis. These enzymes are present in high activities in Derbesia, the mutant, and the parthogenetic form but are absent or present only with very small activities in Halicystis. From these results we conclude that the differences in morphology, cell wall composition and enzyme pattern between the sporophyte (Derbesia) and the gametophyte (Halicystis) are brought about by a relatively stable pattern of gene activity which is changed only during the formation or the out-growth of the zoospores and the formation or fusion of gametes (see discussion).

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Abkürzungen ADPG-Pyr Adenosindiphosphat-glucose-Pyrophosphorylase E.C.2.7.7.b - F-6-P Fructose-6-phosphat - GDPM Guanosindiphosphat-mannose - GDPM-Pyr GDPM-Pyrophosphorylase E.C. 2.7.7.13 - Gluconat-6-P-DH Gluconat-6-phosphat-Dehydrogenase E.C-1.1.1.44 - G-1,6-P Glucose-1,6-diphosphat - G-6-P-DH Glucose-6-phosphat-Dehydrogenase E.C. 1.1.1.49 - M-1-P Mannose-1-phosphat - M-1,6-P Mannose-1,6-diphosphat - PGI Phosphoglucose-Isomerase E.C. 5.3.1.9 - PGM Phosphogluco-Mutase E.C. 2.7.5.1 - PMI Phosphomannose-Isomerase E.C. 5.3.1.8 - PMM Phosphomannose-Mutase - TRAP Triäthanolamin-Puffer - UDPG Uridindiphosphat-glucose - UDPG-DH UDPG-Dehydrogenase E.C. 1.1.1.22 - UDPG-4-Epi UDPG-4-Epimerase E.C. 5.1.3.2 - UDPG-Pyr UDPG-Pyrophosphorylase E.C. 2.7.7.9 - UDPXyl Uridindiphosphat-xylose     

Crystal structure of decaprenylphosphoryl‐β‐ D‐ribose 2'‐epimerase from Mycobacterium smegmatis

Decaprenylphosphoryl‐β‐D ‐ribose 2'‐epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti‐tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C‐terminus and shares homology with FAD‐dependent oxidoreductases of the vanillyl‐alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

BIOSYNTHESIS OF POLY‐3‐HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A cell‐wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC‐849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris‐acetate‐phosphate media containing 10 μg · mL^?1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly‐3‐hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min^?1 · mg protein^?1 to 126 nmol · min^?1 · mg protein^?1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.     

ISOLATION AND CHARACTERIZATION OF PHASE-SPECIFIC COMPLEMENTARY DNAs FROM SPOROPHYTES AND GAMETOPHYTES OF PORPHYRA PURPUREA (RHODOPHYTA) USING SUBTRACTED COMPLEMENTARY DNA LIBRARIES1

The red alga Porphyra purpurea (Roth) C. Agardh has a life cycle that alternates between shell-boring, filamentous sporophytes and free-living, foliose gametophytes. The significant morphological differences between these two phases suggest that many genes should be developmentally regulated and expressed in a phase-specific manner. In this study, we prepared and screened subtracted complementary DNA (cDNA) libraries specific for the sporophyte and gametophyte of P. purpurea. This involved the construction of cDNA libraries from each phase, followed by the removal of common clones through subtractive hybridization. Sampling of the subtracted libraries indicated that 8–10% of the recombinant colonies in each library were specific for the appropriate phase. Of 20 putative phase-specific cDNAs selected from each subtracted library, eight unique clones were obtained for the sporophyte and seven for the gametophyte. After confirming their phase-specificities by hybridization to gametophyte and sporophyte messenger RNA, these 15 phase-specific cDNAs were sequenced, and the deduced amino acid sequences were used to search protein databanks. Two proteins encoded by the sporophyte-specific cDNAs and two by the gametophyte-specific cDNAs were identified by their similarity to databank entries.     

Pathway Engineering of Bacillus subtilis for Enhanced N‐Acetylneuraminic Acid Production via Whole‐Cell Biocatalysis

N‐acetylneuraminic acid (NeuAc) is a common sialic acid that has a wide range of applications in nutraceuticals and pharmaceuticals. However, low production efficiency and high environmental pollution associated with traditional extraction and chemical synthesis methods constrain the supply of NeuAc. Here, a biological approach is developed for food‐grade NeuAc production via whole‐cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis (B. subtilis). Promoters for controlling N‐acetylglucosamine 2‐epimerase (AGE) and NeuAc adolase (NanA) are optimized, yielding 32.84 g L^?1 NeuAc production with a molar conversion rate of 26.55% from N‐acetylglucosamine (GlcNAc). Next, NeuAc production is further enhanced to 46.04 g L^?1, which is 40.2% higher than that of the strain with promoter optimization, by expressing NanA from Staphylococcus hominis instead of NanA from Escherichia coli. To enhance the expression level of ShNanA, the N‐terminal coding sequences of genes with high expression levels are fused to the 5′‐end of the ShNanA gene, resulting in 56.82 g L^?1 NeuAc production. Finally, formation of the by‐product acetoin from pyruvate is blocked by deleting the alsS and alsD genes, resulting in 68.75 g L^?1 NeuAc production with a molar conversion rate of 55.57% from GlcNAc. Overall, a GRAS B. subtilis strain is demonstrated as a whole‐cell biocatalyst for efficient NeuAc production.     

The mitochondrial genome of the brown alga Laminaria digitata: a comparative analysis

We report here the complete sequence of the mitochondrial genome of the brown alga Laminaria digitata (Hudson) J.V. Lamouroux. L. digitata mtDNA is a circular molecule of 38,007?bp (64.9% A?+?T), encoding 63 genes and 3 ORFs and with only 6.7% of non-coding sequences. Based on gene content and order, its overall organization is very similar to that of the mitochondrial genome of Pylaiella littoralis, another brown alga belonging to a different sublineage of the Phaeophyceae. In particular, the two genomes share unusual features, which hence could be unique to brown algae among the heterokont lineage, namely the presence of a rn5 gene, a short nad11 gene, a cox2 gene with a large in-frame insertion and α-proteobacterial-like promoter sequences. On the other hand, L. digitata lacks the sequences which are thought to have been transmitted horizontally to the P. littoralis genome, that is, the group-II introns in the rnl and cox1 genes, and it features only traces of an ancestral T7-like RNA polymerase. Distance phylogenetic trees inferred from concatenated mitochondrial genes confirm that speciation of brown algae occurred recently compared to other heterokonts.     

E. coli galactose-1-phosphate uridyl transferase: N-terminal and C-terminal sequences

Summary A modified procedure for the purification of E. coli galactose-1-phosphate uridyl transferase (E.C. 2.7.6.12) was developed which reproducibly gives pure enzyme. The purified enzyme was shown to be a dimeric protein with a subunit molecular weight of 41,000 and its amino acid composition and content of free sulfhydryl groups were determined. The N-terminal and C-terminal amino acid sequences were found to be NH₂-thr-gln-phe-asn-pro-val-asp and -ser(val leu)-ala-COOH respectively. This N-terminal sequence allowed the identification of the start of the transferase gene in the DNA sequence determined by GRINDLEY. Furthermore it appears to define a nine base intercistronic region between the epimerase and transferase genes.Abbreviations Cyclic AMP Cyclic adenosine 2¹5¹ monophosphate - DPN Diphosphopyridine nucleotide - UDP Uridine diphosphate - EDTA Ethylene diamine tetra acetic acid - SDS sodium dodecyl sulfate - NEM N-ethylmaleimide     

MITOCHONDRIAL DNA PHYLOGENY OF THE SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM,DINOPHYTA)1

Symbiotic dinoflagellates belonging to the genus Symbiodinium (Freudenthal) are found worldwide in association with shallow‐water tropical and subtropical marine invertebrates. Most phylogenetic studies of Symbiodinium have used nuclear rRNA (nrDNA) genes to infer relationships among members of the genus. In this report, we present the first phylogeny of Symbiodinium based on DNA sequences from a mitochondrial protein‐coding gene (cytochrome oxidase subunit I [cox1]). Two principal groups, one comprised of Symbiodinium clade A and the second encompassing Symbiodinium clades B/C/D/E/F, are strongly supported in the cox1 phylogeny. Relationships within Symbiodinium clades B/C/D/E/F, however, are less well resolved compared with phylogenies inferred from nrDNA and chloroplast large subunit (cp23S)‐rDNA genes. Statistical tests between alternative tree topologies verified, with an exception being the position of one controversial member of Symbiodinium clade D, that relationships inferred from cox1 are congruent with those inferred from nrDNA and cp23S‐rDNA. Taken together, the relationships between the major Symbiodinium clades are robust, and there appears to be no evidence of hybridization or differential introgression of nuclear and plastid genomes between clades.     

Characterisation of complementary DNAs from the expressed sequence tag analysis of life cycle stages of Laminaria digitata (Phaeophyceae)

Laminariales (Phaeophyceae, Heterokonta) are characterised by a heteromorphic digenetic life cycle with a filamentous, microscopic gametophyte and a highly evolved, macroscopic sporophyte. With the ultimate goal of comparing gene expression in each life cycle stage, complementary DNA libraries were constructed from sporophytes and gametophytes of Laminaria digitata. A set of ca. 500 expressed sequence tags (EST) was generated from each life history phase, by single-run partial sequencing of randomly picked cDNA clones. Comparison of the EST deduced amino acid sequences with database protein sequences assigned a putative identity for 39% of the 412 gametophyte clones and 48% of the 493 sporophyte clones sequenced thus far. These data represent more than 152 different proteins now probably identified in L. digitata. Several of those newly identified proteins are of interest to our understanding of the molecular physiology of kelps, for example their carbon-concentrating mechanisms, cell wall biosynthesis and halogen metabolism. EST analysis also confirmed that genes with long 3-UTRs are widespread in Laminariales and the study of 5-UTRs allowed the identification of a Kozak consensus sequence, c(A/C)A(A/C)CAUGGc(G/T). Several potential developmentally regulated differences in gene expression are discussed.