Polyglucan Branching Isoenzymes of Algae

The cyanophyte, Oscillatoria princeps, and the enigmatic hot-spring alga, Cyanidium caldarium, contain two branching isoenzymes which can act on amylose and amylopectin, converting these polyglucans into highly branched phytoglycogens. The red alga, Rhodymenia pertusa contains three branching isoenzymes, only one of which is capable of the dual activity of the other algal branching isoenzymes. The other two red algal branching isoenzymes are Q enzymes and can only act on amylose, forming moderately branched amylopectìns. However, when the isoenzymes of all three algae are separated on different concentrations of polyacrylamide gel via electrophoresis, the mobilities of the isoenzymes show that the Q enzymes and the branching enzymes are related and true isoenzymic molecules. They differ only in electrical charges, probably caused by the substitution of amino acid residues in their active peptides. It is possible that if these charge isomers are due merely to amino acid substitutions, the enzymes may have been originally derived from a common catalytic molecule. The study indicates the identity of the branching isoenzymes of Oscillatoria and Cyanidium, and lends further supporting evidence to the cyanophyte origin of the enigmatic alga.     

Glucan and glucosyltransferase isozymes of Glaucocystis nostochinearum

The storage glucan of the alga, Glaucocystis nostochinearum was isolated in dimethyl sulfoxide. The absorption spectrum of its iodine complex was identical with those of other green algae but differed from that of blue-green algae. It was similar to amylopectin, and was much less branched than the phytoglycogen of Cyanophytes. The pattern of glycosyltransferase isozymes involved in the synthesis of this glucan (phosphorylases, synthetases and branching isozymes) was similar to those of Chlorophytes. The branching isozymes of this alga were typical Chlorophycean “Q” enzymes and could only insert branch linkages into linear amylose-like substrates; they were unable to further branch amylopectins, as can the branching isozymes of blue-green algae. If the plastids of this alga are endosymbiotic blue-green algae, then they have lost the ability to form highly branched glucans typical of Cyanophytes.     

COMPOSITION,DISTRIBUTION, AND ABUNDANCE OF DEEP‐WATER (>30 m) MACROALGAE IN CENTRAL CALIFORNIA1

The deep‐water macroalgal assemblage was described at 14 sites off the central California coast during 1999 and 2000 from SCUBA and remotely operated vehicle sampling. The stipitate kelp Pleurophycus gardneri Setchell & Gardner, previously thought to be rare in the region, was abundant from 30 to 45 m, forming kelp beds below the well‐known giant kelp forests. Macroalgae typically formed three broadly overlapping zones usually characterized by one or a few visually dominant taxa: 1) the upper “Pleurophycus zone” (30–45 m) of stipitate kelps and Desmarestia spp. with a high percent cover of corallines, low cover of uncalcified red algae, and rare green algae; 2) a middle “Maripelta zone” (40–55 m) with other uncalcified red algae and infrequent corallines and green algae; and 3) a zone (55–75 m) of infrequent patches of nongeniculate coralline algae. The green alga Palmophyllum umbracola Nelson & Ryan, not previously reported from the Northeast Pacific, was found over the entire geographical range sampled from 35 to 54 m. Year‐round profiles of water column irradiance revealed unexpectedly clear water with an average K₀ of 0.106·m ^{? 1} Received 18 January 2002. Accepted 16 December 2002. . The low percent surface irradiance found at the average lower macroalgal depth limits in this study (0.56% for brown algae, 0.12% for uncalcified red algae, and 0.01% for nongeniculate coralline algae) and lack of large grazers suggest that light controls the lower distributional limits. The ubiquitous distribution, perennial nature, and similar lower depth limits of deep‐water macroalgal assemblages at all sites suggest that these assemblages are a common persistent part of the benthic biota in this region.     

Detection of a Phytochrome-like Protein in Macroalgae

A phytochrome-like protein was detected in extracts from the red algae Corallina elongata and Gelidium sp., from the brown algae Cystoseira abiesmarina and Cystoseira tamariscifolia, and from the green algae Ulva rigida, Enteromorpha compressa and Chara hispida. Relative amounts of the photoreversible protein were determined by measurement of Δ (ΔA) values of the crude extract. SDS gel electrophoresis and immunoblotting with monoclonal antibodies directed to phytochrome from etiolated maize and oat seedlings revealed only one phytochrome-related band with apparent molecular weight of 130 kDa. The absorption difference spectrum after partial purification showed a “normal” absorption band (λ_max = 670 nm) for the Pr form but only a very weak band (λ_max = 705 nm) for the “Pfr form”.     

ULTRASTRUCTURE OF MEIOSIS IN DASYA BAILLOUVIANA (RHODOPHYTA). II. PROMETAPHASE I—TELOPHASE II AND POST-DIVISION NUCLEAR BEHAVIOR1

This is the second of two papers which together are the first comprehensive ultrastructural report of meiosis in a red alga. Many details of the meiotic process in Dasya baillouviana (Gmelin) Montagne are the same as those reported previously for mitotic cells in ceramialian red algae, but several characteristics seem unique to meiotic cells. The nucleus and nucleolus of meiotic cells are larger than those of mitotic cells and large accumulations of smooth ER are often found at the division poles during meiosis 1. The function of the ER accumulations is unknown. Importantly, both interkinesis and a simultaneous division of two separate nuclei during meiosis II was demonstrated. These new observations fail to support earlier speculation on higher red algae for a “uninuclear” meiosis (both nuclear divisions within the same nuclear envelope). However, following meiosis II the four nuclei migrate centripetally and possibly fuse in the center of the tetrasporangium. This post-division nuclear maneuvering is not understood, but our interpretation accounts for the earlier and erroneous impression of “uninuclear” meiosis. Perhaps the most important aspect of meiosis observed in Dasya is its basic adherence to the pattern commonly seen in higher plants and animals. This conservatism of the meiotic process lends further skepticism to the belief that red algae are extremely “primitive” organisms, although they undoubtedly represent a very “ancient” group of eukaryotic plants.     

Diatom Vacuolar 1,6‐β‐Transglycosylases can Functionally Complement the Respective Yeast Mutants

Diatoms are unicellular photoautotrophic algae, which can be found in any aquatic habitat. The main storage carbohydrate of diatoms is chrysolaminarin, a nonlinear β‐glucan, consisting of a linear 1,3‐β‐chain with 1,6‐β‐branches, which is stored in cytoplasmic vacuoles. The metabolic pathways of chrysolaminarin synthesis in diatoms are poorly investigated, therefore we studied two potential 1,6‐β‐transglycosylases (TGS) of the diatom Phaeodactylum tricornutum which are similar to yeast Kre6 proteins and which potentially are involved in the branching of 1,3‐β‐glucan chains by adding d ‐glucose as 1,6‐side chains. We genetically fused the full‐length diatom TGS proteins to GFP and expressed these constructs in P. tricornutum, demonstrating that the enzymes are apparently located in the vacuoles, which indicates that branching of chrysolaminarin may occur in these organelles. Furthermore, we demonstrated the functionality of the diatom enzymes by expressing TGS1 and 2 proteins in yeast, which resulted in a partial complementation of growth deficiencies of a transglycosylase‐deficient ?kre6 yeast strain.     

Substructure of the Cytoplasmic Membrane of Bacillus megaterium

The components of fractions obtained by dialyzing and differential centrifuging the “Ghost” of Bacillus megaterium were analyzed in detail. The compositions of amino acids in the main fractions (Fraction 2 and 3) of the “Ghosts”, were estimated. Fraction 2 was rich in non-polar amino acids, while Fraction 3 was scanty of them. Most of the fatty acids in Fraction 2 were 12-methyl tetradecanoic acid, while in Fraction 3 many kinds of fatty acid were detected.

As for the localization of enzymes, the three enzymes, glucose oxidase, succinic dehydrogenase and reduced nicotinamide-adenine dinucleotide oxidase, which were present in the original “Ghosts”, were mostly observed in Fraction 2, and a very little amount of them was found in the other fractions. Further, Fraction 2 could be dissolved in formic acid and dialysis of the solution brought about reaggregation to form membrane-like structure in the presence of Ca or Mg ion.     

FLORIDOSIDES IN CYANIDIUM CALDARIUM,CYANIDIOSCHYZON MEROLAE AND GALDIERIA SULPHURARIA(RHODOPHYTA,CYANIDIOPHYCEAE)1

Cyanidium caldarium Geitler, Cyanidioschyzon merolae De Luca, Taddei & Varano and Galdieria sulphuraria (Galdieri) Merola are the three thermoacidophilic algae characterized by a chloroplast which is bounded by a single membrane. The presence of this atypical chloroplast made the inclusion of these algae in the Rhodophyta difficult. The discovery in the three algae of floridoside and isofloridoside, typical storage products of red algae, in compatible with their inclusion in the Rhodophyta     

Carotenogenesis diversification in phylogenetic lineages of Rhodophyta

Carotenoid composition is very diverse in Rhodophyta. In this study, we investigated whether this variation is related to the phylogeny of this group. Rhodophyta consists of seven classes, and they can be divided into two groups on the basis of their morphology. The unicellular group (Cyanidiophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae) contained only β‐carotene and zeaxanthin, “ZEA‐type carotenoids.” In contrast, within the macrophytic group (Bangiophyceae, Compsopogonophyceae, and Florideophyceae), Compsopogonophyceae contained antheraxanthin in addition to ZEA‐type carotenoids, “ANT‐type carotenoids,” whereas Bangiophyceae contained α‐carotene and lutein along with ZEA‐type carotenoids, “LUT‐type carotenoids.” Florideophyceae is divided into five subclasses. Ahnfeltiophycidae, Hildenbrandiophycidae, and Nemaliophycidae contained LUT‐type carotenoids. In Corallinophycidae, Hapalidiales and Lithophylloideae in Corallinales contained LUT‐type carotenoids, whereas Corallinoideae in Corallinales contained ANT‐type carotenoids. In Rhodymeniophycidae, most orders contained LUT‐type carotenoids; however, only Gracilariales contained ANT‐type carotenoids. There is a clear relationship between carotenoid composition and phylogenetics in Rhodophyta. Furthermore, we searched open genome databases of several red algae for references to the synthetic enzymes of the carotenoid types detected in this study. β‐Carotene and zeaxanthin might be synthesized from lycopene, as in land plants. Antheraxanthin might require zeaxanthin epoxydase, whereas α‐carotene and lutein might require two additional enzymes, as in land plants. Furthermore, Glaucophyta contained ZEA‐type carotenoids, and Cryptophyta contained β‐carotene, α‐carotene, and alloxanthin, whose acetylenic group might be synthesized from zeaxanthin by an unknown enzyme. Therefore, we conclude that the presence or absence of the four enzymes is related to diversification of carotenoid composition in these three phyla.     

Visual selection of shell colour in two littoral prosobranchs

The rough periwinkle Littorina “saxatilis” exhibits a wide range of shell colours. In current literature it is claimed that these colours are not related to the environment, that they arise randomly as genetic accidents and that they have little positive survival value. In a previous paper it has been shown that, in Wales, “saxatilis” consists of four separate, fully sympatric species. This paper reports an investigation as to whether the colour of two of these species, nigrolineata and rudis, is related to the colour of the background. Because of difficulties in quantitatively describing the colour of the background it was decided to concentrate mainly upon one aspect: whether or not the frequency of red shells is larger upon shores consisting of red sandstone than elsewhere in Wales. In both species the association between red shells and red sandstone is highly significant. All nigrolineata samples in which red was found at a frequency above 15% are from red sandstone. On red sandstone, red shells of this species were found mainly upon sheltered shores, their frequency decreasing and that of white shells increasing with exposure. This may be because barnacles, which occupy the same vertical zone as nigrolineata, are more abundant upon exposed shores. Partly covering the red rock, barnacles create a white background upon which white shells, rather than red, are cryptic. Yellow shells are found mainly on sheltered shores, where the brown alga Fucus is abundant. It was observed that when the tide is in and the algae spread out, a yellow shell situated beneath them is well concealed. Yellow is also found upon barnacles which because of fungal and lichen infections, are dirty yellow. It is suggested that a striped (“nigrolineated”) pattern breaks up the shape of the shell. It also resembles the colour of the dark rock and the dark sutures between barnacle plates. In rudis 80% of the samples containing over 25% red are from red sandstone. Contrast to nigrolineata, in this species the relative frequency of red decreases on sheltered shores. This could be because the brown alga Pelvetia, which occupies the same vertical zone as rudis, is more abundant in sheltered conditions and its colour partly covers over that of the rock beneath. The fact that in both species red shells are more frequent upon red sandstone than elsewhere in the study area, suggests that visual selection is restricting their distribution to the background that they match most. Rock pipits and shore-crabs prey upon winkles. They have colour perception and could be partly responsible for this selection.     

Microbial Community Structure, Pigment Composition, and Nitrogen Source of Red Snow in Antarctica

“Red snow” refers to red-colored snow, caused by bloom of cold-adapted phototrophs, so-called snow algae. The red snow found in Langhovde, Antarctica, was investigated from several viewpoints. Various sizes of rounded red cells were observed in the red snow samples under microscopy. Pigment analysis demonstrated accumulation of astaxanthin in the red snow. Community structure of microorganisms was analyzed by culture-independent methods. In the analyses of small subunit rRNA genes, several species of green algae, fungus, and various phylotypes of bacteria were detected. The detected bacteria were closely related to psychrophilic or psychrotolerant heterotrophic strains, or sequences detected from low-temperature environments. As predominant lineage of bacteria, members of the genus Hymenobacter were consistently detected from samples obtained in two different years. Nitrogen isotopic compositions analysis indicated that the red snow was significantly ¹⁵N-enriched. Based on an estimation of trophic level, it was suggested that primary nitrogen sources of the red snow were supplied from fecal pellet of seabirds including a marine top predator of Antarctica.     

BRIEF INCUBATION OF GAMETANGIA-BEARING CAPS IN ANTIBIOTICS ELIMINATES BRANCHING IN PROGENY OF ACETABULARIA ACETABULUM (CHLOROPHYTA)1

Branching of the stalk of Acetabularia acetabulum L. (Silva) was investigated by inbreeding and by a brief treatment of gametangia with a variety of antibiotics. The position of the branch along the stalk varied, implying that branching was not restricted to any one time in development (base is oldest and apex is youngest). The branching phenotype was not inherited in Mendelian fashion. Although three microscopic structures (“bubbles,”“pustules,” and “scars”) occurred on the stalks of cells that had branched, these structures were not statistically correlated with branching in the population (n=699 cells). However, brief treatment of gametangia with a new antibiotic mixture did eliminate all macro- and microscopic structures associated with branching of the stalk in the subsequent generation. We could not fulfill Koch's postulates or provide clear evidence for the pathogenic nature of cell branching. Our brief antibiotic treatment of gametangaa of Acetabularia acetabulum was rapid, had no adverse effects, and virtually eliminated branching (and any potential pathogens) from laboratory cultures in the subsequent generations. Our method allows biochemical and molecular analyses to proceed uncomplicated by the possible presence of other organisms and provides a clean baseline for the future selection of mutations that may induce heritable branching.     

Isozyme Variation in a Tropical Pioneer Tree Species (Cecropia obtusifolia,Moraceae) with High Contents of Secondary Compounds1

We define 48 allozyme loci for a tropical pioneer tree species, Cecropia obtusifolia Bertol, which has high contents of secondary compounds. Our goals were to find the effects of extraction procedures on artifacts and variation in resolution of enzyme banding patterns; to explore the relationship among the variation of the loci sampled and the enzymes' molecular structure, metabolic function and substrate; to obtain estimates of the genetic variation in this species at Los Tuxtlas rain forest (México) and to explore the variation of allelic frequencies in six successive life-history stages of the species. The resolution of the isozymes bands and the actual banding pattern varied with the type and age of tissue, the collection and storage procedures, the extraction buffer, and other loading and running procedural details. However, artifactual variation was eliminated with a new extraction buffer for species with high contents of secondary compounds. Of the 26 enzymes resolved for C. obtusifolia, we found that enzymes with a greater number of substrates and an oligomeric quaternary structure tended to be more variable than their counterparts, but the relationship was not statistically significant. The proportion of polymorphic systems varied significantly with the metabolic pathway and the function of the enzymes. Enzymes involved in starch synthesis are significantly more variable (p < 0.05) than all others, except those involved in amino acids metabolism and the proportion of polymorphic enzymes is also significantly associated with the hnction of the enzyme, the hydrolases and isomerases are significantly more variable than lyases and oxidoreductases enzymes. The percentage of polymorphic loci for C. obtusifolia was estimated at P = 27.1%. The effective number of alleles was estimated at ne = 1.3 and ne = 2.4 for all loci and only polymorphic ones respectively and the average heterozygosity (H) for all 48 loci was estimated at H = 0.05. Allele frequencies varied throughout the life-cycle of the species, with significant differences for some alleles and loci among some life-cycle stages. “Tree seeds” allele frequencies differ significantly (P < 0.05) from “rainy dispersed seeds” in 7 of 8 loci and from “soil seeds” in Six of eight loci. Allele frequencies of all three seed categories (“tree seeds”, “rainy dispersed seeds”, and “soil seeds”) differed strongly from established individuals (seedlings, juveniles and adults), while allele frequencies of established individuals are relatively similar to one another. Seedling allele frequencies at most loci were also significantly different from those found in seeds collected from trees, seed-rain, and soil. Two alleles (at GOT-2 and FE-2) were only found in soil seeds and one allele (at LAP-2) was only found in seedlings.     

Polyols and UV‐sunscreens in the Prasiola‐clade (Trebouxiophyceae,Chlorophyta) as metabolites for stress response and chemotaxonomy

In many regions of the world, aeroterrestrial green algae of the Trebouxiophyceae (Chlorophyta) represent very abundant soil microorganisms, and hence their taxonomy is crucial to investigate their physiological performance and ecological importance. Due to a lack in morphological features, taxonomic and phylogenetic studies of Trebouxiophycean algae can be a challenging task. Since chemotaxonomic markers could be a great assistance in this regard, 22 strains of aeroterrestrial Trebouxiophyceae were chemically screened for their polyol‐patterns as well as for mycosporine‐like amino acids (MAAs) in their aqueous extracts using RP‐HPLC and LC‐MS. d ‐sorbitol was exclusively detected in members of the Prasiolaceae family. The novel MAA prasiolin and a related compound (“prasiolin‐like”) were present in all investigated members of the Prasiola‐clade, but missing in all other tested Trebouxiophyceae. While prasiolin could only be detected in field material directly after extraction, the “prasiolin‐like” compound present in the other algae was fully converted into prasiolin after 24 h. These findings suggest d ‐sorbitol and prasiolin‐like compounds are suitable chemotaxonomic markers for the Prasiolaceae and Prasiola‐clade, respectively. Additional UV‐exposure experiments with selected strains show that MAA formation and accumulation can be induced, supporting their role as UV‐sunscreen.     

Marine macroalgae as foods for fishes: an evaluation of potential food quality

Synopsis A revitalized view of feeding by herbivorous marine fishes is sought through two questions. First, What characteristics of major taxa of algae identify them as predictably high or low quality foods? Second, are marine algae valuable foods for fishes which do not mechanically disrupt cell walls and do not harbor specialized enzymes or microbes capable of lysing cell walls? Energy, ash and nutrient content of 16 species of marine algae were employed to assess food quality of fleshy red, green, brown and calcareous red algae. On the basis of ash, calories, total protein and total lipid content, fleshy algae should be superior to calcareous algae as foods for fishes; in addition, green algae should be superior to brown algae and brown algae superior to red algae. When the probable digestibility of storage and extracellular carbohydrates is considered, green and red algae are predicted superior to brown algae as food. Two species of damselfishes (Pomacentridae) from the Gulf of California,Eupomacentrus rectifraenum andMicrospathodon dorsalis, eat red and green algae and ignore brown and calcareous algae. They feed, therefore, in a fashion consistent with predictions based only on algal chemistry. These fishes absorb at least 20–24% of the biomass, 57–67% of the protein, 46–56% of the lipid and 37–44% of the carbohydrate contained in algae eaten in the wild. Since these damselfishes do not masticate their food, it appears that herbivorous fishes can digest major fractions of algal nutrients without mechanical destruction of algal cells.     

Structures of Arg‐ and Gln‐type bacterial cysteine dioxygenase homologs

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ～15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg‐type” enzymes) and some having a Gln substituted for this Arg (“Gln‐type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg‐type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln‐type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron‐bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln‐type” CDO enzymes, we conclude that the “Gln‐type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3‐mercaptopropionate dioxygenases.     

Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds

The use of nitrogen‐to‐protein conversion factors (N‐Prot factors) is the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of N‐Prot factors specific to individual species. Experimental data are needed to allow the use of this methodology with seaweeds. The present study was designed to characterize the amino acid composition and to establish specific N‐Prot factors for six green, four brown and nine red marine algae. Mean values for individual amino acids tended to be similar among the three groups, but some differences were found. Green algae tended to show lower percentages of both aspartic acid and glutamic acid than the other two groups of algae. The percentages of both lysine and arginine were higher in red algae, while brown algae tended to show more methionine than green and red algae. The actual protein content of the species, based on the sum of amino acid residues, varied from 10.8% (Chnoospora minima, brown algae) to 23.1% (Aglaothamnion uru‐guayense, red algae) of the dry weight. Nitrogen‐to‐protein conversion factors were established for the species studied, based on the ratio of amino acid residues to total nitrogen, with values ranging from 3.75 (Cryptonemia seminervis, red algae) to 5.72 (Padina gymnospora, brown algae). The relative importance of non‐protein nitrogen is greater in red algae, and consequently lower N‐Prot factors were calculated for these species (average value 4.59). Conversely, protein nitrogen content in both green and brown algae tends to be higher, and average N‐Prot factors were 5.13 and 5.38, respectively. An overall average N‐Prot factor for all species studied of 4.92 ± 0.59 (n = 57) was established. This study confirms that the use of the traditional factor 6.25 is unsuitable for seaweeds, and the use of the N‐Prot factors proposed here is recommended.     

Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan

Chloroplasts are believed to be descendants of ancestral cyanobacteria that had peptidoglycan layer between the outer and the inner membranes. Historically, the glaucophyte Cyanophora paradoxa and the rhizopod Paulinella chromatophora were believed to harbor symbiotic cyanobacteria having peptidoglycan, which were conventionally named “cyanelles”. In addition, the complete set of genes involved in the synthesis of peptidoglycan has been found in the moss Physcomitrella patens and some plants and algae. The presence of peptidoglycan-like structures was demonstrated by a new metabolic labeling technique in P. patens. However, many green algae and all known red algae lack peptidoglycan-related genes. That is the reason why we questioned the origin of peptidoglycan-synthesizing enzymes in the chloroplasts of the green algae and plants. We performed phylogenetic analysis of ten enzymes involved in the synthesis of peptidoglycan exploiting the Gclust homolog clusters and additional genomic data. As expected, all the identified genes encoded in the chromatophore genome of P. chromatophora were closely related to cyanobacterial homologs. In the green algae and plants, only two genes, murA and mraY, were found to be closely related to cyanobacterial homologs. The origins of all other genes were diverse. Unfortunately, the origins of C. paradoxa genes were not clearly determined because of incompleteness of published genomic data. We discuss on the probable evolutionary scenarios to explain the mostly non-cyanobacterial origins of the biosynthetic enzymes of chloroplast peptidoglycan: A plausible one includes extensive multiple horizontal gene transfers during the early evolution of Viridiplantae.