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The oxygenated derivatives of fatty acids, known as oxylipins, are pivotal signaling molecules in animals and terrestrial plants. In animal systems, eicosanoids regulate cell differentiation, immune responses, and homeostasis. In contrast, terrestrial plants use derivatives of C18 and C16 fatty acids as developmental or defense hormones. Marine algae have emerged early in the evolution of eukaryotes as several distinct phyla, independent from the animal and green-plant lineages. The occurrence of oxylipins of the eicosanoid family is well documented in marine red algae, but their biological roles remain an enigma. Here we address the hypothesis that they are involved with the defense mechanisms of the red alga Chondrus crispus. By investigating its association with a green algal endophyte Acrochaete operculata, which becomes invasive in the diploid generation of this red alga, we showed that (1) when challenged by pathogen extracts, the resistant haploid phase of C. crispus produced both C20 and C18 oxylipins, (2) elicitation with pathogen extracts or methyl jasmonate activated the metabolism of C20 and C18 polyunsaturated fatty acids to generate hydroperoxides and cyclopentenones such as prostaglandins and jasmonates, and (3) C20 and C18 hydroperoxides as well as methyl jasmonate did induce shikimate dehydrogenase and Phe ammonialyase activities in C. crispus and conferred an induced resistance to the diploid phase, while inhibitors of fatty acid oxidation reduced the natural resistance of the haploid generation. The dual nature of oxylipin metabolism in this alga suggests that early eukaryotes featured both animal- (eicosanoids) and plant-like (octadecanoids) oxylipins as essential components of innate immunity mechanisms.

iota-Carrageenans are sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of red algae, which auto-associate into crystalline fibers made of aggregates of double-stranded helices. iota-Carrageenases, which constitute family 82 of glycoside hydrolases, fold into a right-handed beta-helix. Here, the structure of Alteromonas fortis iota-carrageenase bound to iota-carrageenan fragments was solved at 2.0A resolution (PDB 1KTW). The enzyme holds a iota-carrageenan tetrasaccharide (subsites +1 to +4) and a disaccharide (subsites -3, -4), thus providing the first direct determination of a 3D structure of iota-carrageenan. Electrostatic interactions between basic protein residues and the sulfate substituents of the polysaccharide chain dominate iota-carrageenan recognition. Glu245 and Asp247 are the proton donor and the base catalyst, respectively. C-terminal domain A, which was highly flexible in the native enzyme structure, adopts a alpha/beta-fold, also found in DNA/RNA-binding domains. In the substrate-enzyme complex, this polyanion-binding module shifts toward the beta-helix groove, forming a tunnel. Thus, from an open conformation which allows for the initial endo-attack of iota-carrageenan chains, the enzyme switches to a closed-tunnel form, consistent with its highly processive character, as seen from the electron-microscopy analysis of the degradation of iota-carrageenan fibers.

A mesophilic, aerobic, non-flagellated, gliding bacterium, forming yellow colonies and designated DsijT, was isolated from a red alga on the sea-shore of Roscoff, Brittany, France. DsijT was selected for its ability to actively degrade both agars and carrageenans. The Gram-negative cells occurred singly or in pairs as long rods. The temperature range for growth was 13-45 degrees C, with an optimum at 35 degrees C. The pH range for growth at 35 degrees C was from 6.0 to 8.5, with an optimum around pH 7.0. The NaCl concentrations required for growth at 35 degrees C and pH 7.0 ranged from 5 to 60 g l(-1), with an optimum around 25 g l(-1). The G+C content of the genomic DNA was 42-43 mol%. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain DsijT is closely related to [Cytophaga] uliginosa DSM 2061T. Phenotypic features, however, allowed DsijT and [Cytophaga] uliginosa strains to be distinguished on the basis of ten traits (spreading behaviour, assimilation of eight compounds and amylase production). Their total protein profiles were also different and DNA-DNA hybridization experiments confirmed that DsijT constitutes a new species, distinct from [Cytophaga] uliginosa. Based on the phenotypic features and the phylogenetic relationships of the Flavobacteriaceae, a new genus designated Zobellia gen. nov. is proposed to include Zobellia galactanovorans gen. nov., sp. nov., while [Cytophaga] uliginosa becomes Zobellia uliginosa comb. nov. The type strain of Zobellia galactanovorans is DsijT (= DSM 12802T = CIP 106680T).

iota-Carrageenases are polysaccharide hydrolases that cleave the beta-1,4 linkages between the d-galactose-4-sulfate and 3, 6-anhydro-d-galactose-2-sulfate residues in the red algal galactans known as iota-carrageenans. We report here on the purification of iota-carrageenase activity from the marine bacterium Zobellia galactanovorans and on the characterization of iota-carrageenase structural genes. Genomic libraries from this latter bacterium as well as from Alteromonas fortis were functionally screened for the presence of iota-carrageenase(+) clones. The Z. galactanovorans and A. fortis iota-carrageenase genes encode homologous proteins of 53.4 and 54.8 kDa, respectively. Based on hydrophobic cluster analysis and on the (1)H NMR monitoring of the products of the overexpressed A. fortis iota-carrageenase, these enzymes appear to form a new family of glycoside hydrolases, unrelated to that of kappa-carrageenases and with an inverting mechanism of hydrolysis. They both feature a 45-amino acid-long N-terminal segment with sequence similarity to the N-terminal region of several other polysaccharidases. In those for which a three-dimensional structure is available, this conspicuous segment, also deemed "glycanase motif" (Chua, J. E. H., Manning, P. A., and Morona, R. (1999) Microbiology (Reading) 145, 1649-1659), corresponds to a strand-helix-strand "cap" that covers the N-terminal end of a common, right-handed beta-helical fold.

The hypothesis that tRNAs are involved in the maturation of the large primary transcripts of Chondrus crispus mitochondrial DNA was addressed by primer extension mapping of the transcript 5' ends of the ten genes that are preceded by tRNA genes in C. crispus mitochondrial genome. Among the 12 tRNAs that were candidates as maturation signals, eight, namely tRNAArg, tRNALys, tRNAAsp, tRNAGln, tRNATrp, tRNAIle, tRNAPhe and tRNAGly, were cleaved internally upon maturation of C. crispus mitochondrial primary transcripts, all of them at the same base, invariant pyrimidine 48. Only four tRNAs departed from this pattern: tRNALeu and tRNACys, which are not maturation signals, tRNAMet, which appears to be excised as a whole from the orf94 primary trancript and tRNAAla, which is cleaved internally at positions other than Y48. Sequence comparisons between the cleaved and the uncleaved tRNAs suggest that their core tertiary structure is involved with their recognition and cleavage. However, the precursor transcripts are also processed at the 5' and 3' ends of the tRNAs to yield tRNA molecules that are stable and functional in translation. This indicates that two different RNA processing mechanisms coexist in C. crispus mitochondria, one required for the production of functional tRNAs and the other for the processing of mRNAs., (Copyright 1999 Academic Press.)

The mitochondrial DNA (mt DNA) of the red alga Chondrus crispus is shown to be transcribed into two large RNA molecules. These primary transcripts are cleaved once, at the level of a tRNA, then the resulting products are processed via multiple maturation events into either mono- or poly-cistronic RNAs. Transcripts were detected for all genes and open reading frames, except for rps11 and orf172. For both transcription units the initiation of transcription was mapped by in vitro RNA capping and primer extension experiments within inverse repeated sequences at the north pole of the molecule. Consistent with primer extension mapping, putative promoter motifs sharing significant similarities with both chicken and Xenopus mitochondrial promoters were found in the C. crispus mitochondrial genome. Altogether C. crispus mitochondrial DNA appears to be transcribed as animal mtDNA is, suggesting that transcription mechanisms in mitochondria are dependent on the overall organization of the mitochondrial genome irrespective of the eukaryotic phylogeny., (Copyright 1998 Academic Press.)

The purpose of this review is to present an account of our current understanding of the structure, organization and evolution of mitochondrial genomes, and to discuss the origin and evolution of mitochondria from the perspective recently provided by the extensive sequencing of various mitochondrial genomes. Mitochondrial-en-coded protein phylogenies are congruent with nuclear phylogenies and strongly support a monophyletic origin of mitochondria. The newly available data from red-algal mitochondrial genomes, in particular, show that the structural and functional diversity of mitochondrial genomes can be accounted for by paralogous evolution. We also discuss the influence of other constraints, such as uniparental inheritance, on the evolution of genome organization in mitochondria.

The entire nucleotide sequence containing the small-subunit ribosomal RNA gene (SSU rRNA) from the mitochondrial genome of Chondrus crispus was determined. To our knowledge, this is the first sequence of a mitochondrial 16S-like rRNA from a red alga. The length of this gene is 1,376 nucleotides. Its secondary structure was constructed and compared with other known secondary structures from eubacteria and from mitochondria of land plants, green and brown algae, and fungi. Phylogenetic trees were built upon SSU rRNA sequence alignment from mitochondria and eubacteria. The results show that rhodophytes and chromophytes provide additional links in the evolution of mitochondria between the green plant lineage and the "nonplant" lineages.

The complete nucleotide sequence of the circular mitochondrial (mt) DNA from the red alga Chondrus crispus was determined (25,836 nucleotides, A+T content 72.1%). Fifty one genes were identified. They include genes encoding three subunits of the cytochrome oxidase (cox1 to 3), apocytochrome b (cob), seven subunits of the NADH dehydrogenase complex (nad1 to 6, nad4L), two ATPase subunits (atp6 and atp9), three ribosomal RNAs (rrn5, srn and lrn), 23 tRNAs and four ribosomal proteins (rps3, rps11, rps12 and rpl16). Two subunits of the succinate dehydrogenase complex (sdhB and sdhC), usually found on nuclear genomes, are also located on the mtDNA of C. crispus. One group IIb intron is inserted in the tRNAIle gene. Six potentially functional open reading frames were identified, four of them having counterparts among green plant mtDNAs. The use of a modified genetic code and the absence of RNA editing, previously reported for the cox3 gene, appears as a general characteristic of this molecule. Mitochondrial genes are encoded on both DNA strands, in two opposite major transcriptional directions, suggesting the existence of two main transcriptional units. Two long and stable stem-loops were identified in intergenic regions, which are believed to be involved with transcription and replication. The main structural features of this genome are compared with the overall organization of mtDNAs and are discussed in view of the evolution of mitochondria.

We have characterized the cDNA and genomic sequences that encode actin from the multicellular red alga Chondrus crispus. Southern-blot analysis indicates that the C. crispus actin gene (ChAc) is present as a single copy. Northern analysis shows that, like the GapA gene, the actin gene is well expressed in gametophytes but weakly in protoplasts. Compared to actin genes of animals, fungi, green plants and oomycetes, that of C. crispus displays a higher evolutionary rate and does not show any of the amino-acid signatures characteristic of the other lineages. As previously described for GapA, ChAc is interrupted by a single intron at the beginning of the coding region. The site of initiation of transcription was characterized by RNAse protection. The promoter region displays a CAAT box but lacks a canonical TATA motif. Other noticeable features, such as a high content of pyrimidines as well as a 14-nt motif found in both the 5'-untranslated region and the intron, were observed.

We present the nucleotide sequence of the gene encoding subunit 3 of cytochrome c oxidase in Chondrus crispus, the first report on a mitochondrial gene from a red alga. Amino acid alignment with homologous proteins shows that tryptophan is specified by UGA, as in the mitochondrial code of most organisms other than green plants. However, phylogenetic analyses of cox3 amino acid and nucleotide sequences indicate that C. crispus COX3 is related to the green-plant mitochondrial lineage. No RNA editing was detected on the corresponding transcript. As the only known photosynthetic eukaryotes that both share an immediate mitochondrial ancestor with green plants and exhibit features characteristic of non-plant mitochondria, ie, a small-sized mitochondrial genome and a modified genetic code, rhodophytes may be thought of as an intermediate evolutionary link at the root of the green-plant mitochondrial lineage.

Algae are a heterogeneous group of photosynthetic eukaryotes traditionally separated into three major subdivisions: rhodophytes, chlorophytes, and chromophytes. The evolutionary origin of rhodophytes or red algae and their links to other photosynthetic and nonphotosynthetic eukaryotes have been a matter of much controversy and speculation. Here we present the first cDNAs of nuclear protein genes from red algae: Those encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Chondrus crispus. A phylogenetic analysis including GAPDH gene sequences from a number of eukaryotic taxa, cyanobacteria, and purple bacteria suggests that chloroplasts and rhodoplasts together form a monophyletic group of cyanobacterial descent and that rhodophytes separated from chlorophytes at about the same time as animals and fungi. The composite GAPDH tree further demonstrates that chloroplast and cytosolic GAPDH genes are closely related to their homologs in cyanobacteria and purple bacteria, respectively, the presumptive ancestors of chloroplasts and mitochondria, thereby firmly establishing the endosymbiotic origin of these nuclear genes and their fixation in eukaryotic cells before the rhodophyte/chlorophyte separation. The present data are in conflict with phylogenetic inferences based on plastid-encoded rbcL sequences supporting a polyphyletic origin of rhodoplasts and chloroplasts. Comparison of rbcL to GAPDH phylogenies suggests that rbcL trees may be misleading because they are composed of branches representing ancient duplicated (paralogous) genes.

Our previous phylogenetic analysis based on cDNA sequences of chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH; genes GapA and GapC, respectively) of the red alga Chondrus crispus suggested that rhodophytes and green plants are sister groups with respect to plastids and mitochondria and diverged at about the same time or somewhat later than animals and fungi. Here we characterize the genomic sequences of genes GapC and GapA of C. crispus with respect to promotor structures, intron/exon organization, genomic complexity, G + C content, CpG suppression and their transcript levels in gametophytes and protoplasts, respectively. To our knowledge this is the first report on nuclear protein genes of red algae. The GapC gene is G + C-rich, contains no introns and displays a number of classic sequence motifs within its promotor region, such as TATA, CAAT, GC boxes and several elements resembling the plant-specific G-box palindrome. The GapA gene has a moderate G+C content, a single CAAT box motif in its promotor region and a single intron of 115 bp near its 5' end. This intron occupies a conserved position corresponding to that of intron 1 in the transit peptide region of chloroplast GAPDH genes (GapA and GapB) of higher plants. It has consensus sequences similar to those of yeast introns and folds into a conspicuous secondary structure of -61.3 kJ. CpG profiles of genes GapC and GapA and their flanking sequences show no significant CpG depletion suggesting that these genomic sequences are not methylated. Genomic Southern blots hybridized with generic and gene specific probes indicate that both genes are encoded by single loci composed of multiple polymorphic alleles. Northern hybridizations demonstrate that both genes are expressed in gametophytes but not in protoplasts where appreciable amounts of transcripts can only be detected for GapC.

A bacterial strain able to degrade various sulfated galactans (carrageenans and agar) was isolated from the marine red alga Delesseria sanguinea. From the cell-free supernatant of cultures grown on crude lambda-carrageenan, a kappa-carrageenase was purified by ammonium sulfate fractionation, gel filtration on Sephacryl S 200 HR and ion-exchange chromatography on DEAE--Sepharose-CL6B. The purified kappa-carrageenase was detected as a single protein upon SDS/PAGE. Its molecular mass was estimated at 40 kDa. Activity was observed against kappa-carrageenan over the pH range 5.0-8.5 and was optimal at pH 7.2 in Tris buffer or 7.0 in Mops buffer. The enzyme activity remained stable at 30 degrees C, but only for up to 1 h at 40 degrees C. Analysis of the degradation products of the kappa-carrageenase by gel filtration and 13C-NMR spectroscopy indicated that the enzyme degrades kappa-carrageenan down to the level of the kappa-neocarratetraose sulfate. The properties of this new enzyme are compared with those of previously characterized carrageenases.