Molecular and ecological evidence for species specificity and coevolution in a group of marine algal-bacterial symbioses.

The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus Prionitis (Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region). Gall formation has been described previously on two species of Prionitis, P. lanceolata (from central California) and P. decipiens (from Peru). This investigation reports gall formation on a third related host, Prionitis filiformis. Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the Roseobacter grouping of the alpha subclass of the division Proteobacteria (99.4 to 98.25% sequence identity among phylotypes). Comparison of symbiont and host molecular phylogenies confirms the presence of three gall-bearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving. The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials. Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host. In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species). Symbiont-host specificity in Prionitis gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation.

Galls on the marine red alga Prionitislanceolata (Halymeniaceae): specific induction and subsequentdevelopment of an algal-bacterial symbiosis.

Gall formation in Prionitis lanceolata is associated with aspecific eubacterium (Proteobacteria [alphasubclass], Rhodobacter grouping), which, typical ofbacterial symbionts, has not yet been cultivated or isolated in pureculture. This investigation tested the hypothesis that P.lanceolata gall formation was caused by the associated eubacteriumusing a species-specific rDNA probe (S-S-P.l.sym-0949-a-A-25) toidentify and assay for symbiont presence during consecutive laboratoryinduction trials. Gall induction was quantified and whole-cell in situhybridization used to determine the relative percentage of symbioticeubacteria in inoculation homogenates. In situ hybridization ofsymbionts in sections allowed localization and monitoring of thismicrobe during gall development. Induction trial results indicate asignificant correlation between bacterial symbiont presence and gallinitiation (P = 0.00005). The gall bacterium comprisedthe majority of the eubacteria hybridized in laboratory inductionhomogenates (85-97%), in galls induced in the laboratoryand in three algal populations in nature. The evidence presented heredemonstrates the causative role of the identified eubacterium in gallinduction and formation. This investigation is significant in theapplication of molecular methods towards understanding the roles ofnoncultivable marine bacteria in marine algal-microbeinteractions.

Molecular characterization of two large DNA plasmids in the red alga Porphyra pulchra.

Five plasmids occur in the red alga Porphyra pulchra. The two larger ones (6859 and 6427 bp) differ in their sequences. The three smaller (1896, 2100, and 2102 bp) have sequences that are similar to one another. These plasmids are circular, double-stranded DNA, present in high copy number, and maintained in this organism through successive generations of laboratory culture. Sequence analysis of the two larger plasmids reveals few prominent structural features, but several potential open reading frames (ORFs) occur, some of which are transcriptionally active. Sequence database comparisons find significant sequence similarity between ORF3 or PP6427 and a 411 amino-acid polypeptide previously characterized in plasmid GC2 from the red alga Gracilaria chilensis. These data support the presence of at least one conserved plasmid coding region in distant orders of red algae. Southern blots of total genomic DNA from other red algae probed with plasmids from P. pulchra demonstrate no hybridization to previously studied Gracilariales species but notable hybridization to several species within the genus Porphyra, although the sizes of detected bands vary.

A 5 S rRNA gene is present in the mitochondrial genome of the protist Reclinomonas americana but is absent from red algal mitochondrial DNA.

Except in the case of land plants, mitochondrial ribosomes apparently lack a 5 S rRNA species, even though this small RNA is a component of all prokaryotic, chloroplast and eukaryotic cytosol ribosomes. In plants, the mitochondrial 5 S rRNA is encoded by mtDNA and differs in sequence from the 5 S rRNA specified by plant nuclear and chloroplast genomes. A distinctive 5 S rRNA component has not been found in the mitochondrial ribosomes of non-plant eukaryotes and, with the notable exception of the chlorophycean alga, Prototheca wickerhamii, a 5 S rRNA gene has not been identified in those non-plant mtDNAs characterized to date. Here, we report the presence of a 5 S rRNA gene in the mtDNA of the heterotrophic flagellate Reclinomonas americana. This unicellular eukaryote is a member of the jakobid flagellates, an early-diverging group of protists that share ultrastructural characteristics with the retortamonads, primitive protists that lack mitochondria. We report sequence data from the mtDNAs of the red algae Porphyra purpurea and Gracilariopsis lemaneiformis, which we use to evaluate a recent claim that a 5 S rRNA gene exists in the mtDNA of a third rhodophyte alga, Chondrus crispus. Our results lead us to the opposite conclusion: that a 5 S rRNA gene is not encoded by red algal mtDNA. In view of the accumulating evidence favoring a monophyletic origin of the mitochondrial genome, it is likely that a 5 S rRNA gene was present in an ancestral proto-mitochondrial genome, and that contemporary mtDNA-encoded 5 S rRNA genes have all descended from this ancestral gene. Considering the highly restricted phylogenetic distribution of identified mtDNA-encoded 5 S rRNA genes, it follows that the mitochondrial 5 S rRNA gene must have been lost multiple times during evolutionary diversification of the eukaryotic lineage.

Fate of Parasite and Host Organelle DNA during Cellular Transformation of Red Algae by Their Parasites.

The transfer of a nucleus into a cytoplasm of a genetically foreign cell and its subsequent multiplication in the cytoplasm of this cell characterize most parasitic red algal species and their interactions with specific red algal hosts. Nuclei enter the host's cytoplasm upon cell fusion of parasite and host cell; here, they replicate, are spread to contiguous host cells, and ultimately are packaged into spores that reinfect other host thalli. In this study, we examined whether the proplastids and mitochondria that occur in these red algal adelphoparasites are acquired from their host or whether they are unique to the parasite and are brought into the host along with the parasite nucleus. To establish their origins and fates, plastid and mitochondrial restriction fragment length polymorphisms (RFLPs) of parasite cells were compared with those of their host plastid and mitochondrial DNA in three host and parasite pairs. For plastids, no RFLP differences were found between hosts and parasites, supporting an earlier conclusion, based on microscopic studies, that the proplastids of parasites are acquired from their hosts. For mitochondria, characteristic RFLP differences were detected between host and parasite for two of the pairs of species but not for the third. Evidence of the evolutionary difference between hosts and their parasites was shown by RFLP differences between nuclear ribosomal repeat regions.

Red algal plasmids.

Five of 21 red algal genera were found to contain circular dsDNA plasmids, typically of two or more sizes per species. Clones of the two plasmids (GL4.4 and GL3.5 kbp), characterizing all isolates of Gracilariopsis lemaneiformis, do not cross-hybridize with each other, with the nuclear, plastid or mitochondrial genomes of G. lemaneiformis, or with any DNA genomes of the other red algae examined. Clones of both plasmids hybridized with discrete bands on Northern blots of total RNA and poly(A)+ RNA. Sequencing of the G. lemaneiformis 3.5 kbp plasmid revealed two potential open reading frames which, when used to probe Northern blots, confirmed the presence of specific transcripts. These autonomously replicating plasmids are present in high copy number per cell and in constant proportion to each other. Their constancy suggests a function of significance to the species. Red algal plasmids may provide useful vectors for transforming economically important red algal species.

The solution to the cytological paradox of isomorphy.

Cells with polyploid nuclei are generally larger than cells of the same organism or species with nonpolyploid nuclei. However, no such change of cell size with ploidy level is observed in those red algae which alternate isomorphic haploid with diploid generations. The results of this investigation reveal the explanation. Nuclear DNA content and other parameters were measured in cells of the filamentous red alga Griffithsia pacifica. Nuclei of the diploid generation contain twice the DNA content of those of the haploid generation. However, all cells except newly formed reproductive cells are multinucleate. The nuclei are arranged in a nearly perfect hexagonal array just beneath the cell surface. When homologous cells of the two generations are compared, although the cell size is nearly identical, each nucleus of the diploid cell is surrounded by a region of cytoplasm (a "domain") nearly twice that surrounding a haploid nucleus. Cytoplasmic domains associated with a diploid nucleus contain twice the number of plastids, and consequently twice the amount of plastid DNA, than is associated with the domain of a haploid nucleus. Thus, doubling of ploidy is reflected in doubling of the size and organelle content of the domain associated with each nucleus. However, cell size does not differ between homologous cells of the two generations, because total nuclear DNA (sum of the DNA in all nuclei in a cell) per cell does not differ. This is the solution to the cytological paradox of isomorphy.

Applications of fluorochromes to pollen biology. I. Mithramycin and 4',6-diamidino-2-phenylindole (DAPI) as vital stains and for quantitation of nuclear DNA.

The two DNA-specific fluorochromes DAPI and mithramycin have been found to be extremely useful dyes in studies of pollen development and growth. Both fluorochromes stain nuclei brilliantly either in fixed or in living tricellular and bicellular angiosperm pollen, thereby permitting rapid scanning for pollen abnormalities and easy observation of nuclear details. These water soluble dyes can be incorporated into the germination medium for studies of pollen germination in vitro, facilitating observation of the movement of generative, sperm and tube nuclei during pollen growth. In fixed pollen, the fluorochromes bind quantitatively with DNA and thus may be used to quantitate ploidy changes and to study cell cycles during pollen development, germination and fertilization.

Transfer of nuclei from a parasite to its host.

During the normal course of infection, nuclei are transferred via secondary pit connections from the parasitic marine red alga Choreocolax to its red algal host Polysiphonia. These "planetic" nuclei are transmitted by being cut off into specialized cells (conjunctor cells) that fuse with an adjacent host cell, thereby delivering parasite nuclei and other cytoplasmic organelles into host cell cytoplasm. Within the foreign cytoplasm, planetic nuclei survive for several weeks and may be active in directing the host cellular responses to infection, since these responses are seen only in host cells containing planetic nuclei. The transfer and long-term survival of a nucleus from one genus into the cytoplasm of another through mechanisms that have evolved in nature challenge our understanding of nuclear-cytoplasmic interactions and our concept of "individual."

Elucidation of fertilization and development in a red alga by quantitative DNA microspectrofluorometry.

Crucial nuclear events throughout the life histories of red algae have eluded researchers. Use of the DNA fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and microspectrofluorometry has now resolved the problems. In the parasitic red alga Choreocolax polysiphoniae (= Leachiella pacifica), the amounts of nuclear DNA during the processes of meiosis and fertilization are documented as well as the transfer of the newly formed zygote nucleus to the adjacent accessory cell. In this organism, the G2 period predominates in the cell cycle of vegatative and reproductive cells; many nondividing vegetative cells become highly polyploid. The results indicate that the use of various fluorochromes combined with microspectrofluorometric measurements of individual nuclei is valuable for study of many developmental problems in lower eukaryotes where the relatively small nuclear DNA content has made study with conventional dyes impossible.