Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge,Rhopaloeides odorabile

Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so‐called sponge‐specific clusters (SCs) or sponge‐ and coral‐specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)‐based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31°C) had no effect on the present and/or active portions of the R. odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two‐thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change.     

Detection and Phylogenetic Analysis of Novel Crenarchaeote and Euryarchaeote 16S Ribosomal RNA Gene Sequences from a Great Barrier Reef Sponge

The presence of Archaea in the Great Barrier Reef marine sponge Rhopaloeides odorabile was investigated by 16S ribosomal RNA community analysis of total DNA extracted from the sponge tissue. The 16S rRNA gene sequences corresponding to group I crenarchaeotes and group II euryarchaeotes were recovered from R. odorabile tissue. The location of archaeal cells within the sponge tissue was investigated using fluorescently labeled oligonucleotide probes. The presence of Archaea was confirmed within all regions of the sponge tissue from R. odorabile, with a significantly higher number of archaeal cells located in the pinacoderm than the mesohyl region. This is the first report of euryarchaeaotes associated with marine sponges. Received April 16, 2001; accepted June 27, 2001     

Anti-actomyosin. Influence on adhesive behaviour of eukaryotic cells and of cuvierian tubules

The effect of antisera against chicken gizzard smooth-muscle actomyosin and against pectoralis striated-muscle actomyosin on adhesive behaviour of eukaryotic cells (from sea urchin embryos and from a silicious sponge) and of Cuvierian tubules has been studied. The results with a sea urchin cells, which require divalent cations for aggregation, showed that antiserum to chicken gizzard smooth-muscle actomyosin inhibited reaggregation of trypsin-treated cells better than mechanically dissociated cells, while anti-chicken pectoralis striated-muscle had no effect. Primary reaggregation of trypsin-dissociated sponge cells, in the presence of calcium and magnesium, is also inhibitable by anti-gizzard smooth-muscle but not by anti-pectoralis striated-muscle. Anti-gizzard smooth-muscle had no effect on secondary reaggregation of sponge cells mediated by a soluble aggregation factor. Anti-gizzard smooth-muscle inhibited Cuvierian tubule adhesion.     

Anti-actomyosin. Influence on adhesive behaviour of eukaryotic cells and of Cuvierian tubules.

The effect of antisera against chicken gizzard smooth-muscle actomyosin and against pectoralis striated-muscle actomyosin on adhesive behaviour of eukaryotic cells (from sea urchin embryos and from a silicious sponge) and of Cuvierian tubules has been studied. The results with sea urchin cells, which require divalent cations for aggregation, showed that antiserum to chicken gizzard smooth-muscle actomyosin inhibited reaggreagation of trypsin-treated cells better than mechanically dissociated cells, while anti-chicken pectoralis striated-muscle had no effect. Primary reaggreagation of trypsin-dissociated sponge cells, in the presence of calcium and magnesium, is also inhibitable by anti-gizzard smooth-muscle but not by anti-pectoralis straited muscle. Anti-gizzard smooth-muscle had no effect on secondary reaggregation of sponge cells mediated by a soluble aggregation factor. Anti-gizzard smooth-muscle inhibited Cuvierian tubule adhesion.     

Aggregation of sponge cells: 22. Species-specific reactivity of a lectin from the sponge Geodia cydonium

Single cells of the marine sponge Geodia cydonium aggregate species-specifically in the presence of a soluble aggregation factor to form large cell clumps. A lectin isolated from the same sponge species does not cause agglutination of Geodia cells but agglutinates only cells from heterologous species (e.g. Tethya lyncurium, Hemimycale columella, Pellina semitubulosa, Cacospongia scalaris, Verongia aerophoba). The process of agglutination is independent of divalent cations (they do not affect the agglutination process at concentrations up to 50 mM), occurs at 2°C, causes a reduction in the viability of the cells and results in an inhibition of programmed syntheses. The observed differences between the properties of cell agglutination (effect of a lectin in a heterologous system) and cell aggregation (effect of an aggregation factor in the homologous system) is discussed. Cell aggregation is dependent upon the presence of an aggregation factor, the presence of cations and an incubation temperature 2̃0°C; cell aggregation results in a stimulation of programmed syntheses. Cell agglutination requires a heterologous macromolecule (e.g. lectin), it is independent of divalent cations and causes inhibition of programmed syntheses in the cells.     

Sulfated polysaccharides from marine sponges: conspicuous distribution among different cell types and involvement on formation of in vitro cell aggregates

Marine sponges (Porifera) display an ancestral type of cell-cell adhesion, based on carbohydrate-carbohydrate interaction. The aim of the present work was to investigate further details of this adhesion by using, as a model, the in vitro aggregation of dissociated sponge cells. Our results showed the participation of sulfated polysaccharides in this cell-cell interaction, as based on the following observations: (1) a variety of sponge cells contained similar sulfated polysaccharides as surface-associated molecules and as intracellular inclusions; (2) ³⁵S-sulfate metabolic labeling of dissociated sponge cells revealed that the majority (two thirds) of the total sulfated polysaccharide occurred as a cell-surface-associated molecule; (3) the aggregation process of dissociated sponge cells demanded the active de novo synthesis of sulfated polysaccharides, which ceased as cell aggregation reached a plateau; (4) the typical well-organized aggregates of sponge cells, known as primmorphs, contained three cell types showing sulfated polysaccharides on their cell surface; (5) collagen fibrils were also produced by the primmorphs in order to fill the extracellular spaces of their inner portion and the external layer surrounding their entire surface. Our data have thus clarified the relevance of sulfated polysaccharides in this system of in vitro sponge cell aggregation. The molecular basis of this system has practical relevance, since the culture of sponge cells is necessary for the production of molecules with biotechnological applications.     

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50–70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a >90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.     

Species-specific aggregation factor in sponges. V. Influence on programmed syntheses.

Isolated cells from the siliceous sponge Geodia cydonium as well as small primary aggregates (diameter: 70 mum) consisting of them show no increase in rates of programmed syntheses and mitotic activity with time. After addition of a highly purified aggregation factor to a culture with primary aggregates which subsequently form secondary aggregates (diameter: larger than 1000 mum), a dramatic increase of DNA, RNA and protein synthesis occurs. Together with this increase, the cells show a high mitotic activity. The values for the mitotic coefficient reach a first maximum 8 h after the beginning of the secondary aggregation process. The stimulation of the mitotic activity of cells during the aggregation factor induced secondary aggregation process can be suppressed by inhibitors of RNA and protein synthesis as well as by a blocker of DNA synthesis. This finding may indicate that cells from the G0-population enter the proliferating cell pool via the G1-phase.     

Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis.

Sublethal doses of gamma radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrophoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena.     

Molecular systematics of sponges (Porifera)

The first application of molecular systematics to sponges was in the 1980s, using allozyme divergence to dis-criminate between conspecific and congeneric sponge populations. Since this time, a fairly large database has been accumulated and, although the first findings seemed to indicate that sponge species were genetically more divergent than those of other marine invertebrates, a recent review of the available dataset indicates that levels of interspecific gene identities in most sponges fall within the normal range found between species of other invertebrates. Nevertheless, some sponge genera have species that are extremely divergent from each other, suggesting a possible polyphyly of these genera. In the 1990s, molecular studies comparing sequences of ribosomal RNA have been used to reappraise the phylogenetic relationships among sponge genera, families, orders and classes. Both the 18S small subunit and the 28S large subunit rRNA genes have been sequenced (41 complete or partial and 75 partial sequences, respectively). Sequences of 18S rRNA show good support for Porifera being true Metazoa, but they are not informative for resolving relationships among genera, families or orders. 28S rRNA domains D1 and D2 appear to be more informative for the terminal nodes and provide resolution for internal topologies in sufficiently closely related species, but the deep nodes between orders or classes cannot be resolved using this molecule. Recently, a more conserved gene, Hsp70, has been used to try to resolve the relationships in the deep nodes. Metazoan monophyly is very well supported. Nevertheless, the divergence between the three classes of Porifera, as well as the divergence between Porifera, Cnidaria and Ctenophora, is not resolved. Research is in progress using other genes such as those of the homeodomain, the tyrosine kinase domain, and those coding for the aggregation factor. For the moment the dataset for these genes is too restricted to resolve the phylogenetic relationships of these phyla. However, whichever the genes, the phylogenies obtained suggest that Porifera could be paraphyletic and that the phylogenetic relationships of most of the families and orders of the Demospongiae have to be reassessed. The Calcarea and Hexactinellida are still to be studied at the molecular level.     

A 12 S precursor to 5.8 S rRNA associated with rat liver nucleolar 28 S rRNA

The pre-rRNA and rRNA components of rat and mouse liver nucleolar RNA were analysed. It was shown that upon denaturation, part of the 32 S pre-rRNA is converted into 28 S rRNA and 12 S RNA. The 12 S RNA from mouse (Mr, 0.36 X 10(6)) is larger than the one from rat (Mr, 0.32 X 10(6). The 12 S RNA chain is intact and resists denaturation treatment. The non-covalent binding of this RNA with nucleolar 28 S rRNA is stronger than that of 5.8 S rRNA with 28 S rRNA. Hybridization with a rat internal-transcribed spacer rDNA fragment identifies 12 S RNA as corresponding to the 5'-end non-conserved segment of 32 S pre-rRNA, including 5.8 S rRNA. The significance of the formation of a 12 S precursor to 5.8 S rRNA in the biogenesis of ribosomes in mammalian cells is discussed.     

Onset of ribosome degradation during cessation of growth in BHK-21/C13 cells.

When BHK-21/C13 cells growing exponentially in 10% serum are transferred to a medium containing only 0.25% serum, cell growth is decreased. After initial changes in RNA synthesis and degradation, protein content of the cultures reaches a plateau and eventually DNA synthesis is arrested. rRNA is relatively stable in exponentially growing cells. Immediately after 'step-down' rRNA degradation commences, but poly(A)-containing RNA does not appear to be degraded any faster than in control cells. Reutilization of RNA precursors has been independently measured and amounts to less than 1%/h for rRNA, insufficient to influence the conclusion that rRNA degradation begins almost immediately after 'step-down'. The degree of reutilization of uridine is much greater for poly(A)-containing RNA than for poly(A)-free RNA.     

Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome

Samples of the sponge Haliclona simulans were collected from Irish waters and subjected to a culture-independent analysis to determine the microbial, polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) diversity. 16S rRNA gene libraries were prepared from total sponge, bacterial enriched sponge and seawater samples. Eight phyla from the Bacteria were detected in the sponge by phylogenetic analyses of the 16S rRNA gene libraries. The most abundant phylum in the total sponge library was the Proteobacteria (86%), with the majority of these clones being from the γ- Proteobacteria (77%); two groups of clones were dominant and together made up 69% of the total. Both of these groups were related to other sponge-derived microbes and comprised novel genera. Within the other bacterial phyla groups of clones representing novel candidate genera within the phyla Verrucomicrobia and Lentisphaerae were also found. Selective enrichment of the bacterial component of the sponge prior to 16S rRNA gene analysis resulted in a 16S rRNA gene library dominated by a novel genus of δ- Proteobacteria , most closely related to the Bdellovibrio . The potential for the sponge microbiota to produce secondary metabolites was also analysed by polymerase chain reaction amplification of PKS and NRPS genes. While no NRPS sequences were isolated seven ketosynthase (KS) sequences were obtained from the sponge metagenome. Analyses of these clones revealed a diverse collection of PKS sequences which were most closely affiliated with PKS from members of the Cyanobacteria , Myxobacteria and Dinoflagellata .     

Inhibition by actinomycin D of ribosomal RNA synthesis in the presynthesis period of the mitotic cycle of Chinese hamster cell cultures]

Using radioautographic technique actinomycin D at a concentration of 0.08 mug/ml was shown to inhibit selectively ribosomal RNA (rRNA) synthesis in monolayer cultures of Chinese hamster cells. The treatment with actinomycin D of cells synchronized by mitotic selection in the beginning of the G1 period causes a delay in the onset of DNA synthesis. However, a similar treatment in the late G1 period does not prevent cells from entering the S-period. The same effect has been produced by 9 mug/ml lucanthone, another inhibitor of rRNA synthesis. The experiments demonstrate a pronounced difference in cell reaction to the depression of rRNA synthesis in early and late G1 period. The data imply that the formation of rRNA, essential for the initiation of DNA synthesis, is accomplished in the first half of the G1 period, while part of rRNA has been already formed in the previous cycle.     

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.     

Repression of rRNA synthesis during slug reconstruction in Dictyostelium discoideum

When cells dissociated from "slugs" are allowed to reaggregate, they reconstruct slugs. During this process, the cells showed a marked decrease in the ratio of labeled RNA lacking poly(A) to labeled total RNA as compared with that of cells in normal slugs. Irrespective of the change in total labeled RNA, the ratio remained low even after 6 h of reconstruction. Sucrose density gradient analysis of RNA showed that the synthesis of high molecular weight rRNA (26S and 17S) was considerably repressed in reconstructed slugs as compared with normal slugs. Polyacrylamide gel electrophoresis of low molecular weight rRNA revealed that the synthesis of 5S rRNA, but not 4S tRNA, was repressed.