Marine gregarine genomes reveal the breadth of apicomplexan diversity with a partially conserved glideosome machinery.

Our current view of the evolutionary history, coding and adaptive capacities of Apicomplexa, protozoan parasites of a wide range of metazoan, is currently strongly biased toward species infecting humans, as data on early diverging apicomplexan lineages infecting invertebrates is extremely limited. Here, we characterized the genome of the marine eugregarine Porospora gigantea, intestinal parasite of Lobsters, remarkable for the macroscopic size of its vegetative feeding forms (trophozoites) and its gliding speed, the fastest so far recorded for Apicomplexa. Two highly syntenic genomes named A and B were assembled. Similar in size (~ 9 Mb) and coding capacity (~ 5300 genes), A and B genomes are 10.8% divergent at the nucleotide level, corresponding to 16-38 My in divergent time. Orthogroup analysis across 25 (proto)Apicomplexa species, including Gregarina niphandrodes, showed that A and B are highly divergent from all other known apicomplexan species, revealing an unexpected breadth of diversity. Phylogenetically these two species branch sisters to Cephaloidophoroidea, and thus expand the known crustacean gregarine superfamily. The genomes were mined for genes encoding proteins necessary for gliding, a key feature of apicomplexans parasites, currently studied through the molecular model called glideosome. Sequence analysis shows that actin-related proteins and regulatory factors are strongly conserved within apicomplexans. In contrast, the predicted protein sequences of core glideosome proteins and adhesion proteins are highly variable among apicomplexan lineages, especially in gregarines. These results confirm the importance of studying gregarines to widen our biological and evolutionary view of apicomplexan species diversity, and to deepen our understanding of the molecular bases of key functions such as gliding, well known to allow access to the intracellular parasitic lifestyle in Apicomplexa.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Alginates along the filament of the brown alga Ectocarpus help cells cope with stress.

Ectocarpus is a filamentous brown alga, which cell wall is composed mainly of alginates and fucans (80%), two non-crystalline polysaccharide classes. Alginates are linear chains of epimers of 1,4-linked uronic acids, ß-D-mannuronic acid (M) and α-L-guluronic acid (G). Previous physico-chemical studies showed that G-rich alginate gels are stiffer than M-rich alginate gels when prepared in vitro with calcium. In order to assess the possible role of alginates in Ectocarpus, we first immunolocalised M-rich or G-rich alginates using specific monoclonal antibodies along the filament. As a second step, we calculated the tensile stress experienced by the cell wall along the filament, and varied it with hypertonic or hypotonic solutions. As a third step, we measured the stiffness of the cell along the filament, using cell deformation measurements and atomic force microscopy. Overlapping of the three sets of data allowed to show that alginates co-localise with the stiffest and most stressed areas of the filament, namely the dome of the apical cell and the shanks of the central round cells. In addition, no major distinction between M-rich and G-rich alginate spatial patterns could be observed. Altogether, these results support that both M-rich and G-rich alginates play similar roles in stiffening the cell wall where the tensile stress is high and exposes cells to bursting, and that these roles are independent from cell growth and differentiation.

Alteromonas fortis sp. nov., a non-flagellated bacterium specialized in the degradation of iota-carrageenan, and emended description of the genus Alteromonas.

Strain 1T, isolated in the 1970s from the thallus of the carrageenophytic red algae, Eucheuma spinosum, collected in Hawaii, USA, was characterized using a polyphasic method. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, ovoid or rod-shaped and grew optimally at 20-25 °C, at pH 6-9 and with 2-4 % NaCl. Strain 1T used the seaweed polysaccharides ι-carrageenan, laminarin and alginic acid as sole carbon sources. The major fatty acids were C16 : 0, C18 : 1 ω7c and summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2OH) with significant amounts (>6 %) of C16 : 0 N alcohol and 10 methyl C17 : 0. The respiratory quinone was Q-8 and major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and an unknown aminolipid. Phylogenetic analyses showed that the bacterium is affiliated to the genus Alteromonas (family Alteromonadaceae, class Gammaproteobacteria). Strain 1T exhibited 16S rRNA gene sequence similarity values of 98.8 and 99.2 % to the type strains of Alteromonas mediterranea and Alteromonas australica respectively, and of 95.2-98.6 % to other species of the genus Alteromonas. The DNA G+C content of strain 1T was determined to be 43.9 mol%. Digital DNA-DNA hybridization predictions by the ANI and GGDC methods between strain 1T and other members of the genus Alteromonas showed values below 83 % and 30 %, respectively. The phenotypic, phylogenetic and genomic analyses show that strain 1T is distinct from species of the genus Alteromonas with validly published names and that it represents a novel species of the genus Alteromonas, for which the name Alteromonasfortis sp. nov. is proposed. The type strain is 1T (=ATCC 43554T=RCC 5933T=CIP 111645T=DSM 106819T).

The brown algal mode of tip growth: Keeping stress under control.

Tip growth has been studied in pollen tubes, root hairs, and fungal and oomycete hyphae and is the most widely distributed unidirectional growth process on the planet. It ensures spatial colonization, nutrient predation, fertilization, and symbiosis with growth speeds of up to 800 µm h-1. Although turgor-driven growth is intuitively conceivable, a closer examination of the physical processes at work in tip growth raises a paradox: growth occurs where biophysical forces are low, because of the increase in curvature in the tip. All tip-growing cells studied so far rely on the modulation of cell wall extensibility via the polarized excretion of cell wall-loosening compounds at the tip. Here, we used a series of quantitative measurements at the cellular level and a biophysical simulation approach to show that the brown alga Ectocarpus has an original tip-growth mechanism. In this alga, the establishment of a steep gradient in cell wall thickness can compensate for the variation in tip curvature, thereby modulating wall stress within the tip cell. Bootstrap analyses support the robustness of the process, and experiments with fluorescence recovery after photobleaching (FRAP) confirmed the active vesicle trafficking in the shanks of the apical cell, as inferred from the model. In response to auxin, biophysical measurements change in agreement with the model. Although we cannot strictly exclude the involvement of a gradient in mechanical properties in Ectocarpus morphogenesis, the viscoplastic model of cell wall mechanics strongly suggests that brown algae have evolved an alternative strategy of tip growth. This strategy is largely based on the control of cell wall thickness rather than fluctuations in cell wall mechanical properties.

Toward Multiscale Modeling of Molecular and Biochemical Events Occurring at Fertilization Time in Sea Urchins.

We review here previous theoretical and experimental works, which aim to model major events that occur at the time of fertilization in the sea urchin. We discuss works that perform experiments and develop hypotheses that link different scales of biological systems such as the intracellular Ca2+ concentration oscillations and the swimming behavior of sperm, the Ca2+ wave propagation and the fertilization membrane elevation of the egg, and the mRNA translational activation and the completion of the first mitotic division of the early embryo. The aim of this review is on one hand, to highlight the value of systems biology for understanding the mechanisms associated with fertilization and early embryonic development in sea urchins. On the other hand, this review attempts to illustrate, for mathematicians and bioinformaticians, the potential that represent these molecular and cellular events for modeling clear physiological processes.

A novel species of the marine cyanobacterium Acaryochloris with a unique pigment content and lifestyle.

All characterized members of the ubiquitous genus Acaryochloris share the unique property of containing large amounts of chlorophyll (Chl) d, a pigment exhibiting a red absorption maximum strongly shifted towards infrared compared to Chl a. Chl d is the major pigment in these organisms and is notably bound to antenna proteins structurally similar to those of Prochloron, Prochlorothrix and Prochlorococcus, the only three cyanobacteria known so far to contain mono- or divinyl-Chl a and b as major pigments and to lack phycobilisomes. Here, we describe RCC1774, a strain isolated from the foreshore near Roscoff (France). It is phylogenetically related to members of the Acaryochloris genus but completely lacks Chl d. Instead, it possesses monovinyl-Chl a and b at a b/a molar ratio of 0.16, similar to that in Prochloron and Prochlorothrix. It differs from the latter by the presence of phycocyanin and a vestigial allophycocyanin energetically coupled to photosystems. Genome sequencing confirmed the presence of phycobiliprotein and Chl b synthesis genes. Based on its phylogeny, ultrastructural characteristics and unique pigment suite, we describe RCC1774 as a novel species that we name Acaryochloris thomasi. Its very unusual pigment content compared to other Acaryochloris spp. is likely related to its specific lifestyle.

Nigritoxin is a bacterial toxin for crustaceans and insects.

The Tetraconata (Pancrustacea) concept proposes that insects are more closely related to aquatic crustaceans than to terrestrial centipedes or millipedes. The question therefore arises whether insects have kept crustacean-specific genetic traits that could be targeted by specific toxins. Here we show that a toxin (nigritoxin), originally identified in a bacterial pathogen of shrimp, is lethal for organisms within the Tetraconata and non-toxic to other animals. X-ray crystallography reveals that nigritoxin possesses a new protein fold of the α/ß type. The nigritoxin N-terminal domain is essential for cellular translocation and likely encodes specificity for Tetraconata. Once internalized by eukaryotic cells, nigritoxin induces apoptotic cell death through structural features that are localized in the C-terminal domain of the protein. We propose that nigritoxin will be an effective means to identify a Tetraconata evolutionarily conserved pathway and speculate that nigritoxin holds promise as an insecticidal protein.

Revision of the Genus Micromonas Manton et Parke (Chlorophyta, Mamiellophyceae), of the Type Species M. pusilla (Butcher) Manton & Parke and of the Species M. commoda van Baren, Bachy and Worden and Description of Two New Species Based on the Genetic and Phenotypic Characterization of Cultured Isolates.

The green picoalgal genus Micromonas is broadly distributed in estuaries, coastal marine habitats and open oceans, from the equator to the poles. Phylogenetic, ecological and genomic analyses of culture strains and natural populations have suggested that this cosmopolitan genus is composed of several cryptic species corresponding to genetic lineages. We performed a detailed analysis of variations in morphology, pigment content, and sequences of the nuclear-encoded small-subunit rRNA gene and the second internal transcribed spacer (ITS2) from strains isolated worldwide. A new morphological feature of the genus, the presence of tip hairs at the extremity of the hair point, was discovered and subtle differences in hair point length were detected between clades. Clear non-homoplasious synapomorphies were identified in the small-subunit rRNA gene and ITS2 spacer sequences of five genetic lineages. These findings lead us to provide emended descriptions of the genus Micromonas, of the type species M. pusilla, and of the recently described species M. commoda, as well as to describe 2 new species, M. bravo and M. polaris. By clarifying the status of the genetic lineages identified within Micromonas, these formal descriptions will facilitate further interpretations of large-scale analyses investigating ecological trends in time and space for this widespread picoplankter.

Redescription and phylogenetic analyses of Durchoniella spp. (Ciliophora, Astomatida) associated with the polychaete Cirriformia tentaculata (Montagu, 1808).

Microscopic and phylogenetic analyses were performed on endocommensal astome ciliates retrieved from the middle intestine of a marine cirratulid polychaete, Cirriformia tentaculata, collected in the bay of Roscoff (English Channel, Northwest French coast) and on the Southwest English coast. Three morphotypes of the astome genus Durchoniella were identified, two corresponding to described species (the type species Durchoniella brasili (Léger and Duboscq, 1904) De Puytorac, 1954 and Durchoniella legeriduboscqui De Puytorac, 1954) while a third morphotype remains undescribed. Their small subunit (SSU) rRNA gene sequences showed at least 97.2% identity and phylogenetic analyses grouped them at the base of the subclass Scuticociliatia (Oligohymenophorea), as a sister lineage to all astomes from terrestrial oligochaete annelids. Ultrastructural examination by transmission electron microscopy and fluorescence in situ hybridization analyses revealed the presence of endocytoplasmic cocci and rod-shaped bacteria surrounded by a very thin membrane. These endocytoplasmic bacteria may play a role in the association between endocommensal astome ciliates and cirratulid polychaetes inhabiting in anoxic coastal sediments.

Temperature, water activity and pH during conidia production affect the physiological state and germination time of Penicillium species.

Conidial germination and mycelial growth are generally studied with conidia produced under optimal conditions to increase conidial yield. Nonetheless, the physiological state of such conidia most likely differs from those involved in spoilage of naturally contaminated food. The present study aimed at investigating the impact of temperature, pH and water activity (aw) during production of conidia on the germination parameters and compatible solutes of conidia of Penicillium roqueforti and Penicillium expansum. Low temperature (5°C) and reduced aw (0.900 aw) during sporulation significantly reduced conidial germination times whereas the pH of the sporulation medium only had a slight effect at the tested values (2.5, 8.0). Conidia of P. roqueforti produced at 5°C germinated up to 45h earlier than those produced at 20°C. Conidia of P. roqueforti and P. expansum produced at 0.900 aw germinated respectively up to 8h and 3h earlier than conidia produced at 0.980 aw. Furthermore, trehalose and mannitol assessments suggested that earlier germination might be related to delayed conidial maturation even though no ultra-structural modifications were observed by transmission electron microscopy. Taken together, these results highlight the importance of considering environmental conditions during sporulation in mycological studies. The physiological state of fungal conidia should be taken into account to design challenge tests or predictive mycology studies. This knowledge may also be of interest to improve the germination capacity of fungal cultures commonly used in fermented foods.

Mechanisms of endoderm formation in a cartilaginous fish reveal ancestral and homoplastic traits in jawed vertebrates.

In order to gain insight into the impact of yolk increase on endoderm development, we have analyzed the mechanisms of endoderm formation in the catshark S. canicula, a species exhibiting telolecithal eggs and a distinct yolk sac. We show that in this species, endoderm markers are expressed in two distinct tissues, the deep mesenchyme, a mesenchymal population of deep blastomeres lying beneath the epithelial-like superficial layer, already specified at early blastula stages, and the involuting mesendoderm layer, which appears at the blastoderm posterior margin at the onset of gastrulation. Formation of the deep mesenchyme involves cell internalizations from the superficial layer prior to gastrulation, by a movement suggestive of ingressions. These cell movements were observed not only at the posterior margin, where massive internalizations take place prior to the start of involution, but also in the center of the blastoderm, where internalizations of single cells prevail. Like the adjacent involuting mesendoderm, the posterior deep mesenchyme expresses anterior mesendoderm markers under the control of Nodal/activin signaling. Comparisons across vertebrates support the conclusion that endoderm is specified in two distinct temporal phases in the catshark as in all major osteichthyan lineages, in line with an ancient origin of a biphasic mode of endoderm specification in gnathostomes. They also highlight unexpected similarities with amniotes, such as the occurrence of cell ingressions from the superficial layer prior to gastrulation. These similarities may correspond to homoplastic traits fixed separately in amniotes and chondrichthyans and related to the increase in egg yolk mass.

Kelps feature systemic defense responses: insights into the evolution of innate immunity in multicellular eukaryotes.

Brown algae are one of the few eukaryotic lineages that have evolved complex multicellularity, together with Opisthokonts (animals, fungi) and Plantae (land plants, green and red algae). In these three lineages, biotic stresses induce similar local defense reactions. Animals and land plants also feature a systemic immune response, protecting the whole organism after an attack on one of its parts. However, the occurrence of systemic defenses has never been investigated in brown algae. We elicited selected parts of the kelp Laminaria digitata and monitored distant, nonchallenged areas of the same individual for subsequent defense reactions. A systemic reaction was detected following elicitation on a distant area, including an oxidative response, an increase in haloperoxidase activities and a stronger resistance against herbivory. Based on experiments with pharmacological inhibitors, the liberation of free fatty acids is proposed to play a key role in systemic signaling, reminiscent of what is known in land plants. This study is the first report, outside the phyla of Opisthokonts and Plantae, of an intraorganism communication leading to defense reactions. These findings indicate that systemic immunity emerged independently at least three times, as a consequence of convergent evolution in multicellular eukaryotic lineages.

Dinomyces arenysensis gen. et sp. nov. (Rhizophydiales, Dinomycetaceae fam. nov.), a chytrid infecting marine dinoflagellates.

Environmental 18S rRNA gene surveys of microbial eukaryotes have recently revealed the diversity of major parasitic agents in pelagic freshwater systems, consisting primarily of chytrid fungi. To date, only a few studies have reported the presence of chydrids in the marine environment and a limited number of marine chytrids have been properly identified and characterized. Here, we report the isolation and cultivation of a marine chytrid from samples taken during a bloom of the toxic dinoflagellate Alexandrium minutum in the Arenys de Mar harbour (Mediterranean Sea, Spain). Cross-infections using cultures and natural phytoplankton communities revealed that this chytrid is only able to infect certain species of dinoflagellates, with a rather wide host range but with a relative preference for Alexandrium species. Phylogenetic analyses showed that it belongs to the order Rhizophydiales, but cannot be included in any of the existing families within this order. Several ultrastructural characters confirmed the placement of this taxon within the Rhizophydiales as well its novelty notably in terms of zoospore structure. This marine chytridial parasitoid is described as a new genus and species, Dinomyces arenysensis, within the Dinomycetaceae fam. nov.

Parvilucifera rostrata sp. nov. (Perkinsozoa), a novel parasitoid that infects planktonic dinoflagellates.

The diversity and ecological roles of protists in marine plankton are still poorly known. In 2011, we made a substantial effort to isolate parasites into cultures during the course of blooms of the toxic microalga Alexandrium minutum (Dinophyceae) in two estuaries (the Penzé and the Rance, Brittany coast, north-west of France). In total, 99 parasitic strains were obtained. Screening of ribosomal internal transcribed spacer regions (including ITS1, 5.8S and ITS2) revealed the existence of two ribotypes. Small subunit and partial large subunit rRNA genes revealed that these two ribotypes belong to different species of the genus Parvilucifera. The first ribotype was tentatively affiliated to the species Parvilucifera infectans, whilst the second represents a new species, Parvilucifera rostrata sp. nov. The new species has several distinct morphological features in the general organization of its zoospore and in the shape and size of processes covering the sporangium. Both Parvilucifera species are generalist parasitoids with similar generation times, and this study thus raises the question of how two parasitoids exploiting similar ecological resources and infection strategies can coexist in the same ecosystem. Taxonomic relationships between Parvilucifera spp. and other closely related marine parasitoids, such as syndinians, are discussed.

ETOILE regulates developmental patterning in the filamentous brown alga Ectocarpus siliculosus.

Brown algae are multicellular marine organisms evolutionarily distant from both metazoans and land plants. The molecular or cellular mechanisms that govern the developmental patterning in brown algae are poorly characterized. Here, we report the first morphogenetic mutant, étoile (etl), produced in the brown algal model Ectocarpus siliculosus. Genetic, cellular, and morphometric analyses showed that a single recessive locus, ETL, regulates cell differentiation: etl cells display thickening of the extracellular matrix (ECM), and the elongated, apical, and actively dividing E cells are underrepresented. As a result of this defect, the overrepresentation of round, branch-initiating R cells in the etl mutant leads to the rapid induction of the branching process at the expense of the uniaxial growth in the primary filament. Computational modeling allowed the simulation of the etl mutant phenotype by including a modified response to the neighborhood information in the division rules used to specify wild-type development. Microarray experiments supported the hypothesis of a defect in cell-cell communication, as primarily Lin-Notch-domain transmembrane proteins, which share similarities with metazoan Notch proteins involved in binary cell differentiation were repressed in etl. Thus, our study highlights the role of the ECM and of novel transmembrane proteins in cell-cell communication during the establishment of the developmental pattern in this brown alga.

Persicivirga ulvanivorans sp. nov., a marine member of the family Flavobacteriaceae that degrades ulvan from green algae.

A rod shaped, Gram-stain-negative, chemo-organotrophic, heterotrophic, strictly aerobic, non-gliding bacterium, designated strain PLR(T), was isolated from faeces of the mollusc Aplysia punctata (Mollusca, Gastropoda) that had been fed with green algae belonging to the genus Ulva. The novel strain was able to degrade ulvan, a polysaccharide extracted from green algae (Chlorophyta, Ulvophyceae). The taxonomic position of strain PLR(T) was investigated by using a polyphasic approach. Strain PLR(T) was dark orange, oxidase-positive, catalase-positive and grew optimally at 25 °C, at pH 7.5 and in the presence of 2.5 % (w/v) NaCl with an oxidative metabolism using oxygen as the electron acceptor. Nitrate could not be used as the electron acceptor. Strain PLR(T) had a Chargaff's coefficient (DNA G+C content) of 35.3 mol%. Phylogenetic analysis based on the sequence of the 16S rRNA gene placed the novel strain in the family Flavobacteriaceae (phylum 'Bacteroidetes'), within a clade comprising Stenothermobacter spongiae, Nonlabens tegetincola, Sandarakinotalea sediminis, Persicivirga xylanidelens and Persicivirga dokdonensis. The closest neighbours of strain PLR(T) were P. xylanidelens and P. dokdonensis, sharing 95.2 and 95.5 % 16S rRNA gene sequence similarity, respectively. Phylogenetic inference and differential phenotypic characteristics demonstrated that strain PLR(T) represents a novel species of the genus Persicivirga, for which the name Persicivirga ulvanivorans sp. nov. is proposed. The type strain is PLR(T) ( = CIP 110082(T) = DSM 22727(T)).

Auxin metabolism and function in the multicellular brown alga Ectocarpus siliculosus.

Ectocarpus siliculosus is a small brown alga that has recently been developed as a genetic model. Its thallus is filamentous, initially organized as a main primary filament composed of elongated cells and round cells, from which branches differentiate. Modeling of its early development suggests the involvement of very local positional information mediated by cell-cell recognition. However, this model also indicates that an additional mechanism is required to ensure proper organization of the branching pattern. In this paper, we show that auxin indole-3-acetic acid (IAA) is detectable in mature E. siliculosus organisms and that it is present mainly at the apices of the filaments in the early stages of development. An in silico survey of auxin biosynthesis, conjugation, response, and transport genes showed that mainly IAA biosynthesis genes from land plants have homologs in the E. siliculosus genome. In addition, application of exogenous auxins and 2,3,5-triiodobenzoic acid had different effects depending on the developmental stage of the organism, and we propose a model in which auxin is involved in the negative control of progression in the developmental program. Furthermore, we identified an auxin-inducible gene called EsGRP1 from a small-scale microarray experiment and showed that its expression in a series of morphogenetic mutants was positively correlated with both their elongated-to-round cell ratio and their progression in the developmental program. Altogether, these data suggest that IAA is used by the brown alga Ectocarpus to relay cell-cell positional information and induces a signaling pathway different from that known in land plants.

Cytokinesis in yeast meiosis depends on the regulated removal of Ssp1p from the prospore membrane.

Intracellular budding is a developmentally regulated type of cell division common to many fungi and protists. In Saccaromyces cerevisiae, intracellular budding requires the de novo assembly of membranes, the prospore membranes (PSMs) and occurs during spore formation in meiosis. Ssp1p is a sporulation-specific protein that has previously been shown to localize to secretory vesicles and to recruit the leading edge protein coat (LEP coat) proteins to the opening of the PSM. Here, we show that Ssp1p is a multidomain protein with distinct domains important for PI(4,5)P(2) binding, binding to secretory vesicles and inhibition of vesicle fusion, interaction with LEP coat components and that it is subject to sumoylation and degradation. We found non-essential roles for Ssp1p on the level of vesicle transport and an essential function of Ssp1p to regulate the opening of the PSM. Together, our results indicate that Ssp1p has a domain architecture that resembles to some extent the septin class of proteins, and that the regulated removal of Ssp1p from the PSM is the major step underlying cytokinesis in yeast sporulation.

Dynamics and compartmentation of the nucleolar processing machinery.

In active nucleoli, machineries involved in the biogenesis of ribosomal RNAs (rRNAs) are compartmentalized. The late rRNA processing proteins are localized in the granular component (GC). Here we investigate the behavior of these proteins when production of 28S is impaired and when this blockage is reversed. The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) provokes dispersion of rDNA clusters and we demonstrate that DRB induces disconnection of the late rRNA processing proteins from the transcription sites. These processing proteins are still associated in independent masses without detectable 28S rRNA, indicating that compartmentation of the late rRNA processing machinery is not necessarily linked to processing activity. Removing DRB reverses this disconnection and promotes rRNA processing. Nucleolar reformation occurs in two successive steps, dynamic recruitment to transcription sites of the processing proteins, followed by rDNA compaction. We demonstrate that both steps are sensitive to temperature, suggesting an energy-dependent process. Traffic of processing proteins analyzed by fluorescence recovery after photobleaching is similar in masses disconnected from transcription sites and in the granular component of the active nucleolus. This suggests that protein dynamics and interactions, and not only their processing activity, determine compartmentation of the nucleolar machineries.