The identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

Random Amplified Polymorphic DNA Profiles as a Tool for the Identification of Acanthamoeba divionensis

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates. RID= ID= <E5>Correspondence to:</E5> A. Ortega-Rivas; <E5>email</E5>: aortega&commat;ull.es Received: 5 August 2002 / Accepted: 23 September 2002     

Tunic morphogenesis in the sea peach (Halocynthia papillosa,Ascidiacea, rochordata): Cellular events,the initiation of twisted fibrous arrangements and spine formation

Summary— The adult tunic of the sea peach (Halocynthia papillosa) shows a high degree of organisation. Tunic morphogenesis was monitored from the onset of tunic secretion until juveniles reached the age of 3 months. While some characteristics of the adult tunic are still missing, like certain types of intratunical cells and striated bodies, its main features have already developed by this time. Crucial events take place at or soon after the onset of metamorphosis (stage M 0). Cuticular spines cover the external surface of the juvenile. At least two types of intratunical cells enter the tunic and the fibrous material adopts a three-dimensional twisted helicoidal architecture. The initiation of this helicoidal arrangement of fibrils directly after stage M 0 is discussed regarding accompanying developmental events. The existence of cells that penetrate the outer compartment of the tunic at the end of larval life is reported for the first time.     

Apoptosis shifts to necrosis via intermediate types of cell death by a mechanism depending on c-myc and bcl-2 expression

Hypoxic and chemical hypoxia (antimycin A) commits cultured rat fibroblasts (Rat-1) towards apoptosis, necrosis or an intermediate form of cell death (aponecrosis) depending on the degree of hypoxia. Aponecrosis also occurs in vivo. Here, we demonstrate that c-myc and bcl-2, two proto-oncogenes known to lower or to enhance, respectively, the apoptotic threshold, also affect the type of cell death: apoptosis shifts to aponecrosis and aponecrosis to necrosis, depending on c-myc or bcl-2 expression and the antimycin A concentration (100–400 M). In cells with basal gene expression, apoptosis shifts to aponecrosis/necrosis at 300 M antimycin A (middle hypoxia). Overexpression of c-myc markedly increases cumulative cell death in response to antimycin A and lowers the antimycin A concentration required to shift apoptosis to aponecrosis/necrosis from 300 M to 100 M (low hypoxia). Overexpression of bcl-2 elicits the opposite effect, decreasing cumulative cell death in response to antimycin A and raising the drug concentration required to shift apoptosis to aponecrosis/necrosis to 400 M (high hypoxia). The passage from one to the other form of cell death involves various aponecrotic features with observed intermediate aspects between apoptosis and necrosis, a progressive increase in necrotic features being correlated with an increase in antimycin A concentration. The mechanism underlying the various effects of c-myc and bcl-2 on cell-death type has been related to the ability of these genes to counteract, to various extents, the ATP decrease occurring in response to different degrees of chemical hypoxia.The first two authors contributed equally to this workThis work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano), CNR/MIUR (Grant Progetto Finalizzato Oncologia), Ente Cassa di Risparmio di Firenze and Ministero dellIstruzione, dellUniversità e della Ricerca (MIUR, Cofin 2003). Andrea Lapucci is supported by a fellowship from the Federazione Italiana per la Ricerca sul Cancro (FIRC, Milano)     

A new cellulosic structure, the tunic cord in the ascidianPolyandrocarpa misakiensis

Summary A specialized structure of tunic cord inPolyandrocarpa misakiensis is investigated by electron microscopy. The tunic cord is a cord-like coiled structure of 5–30 m in diameter and 0.1–9.0 mm in length. The tunic cords originate and elongate from the dorsal tunic, and their termini have a swollen and ornamented structure. Scanning and transmission electron micrographs and the electron diffractogram show that the tunic cords are composed of bundled microfibrils of cellulose I with high crystallinity. The tunic cord is completely surrounded by single-layered epidermal cells, which have been found as the site of cellulose biosynthesis. A number of tunic cords are connected to the internal tunic of the siphon by forming eyelet structures at their termini. These observations suggest that the tunic cords act as a connector between dorsal and internal tunic of the siphon.     

Blue-green algalike cells associated with the tunic of Ciona intestinalis L.

Summary Certain organisms resembling blue-green algae embedded in the tunic of the solitary ascidian Ciona intestinalis L. are described. Their probable symbiotic role as related to the peculiar habitat of this ascidian is suggested.     

Streptococcus bovis as a host for the expression of cloned polysaccharidase genes

The shuttle vector, pVA838, was used to introduce the xynD gene from the cellulolytic rumen anaerobe, Ruminococcus flavefaciens 17, into Streptococcus bovis JB1. Expression of xylanase and -(1,3-1,4)-glucanase activity due to xynD was demonstrated in culture supernatants. SDS-PAGE zymograms revealed the full-length xynD 90 kDa product, together with some proteolytic products. Activities due to the cloned xynD gene, and to the R. flavefaciens 17 endA endoglucanase gene present in the construct pVACMC1, decreased after early growth stages in batch cultures of S. bovis JB1, reflecting the sensitivity of the cloned R. flavefaciens 17 enzymes to inactivation in the presence of accumulated lactic acid. Of xynD activity, 40–80% was detected in the culture supernatant, indicating recognition of the xynD signal peptide by S. bovis JB1.     

Erratum to: Bacterial diversity in toxic<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> blooms off the Orkney Isles and the Firth of Forth

The genetic diversity of the bacterial community associated with Alexandrium tamarense blooms was studied in blooms of the toxic dinoflagellates in the waters around the Orkney Isles and the Firth of Forth (Scotland). For toxin and molecular analysis of the bacterial communities associated with the toxic bloom, water samples were taken in 1998 and 1999 from A. tamarense blooms. The bacterial community structure, as determined by DGGE (denaturing gradient gel electrophoresis) showed clear differences between all three investigated size fractions (dinoflagellate-associated bacteria, attached bacteria and free-living bacteria), with high diversity within each sample. DNA sequence analysis of the dominant and most frequent DGGE bands revealed the dominance of Proteobacteria, mainly of the Roseobacter clade, with similarities of 91–99%. Moreover, DGGE bands occurring at the same position in the gel throughout in most samples corroborate the presence of several specific Proteobacteria of the Roseobacter clade. Overall, 500 bacteria were isolated from the bloom and partly phylogenetically analysed. They were members of two prokaryotic phyla, the Proteobacteria and the Bacteroidetes, related to Proteobacteria of the and subdivisions (Alteromonas, Pseudoalteromonas and Colwellia). All bacteria were tested for the production of sodium channel blocking (SCB) toxins using mouse neuroblastoma assay. No production of SCB toxins was found and high performance liquid chromatography (HPLC) analysis confirmed these results. The content of total paralytic shellfish poisoning (PSP) toxin in the water samples, as measured within the toxic dinoflagellate blooms using HPLC, ranged from 53 to 2191 ng PSP l^–1 in 1998 and from 0 to 478 ng PSP l^–1 in 1999. Changes in PSP toxin content were not accompanied by changes of DGGE band patterns. We therefore presume that the bacterial groups identified in this study were not exclusively associated with toxic A. tamarense, but were generally associated with the phytoplankton.An erratum to this article can be found at Communicated by H.-D. Franke     

Study of the three-genome hybridLycopersicon esculentum Mill. —L. chilense Dun. —L. peruvianum var ‘humifusum’ Mill. and its use as a source for resistance

L. peruvianum var humifusum is reproductively the most isolated of the species of the genusLycopersicon. It can be crossed with the cultivated tomato usingL. chilense as an intermediary. After a series of backcrosses of the three-genome hybrid F₁ (L. esculentum ×L. chilense) ×L. peruvianum var humifusum withL. esculentum, accompanied by selection for resistance to some economically important diseases, several lines were established. One of these lines, Cm 180, which showed resistance toClavibacter michiganensis subsp.michiganensis, was subjected to genetic analysis. This resistance was found to be controlled by a single dominant gene (Cm) that was not allelic to the gene originating fromL. hirsutum f.glabratum. ThisCm gene was genetically mapped on chromosome 4. The germ plasm ofL. peruvianum var humifusum in combination withL. chilense was transferred intoL. esculentum. Different breeding lines possessing resistance to various diseases and pests could be developed from this material.     

Community Structure of Bacteria Associated with Sheaths of Freshwater and Brackish Thioploca Species

Bacterial communities associated with sheaths of Thioploca spp. from two freshwater lakes (Lake Biwa, Japan, and Lake Constance, Germany) and one brackish lake (Lake Ogawara, Japan) were analyzed with denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The comparison between the DGGE band patterns of bulk sediment and Thioploca filaments of Lake Biwa suggested the presence of specific bacterial communities associated with Thioploca sheaths. As members of sheath-associated communities, bacteria belonging to Bacteroidetes were detected from the samples of both freshwater lakes. A DGGE band from Thioploca of Lake Biwa, belonging to candidate division OP8, was quite closely related to another DGGE band detected from that of Lake Constance. In contrast to the case of freshwater lakes, no bacterium of Bacteroidetes or OP8 was detected from Thioploca of Lake Ogawara. However, two DGGE bands from Lake Ogawara, belonging to Chloroflexi, were quite closely related to a DGGE band from Lake Constance. Two DGGE bands obtained from Lake Biwa were closely related to phylogenetically distant dissimilatory Fe(III)-reducing bacteria. Cloning analyses for a dissimilatory sulfite reductase gene were performed on the same samples used for DGGE analysis. The results of the analyses suggest that sheaths of freshwater/brackish Thioploca have little ecological significance for the majority of sulfate reducers.     

Expression of a cellulase gene, celA, from the rumen fungus Neocallimastix patriciarum in Streptococcus bovis by means of promoter fusions

The aerotolerant rumen bacterium, Streptococcus bovis, has been used as a host for expression of genes of eukaryotic origin. The coding regions of the celA cellulase gene from the rumen fungus, Neocallimastix patriciarum, were fused with bacterial promoter/signal peptide regions from the Ruminococcus flavefaciens xynD and S. bovis -(1,3-1,4)-glucanase genes. Fusion cassettes were built into shuttle vector constructs based on pIL253 or pTRW10 and constructs carrying celA were transformed into S. bovis JB1. Active N. patriciarum cellulase was produced in S. bovis with either promoter, although better expression levels were obtained with the native S. bovis -glucanase promoter fragment.     

Phylogenetic diversity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts, USA)

Recent studies have revealed that many marine invertebrates are closely associated with diverse microorganisms, frequently resulting in the production of compounds of biomedical interest. Thus far, ascidians have not been widely examined for the presence of bacterial associations, although the production of secondary metabolites is well documented. In the present study, we examined the gonad of Molgula manhattensis and the tunic surfaces of Botryllus schlosseri, Didemnum sp., and Botrylloides violaceus for the presence of associated bacteria. These ascidians are common inhabitants of the coastal ocean of Cape Cod, Massachusetts. We used denaturing gradient gel electrophoresis (DGGE) as well as cloning and sequencing of 16S rDNA to analyze the microbial communities. There is a strong evidence that spiroplasma-like bacteria colonize the gonad of the solitary ascidian M. manhattensis. The bacteria might be vertically transmitted and may be involved in the production of secondary metabolites that deter predators of the ascidians. The bacterial communities found on the tunic surfaces of the colonial ascidians were found to be more diverse. However, in all cases the bacterial communities were predominated by alpha-proteobacteria. Alpha-proteobacteria related to the obtained sequences have been identified as symbionts in a variety of hosts, suggesting a specific role for these bacteria. However, based on our data it is difficult to differentiate between persistently and only transiently associated bacteria. Overall, this study demonstrates that ascidian species are associated with diverse bacterial populations. Future studies will aim to elucidate the precise relationships between bacteria and ascidians and to identify bioactive compounds that might be produced as a result of these relationships.     

Vegetative and reproductive structure of the extinct Platanus neptuni from the Tertiary of Europe and relationships within the Platanaceae

Platanus neptuni (Ettingshausen) Bek, Holý & Z. Kvaek was a conspicuous warm-temperate to subtropical element of Late Eocene to Late Miocene European floras. In our concept, the P. neptuni plant includes not only globose infructescences upon which the species epithet is based, but also staminate and pistillate inflorescences and distinctive stipulate foliage. The leaves range from simple (P. neptuni morphoforma reussii (Ett.) comb. et stat. nov.) to trifoliolate (P. neptuni morphoforma fraxinifolia (Johnson & Gilmore) comb. et stat. nov.) and sometimes quinquefoliolate (P. neptuni morphoforma hibernica (Johnson & Gilmore) comb. et stat. nov.) with unlobed elliptical to obovate laminae that are uniform in venation, marginal serration, and epidermal structure. Foliar twigs confirm that the leaves are deciduous, with each petiole base enveloping a bud, as in extant Platanus subgen. Platanus. Platanus neptuni differs from extant species of the genus by large peltate glandular trichomes on the fruits and leaves, a prominent circumscissile rim on the stalk below the inflorescence, as well as by the tendency for compound foliage. These characters justify its position within an extinct subgenus of the Platanaceae (Platanus L. subgen. Glandulosa Z. Kvaek, Manchester & Guo). Platanus neptuni was common in mesic humid subtropical forests on volcanogenic subtrates and at sea shores.     

Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata

The solitary ascidian Styela plicata is an introduced species in harbors of temperate and tropical oceans around the world. The invasive potential of this species has been studied through reproductive biology and population genetics but no study has yet examined the microbial diversity associated with this ascidian and its potential role in host ecology and invasiveness. Here, we used 16S rRNA gene tag pyrosequencing and transmission electron microscopy to characterize the abundance, diversity and host-specificity of bacteria associated with 3 Mediterranean individuals of S. plicata. Microscopy revealed low bacterial abundance in the inner tunic and their absence from gonad tissues, while pyrosequencing revealed a high diversity of S. plicata-associated bacteria (284 OTUs from 16 microbial phyla) in the inner tunic. The core symbiont community was small and consisted of 16 OTUs present in all S. plicata hosts. This core community included a recently described ascidian symbiont (Hasllibacter halocynthiae) and several known sponge and coral symbionts, including a strictly anaerobic Chloroflexi lineage. Most recovered bacterial OTUs (79.6 %) were present in single S. plicata individuals and statistical analyses of genetic diversity and community structure confirmed high variability of bacterial communities among host individuals. These results suggest that diverse and variable bacterial communities inhabit the tunic of S. plicata, including environmental and host-associated bacterial lineages that appear to be re-established each host generation. We hypothesize that bacterial communities in S. plicata are dynamic and have the potential to aid host acclimation to new habitats by establishing relationships with beneficial, locally sourced bacteria.     

Bacterial community in the tunic matrix of a colonial ascidian<Emphasis Type="Italic"> Diplosoma migrans</Emphasis>

This paper provides the first information on the morphology of different morphotypes of bacteria in the tunic matrix of the colonial ascidian Diplosoma migrans. Ascidians were collected from waters near Helgoland (German Bight, North Sea). The dominant type is represented by extremely high numbers of long, needle-like rods (length 10–30 µm, width 0.5 µm). The bacteria are motile by means of bipolar monotrichous flagella, generating swift sigmoidal movement. Bacteria are already present during different embryonic stages. It is assumed that they are transferred during sexual propagation from the parental colony to its offspring. As a second morphotype, the tunic harbors screw-like bacteria in low numbers (length 4–10 µm, width 0.5 µm). Besides these conspicuous morphotypes, occasionally motile rods with spore-like globules at one end and additional coccoid forms in large quantities of unknown meaning (possibly spores) were found. The taxonomic status and ecological functions of these differently shaped bacterial groups are unclear.Communicated by H.-D. Franke     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after -ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5 and 3 ends of dTnp1 together with a perfect palindrome located after the 5 inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Microbial community associated with the colonial ascidian Cystodytes dellechiajei

The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.     

Bacterial Diversity in a Marine Methanol-Fed Denitrification Reactor at the Montreal Biodome,Canada

The bacterial biota of a methanol-fed denitrification reactor used to treat seawater at the Montreal Biodome were investigated using culture-dependent and molecular biology methods. The microbiota extracted from the reactor carriers were cultivated on three media. Three isolate types were recovered and their 16S ribosomal DNA (rDNA) genes were determined. The analysis showed that the isolate types were related to -Proteobacteria. They are members of the Hyphomicrobium and Paracoccus genera and the Phyllobacteriaceae family. Uncultured bacteria were identified through a 16S rDNA library generated from total DNA extracted from the microbiota. Clones were screened for different restriction profiles and for different DGGE (denaturing gradient gel electrophoresis) migration profiles. More than 70% of clones have the same restriction profile, and the sequence of representative clones showed a relation with the Methylophaga members of the Piscirickettsia family (-Proteobacteria). Sequences from other profiles were related to bacterial species involved in denitrification. The number of species in the denitrification reactor was estimated at 15. Bacterial colonization on newly added carriers in the denitrification reactor was monitored by PCR-DGGE. The DGGE migration profiles evolved during the first 5 weeks and then remained essentially unchanged. PCR-DGGE was also used to monitor the microbial profiles in various aquarium locations. As expected, bacterial populations differed from one location to another, except for the sand and trickling filters which presented similar DGGE migration profiles.     

Bacterial aggregates in the tentacles of the sea anemone <Emphasis Type="Italic">Metridium senile</Emphasis>

This paper provides first information on organ-like bacterial aggregates in the tentacles of the sea anemone Metridium senile. The specimens were collected from waters near Helgoland (German Bight, North Sea) and the Orkney Islands. Tentacles were prepared for morphological inspection by light and scanning electron microscopy as well as for the phylogenetic analysis of endocytic bacteria. Bacterial aggregates are located in caverns of the tentacles’ epidermis. The aggregates are enwrapped in thin envelopes, which contain coccoid and/or rod-shaped tightly packed bacteria of different division states. Most of the bacterial cells are connected by fine filamentous structures. The phylogenetic determination is based on the sequence data of the 16S rDNA derived from tentacle material. Sequence analysis revealed three different subgroups of intratentacular proteobacteria. The dominant band, detected in all of the samples tested, showed a close relationship (98%) to a gram-negative Endozoicimonas elysicola. Two bands, only detected in tentacles of M. senile from Helgoland were assigned to Pseudomonas saccherophilia (99%), a knallgas bacterium, and to Ralstonia pickettii (100%). The bacteria represent a specific bacterial community. Their DGGE profiles do not correspond to the profiles of the planktonic bacteria generated from seawater close to the habitats of the anemones. The allocation of DNA sequences to the different morphotypes, their isolation, culturing and the elucidation of the physiological functions of intratentacular bacteria are in progress.