First evidence of virus-like particles in the bacterial symbionts of Bryozoa.

Bacteriophage communities associated with humans and vertebrate animals have been extensively studied, but the data on phages living in invertebrates remain scarce. In fact, they have never been reported for most animal phyla. Our ultrastructural study showed for the first time a variety of virus-like particles (VLPs) and supposed virus-related structures inside symbiotic bacteria in two marine species from the phylum Bryozoa, the cheilostomes Bugula neritina and Paralicornia sinuosa. We also documented the effect of VLPs on bacterial hosts: we explain different bacterial 'ultrastructural types' detected in bryozoan tissues as stages in the gradual destruction of prokaryotic cells caused by viral multiplication during the lytic cycle. We speculate that viruses destroying bacteria regulate symbiont numbers in the bryozoan hosts, a phenomenon known in some insects. We develop two hypotheses explaining exo- and endogenous circulation of the viruses during the life-cycle of B. neritina. Finally, we compare unusual 'sea-urchin'-like structures found in the collapsed bacteria in P. sinuosa with so-called metamorphosis associated contractile structures (MACs) formed in the cells of the marine bacterium Pseudoalteromonas luteoviolacea which are known to trigger larval metamorphosis in a polychaete worm.

Pathogenic profile and cytotoxic activity of Aeromonas spp. isolated from Pectinatella magnifica and surrounding water in the South Bohemian aquaculture region.

Pectinatella magnifica is an invasive freshwater bryozoan that has expanded in many localities worldwide, including fishing areas. It contains microbial communities, predominantly consisting of Aeromonas bacteria that are frequently associated with fish infections. The objective of this study was to investigate the potential pathogenicity of Aeromonas spp. associated with P. magnifica and evaluate the health risks for fish. Aeromonas strains were isolated from P. magnifica (101 strains) and from surrounding water (29 strains) in the South Bohemian region and investigated for the presence of 14 virulence-associated genes using PCR. We demonstrated high prevalence of phospholipase GCAT, polar flagellin, enolase, DNAse, aerolysin/cytotoxic enterotoxin, serine protease and heat-stable cytotonic enterotoxin-coding genes. Further, all twelve isolates that were analysed for cytotoxicity against intestinal epithelial cells were found to be cytotoxic. Six of the isolates were also tested as co-cultures composed of pairs. Enhanced cytotoxicity was observed when the pair was composed of strains from different species. In conclusion, P. magnifica is colonized by Aeromonas strains that have a relatively high prevalence of virulence-associated genes and the ability to provoke disease. Results also suggest a possibly increased risk arising from mixed infections.

The Role of Biofilms Developed under Different Anthropogenic Pressure on Recruitment of Macro-Invertebrates.

Microbial biofilms can be key mediators for settlement of macrofoulers. The present study examines the coupled effects of microbial biofilms and local environmental conditions on the composition, structure and functioning of macrofouling assemblages. Settlement of invertebrates over a gradient of human-impacted sites was investigated on local biofilms and on biofilms developed in marine protected areas (MPAs). Special attention was given to the presence of non-indigenous species (NIS), a global problem that can cause important impacts on local assemblages. In general, the formation of macrofouling assemblages was influenced by the identity of the biofilm. However, these relationships varied across levels of anthropogenic pressure, possibly influenced by environmental conditions and the propagule pressure locally available. While the NIS Watersipora subatra seemed to be inhibited by the biofilm developed in the MPA, Diplosoma cf. listerianum seemed to be attracted by biofilm developed in the MPA only under mid anthropogenic pressure. The obtained information is critical for marine environmental management, urgently needed for the establishment of prevention and control mechanisms to minimize the settlement of NIS and mitigate their threats.

Diversity and Dynamics of "Candidatus Endobugula" and Other Symbiotic Bacteria in Chinese Populations of the Bryozoan, Bugula neritina.

Bugula neritina is a common invasive cosmopolitan bryozoan that harbors (like many sessile marine invertebrates) a symbiotic bacterial (SB) community. Among the SB of B. neritina, "Candidatus Endobugula sertula" continues to receive the greatest attention, because it is the source of bryostatins. The bryostatins are potent bioactive polyketides, which have been investigated for their therapeutic potential to treat various cancers, Alzheimer's disease, and AIDS. In this study, we compare the metagenomics sequences for the 16S ribosomal RNA gene of the SB communities from different geographic and life cycle samples of Chinese B. neritina. Using a variety of approaches for estimating alpha/beta diversity and taxonomic abundance, we find that the SB communities vary geographically with invertebrate and fish mariculture and with latitude and environmental temperature. During the B. neritina life cycle, we find that the diversity and taxonomic abundances of the SB communities change with the onset of host metamorphosis, filter feeding, colony formation, reproduction, and increased bryostatin production. "Ca. Endobugula sertula" is confirmed as the symbiont of the Chinese "Ca. Endobugula"/B. neritina symbiosis. Our study extends our knowledge about B. neritina symbiosis from the New to the Old World and offers new insights into the environmental and life cycle factors that can influence its SB communities, "Ca. Endobugula," and bryostatins more globally.

Cultivable bacteria from Pectinatella magnifica and the surrounding water in South Bohemia indicate potential new Gammaproteobacterial, Betaproteobacterial and Firmicutes taxa.

Pectinatella magnifica is a freshwater bryozoan, which has become a subject of scientific interest because of its invasive expansion worldwide. To obtain a comprehensive overview of its influence on environments, information on associated bacteria is needed. In this study, cultivable bacteria associated with P. magnifica were investigated. In total, 253 isolates were selected for preliminary identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and clustered based on repetitive extragenic palindromic-PCR profiles. Among these, 169 strains were selected and identified using 16S rRNA gene comparative analyses. The sequences were grouped into 76 phylotypes and affiliated with 67 species. The majority of isolated bacteria belonged to Gammaproteobacteria, followed by Betaproteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Most strains within the Betaproteobacteria were isolated exclusively from bryozoan colonies. Aeromonas was the genus predominantly isolated from both P. magnifica and the water samples. Based on 16S rDNA similarity values, 15 putative new species belonging to the genera Aeromonas, Aquitalea, Clostridium, Herbaspirillum, Chromobacterium, Chryseobacterium, Morganella, Paludibacterium, Pectobacterium, Rahnella, Rhodoferax and Serratia, and putative new genera belonging to families Clostridiaceae and Sporomusaceae were revealed. The majority of the detected bacteria were species widely distributed in the environments; nevertheless, a possible symbiotic association of two new putative species with P. magnifica cannot be excluded.

Assessment of Chemical Impact of Invasive Bryozoan Pectinatella magnifica on the Environment: Cytotoxicity and Antimicrobial Activity of P. magnifica Extracts.

Pectinatella magnifica, an invasive bryozoan, might significantly affect ecosystem balance due to its massive occurrence in many areas in Europe and other parts of the world. Biological and chemical analyses are needed to get complete information about the impact of the animal on the environment. In this paper, we aimed to evaluate in vitro cytotoxic effects of five extracts prepared from P. magnifica using LDH assay on THP-1 cell line. Antimicrobial activities of extracts against 22 different bacterial strains were tested by microdilution method. Our study showed that all extracts tested, except aqueous portion, demonstrated LD50 values below 100 µg/mL, which indicates potential toxicity. The water extract of P. magnifica with LD50 value of 250 µg/mL also shows potentially harmful effects. Also, an environmental risk resulting from the presence and increasing biomass of potentially toxic benthic cyanobacteria in old colonies should not be underestimated. Toxicity of Pectinatella extracts could be partially caused by presence of Aeromonas species in material, since we found members of these genera as most abundant bacteria associated with P. magnifica. Furthermore, P. magnifica seems to be a promising source of certain antimicrobial agents. Its methanolic extract, hexane, and chloroform fractions possessed selective inhibitory effect on some potential pathogens and food spoiling bacteria in the range of MIC 0.5-10 mg/mL. Future effort should be made to isolate and characterize the content compounds derived from P. magnifica, which could help to identify the substance(s) responsible for the toxic effects of P. magnifica extracts.

Lack of Overt Genome Reduction in the Bryostatin-Producing Bryozoan Symbiont "Candidatus Endobugula sertula".

The uncultured bacterial symbiont "Candidatus Endobugula sertula" is known to produce cytotoxic compounds called bryostatins, which protect the larvae of its host, Bugula neritina The symbiont has never been successfully cultured, and it was thought that its genome might be significantly reduced. Here, we took a shotgun metagenomics and metatranscriptomics approach to assemble and characterize the genome of "Ca Endobugula sertula." We found that it had specific metabolic deficiencies in the biosynthesis of certain amino acids but few other signs of genome degradation, such as small size, abundant pseudogenes, and low coding density. We also identified homologs to genes associated with insect pathogenesis in other gammaproteobacteria, and these genes may be involved in host-symbiont interactions and vertical transmission. Metatranscriptomics revealed that these genes were highly expressed in a reproductive host, along with bry genes for the biosynthesis of bryostatins. We identified two new putative bry genes fragmented from the main bry operon, accounting for previously missing enzymatic functions in the pathway. We also determined that a gene previously assigned to the pathway, bryS, is not expressed in reproductive tissue, suggesting that it is not involved in the production of bryostatins. Our findings suggest that "Ca Endobugula sertula" may be able to live outside the host if its metabolic deficiencies are alleviated by medium components, which is consistent with recent findings that it may be possible for "Ca Endobugula sertula" to be transmitted horizontally. IMPORTANCE: The bryostatins are potent protein kinase C activators that have been evaluated in clinical trials for a number of indications, including cancer and Alzheimer's disease. There is, therefore, considerable interest in securing a renewable supply of these compounds, which is currently only possible through aquaculture of Bugula neritina and total chemical synthesis. However, these approaches are labor-intensive and low-yielding and thus preclude the use of bryostatins as a viable therapeutic agent. Our genome assembly and transcriptome analysis for "Ca Endobugula sertula" shed light on the metabolism of this symbiont, potentially aiding isolation and culturing efforts. Our identification of additional bry genes may also facilitate efforts to express the complete pathway heterologously.

Ecology of cryptic invasions: latitudinal segregation among Watersipora (Bryozoa) species.

Watersipora is an invasive genus of bryozoans, easily dispersed by fouled vessels. We examined Cytochrome c oxidase subunit I haplotypes from introduced populations on the US Pacific coastline to investigate geographic segregation of species and/or haplotypes. In California, the W. subtorquata group fell into three major sub-groups: W. subtorquata clades A and B, and W. "new sp.". W. subtorquata clades A and B were common in southern California south of Point Conception, a recognized biogeographic boundary, whereas further north, W. subtorquata clade A and W. n. sp. were frequent. The southern California region also had colonies of a morphologically distinct species, W. arcuata, also found in southern Australia and Hawaii; COI variation indicates a common ancestral source(s) in these introductions. The distribution of Watersipora-complex lineages on different coastlines is shown to be temperature correlated. Accordingly, pre-exisitng temperature-based adaptations may play a key role in determining invasion patterns.

Bacterial isolates from the bryozoan Membranipora membranacea: influence of culture media on isolation and antimicrobial activity.

From specimens of the bryozoan Membranipora membranacea collected in the Baltic Sea, bacteria were isolated on four different media, which significantly increased the diversity of the isolated groups. All isolates were classified according to 16S rRNA gene sequence analysis and tested for antimicrobial properties using a panel of five indicator strains and six different media. Each medium featured a unique set of isolated phylotypes, and a phylogenetically diverse collection of isolates was obtained. A total of 96 isolates were assigned to 49 phylotypes and 29 genera. Only one-third of the members of these genera had been isolated previously from comparable sources. The isolates were affiliated with Alpha- and Gammaproteobacteria, Bacilli, and Actinobacteria. A comparable large portion of up to 22 isolates, i.e., 15 phylotypes, probably represent new species. Likewise, 47 isolates (approximately 50%) displayed antibiotic activities, mostly against grampositive indicator strains. Of the active strains, 63.8 % had antibiotic traits only on one or two of the growth media, whereas only 12.7 % inhibited growth on five or all six media. The application of six different media for antimicrobial testing resulted in twice the number of positive hits as obtained with only a single medium. The use of different media for the isolation of bacteria as well as the variation of media considered suitable for the production of antibiotic substances significantly enhanced both the number of isolates obtained and the proportion of antibiotic active cultures. Thus the approach described herein offers an improved strategy in the search for new antibiotic compounds.

Bacterial isolates from the bryozoan Membranipora membranacea: influence of culture media on isolation and antimicrobial activity

From specimens of the bryozoan Membranipora membranacea collected in the Baltic Sea, bacteria were isolated on four different media, which significantly increased the diversity of the isolated groups. All isolates were classified according to 16S rRNA gene sequence analysis and tested for antimicrobial properties using a panel of five indicator strains and six different media. Each medium featured a unique set of isolated phylotypes, and a phylogenetically diverse collection of isolates was obtained. A total of 96 isolates were assigned to 49 phylotypes and 29 genera. Only one-third of the members of these genera had been isolated previously from comparable sources. The isolates were affiliated with Alpha- and Gammaproteobacteria, Bacilli, and Actinobacteria. A comparable large portion of up to 22 isolates, i.e., 15 phylotypes, probably represent new species. Likewise, 47 isolates (approximately 50%) displayed antibiotic activities, mostly against grampositive indicator strains. Of the active strains, 63.8 % had antibiotic traits only on one or two of the growth media, whereas only 12.7 % inhibited growth on five or all six media. The application of six different media for antimicrobial testing resulted in twice the number of positive hits as obtained with only a single medium. The use of different media for the isolation of bacteria as well as the variation of media considered suitable for the production of antibiotic substances significantly enhanced both the number of isolates obtained and the proportion of antibiotic active cultures. Thus the approach described herein offers an improved strategy in the search for new antibiotic compounds (AU)

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Phylogenetic diversity and antimicrobial activities of bryozoan-associated bacteria isolated from Mediterranean and Baltic Sea habitats.

To date, only a small number of investigations covering microbe-bryozoa associations have been carried out. Most of them have focused on a few bryozoan species and none have covered the antibacterial activities of associated bacteria. In the current study, the proportion and phylogenetic classification of Bryozoan-associated bacteria with antimicrobial properties were investigated. Twenty-one specimens of 14 different bryozoan species were collected from several sites in the Baltic and the Mediterranean Sea. A total of 340 associated bacteria were isolated, and 101 displayed antibiotic activities. While antibiosis was predominantly directed against Gram-positive test strains, 16S rRNA gene sequencing revealed affiliation of the isolates to Gram-negative classes (Flavobacteria, Alpha- and Gammaproteobacteria). One isolate was related to the Gram-positive Actinobacteria. The sequences were grouped into 27 phylotypes on the basis of similarity values >or=99.5%. A host-specific affiliation was not revealed as members of the same phylotype were derived from different bryozoan species. Site-specific patterns, however, were demonstrated. Strains of the genera Sphingomonas and Alteromonas were exclusively isolated from Mediterranean sites, whereas Shewanella, Marinomonas and Vibrio-related isolates were only from Baltic sites. Although Pseudoalteromonas affiliated strains were found in both habitats, they were separated into respective phylotypes. Isolates with 16S rDNA similarity values <98%, which could possibly represent new species, belonged to the genera Shewanella, Pseudoalteromonas and Tenacibaculum.

Epibiotic Vibrio luminous bacteria isolated from some hydrozoa and bryozoa species.

Luminous bacteria are isolated from both Hydrozoa and Bryozoa with chitinous structures on their surfaces. All the specimens of the examined hydroid species (Aglaophenia kirchenpaueri, Aglaophenia octodonta, Aglaophenia tubiformis, Halopteris diaphana, Plumularia setacea, Ventromma halecioides), observed under blue light excitation, showed a clear fluorescence on the external side of the perisarc (chitinous exoskeleton) around hydrocladia. In the bryozoan Myriapora truncata, luminous bacteria are present on the chitinous opercula. All the isolated luminous bacteria were identified on the basis of both phenotypic and genotypic analysis. The isolates from A. tubiformis and H. diaphana were unambiguously assigned to the species Vibrio fischeri. In contrast, the isolates from the other hydroids, phenotypically assigned to the species Vibrio harveyi, were then split into two distinct species by phylogenetic analysis of 16S rRNA gene sequences and DNA-DNA hybridization experiments. Scanning electron microscopy analysis and results of culture-based and culture-independent approaches enabled us to establish that luminous vibrios represent major constituents of the bacterial community inhabiting the A. octodonta surface suggesting that the interactions between luminous bacteria and the examined hydrozoan and bryozoan species are highly specific. These interactions might have epidemiological as well as ecological implications because of the opportunistic pathogenicity of luminous Vibrio species for marine organisms and the wide-distribution of the hydrozoan and bryozoan functioning as carriers.

Evolutionary relationships of "Candidatus endobugula" bacterial symbionts and their Bugula bryozoan hosts.

Ribosomal gene sequences were obtained from bryozoans in the genus Bugula and their bacterial symbionts; analyses of host and symbiont phylogenetic trees did not support a history of strict cospeciation. Symbiont-derived compounds known to defend host larvae from predation were only detected in two out of four symbiotic Bugula species.

Tenacibaculum adriaticum sp. nov., from a bryozoan in the Adriatic Sea.

A rod-shaped, translucent yellow-pigmented, Gram-negative bacterium, strain B390(T), was isolated from the bryozoan Schizobrachiella sanguinea collected in the Adriatic Sea, near Rovinj, Croatia. 16S rRNA gene sequence analysis indicated affiliation to the genus Tenacibaculum, with sequence similarity levels of 94.8-97.3 % to type strains of species with validly published names. It grew at 5-34 degrees C, with optimal growth at 18-26 degrees C, and only in the presence of NaCl or sea salts. In contrast to other type strains of the genus, strain B390(T) was able to hydrolyse aesculin. The predominant menaquinone was MK-6 and major fatty acids were iso-C(15 : 0), iso-C(15 : 0) 3-OH and iso-C(15 : 1). The DNA G+C content was 31.6 mol%. DNA-DNA hybridization and comparative physiological tests were performed with type strains Tenacibaculum aestuarii JCM 13491(T) and Tenacibaculum lutimaris DSM 16505(T), since they exhibit 16S rRNA gene sequence similarities above 97 %. These data, as well as phylogenetic analyses, suggest that strain B390(T) (=DSM 18961(T) =JCM 14633(T)) should be classified as the type strain of a novel species within the genus Tenacibaculum, for which the name Tenacibaculum adriaticum sp. nov. is proposed.

Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages.

In all probability, natural selection began as ancient marine microorganisms were required to compete for limited resources. These pressures resulted in the evolution of diverse genetically encoded small molecules with a variety of ecological and metabolic roles. Remarkably, many of these same biologically active molecules have potential utility in modern medicine and biomedical research. The most promising of these natural products often derive from organisms richly populated by associated microorganisms (e.g., marine sponges and ascidians), and often there is great uncertainty about which organism in these assemblages is making these intriguing metabolites. To use the molecular machinery responsible for the biosynthesis of potential drug-lead natural products, new tools must be applied to delineate their genetic and enzymatic origins. The aim of this perspective is to highlight both traditional and emerging techniques for the localization of metabolic pathways within complex marine environments. Examples are given from the literature as well as recent proof-of-concept experiments from the authors' laboratories.

Localization of 'Candidatus Endobugula sertula' and the bryostatins throughout the life cycle of the bryozoan Bugula neritina.

'Candidatus Endobugula sertula,' the uncultivated gamma-proteobacterial symbiont of the marine bryozoan Bugula neritina, synthesizes bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of 'E. sertula' or the bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized 'E. sertula' and the bryostatins in multiple stages of the B. neritina life cycle, using a novel bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, 'E. sertula' remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of bryostatin signal with aggregates of 'E. sertula' in buds of ancestrulae suggested new synthesis of bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of 'E. sertula' to regenerated colonies. Furthermore, bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the bryostatins and the 'E. sertula,' we determined how 'E. sertula' is transmitted, and identified shifts in bryostatin localization throughout the life cycle of the host B. neritina.

Alpha-proteobacterial symbionts of marine bryozoans in the genus Watersipora.

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of "W. subtorquata" and "W. arcuata" from several locations along the California coast are actually closely related alpha-Proteobacteria, not mollicutes. We propose the names "Candidatus Endowatersipora palomitas" and "Candidatus Endowatersipora rubus" for the symbionts of "W. subtorquata" and "W. arcuata," respectively.

Isolation of two polyketide synthase gene fragments from the uncultured microbial symbiont of the marine bryozoan Bugula neritina.

"Candidatus Endobugula sertula," the uncultured microbial symbiont of the bryozoan Bugula neritina, produces ecologically and biomedically important polyketide metabolites called bryostatins. We isolated two gene fragments from B. neritina larvae that have high levels of similarity to polyketide synthase genes. These gene fragments are clearly associated with the symbiont and not with the host.

Defense of benthic invertebrates against surface colonization by larvae: a chemical arms race.

Sessile invertebrates evolved in a competitive milieu where space is a limiting resource, setting off an arms race between adults that must maintain clean surfaces and larvae that must locate and attach to a suitable substratum. I review the evidence that invertebrates chemically deter or kill the propagules of fouling animals and protists under natural conditions, and that chemosensory mechanisms may allow larvae to detect and avoid settling on chemically protected organisms. The fouling process is an ecologically complex web of interactions between basibionts, surface-colonizing microbes, and fouling larvae, all mediated by chemical signaling. Host-specific bacterial communities are maintained by many invertebrates, and may inhibit fouling by chemical deterrence of larvae, or by preventing biofilm formation by inductive strains. Larval settlement naturally occurs in a turbulent environment, yet the effects of waterborne versus surface-adsorbed chemical defenses have not been compared in flow, limiting our understanding of how larvae respond to toxic surfaces in the field. The importance of evaluating alternative hypotheses such as mechanical and physical defense is discussed, as is the need for ecologically relevant bioassays that quantify effects on larval behavior and identify compounds likely to play a defensive role in situ.

bryA: an unusual modular polyketide synthase gene from the uncultivated bacterial symbiont of the marine bryozoan Bugula neritina.

"Candidatus Endobugula sertula," the uncultivated bacterial symbiont of Bugula neritina, is the proposed source of the bryostatin family of anticancer compounds. We cloned a large modular polyketide synthase (PKS) gene complex from "Candidatus Endobugula sertula" and characterized one gene, bryA, which we propose is responsible for the initial steps of bryostatin biosynthesis. Typical PKS domains are present. However, acyltransferase domains are lacking in bryA, and beta-ketoacyl synthase domains of bryA cluster with those of PKSs with discrete, rather than integral, acyltransferases. We propose a model for biosynthesis of the bryostatin D-lactate starter unit by the bryA loading module, utilizing atypical domains homologous to FkbH, KR, and DH. The bryA gene product is proposed to synthesize a portion of the pharmacologically active part of bryostatin and may be useful in semisynthesis of clinically useful bryostatin analogs.