Evidence for Vertical Transmission of Bacterial Symbionts from Adult to Embryo in the Caribbean Sponge Svenzea zeai

The Caribbean reef sponge Svenzea zeai was previously found to contain substantial quantities of unicellular photosynthetic and autotrophic microbes in its tissues, but the identities of these symbionts and their method of transfer from adult to progeny are largely unknown. In this study, both a 16S rRNA gene-based fingerprinting technique (denaturing gradient gel electrophoresis [DGGE]) and clone library analysis were applied to compare the bacterial communities associated with adults and embryos of S. zeai to test the hypothesis of vertical transfer across generations. In addition, the same techniques were applied to the bacterial community from the seawater adjacent to adult sponges to test the hypothesis that water column bacteria could be transferred horizontally as sponge symbionts. Results of both DGGE and clone library analysis support the vertical transfer hypothesis in that the bacterial communities associated with sponge adults and embryos were highly similar to each other but completely different from those in the surrounding seawater. Sequencing of prominent DGGE bands and of clones from the libraries revealed that the bacterial communities associated with the sponge, whether adult or embryo, consisted of a large proportion of bacteria in the phyla Chloroflexi and Acidobacteria, while most of the sequences recovered from the community in the adjacent water column belonged to the class Alphaproteobacteria. Altogether, 21 monophyletic sequence clusters, comprising sequences from both sponge adults and embryos but not from the seawater, were identified. More than half of the sponge-derived sequences fell into these clusters. Comparison of sequences recovered in this study with those deposited in GenBank revealed that more than 75% of S. zeai-derived sequences were closely related to sequences derived from other sponge species, but none of the sequences recovered from the seawater column overlapped with those from adults or embryos of S. zeai. In conclusion, there is strong evidence that a dominant proportion of sponge-specific bacteria present in the tissues of S. zeai are maintained through vertical transfer during embryogenesis rather than through acquisition from the environment (horizontal transfer).Besides being the oldest metazoans, sponges are the simplest multicellular animals and possess a low degree of tissue differentiation and coordination (54). Sponges are sessile, filter-feeding organisms that may harbor within their tissues a remarkable array of microorganisms, including bacteria (19, 59, 64), archaea (41), zooxanthellae (22), diatoms (63), and fungi (35). In some cases, microbial consortia can make up to 40 to 60% of the sponge tissue volume (21, 61) and exceed a density of 10⁹ microbial cells per ml of sponge tissue (62), which is several orders of magnitude higher than that found in seawater. Apart from being a source of food (43), bacterial symbionts may participate in the acquisition and transfer of nutrients inside sponges (67, 68), the recycling of insoluble protein (69), the stabilization of the sponge skeleton (44), and the processing of metabolic waste (4, 65). Many antimicrobial compounds have been isolated from sponge bacterial symbionts (24, 47, 53), suggesting the involvement of symbiotic bacteria in sponge chemical defenses. In some cases, bacterial symbionts have been found to be the source of bioactive compounds that were isolated from sponges, which has opened up new research directions in marine natural product chemistry, biotechnology, and pharmaceutical development (18, 23, 40).Based on immunological evidence from the 1980s (66), sponge-bacterium symbioses are thought to have originated in the Precambrian, when bacteria evolved to form a single clade of sponge-specific bacteria that were distinct from isolates found in the surrounding seawater. Since then, many studies have similarly documented a high level of consistency and specificity in sponge-bacterium associations (20, 27, 59). Nevertheless, questions remain about the acquisition and maintenance of symbionts in host sponges. In general, the following two hypotheses have been proposed: (i) a recently metamorphosed sponge selectively retains specific groups of bacteria from the diverse pool of bacteria present in the water column as it begins filter feeding (horizontal transfer) or (ii) specific bacterial strains are transmitted by the maternal sponge to developing embryos and are already present in the metamorphosing sponge (vertical transfer) (58). The first hypothesis requires some recognition of specific microbes by the sponge, perhaps through an innate immune system (36) or other means to distinguish symbiont strains from food bacteria (70).Vertical transfer of bacterial symbionts in sponges was first proposed by Lévi and Porte (29), who demonstrated the presence of bacteria inside the larvae of the sponge Oscarella lobularis. Later, in 1976, Lévi and Lévi (30) studied the transmission of bacteria in the sponge Chondrosia reniformis via sponge oocytes. Since then, vertical transmission of bacterial symbionts via eggs or larvae has been documented for several sponge species, including Tethya citrina (15), Geodia cydonium (50), Stelletta grubii (49), Hippospongia sp. (25), Spongia sp. (25), Halisarca dujardini (10), and Corticium candelabrum (8). However, all of these studies employed transmission and scanning electron microscopy and could only examine the presence of bacteria in maternal sponges, oocytes, or larvae at the morphological level, with no determination of microbial identity. With advances in molecular techniques, Enticknap et al. (9) were the first to report the successful isolation of an alphaproteobacterial symbiont, strain NW001, from both the adult sponge Mycale laxissima and its larvae. They also did a preliminary denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial community in seawater and compared that with the community in the sponge larval sample. However, such a comparison was not extended to the sponge adult, and no solid conclusion can be drawn for the horizontal transfer mechanism of sponge symbionts. More recently, Sharp et al. (52) used fluorescence in situ hybridization (FISH) and clone library techniques to demonstrate the presence of proteobacteria, actinobacteria, and a clade of sponge-associated bacteria in the embryos and mesohyl of the tropical sponge Corticium sp. By clone library and DGGE analyses, Schmitt et al. (48a) identified 28 vertical-transmission clusters in five different Caribbean sponge species and demonstrated that the complex sponge adult microbial community was collectively transmitted through reproductive stages. While these recent studies support the vertical transfer hypothesis, they did not fully address the identities of microbes in the water column surrounding the sponges, which is key to determining whether horizontal transfer may also take place.The Caribbean reef sponge Pseudaxinella zeai was reclassified into a new genus, Svenzea (Demospongiae, Halichondria, Dictyonellidae), in 2002 because it has an unusual skeleton arrangement consisting mainly of short stout styles that are arranged in an isodictyal reticulation (2). It is a viviparous sponge that produces the largest embryos (>1 mm in diameter) and larvae (6 mm long) recorded for the phylum Porifera (45). Svenzea zeai has also been classified as a bacteriosponge because it contains substantial amounts of unicellular photosynthetic and autotrophic microbial symbionts in its tissues (2, 45). Although bacteria were observed in the embryos and larvae of this sponge based on transmission electron microscopy studies (45), neither the direct linkage between the maternal sponge and the propagules nor the identity of the microbial symbionts had been established.In this study, our objective was to examine vertical versus horizontal transfer of bacterial symbionts in Svenzea zeai. This was achieved by comparing the bacterial community profiles of the adults and embryos of the sponge by use of a combination of molecular techniques, including DGGE and clone library analysis. More than one technique was employed to compensate for deficiencies of each technique in revealing bacterial community structure. Additionally, we used the same techniques to examine the bacterial community in the seawater that surrounded the sponge to determine whether horizontal transfer was evident.     

Vertical Transmission of a Phylogenetically Complex Microbial Consortium in the Viviparous Sponge Ircinia felix

Many marine demosponges contain large amounts of phylogenetically complex yet highly sponge-specific microbial consortia within the mesohyl matrix, but little is known about how these microorganisms are acquired by their hosts. Settlement experiments were performed with the viviparous Caribbean demosponge Ircinia felix to investigate the role of larvae in the vertical transmission of the sponge-associated microbial community. Inspections by electron microscopy revealed large amounts of morphologically diverse microorganisms in the center of I. felix larvae, while the outer rim appeared to be devoid of microorganisms. In juveniles, microorganisms were found between densely packed sponge cells. Denaturing gradient gel electrophoresis (DGGE) was performed to compare the bacterial community profiles of adults, larvae, and juvenile sponges. Adults and larvae were highly similar in DGGE band numbers and banding patterns. Larvae released by the same adult individual contained highly similar DGGE banding patterns, whereas larvae released by different adult individuals showed slightly different DGGE banding patterns. Over 200 bands were excised, sequenced, and phylogenetically analyzed. The bacterial diversity of adult I. felix and its larvae was comparably high, while juveniles showed reduced diversity. In total, 13 vertically transmitted sequence clusters, hereafter termed “IF clusters,” that contained sequences from both the adult sponge and offspring (larvae and/or juveniles) were found. The IF clusters belonged to at least four different eubacterial phyla and one possibly novel eubacterial lineage. In summary, it could be shown that in I. felix, vertical transmission of microorganisms through the larvae is an important mechanism for the establishment of the sponge-microbe association.     

Microbial Diversity of the Freshwater Sponge Spongilla lacustris

To provide insight into the phylogenetic bacterial diversity of the freshwater sponge Spongilla lacustris, a 16S rRNA gene libraries were constructed from sponge tissues and from lake water. Restriction fragment length polymorphism (RFLP) analysis of >190 freshwater sponge-derived clones resulted in six major restriction patterns, from which 45 clones were chosen for sequencing. The resulting sequences were affiliated with the Alphaproteobacteria (n = 19), the Actinobacteria (n = 15), the Betaproteobacteria (n = 2), and the Chloroflexi (n = 2) lineages. About half of the sequences belonged to previously described actinobacterial (hgc-I) and betaproteobacterial (beta-II) sequence clusters of freshwater bacteria that were also present in the lake water 16S rRNA gene library. At least two novel, deeply rooting alphaproteobacterial lineages were recovered from S. lacustris that showed <89% sequence similarity to known phylogenetic groups. Electron microscopical observations revealed that digested bacterial remnants were contained within food vacuoles of sponge archaeocytes, whereas the extracellular matrix was virtually free of bacteria. This study is the first molecular diversity study of a freshwater sponge and adds to a growing database on the diversity and community composition of sponge-associated microbial consortia.     

Molecular Mechanistic Insights into Drosophila DHX36-Mediated G-Quadruplex Unfolding: A Structure-Based Model

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New Insights into the Molecular Mechanisms of Evolution: Stress Increases Genetic Diversity

The mechanisms of stress-induced mutagenesis in prokaryotes and realization of reserved (preaccumulated) genetic variation in eukaryotes are considered. In prokaryotes, replication becomes error-prone in stress because of the induction of the SOS response and the inactivation of the mismatch repair system; stress also increases the transposition rate and the efficiency of interspecific gene transfer. In eukaryotes, chaperone HSP90, which restores the native folding of mutant proteins (e.g., signal transduction and morphogenetic proteins) in normal conditions, fails to do so in stress, which leads to abrupt expression of multiple mutations earlier reserved in the corresponding genes. The role of these mechanisms in the evolution of prokaryotes and eukaryotes is discussed.     

New Insights into Microbial Oxidation of Antimony and Arsenic

Sb(III) oxidation was documented in an Agrobacterium tumefaciens isolate that can also oxidize As(III). Equivalent Sb(III) oxidation rates were observed in the parental wild-type organism and in two well-characterized mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting that Sb(III) and As(III) may be biochemical analogs, Sb(III) oxidation is catalyzed by a pathway different than that used for As(III). Sb(III) and As(III) oxidation was also observed for an eukaryotic acidothermophilic alga belonging to the order Cyanidiales, implying that the ability to oxidize metalloids may be phylogenetically widespread.     

Insights into Penicillium roqueforti Morphological and Genetic Diversity

Fungi exhibit substantial morphological and genetic diversity, often associated with cryptic species differing in ecological niches. Penicillium roqueforti is used as a starter culture for blue-veined cheeses, being responsible for their flavor and color, but is also a common spoilage organism in various foods. Different types of blue-veined cheeses are manufactured and consumed worldwide, displaying specific organoleptic properties. These features may be due to the different manufacturing methods and/or to the specific P. roqueforti strains used. Substantial morphological diversity exists within P. roqueforti and, although not taxonomically valid, several technological names have been used for strains on different cheeses (e.g., P. gorgonzolae, P. stilton). A worldwide P. roqueforti collection from 120 individual blue-veined cheeses and 21 other substrates was analyzed here to determine (i) whether P. roqueforti is a complex of cryptic species, by applying the Genealogical Concordance Phylogenetic Species Recognition criterion (GC-PSR), (ii) whether the population structure assessed using microsatellite markers correspond to blue cheese types, and (iii) whether the genetic clusters display different morphologies. GC-PSR multi-locus sequence analyses showed no evidence of cryptic species. The population structure analysis using microsatellites revealed the existence of highly differentiated populations, corresponding to blue cheese types and with contrasted morphologies. This suggests that the population structure has been shaped by different cheese-making processes or that different populations were recruited for different cheese types. Cheese-making fungi thus constitute good models for studying fungal diversification under recent selection.     

微生物分子生态学技术在湖泊微生物多样性研究中的应用

微生物是湖泊生物圈物质循环和能量流动的主要参与者,在湖泊的生态系统中起着重要的作用。但是,湖泊中存在着大量不可培养的细菌,利用传统的培养技术,无法对湖泊微生物的多样性进行深入而全面的研究,而不依赖培养的分子生物学技术的发展为此方面研究开辟了新的路径。微生物分子生态学作为分子生物学与微生物生态学交叉产生的学科,在研究湖泊微生物多样性方面已经得到了广泛的应用。主要综述了变性梯度凝胶电泳(PCR-DGGE)技术,末端限制性酶切片段长度多态性技术(T-RFLP),16SrDNA克隆文库技术等微生物分子生态学技术在研究湖泊微生物多样性方面的应用情况。     

Genetic Diversity and Potential Function of Microbial Symbionts Associated with Newly Discovered Species of Osedax Polychaete Worms

We investigated the genetic diversity of symbiotic bacteria associated with two newly discovered species of Osedax from Monterey Canyon, CA, at 1,017-m (Osedax Monterey Bay sp. 3 “rosy” [Osedax sp. MB3]) and 381-m (Osedax Monterey Bay sp. 4 “yellow collar”) depths. Quantitative PCR and clone libraries of 16S rRNA gene sequences identified differences in the compositions and abundances of bacterial phylotypes associated with the newly discovered host species and permitted comparisons between adult Osedax frankpressi and juveniles that had recently colonized whalebones implanted at 2,891 m. The newly discovered Osedax species hosted Oceanospirillales symbionts that are related to Gammaproteobacteria associated with the previously described O. frankpressi and Osedax rubiplumus (S. K. Goffredi, V. J. Orphan, G. W. Rouse, L. Jahnke, T. Embaye, K. Turk, R. Lee, and R. C. Vrijenhoek, Environ. Microbiol. 7:1369-1378, 2005). In addition, Osedax sp. MB3 hosts a diverse and abundant population of additional bacteria dominated by Epsilonproteobacteria. Ultrastructural analysis of symbiont-bearing root tissues verified the enhanced microbial diversity of Osedax sp. MB3. Root tissues from the newly described host species and O. frankpressi all exhibited collagenolytic enzyme activity, which covaried positively with the abundance of symbiont DNA and negatively with mean adult size of the host species. Members of this unusual genus of bone-eating worms may form variable associations with symbiotic bacteria that allow for the observed differences in colonization and success in whale fall environments throughout the world's oceans.     

Phylogenetic Diversity and Community Structure of the Symbionts Associated with the Coralline Sponge Astrosclera willeyana of the Great Barrier Reef

The coralline sponge Astrosclera willeyana, considered to be a living representative of the reef-building stromatoporoids of the Mesozoic and the Paleozoic periods, occurs widely throughout the Indo-Pacific oceans. We aimed to examine, for the first time, the phylogenetic diversity of the microbial symbionts associated with A. willeyana using molecular methods and to investigate the spatial variability in the sponge-derived microbial communities of A. willeyana from diverse sites along the Great Barrier Reef (GBR). Both denaturing gradient gel electrophoresis (DGGE) analyses of 12 Astrosclera specimens and sequencing of a 16S rRNA gene clone library, constructed using a specimen of A. willeyana from the Yonge Reef (380 clones), revealed the presence of a complex microbial community with high diversity. An assessment of the 16S rRNA gene sequences to the particular phylogenetic groups showed domination of the Chloroflexi (42 %), followed by the Gammaproteobacteria (14 %), Actinobacteria (11 %), Acidobacteria (8 %), and the Deferribacteres (7 %). Of the microbes that were identified, a further 15 % belonged to the Deltaproteobacteria, Alphaproteobacteria, and Nitrospirae genera. The minor phylogenetic groups Gemmatimonadetes, Spirochaetes, Cyanobacteria, Poribacteria, and the Archaea composed 3 % of the community. Over 94 % of the sequences obtained from A. willeyana grouped together with other sponge- or coral-derived sequences, and of these, 72 % formed, with nearest relatives, 46 sponge-specific or sponge–coral clusters, highlighting the uniqueness of the microbial consortia in sponges. The DGGE results showed clear divisions according to the geographical origin of the samples, indicating closer relationships between the microbial communities with respect to their geographic origin (northern vs. southern GBR).     

Vertical Transmission of Diverse Microbes in the Tropical Sponge Corticium sp.

Sponges are host to extremely diverse bacterial communities, some of which appear to be spatiotemporally stable, though how these consistent associations are assembled and maintained from one sponge generation to the next is not well understood. Here we report that a diverse group of microbes, including both bacteria and archaea, is consistently present in aggregates within embryos of the tropical sponge Corticium sp. The major taxonomic groups represented in bacterial 16S rRNA sequences amplified from the embryos are similar to those previously described in a variety of marine sponges. Three selected bacterial taxa, representing proteobacteria, actinobacteria, and a clade including recently described sponge-associated bacteria, were tested and found to be present in all adult samples tested over a 3-year period and in the embryos throughout development. Specific probes were used in fluorescence in situ hybridization to localize cells of the three types in the embryos and mesohyl. This study confirms the vertical transmission of multiple, phylogenetically diverse microorganisms in a marine sponge, and our findings lay the foundation for future work on exploring vertical transmission of specific, yet diverse, microbial assemblages in marine sponges.     

Mechanistic Insights into Interaction of Humic Acid with Silver Nanoparticles

Humic acid (HA) is one of the major components of the natural organic matter present in the environment that alters the fate and behavior of silver nanoparticles (Ag NPs). Transformation of Ag NPs happens upon interaction with HA, thereby, changing both physical and chemical properties. Fluorescence spectroscopy and scanning electron microscopy (SEM) were used to analyze the interaction of Ag NPs with HA. In pH and time-dependent studies, the near field electro dynamical environment of Ag NPs influenced the fluorescence of HA, indicated by fluorescence enhancement. SEM revealed not only morphological changes, but also significant reduction in size of Ag NPs after interaction with HA. Based on these studies, a probable mechanism was proposed for the interaction of HA with Ag NPs, suggesting the possible transformation that these nanoparticles can undergo in the environment.     

New Insights into the Diversity of Marine Picoeukaryotes

Over the last decade, culture-independent surveys of marine picoeukaryotic diversity based on 18S ribosomal DNA clone libraries have unveiled numerous sequences of novel high-rank taxa. This newfound diversity has significantly altered our understanding of marine microbial food webs and the evolution of eukaryotes. However, the current picture of marine eukaryotic biodiversity may be significantly skewed by PCR amplification biases, occurrence of rDNA genes in multiple copies within a single cell, and the capacity of DNA to persist as extracellular material. In this study we performed an analysis of the metagenomic dataset from the Global Ocean Survey (GOS) expedition, seeking eukaryotic ribosomal signatures. This PCR-free approach revealed similar phylogenetic patterns to clone library surveys, suggesting that PCR steps do not impose major biases in the exploration of environmental DNA. The different cell size fractions within the GOS dataset, however, displayed a distinct picture. High protistan diversity in the <0.8 µm size fraction, in particular sequences from radiolarians and ciliates (and their absence in the 0.8–3 µm fraction), suggest that most of the DNA in this fraction comes from extracellular material from larger cells. In addition, we compared the phylogenetic patterns from rDNA and reverse transcribed rRNA 18S clone libraries from the same sample harvested in the Mediterranean Sea. The libraries revealed major differences, with taxa such as pelagophytes or picobiliphytes only detected in the 18S rRNA library. MAST (Marine Stramenopiles) appeared as potentially prominent grazers and we observed a significant decrease in the contribution of alveolate and radiolarian sequences, which overwhelmingly dominated rDNA libraries. The rRNA approach appears to be less affected by taxon-specific rDNA copy number and likely better depicts the biogeochemical significance of marine protists.     

Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex

Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N₁- and N₃-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.     

Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a

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Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.