Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Amphipathic structure of theonellapeptolide-Id, a hydrophobic tridecapeptide lactone from the Okinawa marine sponge Theonella swinhoei

Theonellapeptolide-Id (TNLP), a cyclic tridecapeptide lactone, was crystallized from dimethylformamide-water solution. In the asymmetric unit, two peptide molecules were combined with solvent molecules, and the total molecular weight was over 3000 Dalton. The crystal structure including solvent molecules was finally determined at 0.80 A resolution using synchrotron radiation. The conformations of two independent molecules were similar to each other and were also similar to the previously reported structure (Doi, Ishida, Kobayashi, Deschamps and Flippen-Anderson, 1999, Acta Crystallogr Sect C, 55, 796-798). About 13 hydrated water molecules were found at disordered 19 sites; they were located at a certain region to avoid contact with aliphatic side-chains of peptolide in the crystal. The spatial disposition of the solvent molecules and peptides subsequently caused the formation of the amphipathic layer.     

繁茂膜海绵细胞内微生物多样性的研究

采用海绵组织离散、细胞分离的方法,对繁茂膜海绵细胞进行纯化、胞内微生物DNA提取,构建了繁茂膜海绵细胞内微生物的16SrDNA克隆,对其遗传多样性进行了分析,发现海绵细胞内微生物16SrDNA序列主要归类于紫硫细菌门(Proteobacteria)中的α-亚门、γ-亚门和浮霉菌门(Planctomycetes)等类群。与研磨直接提取海绵组织DNA所得海绵组织中总微生物多样性相比,海绵细胞内存在丰富的浮霉菌(23%),说明浮霉菌主要存在于海绵细胞胞内。     

Biogeography of bacteria associated with the marine sponge Cymbastela concentrica

Recent debate regarding microbial biogeography has focused largely on free-living microbes, yet those microbes associated with host organisms are also of interest from a biogeographical perspective. Marine eukaryotes and associated bacteria should provide ideal systems in which to consider microbial biogeography, as (i) bacteria in seawater should be able to disperse among individuals of the same host species, yet (ii) potential for adaptation to particular hosts (and thus speciation) also exists. We used 16S rDNA-DGGE (denaturing gradient gel electrophoresis) to examine geographic variability in bacterial community composition in the marine sponge Cymbastela concentrica. Denaturing gradient gel electrophoresis banding patterns (and phylogenetic analysis of excised DGGE bands) indicated different communities in Cymbastela concentrica from tropical versus temperate Australia. In contrast, communities were very similar over a 500-km portion of the sponge's temperate range. Variation in bacterial community composition was also considered with respect to ocean current patterns. We speculate that the divergent communities in different parts of the sponge's range provide evidence of endemism attributed to host association, although variation in environmental factors such as light and temperature could also explain the observed results. Interestingly, bacterial communities in seawater varied much less between tropical and temperate locations than did those in C. concentrica, supporting the concept of widespread dispersal among these free-living microbes.     

4-Methylenesterols from Theonella swinhoei sponge are natural pregnane-X-receptor agonists and farnesoid-X-receptor antagonists that modulate innate immunity

We report the isolation and the structural elucidation of a family of polyhydroxylated steroids from the marine sponge Theonella swinhoei. Decodification of interactions of these family with nuclear receptors shows that these steroids are potent agonists of human pregnane-X-receptor (PXR) and antagonists of human farnesoid-X-receptor (FXR) with the putative binding mode to nuclear receptors (NRs) obtained through docking experiments. By using monocytes isolated from transgenic mice harboring hPXR, we demonstrated that swinhosterol B counter-regulates induction of pro-inflammatory cytokines in a PXR-dependent manner. Exposure of CD4(+) T cells to swinhosterol B upregulates the expression of IL-10 causing a shift toward a T cells regulatory phenotype in a PXR dependent manner. These results pave the way to development of a dual PXR agonist/FXR antagonist with a robust immunomodulatory activity and endowed with the ability to modulate the expression of bile acid-regulated genes in the liver.     

Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp.

The microbial community associated with a marine sponge (Haliclona sp.) collected from Tateyama city, Japan was studied using 16S rRNA gene clone libraries. Two DNA templates were prepared using methods recommended for Gram-positive and Gram-negative bacteria in the Qiagen kit manual. From each DNA template, two 16S rRNA genes were PCR amplified, using the combination of universal bacterial primer 27f and primers 1385r and 1492r, respectively. A total of 347 clones were sequenced and compared with those available in DNA data banks. These sequences were members of ten bacterial phyla. Interestingly, more than 30 % of the clones represent novel sequences. A comparison of these sequences with sequences in a library prepared from DNA extracted from the surrounding water shows minimum DNA contamination. Taxonomically, the highest diversity was detected in the clone library prepared using a combination of primers 27f and 1492r and DNA isolated using the Gram-positive bacteria protocol. The potential of Haliclona sp.-associated bacteria to produce secondary metabolites was studied by cloning and sequencing the polyketide synthase (PKS, type 1) gene using the same DNA samples. Analysis of partial sequences derived from the sponge metagenome revealed 27 unique ketosynthase domains of PKS type I. This study suggests strongly that this Haliclona sp. plays host to diverse novel bacteria with a potential to produce novel polyketides.     

Antimicrobial activity of bacteria from marine sponge Suberea mollis and bioactive metabolites of Vibrio sp. EA348

Discovery of potential bioactive metabolites from sponge-associated bacteria have gained attraction in recent years. The current study explores the potential of sponge (Suberea mollis) associated bacteria against bacterial and fungal pathogens. Sponge samples were collected from Red sea in Obhur region, Jeddah, Saudi Arabia. Of 29 isolated bacteria belong to four different classes i.e. Firmicutes (62%), γ-Proteobacteria (21%), α-Proteobacteria (10%) and Actinobacteria (7%). Among them nineteen (65%) bacterial strains showed antagonistic activity against oomycetes and only 3 (10%) bacterial strains were active against human pathogenic bacteria tested. Most bioactive genera include Bacillus (55%), Pseudovibrio (13%) and Ruegeria (10%). Enzyme production (protease, lipase, amylase, cellualse) was identified in 12 (41%) bacterial strains where potential strains belonging to γ-Proteobacteria and Firmicutes groups. Production of antimicrobial metabolites and hydrolysates in these bacteria suggest their potential role in sponge against pathogens. Further bioactive metabolites from selected strain of Vibrio sp. EA348 were identified using LC-MS and GC–MS analyses. We identified many active metabolites including antibiotics such as Amifloxacin and fosfomycin. Plant growth hormones including Indoleacetic acid and Gibberellin A3 and volatile organic compound such as methyl jasmonate were also detected in this strain. Our results highlighted the importance of marine bacteria inhabiting sponges as potential source of antimicrobial compounds and plant growth hormones of pharmaceutical and agricultural significance.     

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.     

Antimicrobial activity of surface attached marine bacteria in biofilms

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an ‘inducer’ bacterium stimulated the production of antimicrobial molecules by ‘producer’ bacteria that were active against the ‘inducer’ bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.     

Antimicrobial activity of marine sponge Clathria indica (Dendy, 1889)

Sponges are sessile filter feeders that have developed efficient defense mechanisms against foreign invaders such as viruses, bacteria or eukaryotic organisms. Antimicrobial peptides are known as major components of the innate immune defense system in marine invertebrates. The aim of the present work was to study the antimicrobial properties of the Indian sponge Clathria indica with special reference to the identification of antimicrobial peptides. Crude methanolic extract and its chloroform, n-butanol and aqueous fractions were tested against 16 human pathogens which include eleven bacteria with four of them being multidrug resistant and five pathogenic fungi. All fractions showed effective antibacterial activity against common and multidrug-resistant Salmonella typhi and antifungal activity against C. albicans and C. neoformans. However, they were ineffective against Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. Chloroform fraction being the most potent among the fractions tested on chemical investigation was indicative of the presence of peptides as evidenced by ninhydrin positive spots on TLC and presence of peptide bonds by NMR. Its ESI-MS showed presence of several peptides in the range of m/z 850 to 980. Structure of three peptides has been tentatively assigned by ESI-MS/MS or tandem mass analysis, on the basis of the amino acid sequence established. The results clearly show that the sponge C. indica represent an interesting source of marine invertebrates-derived antimicrobial peptides in the development of new strategies to treat various infectious diseases.     

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.     

Isoswinholide B and swinholide K,potently cytotoxic dimeric macrolides from Theonella swinhoei

Chemical investigation of an Indonesian specimen of Theonella swinhoei afforded the new dimeric macrolides isoswinholide B (5) and swinholide K (6), along with the known swinholides A (1), B (2) and D (3) and isoswinholide A (4). Isoswinholide B showed an unprecedented 21/19′ lactonization pattern, while swinholide K included an sp² methylene attached at C-4 and an additional oxymethine group at C-5, whose configuration has been determined through application of J-based configuration analysis. The isolated swinholides (1–6), with the exception of isoswinholide B, showed a cytotoxic activity on HepG2 (hepatocarcinoma cell line) in the nanomolar range.     

Koshikamide A2, a cytotoxic linear undecapeptide isolated from a marine sponge of Theonella sp

Koshikamide A2 (2) was isolated as a cytotoxic metabolite from a marine sponge of Theonella sp. Its structure was elucidated to be a linear undecapeptide by spectroscopic and chemical methods, together with enzymatic conversion to known koshikamide A1 (1). The new peptide moderately inhibited the growth of P388 murine leukemia cells.     

Tokaramide A, a new cathepsin B inhibitor from the marine sponge Theonella aff. mirabilis

A new cathepsin B inhibitor, tokaramide A (1) has been isolated from the marine sponge Theonella aff. mirabilis. Its structure was determined by spectroscopic and chemical methods. Tokaramide A inhibits cathepsin B with an IC₅₀ value of 29.0 ng/mL.     

Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches

Diverse ketosynthase (KS) genes were retrieved from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata. Bacterial isolation and metagenomic approaches were employed. Phylogenetic analysis of 16S rRNA of culturable sponge-associated bacterial communities comprised eight groups over four phyla. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacterial, cyanobacterial and trans-AT type). Metagenomic DNA of the sponge microbial community was extracted to explore community KS genes by two approaches: direct amplification of KS domains and construction of fosmid libraries for KS domain screening. Five KS domains were retrieved from polymerase chain reaction (PCR) amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains found using multiplex PCR screening. Analysis of selected polyketide synthase (PKS) from one fosmid showed that the PKS consists of two modules. Open reading frames located up- and downstream of the PKS displayed similarity with membrane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect KS domains found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with KS domains originating from metagenomes derived from other sponge species from other geographical regions.     

Screening of rifamycin producing marine sponge bacteria by LC-MS-MS

HPLC-MS-MS has been used for the identification and characterisation of rifamycin B and rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain rifamycin thus an alternate actinobacterial source of rifamycin was established.     

Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.     

Marine bacteria associated with sponge as source of cyclic peptides

Two bacteria associated with the marine sponge Ircinia variabilis were isolated using commercial and experimental media. The use of media containing marine derived proteins improved the growth of both isolated bacteria, showing that marine bacteria need of marine derived proteins for a better growth. The composition of free and total fatty acids of both strains cultivated under different carbon source was investigated. Several diketopiperazines were isolated from both bacteria and the hypothesis of their role in the bacterial-spongy interaction is discussed.     

Radiolaria associated with large diversity of marine alveolates

We have isolated cells of unculturable radiolarians from marine coastal waters. Individual cells were subjected to single cell whole genome amplification (SCWGA) and gene-targeted PCR. Using this approach we recover a surprisingly large diversity of sequences related to the enigmatic marine alveolate groups 1 and 2 (MALV I and MALV II) that most likely represent intracellular symbionts or parasites of the radiolarian cells. 18S rDNA phylogeny of the MALV sequences reveals 4 distinct clades of radiolarian associates here named Radiolarian Associated Sequences (RAS) 1-4. One clade of both phaeodarian and radiolarian associates and one clade of only phaeodarian associates are also identified. The MALV sequences cluster according to host type, i.e. sequences from associates identified in radiolarians, fish, copepods, ciliates or dinoflagellates are not intermixed but separated into distinct clades. This implies several independent colonizations of host lineages and links a large diversity of MALV to radiolarian-associated species. This demonstrates that radiolarians may be an important reservoir for MALV, making them a key group for understanding the impact of intracellular symbionts on the marine ecosystem. This study shows that applying SCWGA on unculturable cells is a promising approach to study the vast diversity and interactions of intracellular eukaryote organisms.     

Antidiatom activity of marine bacteria associated with sponges from San Juan Island,Washington

Crude extracts of 52 marine bacteria associated with sponges, which were collected from the sea near San Juan Island, Washington, USA, were screened using diatom attachment assays against Amphora sp., Nitzschia closterium, Sellaphora sp. and Stauroneis sp. to investigate their antidiatom activities. Among these samples, five expressed strong anti-adhesion effects on all four tested diatoms. There was no negative effect observed from those five active samples on the growth of Amphora sp. Those five active samples were prepared from respective isolates, which all belonged to the genus Bacillus based on 16S rRNA gene sequencing analysis. The results of present study indicate that Bacillus may play important roles for sponges’ chemical defence against biofouling of diatoms and that the metabolites of Bacillus may be a potential source of natural antifouling compounds.