Trikoramide A, a Prenylated Cyanobactin from the Marine Cyanobacterium Symploca hydnoides.

A new cyclic decapeptide, trikoramide A (1), has been isolated from samples of the marine cyanobacterium Symploca hydnoides, collected from Bintan Island, Indonesia. Trikoramide A (1) is a C-prenylated cyclotryptophan-containing cyanobactin. Its planar structure was deduced by 1D and 2D NMR spectroscopy as well as HR-MS/MS data. In addition, its absolute configuration was determined by Marfey's method and 2D NOESY NMR spectroscopic analysis. Compound 1 possessed cytotoxicity against the MOLT-4 and AML2 cancer cell lines with IC50 values of 4.8 and 8.2 µM, respectively.

Targeted Enrichment of Low-Abundance and Uncharacterized Taxon Members in Complex Microbial Community with Primer-Free FISH Probes Designed from Next Generation Sequencing Dataset.

Methods to obtain high-quality assembled genomic information of rare and unclassified member species in complex microbial communities remain a high priority in microbial ecology. Additionally, the supplementation of three-dimensional spatial information that highlights the morphology and spatial interaction would provide additional insights to its ecological role in the community. Fluorescent in-situ hybridization (FISH) coupling with fluorescence-activated cell sorting (FACS) is a powerful tool that enables the detection, visualization, and separation of low-abundance microbial members in samples containing complex microbial compositions. Here, we have described the workflow from designing the appropriate FISH probes from metagenomics or metatranscriptomics datasets to the preparation and treatment of samples to be used in FISH-FACS procedures.

Novel antibacterial proteins from the microbial communities associated with the sponge Cymbastela concentrica and the green alga Ulva australis.

The functional metagenomic screening of the microbial communities associated with a temperate marine sponge and a green alga identified three novel hydrolytic enzymes with antibacterial activities. The results suggest that uncultured alpha- and gammaproteobacteria contain new classes of proteins that may be a source of antibacterial agents.

Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion.

Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information.

Primer-free FISH probes from metagenomics/metatranscriptomics data permit the study of uncharacterised taxa in complex microbial communities.

Methods for the study of member species in complex microbial communities remain a high priority, particularly for rare and/or novel member species that might play an important ecological role. Specifically, methods that link genomic information of member species with its spatial structure are lacking. This study adopts an integrative workflow that permits the characterisation of previously unclassified bacterial taxa from microbiomes through: (1) imaging of the spatial structure; (2) taxonomic classification and (3) genome recovery. Our study attempts to bridge the gaps between metagenomics/metatranscriptomics and high-resolution biomass imaging methods by developing new fluorescence in situ hybridisation (FISH) probes-termed as R-Probes-from shotgun reads that harbour hypervariable regions of the 16S rRNA gene. The sample-centric design of R-Probes means that probes can directly hybridise to OTUs as detected in shotgun sequencing surveys. The primer-free probe design captures larger microbial diversity as compared to canonical probes. R-Probes were designed from deep-sequenced RNA-Seq datasets for both FISH imaging and FISH-Fluorescence activated cell sorting (FISH-FACS). FISH-FACS was used for target enrichment of previously unclassified bacterial taxa prior to downstream multiple displacement amplification (MDA), genomic sequencing and genome recovery. After validation of the workflow on an axenic isolate of Thauera species, the techniques were applied to investigate two previously uncharacterised taxa from a tropical full-scale activated sludge community. In some instances, probe design on the hypervariable region allowed differentiation to the species level. Collectively, the workflow can be readily applied to microbiomes for which shotgun nucleic acid survey data is available.

Global transcriptomic responses of Escherichia coli K-12 to volatile organic compounds.

Volatile organic compounds (VOCs) are commonly used as solvents in various industrial settings. Many of them present a challenge to receiving environments, due to their toxicity and low bioavailability for degradation. Microorganisms are capable of sensing and responding to their surroundings and this makes them ideal detectors for toxic compounds. This study investigates the global transcriptomic responses of Escherichia coli K-12 to selected VOCs at sub-toxic levels. Cells grown in the presence of VOCs were harvested during exponential growth, followed by whole transcriptome shotgun sequencing (RNAseq). The analysis of the data revealed both shared and unique genetic responses compared to cells without exposure to VOCs. Results suggest that various functional gene categories, for example, those relating to Fe/S cluster biogenesis, oxidative stress responses and transport proteins, are responsive to selected VOCs in E. coli. The differential expression (DE) of genes was validated using GFP-promoter fusion assays. A variety of genes were differentially expressed even at non-inhibitory concentrations and when the cells are at their balanced-growth. Some of these genes belong to generic stress response and others could be specific to VOCs. Such candidate genes and their regulatory elements could be used as the basis for designing biosensors for selected VOCs.

A polyphasic approach to the exploration of collagenolytic activity in the bacterial community associated with the marine sponge Cymbastela concentrica.

Collagen is an important, extracellular structural protein for metazoans and provides a rich nutrient source for bacteria that possess collagen-degrading enzymes. In a symbiotic host system, collagen degradation could benefit the bacteria, but would be harmful for the eukaryotic host. Using a polyphasic approach, we investigated the presence of collagenolytic activity in the bacterial community hosted by the marine sponge Cymbastela concentrica. Functional screening for collagenase activity using metagenomic library clones (227 Mbp) and cultured isolates of sponge's bacterial community, as well as bioinformatic analysis of metagenomic shotgun-sequencing data (106,679 predicted genes) were used. The results show that the abundant members of the bacterial community contain very few genes encoding for collagenolytic enzymes, while some low-abundance sponge isolates possess collagenolytic activities. These findings indicate that collagen is not a preferred nutrient source for the majority of the members of the bacterial community associated with healthy C. concentrica, and that some low-abundance bacteria have collagenase activities that have the potential to harm the sponge through tissue degradation.

Functional genomic signatures of sponge bacteria reveal unique and shared features of symbiosis.

Sponges form close relationships with bacteria, and a remarkable phylogenetic diversity of yet-uncultured bacteria has been identified from sponges using molecular methods. In this study, we use a comparative metagenomic analysis of the bacterial community in the model sponge Cymbastela concentrica and in the surrounding seawater to identify previously unrecognized genomic signatures and functions for sponge bacteria. We observed a surprisingly large number of transposable insertion elements, a feature also observed in other symbiotic bacteria, as well as a set of predicted mechanisms that may defend the sponge community against the introduction of foreign DNA and hence contribute to its genetic resilience. Moreover, several shared metabolic interactions between bacteria and host include vitamin production, nutrient transport and utilization, and redox sensing and response. Finally, an abundance of protein-protein interactions mediated through ankyrin and tetratricopeptide repeat proteins could represent a mechanism for the sponge to discriminate between food and resident bacteria. These data provide new insight into the evolution of symbiotic diversity, microbial metabolism and host-microbe interactions in sponges.

Biofilm formation and phenotypic variation enhance predation-driven persistence of Vibrio cholerae.

Persistence of the opportunistic bacterial pathogen Vibrio cholerae in aquatic environments is the principal cause for seasonal occurrence of cholera epidemics. This causality has been explained by postulating that V. cholerae forms biofilms in association with animate and inanimate surfaces. Alternatively, it has been proposed that bacterial pathogens are an integral part of the natural microbial food web and thus their survival is constrained by protozoan predation. Here, we report that both explanations are interrelated. Our data show that biofilms are the protective agent enabling V. cholerae to survive protozoan grazing while their planktonic counterparts are eliminated. Grazing on planktonic V. cholerae was found to select for the biofilm-enhancing rugose phase variant, which is adapted to the surface-associated niche by the production of exopolymers. Interestingly, grazing resistance in V. cholerae biofilms was not attained by exopolymer production alone but was accomplished by the secretion of an antiprotozoal factor that inhibits protozoan feeding activity. We identified that the cell density-dependent regulator hapR controls the production of this factor in biofilms. The inhibitory effect of V. cholerae biofilms was found to be widespread among toxigenic and nontoxigenic isolates. Our results provide a mechanistic explanation for the adaptive advantage of surface-associated growth in the environmental persistence of V. cholerae and suggest an important contribution of protozoan predation in the selective enrichment of biofilm-forming strains in the out-of-host environment.