Functional guilds and drivers of diversity in seaweed-associated bacteria.

Comparisons of functional and taxonomic profiles from bacterial communities in different habitats have suggested the existence of functional guilds composed of taxonomically or phylogenetically distinct members. Such guild membership is, however, rarely defined and the factors that drive functional diversity in bacteria remain poorly understood. We used seaweed-associated bacteria as a model to shed light on these important aspects of community ecology. Using a large dataset of over 1300 metagenome-assembled genomes from 13 seaweed species we found substantial overlap in the functionality of bacteria coming from distinct taxa, thus supporting the existence of functional guilds. This functional equivalence between different taxa was particularly pronounced when only functions involved in carbohydrate degradation were considered. We further found that bacterial taxonomy is the dominant driver of functional differences between bacteria and that seaweed species or seaweed type (i.e. brown, red and green) had relatively stronger impacts on genome functionality for carbohydrate-degradation functions when compared to all other cellular functions. This study provides new insight into the factors underpinning the functional diversity of bacteria and contributes to our understanding how community function is generated from individual members.

Not all parents are the same: Diverse strategies of symbiont transmission in seaweeds.

Different marine seaweed species have been shown to harbour specific bacterial communities, however, the extent to which vertical symbiont transmission from parents to offspring contributes to host-specificity is unclear. Here we use fluorescence and electron microscopy as well as 16S rRNA gene-based community analysis to investigate symbiont transmission in members of the three major seaweed groups (green Chlorophyta, red Rhodophyta and brown Phaeophyceae). We found seaweeds employ diverse strategies to transfer symbionts to their progeny. For instance, the green Ulva australis does not appear to have the capacity for vertical transmission. In contrast, the brown Phyllospora comosa adopts a non-selective vertical transmission. The red Delisea pulchra demonstrates weak selectivity in symbiont transmission, while the brown Hormosira banksii exhibits a strongly selective symbiont transfer. Mucilage on the gametes appears to facilitate vertical transmission and transferred bacteria have predicted properties that could support early development of the seaweeds. Previous meta-analysis has indicated that vertical transmission is rare in aquatic compared to terrestrial environments, however, our results contribute to the growing evidence that this might not be the case and that instead vertical transmission with various degrees of symbiont selection occurs in the ecologically important group of seaweeds.

Differential priority effects impact taxonomy and functionality of host-associated microbiomes.

Most multicellular eukaryotes host complex communities of microorganisms, but the factors that govern their assembly are poorly understood. The settlement of specific microorganisms may have a lasting impact on community composition, a phenomenon known as the priority effect. Priority effects of individual bacterial strains on a host's microbiome are, however, rarely studied and their impact on microbiome functionality remains unknown. We experimentally tested the effect of two bacterial strains (Pseudoalteromonas tunicata D2 and Pseudovibrio sp. D323) on the assembly and succession of the microbial communities associated with the green macroalga Ulva australis. Using 16S rRNA gene sequencing and qPCR, we found that both strains exert a priority effect, with strain D2 causing initially strong but temporary taxonomic changes and strain D323 causing weaker but consistent changes. Consistent changes were predominately facilitatory and included taxa that may benefit the algal host. Metagenome analyses revealed that the strains elicited both shared (e.g., depletion of type III secretion system genes) and unique (e.g., enrichment of antibiotic resistance genes) effects on the predicted microbiome functionality. These findings indicate strong idiosyncratic effects of colonizing bacteria on the structure and function of host-associated microbial communities. Understanding the idiosyncrasies in priority effects is key for the development of novel probiotics to improve host condition.

Novel Nematode-Killing Protein-1 (Nkp-1) from a Marine Epiphytic Bacterium Pseudoalteromonas tunicata.

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Combating Parasitic Nematode Infections, Newly Discovered Antinematode Compounds from Marine Epiphytic Bacteria.

Parasitic nematode infections cause debilitating diseases and impede economic productivity. Antinematode chemotherapies are fundamental to modern medicine and are also important for industries including agriculture, aquaculture and animal health. However, the lack of suitable treatments for some diseases and the rise of nematode resistance to many available therapies necessitates the discovery and development of new drugs. Here, marine epiphytic bacteria represent a promising repository of newly discovered antinematode compounds. Epiphytic bacteria are ubiquitous on marine surfaces where they are under constant pressure of grazing by bacterivorous predators (e.g., protozoans and nematodes). Studies have shown that these bacteria have developed defense strategies to prevent grazers by producing toxic bioactive compounds. Although several active metabolites against nematodes have been identified from marine bacteria, drug discovery from marine microorganisms remains underexplored. In this review, we aim to provide further insight into the need and potential for marine epiphytic bacteria to become a new source of antinematode drugs. We discuss current and emerging strategies, including culture-independent high throughput screening and the utilization of Caenorhabditis elegans as a model target organism, which will be required to advance antinematode drug discovery and development from marine microbial sources.

Diversity and Distribution of Bacteria Producing Known Secondary Metabolites.

There is an increasing interest in the utilisation of marine bioactive compounds as novel biopharmaceuticals and agrichemicals; however, little is known about the environmental distribution for many of these molecules. Here, we aimed to elucidate the environmental distribution and to detect the biosynthetic gene clusters in environmental samples of four bioactive compounds, namely violacein, tropodithietic acid (TDA), tambjamine and the antibacterial protein AlpP. Our database analyses revealed high bacterial diversity for AlpP and violacein producers, while TDA-producing bacteria were mostly associated with marine surfaces and all belonged to the roseobacter group. In contrast, the tambjamine cluster was only found in the genomes of two Pseudoalteromonas species and in one terrestrial species belonging to the Cupriavidus genus. Using a PCR-based screen of different marine samples, we detected TDA and violacein genes associated with the microbiome of Ulva and Protohyale niger and tambjamine genes associated with Nodilittorina unifasciata; however, alpP was not detected. These results highlight the variable distribution of the genes encoding these four bioactive compounds, including their detection from the surface of multiple marine eukaryotic hosts. Determining the natural distribution of these gene clusters will help to understand the ecological importance of these metabolites and the bacteria that produce them.

Correction: Antinematode Activity of Violacein and the Role of the Insulin/IGF-1 Pathway in Controlling Violacein Sensitivity in Caenorhabditis elegans.

[This corrects the article DOI: 10.1371/journal.pone.0109201.].

Caenorhabditis elegans employs innate and learned aversion in response to bacterial toxic metabolites tambjamine and violacein.

Bacteriovorus eukaryotes such as nematodes are one of the major natural predators of bacteria. In their defense bacteria have evolved a number of strategies to avoid predation, including the production of deterrent or toxic metabolites, however little is known regarding the response of predators towards such bacterial defenses. Here we use the nematode C. elegans as a model to study a predators' behavioral response towards two toxic bacterial metabolites, tambjamine YP1 and violacein. We found that C. elegans displays an innate avoidance behavior towards tambjamine YP1, however requires previous exposure to violacein before learning to avoid this metabolite. The learned avoidance of violacein is specific, reversible, is mediated via the nematode olfactory apparatus (aversive olfactory learning) and is reduced in the absence of the neurotransmitter serotonin. These multiple strategies to evade bacterial toxic metabolites represent a valuable behavioral adaptation allowing bacteriovorus predators to distinguish between good and bad food sources, thus contributing to the understanding of microbial predator-prey interactions.

Antinematode activity of Violacein and the role of the insulin/IGF-1 pathway in controlling violacein sensitivity in Caenorhabditis elegans.

The purple pigment violacein is well known for its numerous biological activities including antibacterial, antiviral, antiprotozoan, and antitumor effects. In the current study we identify violacein as the antinematode agent produced by the marine bacterium Microbulbifer sp. D250, thereby extending the target range of this small molecule. Heterologous expression of the violacein biosynthetic pathway in E. coli and experiments using pure violacein demonstrated that this secondary metabolite facilitates bacterial accumulation in the nematode intestine, which is accompanied by tissue damage and apoptosis. Nematodes such as Caenorhabditis elegans utilise a well-defined innate immune system to defend against pathogens. Using C. elegans as a model we demonstrate the DAF-2/DAF-16 insulin/IGF-1 signalling (IIS) component of the innate immune pathway modulates sensitivity to violacein-mediated killing. Further analysis shows that resistance to violacein can occur due to a loss of DAF-2 function and/or an increased function of DAF-16 controlled genes involved in antimicrobial production (spp-1) and detoxification (sod-3). These data suggest that violacein is a novel candidate antinematode agent and that the IIS pathway is also involved in the defence against metabolites from non-pathogenic bacteria.