Stephen A. Jackson

Marine Metagenomics: New Tools for the Study and Exploitation of Marine Microbial Metabolism

The marine environment is extremely diverse, with huge variations in pressure and temperature. Nevertheless, life, especially microbial life, thrives throughout the marine biosphere and microbes have adapted to all the divergent environments present. Large scale DNA sequence based approaches have recently been used to investigate the marine environment and these studies have revealed that the oceans harbor unprecedented microbial diversity. Novel gene families with representatives only within such metagenomic datasets represent a large proportion of the ocean metagenome. The presence of so many new gene families from these uncultured and highly diverse microbial populations represents a challenge for the understanding of and exploitation of the biology and biochemistry of the ocean environment. The application of new metagenomic and single cell genomics tools offers new ways to explore the complete metabolic diversity of the marine biome.

Evidence of a Putative Deep Sea Specific Microbiome in Marine Sponges

The microbiota of four individual deep water sponges, Lissodendoryx diversichela, Poecillastra compressa, Inflatella pellicula, and Stelletta normani, together with surrounding seawater were analysed by pyrosequencing of a region of the 16S rRNA gene common to Bacteria and Archaea. Due to sampling constraints at depths below 700 m duplicate samples were not collected. The microbial communities of L. diversichela, P. compressa and I. pellicula were typical of low microbial abundance (LMA) sponges while S. normani had a community more typical of high microbial abundance (HMA) sponges. Analysis of the deep sea sponge microbiota revealed that the three LMA-like sponges shared a set of abundant OTUs that were distinct from those associated with sponges from shallow waters. Comparison of the pyrosequencing data with that from shallow water sponges revealed that the microbial communities of all sponges analysed have similar archaeal populations but that the bacterial populations of the deep sea sponges were distinct. Further analysis of the common and abundant OTUs from the three LMA-like sponges placed them within the groups of ammonia oxidising Archaea (Thaumarchaeota) and sulphur oxidising γ-Proteobacteria (Chromatiales). Reads from these two groups made up over 70% of all 16S rRNA genes detected from the three LMA-like sponge samples, providing evidence of a putative common microbial assemblage associated with deep sea LMA sponges.

Metagenomics for the discovery of novel biosurfactants of environmental interest from marine ecosystems

Research focused on the search for new biosurfactants aims to replace chemical surfactants, which while being cost-effective are ecologically undesirable. Metagenomics can lead to discovery of novel biosurfactants, tackling issues of low production yields. Recent successes include the heterologous production of biosurfactants. The dearth of biosurfactants discovered to date through metagenomics is puzzling given that good screening systems and heterologous host systems are available.

Archaea Appear to Dominate the Microbiome of Inflatella pellicula Deep Sea Sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.

Study of the performance of a thermophilic biological methanation system

This study investigated the operation of ex-situ biological methanation at two thermophilic temperatures (55°C and 65°C). Methane composition of 85-88% was obtained and volumetric productivities of 0.45 and 0.4LCH4/Lreactor were observed at 55°C and 65°C after 24h respectively. It is postulated that at 55°C the process operated as a mixed culture as the residual organic substrates in the starting inoculum were still available. These were consumed prior to the assessment at 65°C; thus the methanogens were now dependent on gaseous substrates CO2 and H2. The experiment was repeated at 65°C with fresh inoculum (a mixed culture); methane composition and volumetric productivity of 92% and 0.46LCH4/Lreactor were achieved in 24h. Methanothermobacter species represent likely and resilient candidates for thermophilic biogas upgrading.

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa.

Methanosarcina Play an Important Role in Anaerobic Co-Digestion of the Seaweed Ulva lactuca: Taxonomy and Predicted Metabolism of Functional Microbial Communities.

Macro-algae represent an ideal resource of third generation biofuels, but their use necessitates a refinement of commonly used anaerobic digestion processes. In a previous study, contrasting mixes of dairy slurry and the macro-alga Ulva lactuca were anaerobically digested in mesophilic continuously stirred tank reactors for 40 weeks. Higher proportions of U. lactuca in the feedstock led to inhibited digestion and rapid accumulation of volatile fatty acids, requiring a reduced organic loading rate. In this study, 16S pyrosequencing was employed to characterise the microbial communities of both the weakest (R1) and strongest (R6) performing reactors from the previous work as they developed over a 39 and 27-week period respectively. Comparing the reactor communities revealed clear differences in taxonomy, predicted metabolic orientation and mechanisms of inhibition, while constrained canonical analysis (CCA) showed ammonia and biogas yield to be the strongest factors differentiating the two reactor communities. Significant biomarker taxa and predicted metabolic activities were identified for viable and failing anaerobic digestion of U. lactuca. Acetoclastic methanogens were inhibited early in R1 operation, followed by a gradual decline of hydrogenotrophic methanogens. Near-total loss of methanogens led to an accumulation of acetic acid that reduced performance of R1, while a slow decline in biogas yield in R6 could be attributed to inhibition of acetogenic rather than methanogenic activity. The improved performance of R6 is likely to have been as a result of the large Methanosarcina population, which enabled rapid removal of acetic acid, providing favourable conditions for substrate degradation.

Rhizobium metallidurans sp. nov., a symbiotic heavy metal resistant bacterium isolated from the Anthyllis vulneraria Zn-hyperaccumulator.

A Gram-stain-negative, aerobic, rod-shaped, non-spore-forming bacterium (ChimEc512(T)) was isolated from 56 host seedlings of the hyperaccumulating Anthyllis vulneraria legume, which was on an old zinc mining site at Les Avinières, Saint-Laurent-Le-Minier, Gard, South of France. On the basis of 16S rRNA gene sequence similarities, strain ChimEc512(T) was shown to belong to the genus Rhizobium and to be most closely related to Rhizobium endophyticum CCGE 2052(T) (98.4%), Rhizobium tibeticum CCBAU 85039(T) (98.1%), Rhizobium grahamii CCGE 502(T) (98.0%) and Rhizobium mesoamericanum CCGE 501(T) (98.0%). The phylogenetic relationships of ChimEc512(T) were confirmed by sequencing and analyses of recA and atpD genes. DNA-DNA relatedness values of strain ChimEc512(T) with R. endophyticum CCGE 2052(T), R. tibeticum CCBAU 85039(T), R. mesoamericanum CCGE 52(T), Rhizobium grahamii CCGE 502(T), Rhizobium etli CCBAU 85039(T) and Rhizobium radiobacter KL09-16-8-2(T) were 27, 22, 16, 18, 19 and 11%, respectively. The DNA G+C content of strain ChimEc512(T) was 58.9 mol%. The major cellular fatty acid was C18 : 1ω7c, characteristic of the genus Rhizobium . The polar lipid profile included phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine and moderate amounts of aminolipids, phospholipid and sulfoquinovosyl diacylglycerol. Although ChimEc512(T) was able to nodulate A. vulneraria, the nodC and nifH genes were not detected by PCR. The rhizobial strain was tolerant to high concentrations of heavy metals: up to 35 mM Zn and up to 0.5 mM Cd and its growth kinetics was not impacted by Zn. The results of DNA-DNA hybridizations and physiological tests allowed genotypic and phenotypic differentiation of strain ChimEc512(T) from species of the genus Rhizobium with validly published names. Strain ChimEc512(T), therefore, represents a novel species, for which the name Rhizobium metallidurans sp. nov. is proposed, with the type strain ChimEc512(T) ( =DSM 26575 = CIP 110550(T)).

Maribacter spongiicola sp. nov. and Maribacter vaceletii sp. nov., isolated from marine sponges, and emended description of the genus Maribacter.

Two Gram-stain-negative, rod-shaped, orange, catalase- and oxidase-positive, non-motile bacteria, designated W13M1A(T) and W15M10(T), were isolated from the marine sponges Suberites carnosus and Leucosolenia sp., respectively, which were sampled from Lough Hyne, Co. Cork, Ireland. Analysis of the 16S rRNA gene sequences of these isolates revealed that they are members of the genus Maribacter, in the family Flavobacteriaceae of the phylum Bacteroidetes. The type strain most closely related to strain W13M1A(T) is Maribacter forsetii DSM 18668(T) with a gene sequence similarity of 96.5%. The closest related type strain to strain W15M10(T) is Maribacter orientalis DSM 16471(T) with a gene sequence similarity of 98.3%. Phylogenetic inference and phenotypic data combined indicate that the isolates represent two novel species of the genus Maribacter, for which the names Maribacter spongiicola sp. nov. with type strain W15M10(T) ( = NCIMB 14725(T) = DSM 25233(T)) and Maribacter vaceletii sp. nov. with type strain W13M1A(T) ( = NCIMB 14724(T) = DSM 25230(T)), are proposed.

Diversity of Natural Product Biosynthetic Genes in the Microbiome of the Deep Sea Sponges Inflatella pellicula, Poecillastra compressa, and Stelletta normani.

Three different deep sea sponge species, Inflatella pellicula, Poecillastra compressa, and Stelletta normani comprising seven individual samples, retrieved from depths of 760–2900 m below sea level, were investigated using 454 pyrosequencing for their secondary metabolomic potential targeting adenylation domain and ketosynthase domain sequences. The data obtained suggest a diverse microbial origin of nonribosomal peptide synthetases and polyketide synthase fragments that in part correlates with their respective microbial community structures that were previously described and reveals an untapped source of potential novelty. The sequences, especially the ketosynthase fragments, display extensive clade formations which are clearly distinct from sequences hosted in public databases, therefore highlighting the potential of the microbiome of these deep sea sponges to produce potentially novel small-molecule chemistry. Furthermore, sequence similarities to gene clusters known to be involved in the production of many classes of antibiotics and toxins including lipopeptides, glycopeptides, macrolides, and hepatotoxins were also identified.