Conservation of Genomic Information in Multiple Displacement Amplified Low-Quantity Metagenomic Material from Marine Invertebrates.

Marine invertebrate microbiomes have been a rich source of bioactive compounds and interesting genomic features. In cases where the achievable amounts of metagenomic DNA are too low for direct sequencing, multiple displacement amplification (MDA) can be used for whole genome amplification. However, MDA has known limitations which can affect the quality of the resulting genomes and metagenomes. In this study, we evaluated the conservation of biosynthetic gene clusters (BGCs) and enzymes in MDA products from low numbers of prokaryotic cells (estimated 2-850). Marine invertebrate microbiomes collected from Arctic and sub-Arctic areas served as source material. The cells were separated from the host tissue, lysed, and directly subjected to MDA. The MDA products were sequenced by Illumina sequencing. Corresponding numbers of bacteria from a set of three reference strains were treated the same way. The study demonstrated that useful information on taxonomic, BGC, and enzyme diversities was obtainable from such marginal quantities of metagenomic material. Although high levels of assembly fragmentation resulted in most BGCs being incomplete, we conclude that this genome mining approach has the potential to reveal interesting BGCs and genes from hard-to-reach biological sources.

Seafloor deposition of water-based drill cuttings generates distinctive and lengthy sediment bacterial community changes.

The spatial extent and persistence of bacterial change caused by deposition of water-based drill cuttings on the seafloor were explored by a community-wide approach. Ten centimeter sediment cores were sampled along transects extending from ≤15 m to 250 m from three nearby drilling sites in the southern Barents Sea. Eight months, 8 years and 15 years, respectively, had passed since the completion of the drillings. At locations heavily affected by drill cuttings, the two most recent sites showed distinct, corresponding deviances from native Barents Sea bacterial community profiles. Otherwise marginal groups, including Mollicutes and Clostridia, showed significant increases in relative abundance. Beyond 100 m from the boreholes the microbiotas appeared undisturbed, as they did at any distance from the 15-years old borehole. The extent of the biological distortion, as indicated by the present microbial study, agreed with previously published macrofaunal surveys at the same drilling sites.

Vibrio echinoideorum sp. nov., isolated from an epidermal lesion on the test of a green sea urchin (Strongylocentrotus droebachiensis).

A Gram-stain-negative, facultatively anaerobic Vibrio strain, designated NFH.MB010T, was isolated from an epidermal lesion on the test (hard shell skeleton) of a green sea urchin (Strongylocentrotus droebachiensis) collected from northern Norway. Cells of strain NFH.MB010T were rod shaped and motile by means of a single, long polar flagellum. Growth was observed at 1-5% NaCl (w/v) and at 4 °C, but not above 28 °C. Phylogenetic analyses based on eight-gene multilocus sequence analysis (16S rRNA, atpA, gyrB, mreB, pyrH, recA, rpoA and rpoD) suggested novelty at the species level. In silico DNA-DNA hybridization and orthologous average nucleotide identity estimates showed percentage genomic resemblances to its closest relative, Vibrio splendidus, that were well below the established same species threshold values. Phenotypically, utilization of glycogen and gentiobiose, inability of acetoin production, and undetectable valine arylamidase and trypsin activity discriminated strain NFH.MB010T from the closely related reference strains. Protein spectra generated by maldi-tof mass spectrometry further consolidated the species level uniqueness of strain NFH.MB010T. Based on the described polyphasic approach, strain NFH.MB010T therefore appears as a novel species within the Splendidus clade of the genus Vibrio, and the name Vibrio echinoideorum sp. nov. is proposed, with NFH.MB010T (=DSM 107264T=LMG 30656T) as the type strain.

Perturbation of seafloor bacterial community structure by drilling waste discharge.

Offshore drilling operations result in the generation of drill cuttings and localized smothering of the benthic habitats. This study explores bacterial community changes in the in the upper layers of the seafloor resulting from an exploratory drilling operation at 1400m water depth on the Barents Sea continental slope. Significant restructurings of the sediment microbiota were restricted to the sampling sites notably affected by the drilling waste discharge, i.e. at 30m and 50m distances from the drilling location, and to the upper 2cm of the seafloor. Three bacterial groups, the orders Clostridiales and Desulfuromonadales and the class Mollicutes, were almost exclusively confined to the upper two centimeters at 30m distance, thereby corroborating an observed increase in anaerobicity inflicted by the drilling waste deposition. The potential of these phylogenetic groups as microbial bioindicators of the spatial extent and persistence of drilling waste discharge should be further explored.

Microbial trimethylamine-N-oxide as a disease marker: something fishy?

Production of trimethylamine-N-oxide (TMAO) via the gut microbiota has recently been proposed as an important pathophysiological mechanism linking ingestion of 'unhealthy foods', such as beef (containing carnitine) and eggs (containing choline), and the development of atherosclerosis. Hence, TMAO has gained attention as a novel biomarker for cardiovascular disease. However, fish and seafood contain considerable amounts of TMAO and are generally accepted as cardioprotective: a puzzling paradox that seems to have been neglected. We suspect that the TMAO story may be a red herring.

Biological surface-active compounds from marine bacteria.

Surface-active compounds (SACs) are widely used in different industries as well as in many daily consumption products. However, with the increasing concern for their environmental acceptability, attention has turned towards biological SACs which are biodegradable, less toxic and more environmentally friendly. In this work, 176 marine hydrocarbon-degrading bacterial isolates from petroleum-contaminated sites along the Norwegian coastline were isolated and screened for their capacity to produce biological SACs. Among them, 18 isolates were capable of reducing the surface tension of the culture medium by at least 20 mN m(-1) and/or capable of maintaining more than 40% of the emulsion volume after 24 h when growing on glucose or kerosene as carbon and energy source. These isolates were members of the genera Pseudomonas, Pseudoalteromonas, Rhodococcus, Catenovulum, Cobetia, Glaciecola, Serratia, Marinomonas and Psychromonas. Two isolates, Rhodococcus sp. LF-13 and Rhodococcus sp. LF-22, reduced surface tension of culture medium by more than 40 mN m(-1) when growing on kerosene, n-hexadecane or rapeseed oil. The biosurfactants were produced by resting cells of the two Rhodococcus strains suggesting the biosynthesis of the biosurfactants was not necessarily associated with their growth on hydrocarbons.

Polar front associated variation in prokaryotic community structure in Arctic shelf seafloor.

Spatial variations in composition of marine microbial communities and its causes have largely been disclosed in studies comprising rather large environmental and spatial differences. In the present study, we explored if a moderate but temporally permanent climatic division within a contiguous arctic shelf seafloor was traceable in the diversity patterns of its bacterial and archaeal communities. Soft bottom sediment samples were collected at 10 geographical locations, spanning spatial distances of up to 640 km, transecting the oceanic polar front in the Barents Sea. The northern sampling sites were generally colder, less saline, shallower, and showed higher concentrations of freshly sedimented phytopigments compared to the southern study locations. Sampling sites depicted low variation in relative abundances of taxa at class level, with persistent numerical dominance by lineages of Gamma- and Deltaproteobacteria (57-66% of bacterial sequence reads). The Archaea, which constituted 0.7-1.8% of 16S rRNA gene copy numbers in the sediment, were overwhelmingly (85.8%) affiliated with the Thaumarchaeota. Beta-diversity analyses showed the environmental variations throughout the sampling range to have a stronger impact on the structuring of both the bacterial and archaeal communities than spatial effects. While bacterial communities were significantly influenced by the combined effect of several weakly selective environmental differences, including temperature, archaeal communities appeared to be more uniquely structured by the level of freshly sedimented phytopigments.

Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library.

A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K m and k cat of 39 µM and 25.8 s(-1), respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/ß-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80-114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.

The microbial diversity of Polar environments is a fertile ground for bioprospecting.

The term bioprospecting has been adopted for systematic searches in nature for new bioactive compounds, genes, proteins, microorganisms and other products with potential for commercial use. Much effort has been focused on microorganisms able to thrive under harsh conditions, including the Polar environments. Both the lipid and protein cellular building blocks of Polar microorganisms are shaped by their adaptation to the permanently low temperatures. In addition, strongly differing environments, such as permafrost, glaciers and sea ice, have contributed to additional functional diversity. Emerging massive-parallel sequencing technologies have revealed the existence of a huge, hitherto unseen diversity of low-abundance phylotypes--the rare biosphere--even in the Polar environments. This realization has further strengthened the need to employ cultivation-independent approaches, including metagenomics and single-cell genomic sequencing, to get comprehensive access to the genetic diversity of microbial communities for bioprospecting purposes. In this review, we present an updated snapshot of recent findings on the molecular basis for adaptation to the cold and the phylogenetic diversities of different Polar environments. Novel approaches in bioprospecting are presented and we conclude by showing recent bioprospecting outcomes in terms of new molecules patented or applied by some biotech companies.

Persistence and biodegradation of kerosene in high-arctic intertidal sediment.

A kerosene type hydrocarbon fraction (equivalent to 7 L m(-2)) was added to enclosures in the surface layer of high-arctic intertidal beach sediment. The experimental spill was repeated in two consecutive years in the period July-September. The rate and extent of hydrocarbon removal and the accompanying bacterial response were monitored for 79 days (2002) and 78 days (2003). The bulk of added kerosene, i.e. 94-98%, was lost from the upper 5 cm layer by putatively abiotic processes within 2 days and a residual fraction in the range 0.6-1.2mg per g dry sediment was stably retained. Concomitant addition of oleophilic fertilizer led to higher initial retention, as 24% of the kerosene remained after 2 days in the presence of a modified, cold-climate adapted version of the well-known Inipol EAP 22 bioremediation agent. In these enclosures, which showed an increase in hydrocarbon-degrader counts from 6.5 x 10(3) to 4.1 x 10(7) per g dry sediment within 8 days, a 17% contribution by biodegradation to subsequent hydrocarbon removal was estimated. Stimulation in hydrocarbon-degrader counts in fertilizer-alone control enclosures was indistinguishable from the stimulation observed with both kerosene and fertilizer present, suggesting that the dynamics in numbers of hydrocarbon-degrading bacteria was primarily impacted by the bioremediation agent.

Torularhodin and torulene are the major contributors to the carotenoid pool of marine Rhodosporidium babjevae (Golubev).

A carotenoid-producing yeast strain, isolated from the sub-arctic, marine copepod Calanus finmarchicus, was identified as Rhodosporidium babjevae (Golubev) according to morphological and biochemical characteristics and phylogenetic inference from the small-subunit ribosomal RNA gene sequence. The total carotenoids content varied with cultivation conditions in the range 66-117 microg per g dry weight. The carotenoid pool, here determined for the first time, was dominated by torularhodin and torulene, which collectively constituted 75-91% of total carotenoids under various regimes of growth. Beta-carotene varied in the range 5-23%. A high-peptone/low-yeast extract (weight ratio 38:1) marine growth medium favoured the production of torularhodin, the carotenoid at highest oxidation level, with an average of 63% of total carotenoids. In standard yeast medium (YM; ratio 1.7:1), torularhodin averaged 44%, with increased proportions of the carotenes, torulene and beta-carotene. The anticipated metabolic precursor gamma-carotene (beta,psi-carotene) constituted a minor fraction (