A new species and a new subspecies of the stargazer genusGnathagnus from Northwestern Australia

Heretofore the genusGnathagnus (Uranoscopidae) has been composed of three species:G. elongatus (Temminck et Schlegel) from northern Australia to Japan,G. innotabilis (Waite) from Australasia, andG. egregius (Jordan et Thompson) from the western Atlantic Ocean. In this paper, a new species and a new subspecies from northwestern Australia are described and a key to the known species and subspecies of the genus is presented.     

Hemoglobin from a deep-sea hydrothermal-vent copepod

Deep-sea hydrothermal-vent fauna live in a highly variable environment where oxygen levels can be very low, and carbon dioxide and sulfide can reach high concentrations (1). These conditions are harsh for most aerobic metazoans, yet copepods can be abundant at hydrothermal vents. Here we report the structure and functional properties of hemoglobin extracted from the copepod Benthoxynus spiculifer, which was found in large numbers in a paralvinellid/gastropod community collection made during a cruise to the Juan de Fuca Ridge in 1998. Although hemoglobin has been reported in some littoral copepods (2), this is the first study of the structure and functional properties of copepod hemoglobin. Hemoglobin represents about 60% of the total soluble proteins extracted from B. spiculifer, and although it imparts a red color to the copepod, it does not provide a significant storage pool of oxygen. It is a 208-kDa protein, composed of 14 globin chains--7 of 14.3 kDa and 7 of 15.2 kDa. The hemoglobin has a very high and temperature-sensitive oxygen affinity, with no cooperativity or Bohr effect. These properties are adaptive for an animal living in a low-oxygen environment in which the primary function of the hemoglobin is most likely oxygen acquisition to support aerobic respiration.     

Proteomic analysis of stargazer mutant mouse neuronal proteins involved in absence seizure

The stargazer ( stg ) mutant mouse, having mutation in stargazin, the calcium channel γ2 subunit, exhibited several neurological disorders including spontaneous absence seizure, cerebellar ataxia, and head tossing. To understand the molecular pathogenic mechanism of the absence seizure resulted from the loss of stargazin function, the thalamic proteomes between control mouse and stg mouse were compared. We identified 12 proteins expressed differentially (> 1.6-fold) by fluorescence two-dimensional difference gel electrophoresis and tandem mass spectrometry. Six of them are involved in basic metabolism including energy metabolism, three in stress response, two in axonal growth regulation, and one in the endoplasmic reticulum processing. All except mortalin showed decreased level of expression in stg mouse. Two stress-related proteins, mouse stress induced phosphoprotein 1 and peroxiredoxin 6 exhibited reduced levels of expression in stg mouse, while the level of another stress protein, mortalin was increased. Analysis of oxidative protein carbonylation in thalamic proteome of stg mouse showed higher level of carbonylated proteins in stg mouse than in control mouse. Interestingly, down-regulation of stress protein mouse stress induced phosphoprotein 1, metabolic enzyme isovaleryl-CoA dehydrogenase, and the two in neuronal axon growth, collapsin response mediator protein 2 and fascin homolog 1 coincides with the results of our previous study on γ-butyrolactone-induced transient absence seizure. Our results suggest that the pathogenesis mechanism underlying absence seizure may involve the molecular events contributed by these proteins.     

The commonness of rarity in a deep-sea taxon

The generality and drivers of rarity, defined along the axes of geographic range, population size and habitat specificity, have received considerable scientific attention for well over a century. Yet, studies that examine rarity holistically among these three attributes are limited, especially among invertebrate and marine taxa. The perceived paradox of deep-sea species, with often low population size but large geographic ranges, remains poorly resolved and understood. Here I assess seven forms of rarity and their drivers in deep-sea bivalves across the Atlantic Ocean. Rarity appears to be a common trait among deep-sea bivalves, with nearly 85% of the species exhibiting some form of rarity. Bivalves also showed a strong bimodal pattern of very common and very rare species. Geographic range, population size and habitat specificity were all heavily right skewed. Taxonomic superfamilies, body size, energy availability, temperature, depth and latitude, all significantly predicted geographic range, population size and habitat specificity. In a few cases, these patterns were counter to theoretical expectations. The drivers of rarity appear to be predictable from knowledge of the intrinsic biological and extrinsic environmental context of the species. These findings have major implications for deep-sea conversation, especially as anthropogenic threats are increasing.     

Membrane lipids of a psychrophilic and barophilic deep-sea bacterium

In a psychrophilic and barophilic marine bacterial isolate of the genusAlteromonas, the ratio of total unsaturated versus saturated fatty acids in the membrane lipids increased when the organism was grown at increasing hydrostatic pressures and decreasing temperatures. This regulatory capacity, as well as the presence of relatively large amounts of 20:5 polyunsaturated fatty acid, appear to be functional in maintaining membrane fluidity within a range of pressures distinctly below and above the specific optimum and at typical deep sea temperatures.     

深海微生物多样性

海洋面积约占地球总面积的70％,平均深度3,800 m,海底平均压力38 MPa,海水以下更是包含有物理化学性质迥异的多种地质结构,例如海洋沉积物、洋壳、热液口以及冷泉等.这些性质迥异的地质结构环境造就了丰富的生物多样性,构成了地球上最大的微生物生态系统.深海海水中最主要的微生物类群是α-,γ-变形菌(Alpha-&Gammaproteobacteria),以及海洋古菌群I(Marine Group I).深海沉积物中微生物含量与有机物含量和距离大陆板块的距离相关,以异养微生物为主.深海冷泉区富集了厌氧甲烷氧化古菌ANME和硫酸盐还原菌(Deltaproteobacteria);深海热液区由于具有化学物质的多样性和快速的动态变化而导致形成微生物的高度多样性.洋壳主要由基性、超基性岩构成,含有丰富的矿物,其中不乏参与铁、锰、硫等关键代谢反应的化能自养微生物.同时,由于环境中99％以上的微生物没有已培养的亲缘种,因此对深海微生物的多样性、生理功能特性以及生物地球化学作用的理解和研究仍然存在巨大的挑战.本文将尝试从不同的深海环境分区来综述深海海水、沉积物、洋壳,以及冷泉区和热液口等特殊生态环境中微生物的分布和多样性.     

Metabolic activities of the intestinal microflora of a deep-sea invertebrate

The intestinal microflora of deep-sea amphipods, in enrichment culture employing starch, urea, and N-acetyl-d-glucosamine and when examined under simulated in situ conditions, exhibited growth rates and substrate conversion approximately equal to, or greater than, atmospheric controls during short-term incubation. These observations are significant since these microorganisms may play an important role in biodegradation in the deep sea.     

Unexpectedly high mutation rate of a deep-sea hyperthermophilic anaerobic archaeon

Deep-sea hydrothermal vents resemble the early Earth, and thus the dominant Thermococcaceae inhabitants, which occupy an evolutionarily basal position of the archaeal tree and take an obligate anaerobic hyperthermophilic free-living lifestyle, are likely excellent models to study the evolution of early life. Here, we determined that unbiased mutation rate of a representative species, Thermococcus eurythermalis, exceeded that of all known free-living prokaryotes by 1-2 orders of magnitude, and thus rejected the long-standing hypothesis that low mutation rates were selectively favored in hyperthermophiles. We further sequenced multiple and diverse isolates of this species and calculated that T. eurythermalis has a lower effective population size than other free-living prokaryotes by 1-2 orders of magnitude. These data collectively indicate that the high mutation rate of this species is not selectively favored but instead driven by random genetic drift. The availability of these unusual data also helps explore mechanisms underlying microbial genome size evolution. We showed that genome size is negatively correlated with mutation rate and positively correlated with effective population size across 30 bacterial and archaeal lineages, suggesting that increased mutation rate and random genetic drift are likely two important mechanisms driving microbial genome reduction. Future determinations of the unbiased mutation rate of more representative lineages with highly reduced genomes such as Prochlorococcus and Pelagibacterales that dominate marine microbial communities are essential to test these hypotheses.Subject terms: Archaea, Population genetics

One theory for the origin of life is that the last universal common ancestor was an anaerobic hyperthermophilic organism inhabiting the deep-sea hydrothermal vents, as these environments display a few characteristics paralleling the early Earth [1]. While hydrothermal vents vary with chemical parameters, they all share a high temperature zone near the black chimney with anaerobic fluid from it. In the past decades, great efforts were made to understand the metabolic strategies deep-sea hyperthermophiles use to conserve energy and cope with physicochemical stresses, and to appreciate the molecular mechanisms leading to the stabilization of nucleic acids and proteins at exceedingly high temperatures [2, 3]. However, little is known whether they have a low or high intrinsic (i.e., not selected by environmental pressure) rate to change their genetic background information and whether this intrinsic potential itself is a result of selection shaped by these unique habitats.A previous population genomic analysis showed that protein sequences are under greater functional constraints in thermophiles than in mesophiles, suggesting that mutations are functionally more deleterious in thermophiles than in mesophiles [4]. This explanation is also supported by experimental assays showing nearly neutral mutations in temperate conditions become strongly deleterious at high temperature [5]. Furthermore, fluctuation tests on a hyperthermophilic archeaon Sulfolobus acidocaldarius [6] and a hyperthermophilic bacterium Thermus thermophilus [7] consistently showed that hyperthermophiles have much lower mutation rate compared to mesophiles. This appears to support the hypothesis that selection favors high replication fidelity at high temperature [5].Nevertheless, mutation rates measured using fluctuation experiments based on reporter loci are known to be biased, since the mutation rate of the organism is extrapolated from a few specific nonsynonymous mutations enabling survival in an appropriate selective medium, which renders the results susceptible to uncertainties associated with the representativeness of these loci and to inaccuracies of the assumptions made in extrapolation methods [8–10]. These limitations are avoided by the mutation accumulation (MA) experiment followed by whole-genome sequencing (WGS) of derived lines. In the MA part, multiple independent MA lines initiated from a single progenitor cell each regularly pass through a single-cell bottleneck, usually by transferring on solid medium. As the effective population size (N_e) becomes one, selection is unable to eliminate all but the lethal mutations, rendering the MA/WGS an approximately unbiased method to measure the spontaneous mutation rate [11].Members of the free-living anaerobic hyperthermophilic archaeal family Thermococcaceae are among the dominant microbial lineages in the black-smoker chimney at Guaymas Basin [12] and other deep-sea hydrothermal vents [13, 14]. This family only contains three genera: Thermococcus, Pyrococcus and Palaeococcus. In this study, the MA/WGS procedure was applied to determine the unbiased spontaneous mutation rate of a representative member Thermococcus eurythermalis A501, a conditional pizeophilic archaeon which can grow equally well from 0.1 MPa to 30 MPa at 85 °C [15, 16]. The MA lines were propagated at this optimal temperature on plates with gelrite which tolerates high temperature, and the experiment was performed under normal air pressure and in strictly anaerobic condition (Fig. 1A–D). To the best of our knowledge, this is the first report of unbiased mutation rate of a hyperthermophile and an obligate anaerobe.Open in a separate windowFig. 1Experimental determination of the unbiased mutation rate of the Thermococcus eurythermalis A501 is challenging because this archaeon has unusual physiology (i.e., obligate anaerobic and obligate hyperthermophilic).A The preparation of anaerobic high temperature tolerant gelrite plate. After sterilization and polysulfide addition via syringe, the plates are made in an anaerobic chamber. B The incubation of the strain T. eurythermalis A501 at 85 °C in liquid medium. C The initiation of mutation accumulation (MA) by spreading cells from a single founding colony to 100 lines. Plates are placed in an anaerobic jar for incubation in strictly anaerobic condition at 85 °C. D The MA process followed by whole-genome sequencing and data analysis. Single colony of each line is transferred to a new plate for N times (here N = 20). E Base-substitution mutations and insertion/deletion mutations across the whole genome of T. eurythermalis. The dashed vertical line separates the chromosome and plasmid. The height of each bar represents the number of base-substitution mutations across all MA lines within 10 kbp window. Green and red triangles denote insertion and deletion, respectively. The locus tags of the 14 genes with statistical enrichment of mutations are shown.Our MA experiment allowed accumulation of mutations over 314 cell divisions (after correcting the death rate (Table S1) [17]) in 100 independent lines initiated from a single founder colony and passed through a single cell bottleneck every day. By sequencing genomes of 96 survived lines at the end of the MA experiment, we identified 544 base-substitution mutations over these lines (Table S2), which translates to an average mutation rate (µ) of 85.01 × 10⁻¹⁰ per cell division per nucleotide site (see Supplementary information). The ratio of accumulated nonsynonymous to synonymous mutations (371 vs 107) did not differ from the ratio of nonsynonymous to synonymous sites (1,485,280 vs 403,070) in the A501 genome (χ² test; p > 0.05). Likewise, there was no difference of the accumulated mutations between intergenic (65) and protein-coding sites (478) (χ² test; p > 0.05). These are evidence for minimal selective elimination of deleterious mutations during the MA process. In general, the mutations were randomly distributed along the chromosome and the plasmid, though 86 base-substitution mutations fell into 14 genes which showed significant enrichment of mutations (bootstrap test; p < 0.05 for each gene) and 52 out of the 86 base-substitution mutations were found in five genes (TEU_RS04685 and TEU_RS08625-08640 gene cluster) (Fig. 1E, Table S3). A majority of mutations in these five genes may have inactivated these genes (38 out of 71 in the former gene and 33 out of 43 in the latter gene cluster) either by nonsense mutation or insertion-deletion (INDEL) mutation. The phenomenon of mutation clustering is not unique to this organism; it was reported in another MA study with the yeast Schizosaccharomyces pombe, and these genomic regions may represent either mutational hotspots or that mutations confer selective advantages under experimental conditions [18]. The TEU_RS04685 encodes the beta subunit of the sodium ion-translocating decarboxylase which is an auxiliary pathway for ATP synthesis by generating sodium motive force via decarboxylation [19], and the TEU_RS08625-08640 encodes a putative nucleoside ABC transporter. These genes appear to be important for energy conservation in the highly fluctuating deep-sea hydrothermal fluids. Under the culture conditions in which peptides and amino acids were stably and sufficiently supplied (see the TRM medium recipe in Supplementary information), however, these genes may be dispensable because peptides and amino acids are the preferred carbon and energy sources for T. eurythermalis [15]. On the other hand, some of these genes (e.g., TEU_RS08625) were shown to be upregulated under alkaline stress [16], and thus may be similarly induced under the culture condition in which pH is elevated compared to the vents. Besides, the laboratory condition differed from the vents in a number of other physicochemical features including hydrostatic pressure (0.1 MPa during the MA process versus 20 MPa in situ), temperature and salinity, which likely imposed additional selective pressures on the mutation accumulation processes. Taken together, deleting these genes were likely translated to a net fitness gain and were thus driven by selection. Removing these mutations led to a spontaneous mutation rate of 71.57 × 10⁻¹⁰ per cell division per site for T. eurythermalis A501. After removing the mutations in these 14 genes, both the accumulated mutations at nonsynonymous sites (288) relative to those (104) at synonymous sites (χ² test; p = 0.014) and the accumulated mutations at intergenic regions (65) relative to protein-coding regions (392) (χ² test; p = 0.013) showed marginally significant differences.To date, over 20 phylogenetically diverse free-living bacterial species and two archaeal species isolated from various environments have been assayed with MA/WGS, and their mutation rates vary from 0.79 × 10⁻¹⁰ to 97.80 × 10⁻¹⁰ per cell division per site [20]. The only prokaryote that displays a mutation rate (97.80 × 10⁻¹⁰ per cell division per site) comparable to A501 is Mesoplasma florum L1 [21], a host-dependent wall-less bacterium with highly reduced genome (~700 genes). Our PCR validation of randomly chosen 20 base-substitution mutations from two MA lines displaying highest mutation rates and of all nine INDEL mutations involving >10 bp changes across all lines (Table S2) indicates that the calculated high mutation rate did not result from false bioinformatics predictions.The extremely high mutation rate of T. eurythermalis is unexpected. One potential explanation in line with the “mutator theory” [22–24] is that high mutation rate may allow the organisms to gain beneficial mutations more rapidly and thus is selectively favored in deep-sea hydrothermal vents where physicochemical parameters are highly fluctuating. Alternatively, high mutation rate is the result of random genetic drift according to the “drift-barrier model” [21]. In this model, increased mutation rates are associated with increased load of deleterious mutations, so natural selection favors lower mutation rates. On the other hand, increased improvements of replication fidelity come at an increased cost of investments in DNA repair activities. Therefore, natural selection pushes the replication fidelity to a level that is set by genetic drift, and further improvements are expected to reduce the fitness advantages [11, 21]. These two explanations for the high mutation rate of T. eurythermalis are mutually exclusive, and resolving them requires the calculation of the power of genetic drift, which is inversely proportional to N_e.A common way to calculate N_e for a prokaryotic population is derived from the equation π_S = 2 × N_e × µ, where π_S represents the nucleotide diversity at synonymous (silent) sites among randomly sampled members of a panmictic population [25]. We therefore sequenced genomes of another eight T. eurythermalis isolates available in our culture collections. Like T. eurythermalis A501, these additional isolates were collected from the same cruise but varying at the water depth from 1987 m to 2009 m at Guaymas Basin. They differ by only up to 0.135% in the 16S rRNA gene sequence and share a minimum whole-genome average nucleotide identity (ANI) of 95.39% (Table S4), and thus fall within an operationally defined prokaryotic species typically delineated at 95% ANI [26]. Population structure analysis with PopCOGenT [27] showed that these isolates formed a panmictic population and that two of them were repetitive as a result of clonal descent (see Supplementary information). Using the median value of π_S = 0.083 across 1628 single-copy orthologous genes shared by the seven non-repetitive genomes, we calculated the N_e of T. eurythermalis to be 5.83 × 10⁶.Next, we collected the unbiased mutation rate of other prokaryotic species determined with the MA/WGS strategy from the literature [11, 28–30]. While the N_e data were also provided from those studies, the isolates used to calculate the N_e were identified based on their membership of either an operationally defined species (e.g., ANI at 95% cutoff) or a phenotypically characterized species (e.g., many pathogens), which often create a bias in calculating N_e [25]. We therefore again employed PopCOGenT to delineate panmictic populations from those datasets and re-calculated N_e accordingly. There was a significant negative linear relationship between µ and N_e on a logarithmic scale (dashed gray line in Fig. 2A [r² = 0.83, slope = −0.85, s.e.m. = 0.09, p < 0.001]) according to a generalized linear model (GLM) regression. This relationship cannot be explained by shared ancestry, as confirmed by phylogenetic generalized least square (PGLS) regression analysis (solid blue line in Fig. 2A [r² = 0.81, slope = −0.81, s.e.m. = 0.09, p < 0.001]). The nice fit of T. eurythermalis to the regression line validated the drift-barrier hypothesis. This is evidence that the high mutation rate of T. eurythermalis is driven by genetic drift rather than by natural selection.Open in a separate windowFig. 2The scaling relationship involving the base-substitution mutation rate per cell division per site (µ), the estimated effective population size (N_e), and genome size across 28 bacterial and two archaeal species.All three traits’ values were logarithmically transformed. The mutation rates of these species are all determined with the mutation accumulation experiment followed by whole-genome sequencing of the mutant lines. The mutation rate of species numbered 1–29 (blue) is collected from literature and that of the species 30 (red) is determined in the present study. Among the numbered species shown in the figure, the species #6 Haloferax volcanii is facultative anaerobic halophilic archaeon, and the species #30 is an obligate anaerobic hyperthermophilic archaeon. A The scaling relationship between µ and N_e. B The scaling relationship between µ and genome size. C The scaling relationship between genome size and N_e. Numbered data points 21–29 are not shown in A and C because of the lack of population dataset for estimation of N_e. The dashed gray lines and blue lines represent the generalized linear model (GLM) regression and the phylogenetic generalized least square (PGLS) regression, respectively. The Bonferroni adjusted outlier test for the GLM regression show that #7 Janthinobacterium lividum is an outlier in the scaling relationship between µ and N_e, and #9 Mesoplasma florum is an outlier in the scaling relationship between genome size and N_e. No outlier was identified in the PGLS regression results.As stated in the drift-barrier theory, high mutation rate is associated with a high load of deleterious mutations. In the absence of back mutations, recombination becomes an essential mechanism in eliminating deleterious mutations [31]. In support of this argument, the ClonalFrameML analysis [32] shows that members of the T. eurythermalis population recombine frequently, with a high ratio of the frequency of recombination to mutation (ρ/θ = 0.59) and a high ratio of the effect of recombination to mutation (r/m = 5.76). In fact, efficient DNA incorporation to Thermococcaceae genomes from external sources has been well documented experimentally [33, 34]. A second potentially important mechanism facilitating T. eurythermalis adaptation at high temperature is strong purifying selection at the protein sequence level, as protein sequences in thermophiles are generally subjected to stronger functional constraints compared to those in mesophiles [4, 35].Our result of the exceptionally high mutation rate of a free-living archaeon is a significant addition to the available collection of the MA/WGS data (Table S5), in which prokaryotic organisms with very high mutation rate have only been known for a host-dependent bacterium (Mesoplasma florum L1) with unusual biology (e.g., cell wall lacking). The availability of these two deeply branching (one archaeal versus the other bacterial) organisms adopting opposite lifestyles (one free-living versus the other host-restricted; one hyperthermophilic versus the other mesophilic; one obligate anaerobic versus the other facultative anaerobe), along with other phylogenetically and ecologically diverse prokaryotic organisms displaying low and intermediate mutation rates, provides an opportunity to help illustrate mechanisms potentially driving genome size evolution across prokaryotes. We found a negative linear relationship (dashed gray line in Fig. 2B [r² = 0.49, slope = −1.66, s.e.m. = 0.32, p < 0.001]) between genome size and base-substitution mutation rate, which is consistent with the hypothesis that increased mutation rate drives microbial genome reduction. We also showed a positive linear relationship (dashed gray line in Fig. 2C [r² = 0.47, slope = 0.24, s.e.m. = 0.06, p < 0.001]) between genome size and N_e, which suggests that random genetic drift drives genome reduction across prokaryotes. These correlations remain robust when the data were analyzed as phylogenetically independent contrasts (blue solid lines in Fig. 2B [r² = 0.47, slope = −1.75, s.e.m. = 0.34, p < 0.001] and in Fig. 2C [r² = 0.45, slope = 0.25, s.e.m. = 0.06, p < 0.001]). Our results are consistent with recent studies which employed mathematical modeling and/or comparative sequence analyses to show random genetic drift [36] and increased mutation rate [37] driving genome reduction across diverse bacterial lineages including both free-living and host-dependent bacteria. One benefit of the present study is that it directly measures µ and N_e, as compared to those recent advances which relied on proxies for these metrics (e.g., using the ratio of nonsynonymous substitution rate to synonymous substitution rate to represent N_e) to infer mechanisms of genome reduction.Despite this advantage, there are important caveats to our conclusions related to the mechanisms of genome reduction. The correlation analyses performed here are inspired by Lynch and colleagues’ work, who had great success explaining eukaryotic genome expansion with genetic drift [11, 38]. However, there are a few key differences of genomic features between prokaryotes and eukaryotes, which makes it more difficult to explain the correlation observed in prokaryotes. Importantly, genome sizes of eukaryotes can vary over several orders of magnitude, whereas those of free-living prokaryotes differ by only an order of magnitude [11], so there is much less variability to explain in prokaryotes. Moreover, eukaryotic genomes experience dramatic expansions of transposable elements which are often considered as genomic parasites, whereas prokaryotic genomes including those large ones are usually depleted with transposable elements and their genome size variations are largely driven by gene content [39]. Aside from these conceptual difficulties, the plots (Fig. 2B, C) are poorly populated with typical free-living species carrying small genomes such as the Prochlorococcus (mostly 1.6–8 Mb) and Pelagibacterales (1.3–1.5 Mb), which dominate the photosynthetic and heterotrophic microbial communities, respectively, in the ocean [40]. It has been generally postulated that bacterial species in these lineages have very large N_e [39–41], though there has been little direct evidence for it [42, 43]. If confirmed through the measurement of the unbiased mutation rate (µ) followed by the calculation of N_e based on µ, it might compromise the linear relationship between genome size and N_e observed here (Fig. 2C). It is also not necessarily appropriate to translate correlations to causal relationships. For example, the correlation between increased mutation rates and decreased genome sizes (Fig. 2B) does not necessarily mean that increased mutation rate drives genome reduction. This is because high mutation rates are observed in species with small N_e. Given that deletion bias is commonly found in prokaryotes [44, 45], genome reduction can be easily explained by increased fixation of deletional mutations in species with smaller N_e. High mutation rates in these species are simply the result of random genetic drift as explained by the drift-barrier theory, and they may have a limited role in driving genome reduction.Whereas our analysis based on the available data did not support natural selection as a universal mechanism driving genome reduction across prokaryotes (Fig. 2B, C), it does not mean that selection has no role in genome reduction of a particular taxon. In the case of thermophiles, proponents for selection acting to reduce genomes explained that genome size, due to its positive correlation with cell volume, may be an indirect target of selection which strongly favors smaller cell volume [35]. The underlying principle is that high temperature requires cells to increase the lipid content and change the lipid composition of the cell membranes, which consumes a large part of the cellular energy, and thus lower cell volume is selectively favored at high temperature [35]. Our calculations of a relatively small N_e in T. eurythermalis does not necessarily contradict with this selective argument, given that the fitness gained by decreasing cell volume and thus reducing genome size is large enough to overcome the power of random genetic drift. On the other hand, our data strongly indicate that neutral forces dictate the genome evolution of T. eurythermalis, and they are not negligible with regard to its genome reduction process. The significantly more deletion over insertion events (t test; 95 versus 37 events with p < 0.001 and 48 versus 20 events with p < 0.05 before and after removing the 14 genes enriched in mutations, respectively) and the significantly more nucleotides involved in deletions over insertions (t test; 433 versus 138 bp with p < 0.05 and 386 versus 121 bp with p < 0.001 before and after removing the 14 genes enriched in mutations, respectively) suggest that the deletion bias, combined with increased chance fixation of deletion mutants due to low N_e, is a potentially important neutral mechanism giving rise to the small genomes of T. eurythermalis (2.12 Mbp).The globally distributed deep-sea hydrothermal vents are microbe-driven ecosystems, with no known macroorganisms surviving at the vent fluids. Sample collections, microbial isolations, and laboratory propagations of mutation lines at hot and anoxic conditions are challenging. In the present study, we determined that T. eurythermalis, and perhaps Thermococcaceae in general, has a highly increased mutation rate and a highly decreased effective population size compared to all other known free-living prokaryotic lineages. While it remains to be tested whether this is a common feature among the vents’ microbes, the present study nevertheless opens a new avenue for investigating the hyperthemophile ecology and evolution in the deep sea.     

Benthic taxa as potential indicators of a deep-sea oil spill

The effect of the Deepwater Horizon oil spill on benthic macrofauna in the deep-sea Gulf of Mexico was measured in September–October 2010. Macrofauna community diversity and abundance were lowest closest to the wellhead and increased with distance from the wellhead up to 10 km. The macrofauna loss was primarily in surface sediments, which could be due to the deposition of oil and other toxic chemicals. Crustacean taxa appeared to be sensitive to the deep-sea blowout. Polychaete taxa varied in their sensitivity, but Dorvilleidae which is often associated with organic enrichment, was responsible for the largest amount of dissimilarity between stations close and far from the wellhead. Several other taxa were classified as sensitive or tolerant to the deep-sea blowout by comparing their distributions among impacted and non-impacted zones. The macrobenthic communities in the deep Gulf of Mexico exhibit a toxic response to the blowout on the Deepwater Horizon well, and this is correlated with barium and petroleum hydrocarbons.     

Characterization of a lysin from deep-sea thermophilic bacteriophage GVE2

Thermostable enzymes from thermophiles have attracted extensive studies. However, little is known about thermophilic lysin of bacteriophage obtained from deep-sea hydrothermal vent. In this study, a lysin from deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) was characterized for the first time. It was found that the GVE2 lysin was highly homologous with N-acetylmuramoyl-L-alanine amidases. After expression in Escherichia coli, the recombinant GVE2 lysin was purified. The recombinant lysin was active over a range of temperature from 40 °C to 80 °C, with an optimum at 60 °C. Its optimal pH was 6.0, and it was stable over a wide range of pH from 4.0 to 10.0. The lysin was highly active when some enzyme inhibitors or detergents (phenylmethylsulfonyl fluoride, Tween 20, Triton X-100, and chaps) were used. However, it was strongly inhibited by sodium dodecyl sulfate and ethylene diamine tetraacetic acid. Its enzymatic activity could be slightly stimulated in the presence of Na⁺ and Li⁺. But the metal ions Mg²⁺, Ba²⁺, Zn²⁺, Fe³⁺, Ca²⁺, and Mn²⁺ at concentrations of 1 or 10 mM showed inhibitions to the lysin activity. Our study demonstrated the first characterization of lysin from deep-sea thermophilic bacteriophage.     

Mercury adaptation among bacteria from a deep-sea hydrothermal vent

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9 degrees N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 microM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45 degrees C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65 degrees C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.     

Experimental ecology at deep-sea hydrothermal vents: a perspective

In situ and laboratory experiments conducted over the past quarter of a century have greatly increased our understanding of the ecology of deep-sea hydrothermal systems. Early experiments suggested that chemosynthetic primary production constituted the principal source of organic matter for biological communities associated with vents, although subsequent studies have revealed many complexities associated with interactions between microbes and higher organisms inhabiting these ecosystems. A diversity of host-microbial symbiont relationships has been identified and experimental studies have revealed the exquisite physiological adaptations within the giant tubeworm, Riftia pachyptila, for the uptake, fixation, and assimilation of carbon. In vitro experiments demonstrated the unusual sulfide binding properties of tubeworm hemoglobin that prevent inhibition of the cytochrome-c oxidase enzyme system during transport of sulfide to symbiont-bearing tissues. Studies of respiration and growth of several species of vent organisms conducted over the past two decades transformed earlier views that low metabolism and slow growth are characteristics of all organisms inhabiting all deep-sea environments. Results of recent experiments suggest that metabolic rates correlate with the degree of mobility of the organisms rather than with any specific attribute of the deep-sea environment itself, and growth rates of certain vent organisms (e.g., R. pachyptila) were found to be among the highest in any marine environments. While extreme thermal tolerance has been suggested as characteristic of certain vent fauna (e.g., alvinellid polychaetes and alvinocarid shrimp), the majority of vent metazoans live at temperatures below 20 °C and additional experiments are necessary to reconcile field experiments documenting thermal tolerance in situ, thermal tolerance in vivo, and thermal sensitivity of biochemical constituents of vent organisms. Transplantation and clearance experiments, as well as in situ characterization of vent fluid chemistry, have greatly increased our understanding of organism–environment interactions. Early analyses of metazoan egg size and larval morphology, coupled with in vivo larval culture experiments, available physical oceanographic data, and genetic studies of gene flow, have contributed greatly to our understanding of mechanisms of dispersal between widely separated vent sites. The documentation of invertebrate colonization and succession of new vents following a volcanic eruption, and a series of manipulative field experiments, provide considerable insights into the relative roles of abiotic conditions and biotic interactions in structuring vent communities. Recent and emerging technological developments, such as in situ chemical analyzers, observatory approaches, and laboratory-based pressure culture systems, should provide invaluable new experimental tools for tackling many remaining questions concerning the ecology of deep-sea hydrothermal systems.     

Direct observations of the association between a deep-sea fish and a giant scyphomedusa

This is a report of evidence of a close symbiotic relationship between the scyphomedusa, Stygiomedusa gigantea and the fish, Thalassobathia pelagica. Images from remotely operated vehicles (ROV) were obtained of the fish swimming on and around the large scyphomedusa. This is the first ever documented symbiosis between an Ophidiform fish and a medusa.     

Direct observations of the association between a deep-sea fish and a giant scyphomedusa

This is a report of evidence of a close symbiotic relationship between the scyphomedusa, Stygiomedusa gigantea and the fish, Thalassobathia pelagica. Images from remotely operated vehicles (ROV) were obtained of the fish swimming on and around the large scyphomedusa. This is the first ever documented symbiosis between an Ophidiform fish and a medusa.     

Bacterial group II introns in a deep-sea hydrothermal vent environment

Group II introns are catalytic RNAs and mobile retrotransposable elements known to be present in the genomes of some nonmarine bacteria and eukaryotic organelles. Here we report the discovery of group II introns in a bacterial mat sample collected from a deep-sea hydrothermal vent near 9 degrees N on the East Pacific Rise. One of the introns was shown to self-splice in vitro. This is the first example of marine bacterial introns from molecular population structure studies of microorganisms that live in the proximity of hydrothermal vents. These types of mobile genetic elements may prove useful in improving our understanding of bacterial genome evolution and may serve as valuable markers in comparative studies of bacterial communities.     

Antifouling diketopiperazines produced by a deep-sea bacterium, Streptomyces fungicidicus

Modern antifouling coatings use heavy metals and toxic organic molecules to prevent biofouling, the undesirable growth of marine organisms on man-made substrata. In an ongoing survey of deep-sea microorganisms aimed at finding low toxic antifouling metabolites, an actinomycete bacterium was isolated from the Pacific sediment at the depth of about 5000 m. The bacterium was closely related to Streptomyces fungicidicus (99% similarity) according to 16S ribosomal RNA sequence information. The spent culture medium of this bacterium inhibited barnacle larval attachment. Bioassay-guided fractionation was employed to isolate antifouling compounds. The ethyl acetate extract was fractionated by using an open silica gel column. Active fractions were further purified on a HPLC C18 column. Five diketopiperazines, cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Pro), cyclo-(L-Trp-L-Pro), and cyclo-(L-Leu-L-Val) were isolated for the first time from a deep sea bacterium, and the structures of the compounds were elucidated by nuclear magnetic resonance spectroscopy and mass spectrometry. The pure diketopiperazines were tested for antilarval activity using the barnacle Balanus amphitrite. Effective concentrations that inhibited 50% larval attachment (EC50) after 24 h ranged from 0.10- 0.27 mM. The data suggest that diketopiperazines and other compounds from deep-sea bacteria may be used as novel antifoulants.     

Impacts on the deep-sea ecosystem by a severe coastal storm

Major coastal storms, associated with strong winds, high waves and intensified currents, and occasionally with heavy rains and flash floods, are mostly known because of the serious damage they can cause along the shoreline and the threats they pose to navigation. However, there is a profound lack of knowledge on the deep-sea impacts of severe coastal storms. Concurrent measurements of key parameters along the coast and in the deep-sea are extremely rare. Here we present a unique data set showing how one of the most extreme coastal storms of the last decades lashing the Western Mediterranean Sea rapidly impacted the deep-sea ecosystem. The storm peaked the 26(th) of December 2008 leading to the remobilization of a shallow-water reservoir of marine organic carbon associated with fine particles and resulting in its redistribution across the deep basin. The storm also initiated the movement of large amounts of coarse shelf sediment, which abraded and buried benthic communities. Our findings demonstrate, first, that severe coastal storms are highly efficient in transporting organic carbon from shallow water to deep water, thus contributing to its sequestration and, second, that natural, intermittent atmospheric drivers sensitive to global climate change have the potential to tremendously impact the largest and least known ecosystem on Earth, the deep-sea ecosystem.