Taxonomic diversity of microbial communities in sub-seafloor hydrothermal sediments of the active Santorini-Kolumbo volcanic field.

Introduction: Active hydrothermal vents of volcanic origin provide a remarkable manifestation of life on Earth under extreme conditions, which may have consequences for our understanding of habitability on other terrestrial bodies as well. Methods: Here, we performed for the first time Illumina sequencing of bacterial and archaeal communities on sub-seafloor samples collected from the Santorini-Kolumbo volcanic field. A total of 19 (3-m long) gravity corers were collected and processed for microbial community analysis. Results: From a total of 6,46,671 produced V4 sequences for all samples, a total of 10,496 different Operational Taxonomic Units (OTUs) were identified that were assigned to 40 bacterial and 9 archaeal phyla and 14 candidate divisions. On average, the most abundant phyla in all samples were Chloroflexi (Chloroflexota) (24.62%), followed by Proteobacteria (Pseudomonadota) (11.29%), Firmicutes (Bacillota) (10.73%), Crenarchaeota (Thermoproteota) (8.55%), and Acidobacteria (Acidobacteriota) (8.07%). At the genus level, a total of 286 known genera and candidate genera were mostly dominated by members of Bacillus, Thermoflexus, Desulfatiglans, Pseudoalteromonas, and Pseudomonas. Discussion: In most of the stations, the Chao1 values at the deeper layers were comparable to the surface sediment samples denoting the high diversity in the subsurface of these ecosystems. Heatmap analysis based on the 100 most abundant OTUs, grouped the sampling stations according to their geographical location, placing together the two hottest stations (up to 99°C). This result indicates that this specific area within the active Kolumbo crater create a distinct niche, where microorganisms with adaptation strategies to withstand heat stresses can thrive, such as the endospore-forming Firmicutes.

Genomic adaptation of Pseudomonas strains to acidity and antibiotics in hydrothermal vents at Kolumbo submarine volcano, Greece.

Although the rise of antibiotic and multidrug resistant bacteria is one of the biggest current threats to human health, our understanding of the mechanisms involved in antibiotic resistance selection remains scarce. We performed whole genome sequencing of 21 Pseudomonas strains, previously isolated from an active submarine volcano of Greece, the Kolumbo volcano. Our goal was to identify the genetic basis of the enhanced co-tolerance to antibiotics and acidity of these Pseudomonas strains. Pangenome analysis identified 10,908 Gene Clusters (GCs). It revealed that the numbers of phage-related GCs and sigma factors, which both provide the mechanisms of adaptation to environmental stressors, were much higher in the high tolerant Pseudomonas strains compared to the rest ones. All identified GCs of these strains were associated with antimicrobial and multidrug resistance. The present study provides strong evidence that the CO2-rich seawater of the volcano associated with low pH might be a reservoir of microorganisms carrying multidrug efflux-mediated systems and pumps. We, therefore, suggest further studies of other extreme environments (or ecosystems) and their associated physicochemical parameters (or factors) in the rise of antibiotic resistance.

Microbial strains isolated from CO2-venting Kolumbo submarine volcano show enhanced co-tolerance to acidity and antibiotics.

As ocean acidification intensifies, there is growing global concern about the impacts that future pH levels are likely to have on marine life and ecosystems. By analogy, a steep decrease of seawater pH with depth is encountered inside the Kolumbo submarine volcano (northeast Santorini) as a result of natural CO2 venting, making this system ideal for ocean acidification research. Here, we investigated whether the increase of acidity towards deeper layers of Kolumbo crater had any effect on relevant phenotypic traits of bacterial isolates. A total of 31 Pseudomonas strains were isolated from both surface- (SSL) and deep-seawater layers (DSL), with the latter presenting a significantly higher acid tolerance. In particular, the DSL strains were able to cope with H+ levels that were 18 times higher. Similarly, the DSL isolates exhibited a significantly higher tolerance than SSL strains against six commonly used antibiotics and As(III). More importantly, a significant positive correlation was revealed between antibiotics and acid tolerance across the entire set of SSL and DSL isolates. Our findings imply that Pseudomonas species with higher resilience to antibiotics could be favored by the prospect of acidifying oceans. Further studies are required to determine if this feature is universal across marine bacteria and to assess potential ecological impacts.

A simple cleanup method for the removal of humic substances from soil protein extracts using aluminum coagulation.

Soil proteomics, the large-scale characterization of the entire protein complement in soils, provides a promising approach for deciphering the role of microbial functioning in terrestrial ecosystems. However, the extraction of soil proteins in sufficient quantities and of adequate purity remains a challenging task mainly due to the co-extraction of interfering humic substances. Up to now, the treatment of soil extracts with liquid phenol has been the "gold standard" for reducing humics, while the NoviPure cleanup kit was recently launched as a non-toxic approach. The present study describes an alternative method for delivering high-purity proteins based on humic coagulation with trivalent aluminum ions (Al3+). Various experimental parameters were optimized individually in order to maximize protein yield and diminish co-extracted humics. The optimized method was applied on a set of soil samples with diverse physicochemical characteristics and a comparison with the other two techniques was conducted. The amount of residual humics resulting from Al3+-based method was 26 and 35% higher than that from phenol treatment and NoviPure Kit, respectively, but these differences were of marginal statistical significance. With regard to extracted proteins, the average yields of the three methods were comparable, without showing any statistically significant differences. Overall, humic coagulation with Al3+ offers comparable cleanup performance in terms of protein yield and purity, but it is less toxic and less complex than the phenol-partitioning method, whereas it is far less expensive than the NoviPure Kit. The new technique is expected to facilitate the implementation of proteomic studies in soils.

Correction to: Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano, Hellenic Volcanic Arc.

In the original publication there is a mistake in the supplementary material. The correct supplementary material is provided in this correction article.

Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano, Hellenic Volcanic Arc.

Over the last decades, there has been growing interest about the ecological role of hydrothermal sulfide chimneys, their microbial diversity and associated biotechnological potential. Here, we performed dual-index Illumina sequencing of bacterial and archaeal communities on active and inactive sulfide chimneys collected from the Kolumbo hydrothermal field, situated on a geodynamic convergent setting. A total of 15,701 OTUs (operational taxonomic units) were assigned to 56 bacterial and 3 archaeal phyla, 133 bacterial and 16 archaeal classes. Active chimney communities were dominated by OTUs related to thermophilic members of Epsilonproteobacteria, Aquificae and Deltaproteobacteria. Inactive chimney communities were dominated by an OTU closely related to the archaeon Nitrosopumilus sp., and by members of Gammaproteobacteria, Deltaproteobacteria, Planctomycetes and Bacteroidetes. These lineages are closely related to phylotypes typically involved in iron, sulfur, nitrogen, hydrogen and methane cycling. Overall, the inactive sulfide chimneys presented highly diverse and uniform microbial communities, in contrast to the active chimney communities, which were dominated by chemolithoautotrophic and thermophilic lineages. This study represents one of the most comprehensive investigations of microbial diversity in submarine chimneys and elucidates how the dissipation of hydrothermal activity affects the structure of microbial consortia in these extreme ecological niches.

Kolumbo submarine volcano (Greece): An active window into the Aegean subduction system.

Submarine volcanism represents ~80% of the volcanic activity on Earth and is an important source of mantle-derived gases. These gases are of basic importance for the comprehension of mantle characteristics in areas where subaerial volcanism is missing or strongly modified by the presence of crustal/atmospheric components. Though, the study of submarine volcanism remains a challenge due to their hazardousness and sea-depth. Here, we report (3)He/(4)He measurements in CO2-dominated gases discharged at 500 m below sea level from the high-temperature (~220 °C) hydrothermal system of the Kolumbo submarine volcano (Greece), located 7 km northeast off Santorini Island in the central part of the Hellenic Volcanic Arc (HVA). We highlight that the mantle below Kolumbo and Santorini has a (3)He/(4)He signature of at least 7.0 Ra (being Ra the (3)He/(4)He ratio of atmospheric He equal to 1.39×10(-6)), 3 Ra units higher than actually known for gases-rocks from Santorini. This ratio is also the highest measured across the HVA and is indicative of the direct degassing of a Mid-Ocean-Ridge-Basalts (MORB)-like mantle through lithospheric faults. We finally highlight that the degassing of high-temperature fluids with a MORB-like (3)He/(4)He ratio corroborates a vigorous outgassing of mantle-derived volatiles with potential hazard at the Kolumbo submarine volcano.

Metagenomic investigation of the geologically unique Hellenic Volcanic Arc reveals a distinctive ecosystem with unexpected physiology.

Hydrothermal vents represent a deep, hot, aphotic biosphere where chemosynthetic primary producers, fuelled by chemicals from Earth's subsurface, form the basis of life. In this study, we examined microbial mats from two distinct volcanic sites within the Hellenic Volcanic Arc (HVA). The HVA is geologically and ecologically unique, with reported emissions of CO2 -saturated fluids at temperatures up to 220°C and a notable absence of macrofauna. Metagenomic data reveals highly complex prokaryotic communities composed of chemolithoautotrophs, some methanotrophs, and to our surprise, heterotrophs capable of anaerobic degradation of aromatic hydrocarbons. Our data suggest that aromatic hydrocarbons may indeed be a significant source of carbon in these sites, and instigate additional research into the nature and origin of these compounds in the HVA. Novel physiology was assigned to several uncultured prokaryotic lineages; most notably, a SAR406 representative is attributed with a role in anaerobic hydrocarbon degradation. This dataset, the largest to date from submarine volcanic ecosystems, constitutes a significant resource of novel genes and pathways with potential biotechnological applications.

Biodiversity, Anti-Trypanosomal Activity Screening, and Metabolomic Profiling of Actinomycetes Isolated from Mediterranean Sponges.

Marine sponge-associated actinomycetes are considered as promising sources for the discovery of novel biologically active compounds. In the present study, a total of 64 actinomycetes were isolated from 12 different marine sponge species that had been collected offshore the islands of Milos and Crete, Greece, eastern Mediterranean. The isolates were affiliated to 23 genera representing 8 different suborders based on nearly full length 16S rRNA gene sequencing. Four putatively novel species belonging to genera Geodermatophilus, Microlunatus, Rhodococcus and Actinomycetospora were identified based on a 16S rRNA gene sequence similarity of < 98.5% to currently described strains. Eight actinomycete isolates showed bioactivities against Trypanosma brucei brucei TC221 with half maximal inhibitory concentration (IC50) values <20 µg/mL. Thirty four isolates from the Milos collection and 12 isolates from the Crete collection were subjected to metabolomic analysis using high resolution LC-MS and NMR for dereplication purposes. Two isolates belonging to the genera Streptomyces (SBT348) and Micromonospora (SBT687) were prioritized based on their distinct chemistry profiles as well as their anti-trypanosomal activities. These findings demonstrated the feasibility and efficacy of utilizing metabolomics tools to prioritize chemically unique strains from microorganism collections and further highlight sponges as rich source for novel and bioactive actinomycetes.

Pyrosequencing analysis of microbial communities reveals dominant cosmopolitan phylotypes in deep-sea sediments of the eastern Mediterranean Sea.

The deep eastern basin of the Mediterranean Sea is considered to be one of the world's most oligotrophic areas in the world. Here we performed pyrosequenicng analysis of bacterial and archaeal communities in oxic nutrient-poor sediments collected from the eastern Mediterranean at 1025-4393 m depth. Microbial communities were surveyed by targeting the hypervariable V5-V6 regions of the 16S ribosomal RNA gene using bar-coded pyrosequencing. With a total of 13,194 operational taxonomic units (OTUs) or phylotypes at 97% sequence similarities, the phylogenetic affiliation of microbes was assigned to 23 bacterial and 2 archaeal known phyla, 23 candidate divisions at the phylum level and distributed into 186 families. It was further revealed that the microbial consortia inhabiting all sampling sites were highly diverse, but dominated by phylotypes closely related to members of the genus Pseudomonas and Marine Group I archaea. Such pronounced and widespread enrichment probably manifests the cosmopolitan character of these species and raises questions about their metabolic adaptation to the physical stressors and low nutrient availability of the deep eastern Mediterranean Sea.

Distribution of aliphatic hydrocarbons, polycyclic aromatic hydrocarbons and organochlorinated pollutants in deep-sea sediments of the Southern Cretan margin, Eastern Mediterranean Sea: a baseline assessment.

Deep sediments from the southern Cretan margin were analyzed to establish baseline levels for various types of organic pollutants before the anticipated intensification of anthropogenic activities. The total concentration of aliphatic hydrocarbons (ΣAH:326-3758ngg(-1), dry weight) was similar to those reported for deep sediments of the western Mediterranean Sea, while considerably lower levels were measured for polycyclic aromatic hydrocarbons (ΣPAH:9-60ngg(-1)). Source-diagnostic ratios suggested that the aliphatic hydrocarbons in sediments were mainly of terrestrial biogenic origin, while polycyclic aromatic hydrocarbons stemmed from the deposition of long-range transported combustion aerosols. Among the organochlorinated compounds analyzed, ß-hexachlorocyclohexane (ß-HCH:222-7052pgg(-1)), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT:37-2236pgg(-1)) and polychlorinated biphenyls (ΣPCB:38-1182pgg(-1)) showed the highest abundance in sediments. The presence of HCHs and PCBs was attributed to historical inputs that have undergone extensive weathering, whereas an ongoing fresh input was suggested for p,p'-DDT. Multiple linear regression analysis revealed that the levels of the various pollutants in sediments were controlled by different factors, but with organic carbon content playing a prominent role in most cases.

New insights into hydrothermal vent processes in the unique shallow-submarine arc-volcano, Kolumbo (Santorini), Greece.

We report on integrated geomorphological, mineralogical, geochemical and biological investigations of the hydrothermal vent field located on the floor of the density-stratified acidic (pH ~ 5) crater of the Kolumbo shallow-submarine arc-volcano, near Santorini. Kolumbo features rare geodynamic setting at convergent boundaries, where arc-volcanism and seafloor hydrothermal activity are occurring in thinned continental crust. Special focus is given to unique enrichments of polymetallic spires in Sb and Tl (±Hg, As, Au, Ag, Zn) indicating a new hybrid seafloor analogue of epithermal-to-volcanic-hosted-massive-sulphide deposits. Iron microbial-mat analyses reveal dominating ferrihydrite-type phases, and high-proportion of microbial sequences akin to "Nitrosopumilus maritimus", a mesophilic Thaumarchaeota strain capable of chemoautotrophic growth on hydrothermal ammonia and CO2. Our findings highlight that acidic shallow-submarine hydrothermal vents nourish marine ecosystems in which nitrifying Archaea are important and suggest ferrihydrite-type Fe(3+)-(hydrated)-oxyhydroxides in associated low-temperature iron mats are formed by anaerobic Fe(2+)-oxidation, dependent on microbially produced nitrate.

Carbon and chlorine isotope fractionation during microbial degradation of tetra- and trichloroethene.

Two-dimensional compound-specific isotope analysis (2D-CSIA), combining stable carbon and chlorine isotopes, holds potential for monitoring of natural attenuation of chlorinated ethenes (CEs) in contaminated soil and groundwater. However, interpretation of 2D-CSIA data sets is challenged by a shortage of experimental Cl isotope enrichment factors. Here, isotope enrichments factors for C and Cl (i.e., εC and εCl) were determined for biodegradation of tetrachloroethene (PCE) and trichloroethene (TCE) using microbial enrichment cultures from a heavily CE-contaminated aquifer. The obtained values were εC = -5.6 ± 0.7‰ (95% CI) and εCl = -2.0 ± 0.5‰ for PCE degradation and εC = -8.8 ± 0.2‰ and εCl = -3.5 ± 0.5‰ for TCE degradation. Combining the values for both εC and εCl yielded mechanism-diagnostic εCl/εC ratios of 0.35 ± 0.11 and 0.37 ± 0.11 for the degradation of PCE and TCE, respectively. Application of the obtained εC and εCl values to a previously investigated field site gave similar estimates for the fraction of degraded contaminant as in the previous study, but with a reduced uncertainty in assessment of the natural attenuation. Furthermore, 16S rRNA gene clone library analyses were performed on three samples from the PCE degradation experiments. A species closely related to Desulfitobacterium aromaticivorans UKTL dominated the reductive dechlorination process. This study contributes to the development of 2D-CSIA as a tool for evaluating remediation strategies of CEs at contaminated sites.

Phylogenetic diversity of sediment bacteria from the deep Northeastern Pacific Ocean: a comparison with the deep Eastern Mediterranean Sea.

The variability of bacterial community composition and diversity was studied by comparative analysis of five 16S rRNA gene clone libraries from deep-sea sediments (water column depth: 4000 m) of the Northeastern Pacific Ocean and Eastern Mediterranean Sea. This is the first comparison of the bacterial communities living in these deep-sea ecosystems. The estimated chlorophyll a, organic carbon, and C/N ratio provided evidence of significant differences in the trophic state of the sediments between the Northeastern Pacific Ocean and the much warmer Eastern Mediterranean Sea. A diverse range of 16S rRNA gene phylotypes was found in the sediments of both regions. These were represented by 11 different taxonomic groups, with Gammaproteobacteria predominating in the Northeastern Pacific Ocean sediments and Acidobacteria in the Eastern Mediterranean microbial community. In addition, several 16S rRNA gene phylotypes only distantly related to any of the previously identified sequences (non-affiliated rRNA genes) represented a significant fraction of the total sequences. The potential diversity at the two sites differs but remains largely unexplored and remains of continuing scientific interest.

Phylogenetic diversity of sediment bacteria from the deep Northeastern Pacific Ocean: a comparison with the deep Eastern Mediterranean Sea

The variability of bacterial community composition and diversity was studied by comparative analysis of five 16S rRNA gene clone libraries from deep-sea sediments (water column depth: 4000 m) of the Northeastern Pacific Ocean and Eastern Mediterranean Sea. This is the first comparison of the bacterial communities living in these deep-sea ecosystems. The estimated chlorophyll a, organic carbon, and C/N ratio provided evidence of significant differences in the trophic state of the sediments between the Northeastern Pacific Ocean and the much warmer Eastern Mediterranean Sea. A diverse range of 16S rRNA gene phylotypes was found in the sediments of both regions. These were represented by 11 different taxonomic groups, with Gammaproteobacteria predominating in the Northeastern Pacific Ocean sediments and Acidobacteria in the Eastern Mediterranean microbial community. In addition, several 16S rRNA gene phylotypes only distantly related to any of the previously identified sequences (non-affiliated rRNA genes) represented a significant fraction of the total sequences. The potential diversity at the two sites differs but remains largely unexplored and remains of continuing scientific interest (AU)

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Sulfur cycling and methanogenesis primarily drive microbial colonization of the highly sulfidic Urania deep hypersaline basin.

Urania basin in the deep Mediterranean Sea houses a lake that is >100 m deep, devoid of oxygen, 6 times more saline than seawater, and has very high levels of methane and particularly sulfide (up to 16 mM), making it among the most sulfidic water bodies on Earth. Along the depth profile there are 2 chemoclines, a steep one with the overlying oxic seawater, and another between anoxic brines of different density, where gradients of salinity, electron donors and acceptors occur. To identify and differentiate the microbes and processes contributing to the turnover of organic matter and sulfide along the water column, these chemoclines were sampled at a high resolution. Bacterial cell numbers increased up to a hundredfold in the chemoclines as a consequence of elevated nutrient availability, with higher numbers in the upper interface where redox gradient was steeper. Bacterial and archaeal communities, analyzed by DNA fingerprinting, 16S rRNA gene libraries, activity measurements, and cultivation, were highly stratified and metabolically more active along the chemoclines compared with seawater or the uniformly hypersaline brines. Detailed analysis of 16S rRNA gene sequences revealed that in both chemoclines delta- and epsilon-Proteobacteria, predominantly sulfate reducers and sulfur oxidizers, respectively, were the dominant bacteria. In the deepest layers of the basin MSBL1, putatively responsible for methanogenesis, dominated among archaea. The data suggest that the complex microbial community is adapted to the basin's extreme chemistry, and the elevated biomass is driven largely by sulfur cycling and methanogenesis.

Phylogenetic diversity of sediment bacteria from the southern Cretan margin, Eastern Mediterranean Sea.

This study is the first culture-independent report on the regional variability of bacterial diversity in oxic sediments from the unexplored southern Cretan margin (SCM). Three main deep basins (water column depths: 2670-3603m), located at the mouth of two submarine canyons (Samaria Gorge and Paximades Channel) and an adjacent slope system, as well as two shallow upper-slope stations (water column depths: 215 and 520m), were sampled. A total of 454 clones were sequenced and the bacterial richness, estimated through five clone libraries using rarefaction analysis, ranged from 71 to 296 unique phylotypes. The average sequence identity of the retrieved Cretan margin sequences compared to the >1,000,000 known rRNA sequences was only 93.5%. A diverse range of prokaryotes was found in the sediments, which were represented by 15 different taxonomic groups at the phylum level. The phylogenetic analysis revealed that these new sequences grouped with the phyla Acidobacteria, Planctomycetes, Actinobacteria, Gamma-, Alpha- and Delta-proteobacteria. Only a few bacterial clones were affiliated with Chloroflexi, Bacteroidetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Nitrospirae, Beta-proteobacteria, Lentisphaerae and Dictyoglomi. A large fraction of the retrieved sequences (12%) did not fall into any taxonomic division previously characterized by molecular criteria, whereas four novel division-level lineages, termed candidate division SCMs, were identified. Bacterial community composition demonstrated significant differences in comparison to previous phylogenetic studies. This divergence was mainly triggered by the dominance of Acidobacteria and Actinobacteria and reflected a bacterial community different from that currently known for oxic and pristine marine sediments.

Particle size distribution of airborne microorganisms and pathogens during an Intense African dust event in the eastern Mediterranean.

BACKGROUND: The distribution of microorganisms, and especially pathogens, over airborne particles of different sizes has been ignored to a large extent, but it could have significant implications regarding the dispersion of these microorganisms across the planet, thus affecting human health. OBJECTIVES: We examined the microbial quality of the aerosols over the eastern Mediterranean region during an African storm to determine the size distribution of microorganisms in the air. METHODS: We used a five-stage cascade impactor for bioaerosol collection in a coastal city on the eastern Mediterranean Sea during a north African dust storm. Bacterial communities associated with aerosol particles of six different size ranges were characterized following molecular culture-independent methods, regardless of the cell culturability (analysis of 16S rRNA genes). RESULTS: All 16S rDNA clone libraries were diverse, including sequences commonly found in soil and marine ecosystems. Spore-forming bacteria such as Firmicutes dominated large particle sizes (> 3.3 microm), whereas clones affiliated with Actinobacteria (found commonly in soil) and Bacteroidetes (widely distributed in the environment) gradually increased their abundance in aerosol particles of reduced size (< 3.3 microm). A large portion of the clones detected at respiratory particle sizes (< 3.3 microm) were phylogenetic neighbors to human pathogens that have been linked to several diseases. CONCLUSIONS: The presence of aerosolized bacteria in small size particles may have significant implications to human health via intercontinental transportation of pathogens.

Carbon speciation and composition of natural microbial communities in polluted and pristine sediments of the Eastern Mediterranean Sea.

Sediment samples collected from polluted (Augusta Bay) and pristine regions of the Eastern Mediterranean Sea (South Ionian Sea, Thracian Sea) were analyzed for black carbon, aliphatic hydrocarbons and phospholipid ester-linked fatty acids (PLFA). The aim of the study was to investigate the anthropogenic and biogenic inputs into the Eastern Mediterranean Sea and to evaluate the effects of refractory organic matter (e.g. black carbon) and the level of hydrocarbon contamination on benthic microbial community composition. Black carbon, generally considered to be chemically and biologically inert, comprised a significant but highly variable fraction of the sedimentary carbon pool in the analyzed sediments with a ratio to total organic carbon ranging from 0.02 to 0.66. Principal component analysis of the chemical characteristics of the sediments (organic carbon content, black carbon, bioavailable organic carbon, chlorophyll a, phaeopigments, aliphatic hydrocarbons) revealed clustering of samples along a gradient from the most productive and contaminated region of Augusta Bay to the carbon-poor and pristine sediments of the Thracian Sea. PLFA analysis revealed that gram-negative bacteria and microeukaryotes were most abundant in Augusta Bay and in the most impacted station of the Thracian Sea. The high levels of branched and odd-chain fatty acids recorded for these stations is probably linked to the elevated amounts of hydrocarbons at these stations; e.g. microbial communities may have developed the ability to degrade either naturally occurring aliphatic hydrocarbons or hydrocarbons derived from oil contamination.

Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.