Genome sequence of Salinisphaera shabanensis, a gammaproteobacterium from the harsh, variable environment of the brine-seawater interface of the Shaban Deep in the Red Sea

We present the genome of Salinisphaera shabanensis, isolated from a brine-seawater interface and representing a new order within the Gammaproteobacteria. Its adaptations to physicochemical and nutrient availability fluctuations include six genes encoding heavy metal-translocating P-type ATPases and multiple genes involved in iron uptake, siderophore production, and poly-β-hydroxybutyrate synthesis.     

Salinisphaera shabanensis gen. nov., sp. nov., a novel,moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep,Red Sea

A novel, moderately halophilic bacterium was isolated from the brine-seawater interface of the Shaban Deep, northern Red Sea. A polyphasic approach was used for the taxonomic characterization of this isolate, with the phenotypic and phylogenetic data clearly showing the distinctiveness of this bacterium. Cells of isolate E1L3A were Gram-negative, monotrichous cocci that showed a remarkable physiological flexibility, as could be seen by the quite broad growth ranges for oxygen, temperature, NaCl, and, to a smaller degree, pH. In addition, it was able to grow from atmospheric pressure up to 15 MPa, making it a piezotolerant bacterium. Phylogenetically, strain E1L3A represents a new, deeply branching lineage within the gamma-Proteobacteria, as determined by 16S rRNA gene sequence analysis. No close relatives are known so far, with sequence similarity to other cultivated members of the gamma-Proteobacteria being lower than 88%. The creation of the new genus Salinisphaera and the new species Salinisphaera shabanensis (DSM 14853; JCM 11575) for this new and highly versatile microorganism is therefore proposed.     

Microbial diversity of the brine-seawater interface of the Kebrit Deep, Red Sea, studied via 16S rRNA gene sequences and cultivation methods.

The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other Halanaerobium representatives, showing unusually large amounts of Delta7 and Delta11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus Halanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface.     

Unique Prokaryotic Consortia in Geochemically Distinct Sediments from Red Sea Atlantis II and Discovery Deep Brine Pools

The seafloor is a unique environment, which allows insights into how geochemical processes affect the diversity of biological life. Among its diverse ecosystems are deep-sea brine pools - water bodies characterized by a unique combination of extreme conditions. The 'polyextremophiles' that constitute the microbial assemblage of these deep hot brines have not been comprehensively studied. We report a comparative taxonomic analysis of the prokaryotic communities of the sediments directly below the Red Sea brine pools, namely, Atlantis II, Discovery, Chain Deep, and an adjacent brine-influenced site. Analyses of sediment samples and high-throughput pyrosequencing of PCR-amplified environmental 16S ribosomal RNA genes (16S rDNA) revealed that one sulfur (S)-rich Atlantis II and one nitrogen (N)-rich Discovery Deep section contained distinct microbial populations that differed from those found in the other sediment samples examined. Proteobacteria, Actinobacteria, Cyanobacteria, Deferribacteres, and Euryarchaeota were the most abundant bacterial and archaeal phyla in both the S- and N-rich sections. Relative abundance-based hierarchical clustering of the 16S rDNA pyrotags assigned to major taxonomic groups allowed us to categorize the archaeal and bacterial communities into three major and distinct groups; group I was unique to the S-rich Atlantis II section (ATII-1), group II was characteristic for the N-rich Discovery sample (DD-1), and group III reflected the composition of the remaining sediments. Many of the groups detected in the S-rich Atlantis II section are likely to play a dominant role in the cycling of methane and sulfur due to their phylogenetic affiliations with bacteria and archaea involved in anaerobic methane oxidation and sulfate reduction.     

Prokaryotic diversity,composition structure,and phylogenetic analysis of microbial communities in leachate sediment ecosystems

In order to obtain insight into the prokaryotic diversity and community in leachate sediment, a culture-independent DNA-based molecular phylogenetic approach was performed with archaeal and bacterial 16S rRNA gene clone libraries derived from leachate sediment of an aged landfill. A total of 59 archaeal and 283 bacterial rDNA phylotypes were identified in 425 archaeal and 375 bacterial analyzed clones. All archaeal clones distributed within two archaeal phyla of the Euryarchaeota and Crenarchaeota, and well-defined methanogen lineages, especially Methanosaeta spp., are the most numerically dominant species of the archaeal community. Phylogenetic analysis of the bacterial library revealed a variety of pollutant-degrading and biotransforming microorganisms, including 18 distinct phyla. A substantial fraction of bacterial clones showed low levels of similarity with any previously documented sequences and thus might be taxonomically new. Chemical characteristics and phylogenetic inferences indicated that (1) ammonium-utilizing bacteria might form consortia to alleviate or avoid the negative influence of high ammonium concentration on other microorganisms, and (2) members of the Crenarchaeota found in the sediment might be involved in ammonium oxidation. This study is the first to report the composition of the microbial assemblages and phylogenetic characteristics of prokaryotic populations extant in leachate sediment. Additional work on microbial activity and contaminant biodegradation remains to be explored.     

Abundance and diversity of surface zooplankton in the Gulf of Aqaba, Red Sea, Egypt

Surface zooplankton were studied in Egyptian coastal watersof the Gulf of Aqaba, from bimonthly samples from July 1994to May 1995. Species diversity, numerical abundance and dynamicswere analysed for each taxon, at six sites, inside three Protectorates.A total of 62 taxa and species were identified. At all sites,copepods were predominant in the standing crop with an averageof 1945 ind. M^–3 and formed {small tilde}75.5%, numerically,of the total zooplankton community. The meroplanktonic larvaeoccupied the second rank and they constituted {small tilde}19.7%of the total zooplankton. Seasonally, the main peak of zooplanktonabundance was recorded in winter (January) with an average of3510 ind. M^–3 while September was characterized by thelowest density (1906 ind. m^–3 The relatively higher diversityvalues were recorded at Ras Mohammed Protectorate and a progressivedecline in diversity was observed northward.     

Coupling of functional gene diversity and geochemical data from environmental samples

Genomic techniques commonly used for assessing distributions of microorganisms in the environment often produce small sample sizes. We investigated artificial neural networks for analyzing the distributions of nitrite reductase genes (nirS and nirK) and two sets of dissimilatory sulfite reductase genes (dsrAB1 and dsrAB2) in small sample sets. Data reduction (to reduce the number of input parameters), cross-validation (to measure the generalization error), weight decay (to adjust model parameters to reduce generalization error), and importance analysis (to determine which variables had the most influence) were useful in developing and interpreting neural network models that could be used to infer relationships between geochemistry and gene distributions. A robust relationship was observed between geochemistry and the frequencies of genes that were not closely related to known dissimilatory sulfite reductase genes (dsrAB2). Uranium and sulfate appeared to be the most related to distribution of two groups of these unusual dsrAB-related genes. For the other three groups, the distributions appeared to be related to pH, nickel, nonpurgeable organic carbon, and total organic carbon. The models relating the geochemical parameters to the distributions of the nirS, nirK, and dsrAB1 genes did not generalize as well as the models for dsrAB2. The data also illustrate the danger (generating a model that has a high generalization error) of not using a validation approach in evaluating the meaningfulness of the fit of linear or nonlinear models to such small sample sizes.     

Targeted recovery of novel phylogenetic diversity from next-generation sequence data

Next-generation sequencing technologies have led to recognition of a so-called ‘rare biosphere''. These microbial operational taxonomic units (OTUs) are defined by low relative abundance and may be specifically adapted to maintaining low population sizes. We hypothesized that mining of low-abundance next-generation 16S ribosomal RNA (rRNA) gene data would lead to the discovery of novel phylogenetic diversity, reflecting microorganisms not yet discovered by previous sampling efforts. Here, we test this hypothesis by combining molecular and bioinformatic approaches for targeted retrieval of phylogenetic novelty within rare biosphere OTUs. We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample. Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities. These divergent target sequences correspond to (a) a previously unknown lineage within the BRC1 candidate phylum, (b) a sister group to the early diverging and currently recognized monospecific Cyanobacteria Gloeobacter, a genus containing multiple plesiomorphic traits and (c) a highly divergent lineage phylogenetically resolved within mitochondria. A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes. The results demonstrate this sequence analysis and retrieval pipeline as applicable for exploring underrepresented phylogenetic novelty and recovering taxa that may represent significant steps in bacterial evolution.     

Deep chlorophyll a maxima in the Red Sea observed by in-vivo flash fluorometry

An investigation of the hydrography and the population, production and biomass of plankton in the Red Sea, carried out during the METEOR cruise in summer 1987, aimed to describe the ecosystem characteristics during the SW monsoon period. Vertical profiles of in-vivo chlorophyll fluorescence intensity, measured in the presence of chlorphenyl-dimethyl-urea (CMU), are presented. Variations in the fluorescence pattern were observed and assumed to be due to the influence of a reef and surface influx of nutrient rich water from the Gulf of Aden into the Red Sea. This northward influx was driven by SE winds, caused by an unusual northward shift of the innertropical convergence zone up to 20°N in summer 1987. Integrated chlorophyll a values were calculated from fluorescence data. They showed a slight increase from north to south and higher pigment contents in August (8.7–20.2 mg m^–2) than in July (3.3–9.0 mg m^–2), the latter was attributed to the above mentioned influx. Calibration of the fluorescence measurements using cultures of a green alga and cyanobacterium indicated that there may have been an underestimate of the contribution of Oscillatoria populations to the chlorophyll a concentration of the samples. Fluorescence peaks were recorded in the lower part of the euphotic zone, indicating a deep maximum of phytoplankton and/or an increase in chlorophyll fluorescence per unit biomass at these depths.     

Flexistipes sinusarabici,a novel genus and species of eubacteria occurring in the Atlantis II Deep brines of the Red Sea

Four isolates of a gram-negative flexible bacterium have been obtained from brine water samples of the Atlantis II Deep of the Red Sea at a depth of 2000 m. One isolate (MAS 10) was studied in detail. Cells are nonmotile, flexible rods, measuring about 0.3 m in width and 5 to 50 m in length. The new organisms are heterotrophs growing anaerobically on yeast extract, meat extract, peptone, tryptone, and, less efficiently, on acetate and casamino acids. Growth occurs between 30% and 53°C at pH 6 to 8 in the presence of at least 3% NaCl. The shortest doubling time is 8.5 h under optimal growth conditions. Cells are sensitive to the antibiotics penicillin, ampicillin, vancomycin, and streptomycin, but resistant to tetracyclin and rifamipicin. The GC-content of the DNA is 39 mol%. Based on their 16S rRNA the new isolates group with the general cluster of eubacterial phyla. Since they show no specific relationship to any of them, a new genus is described, which is named Flexistipes, the flexible stick. Type species is Flexistipes sinusarabici strain MAS 10 (DSM 4947).     

Novel thermostable antibiotic resistance enzymes from the Atlantis II Deep Red Sea brine pool

The advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence‐based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3′‐aminoglycoside phosphotransferase [APH(3′)] and a class A beta‐lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3′) was active in vivo. Interestingly, APH(3′) proved to be thermostable (T_m = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a T_m = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.     

Symbiodiniaceae diversity of Palythoa tuberculosa in the central and southern Red Sea influenced by environmental factors

Sea surface temperatures (SST) and chlorophyll a concentrations (Chl a) in the southern Red Sea have wide variations based on distance from the coast. To understand how these variations can affect the diversity of symbionts hosted by reef-associated organisms, we conducted a study in the central and southern Red Sea to examine the diversity of Symbiodiniaceae hosted by the zooxanthellate zoantharian Palythoa tuberculosa at different distances from the coast: offshore (FBO), midshelf (FBM) and inshore (FBI) of Farasan Banks, and inshore at Thuwal (TI). Genomic DNA was extracted from 198 specimens, followed by amplification of the ribosomal DNA internal transcribed spacer 2 (ITS-2) and noncoding region of the chloroplast plastid minicircle (psbA^ncr). Durusdinium and six lineages of Cladocopium (Pt-1-a, Pt-1-b, Pt-1-c, Pt-1-d, Pt-3-a, Pt-3-b) were identified based on sequences of the two marker regions. Changes in composition of Symbiodiniaceae lineages were observed from FBI (high SST, high Chl a) to FBO (low SST, low Chl a). Molecular variance analyses showed that distance from coast was the most likely predictor of differences in Cladocopium lineages. Multinomial logistic regression analysis showed a transition among different Cladocopium lineages as SST increased. One Cladocopium lineage, Pt-1-b, demonstrated higher prevalences at high SSTs and increased in prevalences at the same rate as thermotolerant Durusdinium. Additionally, Cladocopium lineage Pt-3-a had a high affinity to low Chl a concentrations. This study demonstrates that environmental variations in SSTs and Chl a concentrations are significant predictors for the diversity of dominant Symbiodiniaceae within individual host P. tuberculosa colonies. We theorize that flexibility with different lineages of Symbiodiniaceae allows generalist P. tuberculosa to live across a wide range of environments in the southern Red Sea.

     

Taxonomy of Oncaeidae (Copepoda,Poecilostomatoida) from the Red Sea. III. Morphology and phylogenetic position of Oncaea subtilis Giesbrecht, 1892

Both sexes of Oncaea subtilis Giesbrecht, 1892, a small oncaeid species commonly occurring in temperate and tropical regions, are redescribed on the basis of material from the Red Sea. It is placed in a new monotypic genus, Monothula, on the basis of the loss of the outer spine on the third segment of the endopod of legs 2–4, and the presence of a single dorsal egg-sac, which is attached medially to the genital double-somite. The latter character is unique among oncaeids. The geographical distribution of M. subtilis comb. nov. is reviewed, and additional taxonomic data based on material from the eastern Mediterranean, the Arabian Sea and the eastern Indian Ocean are presented. The phylogenetic relationships of M. subtilis within the Oncaeidae are discussed.     

Drivers of bacterial diversity dynamics in permeable carbonate and silicate coral reef sands from the Red Sea

Permeable sediments and associated microbial communities play a fundamental role in nutrient recycling within coral reef ecosystems by ensuring high levels of primary production in oligotrophic environments. A previous study on organic matter degradation within biogenic carbonate and terrigenous silicate reef sands in the Red Sea suggested that observed sand-specific differences in microbial activity could be caused by variations in microbial biomass and diversity. Here, we tested this hypothesis by comparing bacterial abundance and community structure in both sand types, and by further exploring the structuring effects of time (season) and space (sediment depth, in/out-reef). Changes in bacterial community structure, as determined via automated ribosomal intergenic spacer analysis (ARISA), were primarily driven by sand mineralogy at specific seasons, sediment depths and reef locations. By coupling ARISA with 16S-ITS rRNA sequencing, we detected significant community shifts already at the bacterial class level, with Proteobacteria (Gamma-, Delta-, Alpha-) and Actinobacteria being prominent members of the highly diverse communities. Overall, our findings suggest that reef sand-associated bacterial communities vary substantially with sand type. Especially in synergy with environmental variation over time and space, mineralogical differences seem to play a central role in maintaining high levels of bacterial community heterogeneity. The local co-occurrence of carbonate and silicate sands may thus significantly increase the availability of microbial niches within a single coral reef ecosystem.     

Preliminary studies of cyanobacteria, picoplankton, and virioplankton in the Salton Sea with special attention to phylogenetic diversity among eight strains of filamentous cyanobacteria

Cyanobacterial diversity in the Salton Sea, a high-salinity, eutrophic lake in Southern California, was investigated using a combination of molecular and morphological approaches. Representatives of a total of 10 described genera (Oscillatoria, Spirulina, Arthrospira, Geitlerinema, Lyngbya, Leptolyngbya, Calothrix, Rivularia, Synechococcus, Synechocystis) were identified in the samples; additionally, the morphology of two cultured strains do not conform to any genus recognized at present by the bacteriological system. Genetic analysis, based on partial 16S rRNA sequences suggested considerable cryptic genetic variability among filamentous strains of similar or identical morphology and showed members of the form-genus Geitlerinema to be distributed among three major phylogenetic clades of cyanobacteria. Cyanobacterial mats, previously described from the Sea were, in fact, composed of both filamentous cyanobacteria and a roughly equivalent biomass of the sulfur-oxidizing bacterium Beggiatoa, indicating their formation in sulfide rich regions of the lake. Flow cytometric analysis of the water samples showed three striking differences between samples from the Salton Sea and representative marine waters: (1) phycoerythrin-containing unicells, while abundant, were much less abundant in the Salton Sea than they were in typical continental shelf waters, (2) Prochlorococcus appears to be completely absent, and (3) small (3–5 m) eukaryotic algae were more abundant in the Salton Sea than in typical neritic waters by one-to-two orders-of-magnitude. Based on flow cytometric analysis, heterotrophic bacteria were more than an order of magnitude more abundant in the Salton Sea than in seawater collected from continental shelf environments. Virus particles were more abundant in the Salton Sea than in typical neritic waters, but did not show increases proportionate with the increase in bacteria, picocyanobacteria, or eukaryotic algae.     

Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea

In this study, we report on first 16S rRNA gene sequences from highly saline brine sediments taken at a depth of 1,515 m in the Kebrit Deep, northern Red Sea. Microbial DNA extracted directly from the sediments was subjected to PCR amplification with primers specific for bacterial and archaeal 16S rRNA gene sequences. The PCR products were cloned, and a total of 11 (6 bacterial and 5 archaeal) clone types were determined by restriction endonuclease digestion. Phylogenetic analysis revealed that most of the cloned sequences were unique, showing no close association with sequences of cultivated organisms or sequences derived from environmental samples. The bacterial clone sequences form a novel phylogenetic lineage (KB1 group) that branches between the Aquificales and the Thermotogales. The archaeal clone sequences group within the Euryarchaeota. Some of the sequences cluster with the group II and group III uncultivated archaea sequence clones, while two clone groups form separate branches. Our results suggest that hitherto unknown archaea and bacteria may thrive in highly saline brines of the Red Sea under extreme environmental conditions. Received: 5 February 1999 / Accepted: 14 July 1999     

A Novel Mercuric Reductase from the Unique Deep Brine Environment of Atlantis II in the Red Sea

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg²⁺, and efficiently detoxifies Hg²⁺ in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.