André Antunes

Microbiology of the Red Sea (and other) deep‐sea anoxic brine lakes

The Red Sea harbours approximately 25 deep-sea anoxic brine pools. They constitute extremely unique and complex habitats with the conjugation of several extreme physicochemical parameters rendering them some of the most inhospitable environments on Earth. After 50 years of research mostly driven by chemists, geophysicists and geologists, the microbiology of the brines has been receiving increased interest in the last decade. Recent molecular and cultivation-based studies have provided us with a first glimpse on the enormous biodiversity of the local microbial communities, the identification of several new taxonomic groups, and the isolation of novel extremophiles that thrive in these environments. This review presents a general overview of these unusual biotopes and compares them with other similar environments in the Mediterranean Sea and the Gulf of Mexico, with a focus on their microbial ecology.

Halorhabdus tiamatea sp. nov., a non-pigmented, extremely halophilic archaeon from a deep-sea, hypersaline anoxic basin of the Red Sea, and emended description of the genus Halorhabdus.

An extremely halophilic archaeon was isolated from a sample of the brine-sediment interface of the Shaban Deep in the northern Red Sea. Phylogenetic analysis of the 16S rRNA gene sequence revealed a close proximity to Halorhabdus utahensis (99.3%), the sole species of the genus Halorhabdus. Strain SARL4B(T) formed non-pigmented colonies and showed optimum growth at 45 degrees C, in 27% (w/v) NaCl and at pH 6.5-7.0. This organism utilized a few complex substrates, such as yeast extract and starch, for growth. Strain SARL4B(T) grew under anaerobic and microaerophilic conditions but grew extremely poorly under aerobic conditions. The ether lipids were diphytanyl derivatives. The DNA G+C content of the type strain was 61.7 mol%. On the basis of the phylogenetic data and physiological and biochemical characteristics, strain SARL4B(T) represents a novel species of the genus Halorhabdus, for which the name Halorhabdus tiamatea is proposed. The type strain is SARL4B(T) (=DSM 18392(T)=JCM 14471(T)). An emended description of the genus Halorhabdus is also proposed.

Biogeography of pelagic bacterioplankton across an antagonistic temperature-salinity gradient in the Red Sea.

The Red Sea is a unique marine ecosystem with contrasting gradients of temperature and salinity along its north‐to‐south axis. It is an extremely oligotrophic environment that is characterized by perpetual year‐round water column stratification, high annual solar irradiation, and negligible riverine and precipitation inputs. In this study, we investigated whether the contemporary environmental conditions shape community assemblages by pyrosequencing 16S rRNA genes of bacteria in surface water samples collected from the northeastern half of this water body. A combined total of 1855 operational taxonomic units (OTUs) were recovered from the ‘small‐cell’ and ‘large‐cell’ fractions. Here, a few major OTUs affiliated with Cyanobacteria and Proteobacteria accounted for ∼93% of all sequences, whereas a tail of ‘rare’ OTUs represented most of the diversity. OTUs allied to Surface 1a/b SAR11 clades and Prochlorococcus related to the high‐light‐adapted (HL2) ecotype were the most widespread and predominant sequence types. Interestingly, the frequency of taxa that are typically found in the upper mesopelagic zone was significantly elevated in the northern transects compared with those in the central, presumably as a direct effect of deep convective mixing in the Gulf of Aqaba and water exchange with the northern Red Sea. Although temperature was the best predictor of species richness across all major lineages, both spatial and environmental distances correlated strongly with phylogenetic distances. Our results suggest that the bacterial diversity of the Red Sea is as high as in other tropical seas and provide evidence for fundamental differences in the biogeography of pelagic communities between the northern and central regions.

INDIGO – INtegrated Data Warehouse of MIcrobial GenOmes with Examples from the Red Sea Extremophiles

Background The next generation sequencing technologies substantially increased the throughput of microbial genome sequencing. To functionally annotate newly sequenced microbial genomes, a variety of experimental and computational methods are used. Integration of information from different sources is a powerful approach to enhance such annotation. Functional analysis of microbial genomes, necessary for downstream experiments, crucially depends on this annotation but it is hampered by the current lack of suitable information integration and exploration systems for microbial genomes. Results We developed a data warehouse system (INDIGO) that enables the integration of annotations for exploration and analysis of newly sequenced microbial genomes. INDIGO offers an opportunity to construct complex queries and combine annotations from multiple sources starting from genomic sequence to protein domain, gene ontology and pathway levels. This data warehouse is aimed at being populated with information from genomes of pure cultures and uncultured single cells of Red Sea bacteria and Archaea. Currently, INDIGO contains information from Salinisphaera shabanensis, Haloplasma contractile, and Halorhabdus tiamatea - extremophiles isolated from deep-sea anoxic brine lakes of the Red Sea. We provide examples of utilizing the system to gain new insights into specific aspects on the unique lifestyle and adaptations of these organisms to extreme environments. Conclusions We developed a data warehouse system, INDIGO, which enables comprehensive integration of information from various resources to be used for annotation, exploration and analysis of microbial genomes. It will be regularly updated and extended with new genomes. It is aimed to serve as a resource dedicated to the Red Sea microbes. In addition, through INDIGO, we provide our Automatic Annotation of Microbial Genomes (AAMG) pipeline. The INDIGO web server is freely available at http://www.cbrc.kaust.edu.sa/indigo.

Leuconostoc ficulneum sp. nov., a novel lactic acid bacterium isolated from a ripe fig, and reclassification of Lactobacillus fructosus as Leuconostoc fructosum comb. nov.

An isolate, designated strain FS-1T, was recovered from a ripe fig. Phylogenetic analysis of the 16S rRNA genes and DNA-DNA reassociation values showed that the organism represented a novel species of the genus Leuconostoc closely related to Lactobacillus fructosus. The novel isolate could be distinguished from the type strain of Lactobacillus fructosus by the fatty acid composition and several phenotypic and growth characteristics. In strain FS-1T, 18:1 delta9 (18:1omega9c) was present in relatively large amounts whilst, in Lactobacillus fructosus, this fatty acid was a minor component. Strain FS-1T and Lactobacillus fructosus produced acid in API 50CHL microtubes from glucose, fructose and mannitol within 48 h, whereas only strain FS-1T also fermented trehalose, gluconate, turanose and sucrose after 48 h. Other differences in acid production from carbohydrates also distinguished strain FS-1T from Lactobacillus fructosus. Both organisms were heterofermentative with fructose as a substrate and fermented glucose only in the presence of fructose, as determined by nuclear magnetic resonance studies. Strain FS-1T was catalase-positive. On the basis of the phylogenetic analysis, DNA-DNA reassociation values, physiological and biochemical characteristics and fatty acid composition, the name Leuconostoc ficulneum is proposed for the novel species represented by strain FS-1T, and it is proposed that Lactobacillus fructosus be reclassified in the genus Leuconostoc as Leuconostoc fructosum comb. nov.

Core Microbial Functional Activities in Ocean Environments Revealed by Global Metagenomic Profiling Analyses

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the worlds oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond, proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina.

A halophilic, aerobic Gram-negative bacterium, designated strain CVS-6(T), was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the organism with members of the family Idiomarinaceae. Sequence similarities between CVS-6(T) and the type strains of the species of the genera Pseudidiomarina and Idiomarina ranged from 93.7% to 96.9%. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major cellular fatty acids were 15:0 iso (21.8%), 17:0 iso (12.5%), 17:1 iso omega9c (10.7%), and 16:1 omega7c (10.6%). The DNA G+C content was 51.6 mol%. The species represented by strain CVS-6(T) could be distinguished from the species of the genera Pseudidiomarina and Idiomarina; however, it was not possible to distinguish both genera from each other using the phenotypic or chemotaxonomic characteristics examined. Consequently, we propose that the species classified in the genus Pseudidiomarina should be transferred to the genus Idiomarina. We also propose that, on the basis of physiological and biochemical characteristics, strain CVS-6(T) (=LMG 23123=CIP 108836) represents a new species which we name Idiomarina insulisalsae.

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.

Comparative genomics reveals adaptations of a halotolerant thaumarchaeon in the interfaces of brine pools in the Red Sea.

The bottom of the Red Sea harbors over 25 deep hypersaline anoxic basins that are geochemically distinct and characterized by vertical gradients of extreme physicochemical conditions. Because of strong changes in density, particulate and microbial debris get entrapped in the brine-seawater interface (BSI), resulting in increased dissolved organic carbon, reduced dissolved oxygen toward the brines and enhanced microbial activities in the BSI. These features coupled with the deep-sea prevalence of ammonia-oxidizing archaea (AOA) in the global ocean make the BSI a suitable environment for studying the osmotic adaptations and ecology of these important players in the marine nitrogen cycle. Using phylogenomic-based approaches, we show that the local archaeal community of five different BSI habitats (with up to 18.2% salinity) is composed mostly of a single, highly abundant Nitrosopumilus-like phylotype that is phylogenetically distinct from the bathypelagic thaumarchaea; ammonia-oxidizing bacteria were absent. The composite genome of this novel Nitrosopumilus-like subpopulation (RSA3) co-assembled from multiple single-cell amplified genomes (SAGs) from one such BSI habitat further revealed that it shares ∼54% of its predicted genomic inventory with sequenced Nitrosopumilus species. RSA3 also carries several, albeit variable gene sets that further illuminate the phylogenetic diversity and metabolic plasticity of this genus. Specifically, it encodes for a putative proline-glutamate ‘switch’ with a potential role in osmotolerance and indirect impact on carbon and energy flows. Metagenomic fragment recruitment analyses against the composite RSA3 genome, Nitrosopumilus maritimus, and SAGs of mesopelagic thaumarchaea also reiterate the divergence of the BSI genotypes from other AOA.

Halorhabdus tiamatea: proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep‐sea anoxic brine lake as potential polysaccharide degrader

Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX-2T from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4BT from the Shaban deep-sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments.