Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece

hree halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2–97.1 %. The DNA–DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75 %, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2 %, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5–66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T =NCIMB 14426T =ATCC BAA-1602T)

Complex nitrogen cycling in the sponge Geodia barretti

Marine sponges constitute major parts of coral reefs and deep-water communities. They often harbour high amounts of phylogenetically and physiologically diverse microbes, which are so far poorly characterized. Many of these sponges regulate their internal oxygen concentration by modulating their ventilation behaviour providing a suitable habitat for both aerobic and anaerobic microbes. In the present study, both aerobic (nitrification) and anaerobic (denitrification, anammox) microbial processes of the nitrogen cycle were quantified in the sponge Geodia barretti and possible involved microbes were identified by molecular techniques. Nitrification rates of 566 nmol N cm(-3) sponge day(-1) were obtained when monitoring the production of nitrite and nitrate. In support of this finding, ammonia-oxidizing Archaea (crenarchaeotes) were found by amplification of the amoA gene, and nitrite-oxidizing bacteria of the genus Nitrospira were detected based on rRNA gene analyses. Incubation experiments with stable isotopes ((15)NO(3)(-) and (15)NH(4)(+)) revealed denitrification and anaerobic ammonium oxidation (anammox) rates of 92 nmol N cm(-3) sponge day(-1) and 3 nmol N cm(-3) sponge day(-1) respectively. Accordingly, sequences closely related to 'Candidatus Scalindua sorokinii' and 'Candidatus Scalindua brodae' were detected in 16S rRNA gene libraries. The amplification of the nirS gene revealed the presence of denitrifiers, likely belonging to the Betaproteobacteria. This is the first proof of anammox and denitrification in the same animal host, and the first proof of anammox and denitrification in sponges. The close and complex interactions of aerobic, anaerobic, autotrophic and heterotrophic microbial processes are fuelled by metabolic waste products of the sponge host, and enable efficient utilization and recirculation of nutrients within the sponge-microbe system. Since denitrification and anammox remove inorganic nitrogen from the environment, sponges may function as so far unrecognized nitrogen sinks in the ocean. In certain marine environments with high sponge cover, sponge-mediated nitrogen mineralization processes might even be more important than sediment processes

Effects of sample handling and cultivation bias on the specificity of bacterial communities in keratose marine sponges

Complex and distinct bacterial communities inhabit marine sponges and are believed to be essential to host survival, but our present-day inability to domesticate sponge symbionts in the laboratory hinders our access to the full metabolic breadth of these microbial consortia. We address bacterial cultivation bias in marine sponges using a procedure that enables direct comparison between cultivated and uncultivated symbiont community structures. Bacterial community profiling of the sympatric keratose species Sarcotragus spinosulus and lrcinia variabilis (Dictyoceratida, Irciniidae) was performed by polymerase chain reaction-denaturing gradient gel electrophoresis and 454-pyrosequecing of 16S rRNA gene fragments. Whereas cultivation-independent methods revealed species-specific bacterial community structures in these hosts, cultivation-dependent methods resulted in equivalent community assemblages from both species. Between 15 and 18 bacterial phyla were found in S. spinosulus and I. variabilis using cultivation-independent methods. However, Alphaproteobacteria and Gammaproteobacteria dominated the cultivation-dependent bacterial community. While cultivation-independent methods revealed about 200 and 220 operational taxonomic units (OTUs, 97% gene similarity) in S. spinosulus and I. variabilis, respectively, only 33 and 39 OTUs were found in these species via culturing. Nevertheless, around 50% of all cultured OTUs escaped detection by cultivation-independent methods, indicating that standard cultivation makes otherwise host-specific bacterial communities similar by selectively enriching for rarer and generalist symbionts. This study sheds new light on the diversity spectrum encompassed by cultivated and uncultivated sponge-associated bacteria. Moreover, it highlights the need to develop alternative culturing technologies to capture the dominant sponge symbiont fraction that currently remains recalcitrant to laboratory manipulation.Portuguese Foundation for Science and Technology (FCT) [PTDC/MAR/101431/2008]; FCT [SFRH/BD/60873/2009]; FCT postdoctoral fellowship [SFRII/BPD/62946/2009]; European Regional Development Fund (ERDF) through the COMPETE (Operational Competitiveness) Programme; FCT under the project [PEst-C/MAR/LA0015/2011]; FCT-funded research project [PTDC/BIA-MIC/3865/2012]info:eu-repo/semantics/publishedVersio

Reconstruction of Ribosomal RNA Genes from Metagenomic Data

Direct sequencing of environmental DNA (metagenomics) has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes

Ammonia-oxidizing archaea as main drivers of nitrification in cold-water sponges

The association of archaea with marine sponges was first described 15 years ago and their role in the nitrification process in Mediterranean and tropical sponges has been suggested. Here we explore the occurrence and abundance of potential ammonia-oxidizing archaea (AOA) in four morphologically different cold-water sponges (Phakellia ventilabrum, Geodia barretti, Antho dichotoma and Tentorium semisuberites) from the sublittoral and upper bathyal zone [Correction added on 30 December 2011, after first online publication on 19 December 2011: The term 'mesopelagic zone' has been replaced.] of the Norwegian coast, and relate them to nitrification rates determined in laboratory incubations. Net nitrification rates, calculated from the sum of nitrite and nitrate release during 24 h, were up to 1880 nmol N cm(-3) day(-1); i.e. comparable with those measured in Mediterranean sponges. Furthermore, a high abundance of archaeal cells was determined by fluorescence in situ hybridizations (CARD-FISH) and quantitative PCR, targeting archaeal amoA genes (encoding the alpha subunit of ammonia monooxygenase). AmoA genes as well as amoA transcripts were either exclusively detectable from archaea or were orders of magnitudes higher in abundance than their bacterial counterparts. Phylogenetic analyses of AOA and bacterial nitrite oxidizers (genus Nitrospira) confirmed the presence of specific populations of nitrifying microorganisms in the sponge mesohyl, which either were affiliated with groups detected earlier in marine sponges or were typical inhabitants of cold- and deep-water environments. Estimated cell-specific nitrification rates for P. ventilabrum were 0.6 to 6 fmol N archaeal cell(-1) day(-1), thus comparable with planktonic organisms. Our results identify AOA as the major drivers of nitrification in four cold-water sponges, and indicate that these archaea may be considered as a relevant factor in nitrogen cycling in ocean regions with high sponge biomass