Marine cyanolichens from different littoral zones are associated with distinct bacterial communities.

The microbial diversity and function of terrestrial lichens have been well studied, but knowledge about the non-photosynthetic bacteria associated with marine lichens is still scarce. 16S rRNA gene Illumina sequencing was used to assess the culture-independent bacterial diversity in the strictly marine cyanolichen species Lichina pygmaea and Lichina confinis, and the maritime chlorolichen species Xanthoria aureola which occupy different areas on the littoral zone. Inland terrestrial cyanolichens from Austria were also analysed as for the marine lichens to examine further the impact of habitat/lichen species on the associated bacterial communities. The L. confinis and L. pygmaea communities were significantly different from those of the maritime Xanthoria aureola lichen found higher up on the littoral zone and these latter communities were more similar to those of the inland terrestrial lichens. The strictly marine lichens were dominated by the Bacteroidetes phylum accounting for 50% of the sequences, whereas Alphaproteobacteria, notably Sphingomonas, dominated the maritime and the inland terrestrial lichens. Bacterial communities associated with the two Lichina species were significantly different sharing only 33 core OTUs, half of which were affiliated to the Bacteroidetes genera Rubricoccus, Tunicatimonas and Lewinella, suggesting an important role of these species in the marine Lichina lichen symbiosis. Marine cyanolichens showed a higher abundance of OTUs likely affiliated to moderately thermophilic and/or radiation resistant bacteria belonging to the Phyla Chloroflexi, Thermi, and the families Rhodothermaceae and Rubrobacteraceae when compared to those of inland terrestrial lichens. This most likely reflects the exposed and highly variable conditions to which they are subjected daily.

Author Correction: Surface properties of SAR11 bacteria facilitate grazing avoidance.

In the version of this Letter originally published, the authors incorrectly stated that primers 28F-519R were reported in ref. 54 to underestimate the abundance of SAR11 in the ocean. This statement has now been amended in all versions of the Letter.

Surface properties of SAR11 bacteria facilitate grazing avoidance.

Oceanic ecosystems are dominated by minute microorganisms that play a major role in food webs and biogeochemical cycles 1 . Many microorganisms thrive in the dilute environment due to their capacity to locate, attach to, and use patches of nutrients and organic matter 2,3 . We propose that some free-living planktonic bacteria have traded their ability to stick to nutrient-rich organic particles for a non-stick cell surface that helps them evade predation by mucous filter feeders. We used a combination of in situ sampling techniques and next-generation sequencing to study the biological filtration of microorganisms at the phylotype level. Our data indicate that some marine bacteria, most notably the highly abundant Pelagibacter ubique and most other members of the SAR 11 clade of the Alphaproteobacteria, can evade filtration by slipping through the mucous nets of both pelagic and benthic tunicates. While 0.3 µm polystyrene beads and other similarly-sized bacteria were efficiently filtered, SAR11 members were not captured. Reversed-phase chromatography revealed that most SAR11 bacteria have a much less hydrophobic cell surface than that of other planktonic bacteria. Our data call for a reconsideration of the role of surface properties in biological filtration and predator-prey interactions in aquatic systems.

Distinct Spatial Patterns of SAR11, SAR86, and Actinobacteria Diversity along a Transect in the Ultra-oligotrophic South Pacific Ocean.

Distinct distribution patterns of members of the major bacterial clades SAR11, SAR86, and Actinobacteria were observed across a transect from the Marquesas islands through the ultra-oligotrophic South Pacific Gyre into the Chilean upwelling using 16S rRNA gene sequencing and RNA-DNA fingerprinting. Three different Actinobacteria sequence clusters belonging to "Candidatus Actinomarinidae" were localized in the western half of the transect, one was limited to the gyre deep chlorophyll maximum (DCM) and sequences affiliated to the OCS155 clade were unique to the upwelling. The structure of the surface bacterial community was highly correlated with water mass and remained similar across the whole central gyre (1300 nautical miles). The surface hyperoligotrophic gyre was dominated (>70% of all sequences) by highly diverse SAR11 and SAR86 operational taxonomic units and these communities were significantly different from those in the DCM. Analysis of 16S rRNA fingerprints generated from RNA allowed insights into the potential activity of assigned bacterial groups. SAR11 and Prochlorococcus showed the highest potential activity in all water masses except for the upwelling, accounting together for 65% of the total bacterial 16S rRNA in the gyre surface waters in equal proportions whereas the contribution of SAR11 decreased significantly at the DCM.

Ecological succession leads to chemosynthesis in mats colonizing wood in sea water.

Chemosynthetic mats involved in cycling sulfur compounds are often found in hydrothermal vents, cold seeps and whale falls. However, there are only few records of wood fall mats, even though the presence of hydrogen sulfide at the wood surface should create a perfect niche for sulfide-oxidizing bacteria. Here we report the growth of microbial mats on wood incubated under conditions that simulate the Mediterranean deep-sea temperature and darkness. We used amplicon and metagenomic sequencing combined with fluorescence in situ hybridization to test whether a microbial succession occurs during mat formation and whether the wood fall mats present chemosynthetic features. We show that the wood surface was first colonized by sulfide-oxidizing bacteria belonging to the Arcobacter genus after only 30 days of immersion. Subsequently, the number of sulfate reducers increased and the dominant Arcobacter phylotype changed. The ecological succession was reflected by a change in the metabolic potential of the community from chemolithoheterotrophs to potential chemolithoautotrophs. Our work provides clear evidence for the chemosynthetic nature of wood fall ecosystems and demonstrates the utility to develop experimental incubation in the laboratory to study deep-sea chemosynthetic mats.

A single betaproteobacterium dominates the microbial community of the crambescidine-containing sponge Crambe crambe.

Crambe crambe is a marine sponge that produces high concentrations of the pharmacologically significant pentacyclic guanidine alkaloids (PGAs), Crambescines and Crambescidines. Although bio-mimetic chemical synthesis of PGAs suggests involvement of microorganisms in their biosynthesis, there are conflicting reports on whether bacteria are associated with this sponge or not. Using 16S rRNA gene pyrosequencing we show that the associated bacterial community of C. crambe is dominated by a single bacterial species affiliated to the Betaproteobacteria. Microscopy analysis of sponge tissue sections using a specific probe and in situ hybridization confirmed its dominance in the sponge mesohyl and a single microbial morphology was observed by transmission electron microscopy. If confirmed the presence of a simple bacteria community in C. crambe makes this association a very pertinent model to study sponge-bacteria interactions and should allow further research into the possible implication of bacteria in PGA biosynthesis.

A novel clade of Prochlorococcus found in high nutrient low chlorophyll waters in the South and Equatorial Pacific Ocean.

A novel high-light (HL)-adapted Prochlorococcus clade was discovered in high nutrient and low chlorophyll (HNLC) waters in the South Pacific Ocean by phylogenetic analyses of 16S ribosomal RNA (rRNA) and 16S-23S internal transcribed spacer (ITS) sequences. This clade, named HNLC fell within the HL-adapted Prochlorococcus clade with sequences above 99% similarity to one another, and was divided into two subclades, HNLC1 and HNLC2. The distribution of the whole HNLC clade in a northwest to southeast transect in the South Pacific (HNLC-to-gyre) and two 8°N to 8°S transects in the Equatorial Pacific was determined by quantitative PCR using specific primers targeting ITS regions. HNLC was the dominant HL Prochlorococcus clade (2-9% of bacterial 16S rRNA genes) at the three westernmost stations in the South Pacific but decreased to less than 0.1% at the other stations being replaced by the eMIT9312 ecotype in the hyperoligotrophic gyre. The highest contributions of HNLC Prochlorococcus in both Equatorial Pacific transects along the latitudinal lines of 170°W and 155°W were observed at the southernmost stations, reaching 16 and 6% of bacterial 16S rRNA genes, respectively, whereas eMIT9312 dominated near the Equator. Spearman Rank Order correlation analysis indicated that although both the HNLC clade and eMIT9312 were correlated with temperature, they showed different correlations with regard to nutrients. HNLC only showed significant correlations to ammonium uptake and regeneration rates, whereas eMIT9312 was negatively correlated with inorganic nutrients.

Impact of lower salinity waters on bacterial heterotrophic production and community structure in the offshore NW Mediterranean Sea.

We investigated the impact of water masses originating from freshwater input on bacterial heterotrophic metabolism and community structure at an offshore site in the oligotrophic NW Mediterranean Sea in 2007 and 2008. By combining 16S rRNA gene clone libraries and MICRO-CARD-FISH we determined the dominant operational taxonomic units (OTU) and their contribution to bulk abundance and activity in the presence of buoyant water masses characterized by lower salinity (LSW, < 37.9) and compared these with the winter and spring phytoplankton blooms. We demonstrate that organic matter associated with LSW markedly stimulated bacterial heterotrophic production as determined by [(3) H]-leucine incorporation. The OTUs SAR11-IA, SAR11-IIB, SAR86-I and SAR86-III were dominant in all clone libraries, while the Roseobacter clade and the Bacteroidetes OTU NorSea72 were more specific to the spring phytoplankton bloom. The relative contribution of these OTUs to leucine incorporation varied between 23% and 69% for SAR11, 2% and 17% for Roseobacter and was up to 4% for NorSea72. Together, they accounted for roughly 50% of bulk abundance and leucine incorporation during the four situations investigated. Our results suggest that a few cosmopolitan OTUs respond to different DOM sources in the NW Mediterranean Sea.

Major differences of bacterial diversity and activity inside and outside of a natural iron-fertilized phytoplankton bloom in the Southern Ocean.

One of the first comparisons of a natural iron fertilized bloom with a high-nutrient low-chlorophyll (HNLC) site was undertaken during the Kerguelen ocean and plateau compared study (KEOPS) cruise. To understand better the bacteria-phytoplankton relationship in the context of natural iron fertilization, bacterial diversity and activity was investigated in the bloom and in the adjacent HNLC region by 16S rDNA clone libraries and by single strand conformation polymorphism (SSCP) analysis. Both libraries were dominated by Alphaproteobacteria, Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cluster analysis at 99% sequence similarity yielded several microdiverse clusters and revealed striking differences between the two libraries. In the bloom, the dominant operational taxonomic units (OTUs) were the Roseobacter NAC11-7 cluster, SAR92 and a Cytophaga-Flavobacteria-Bacteroides cluster related to the agg58 group, whereas in the HNLC region, SAR11, Roseobacter RCA and Polaribacter dominated. SSCP analysis of 16S rDNA and 16S rRNA revealed contrasting dynamics of three different Roseobacter OTUs. Roseobacter NAC11-7 and NAC11-6 had higher relative abundances and activities in the bloom compared with the HNLC site and NAC11-6 was only detected at the decline of the bloom concomitant with a shift in phytoplankton composi tion. In contrast, Roseobacter RCA was relatively abundant and active both inside and outside of the bloom. These results suggest that the different OTUs within the Roseobacter group represent functional groups that each play an important role in the cycling of carbon.

High vertical and low horizontal diversity of Prochlorococcus ecotypes in the Mediterranean Sea in summer.

Natural populations of the marine cyanobacterium Prochlorococcus exist as two main ecotypes, inhabiting different layers of the ocean's photic zone. These so-called high light- (HL-) and low light (LL-) adapted ecotypes are both physiologically and genetically distinct. HL strains can be separated into two major clades (HLI and HLII), whereas LL strains are more diverse. Here, we used several molecular techniques to study the genetic diversity of natural Prochlorococcus populations during the Prosope cruise in the Mediterranean Sea in the summer of 1999. Using a dot blot hybridization technique, we found that HLI was the dominant HL group and was confined to the upper mixed layer. In contrast, LL ecotypes were only found below the thermocline. Secondly, a restriction fragment length polymorphism analysis of PCR-amplified pcb genes (encoding the major light-harvesting proteins of Prochlorococcus) suggested that there were at least four genetically different ecotypes, occupying distinct but overlapping light niches in the photic zone. At comparable depths, similar banding patterns were observed throughout the sampled area, suggesting a horizontal homogenization of ecotypes. Nevertheless, environmental pcb gene sequences retrieved from different depths at two stations proved all different at the nucleotide level, suggesting a large genetic microdiversity within those ecotypes.

Molecular ecology of the marine cyanobacterial genera Prochlorococcus and Synechococcus.

Oxygenic photoautotrophs of the genera Synechococcus and Prochlorococcus contribute significantly to primary production and are now widely accepted as the most abundant members of the picophytoplankton in the world's oceans. Since they represent one of the few cultured and representative groups of marine microorganisms, study of their physiology and biochemistry has progressed rapidly since their discovery. The recent and on-going sequencing of the complete genomes of representative strains will further hasten our understanding, and allow a complete interrogation, of the metabolism of these organisms. Moreover, since they inhabit a relatively simple environment they provide an excellent model system to begin to identify the underlying molecular mechanisms which allow their success in water columns with large vertical gradients of light and nutrients. Such work should provide novel insights into the genetic adaptations of these important marine microbes to their environment. We review here molecular ecological methods that are already available or which are currently being developed for these organisms. Such methods allow community structure, growth rate and nutrient status analysis, potentially at the single cell level, and can be used to define the niches, or identify the biotic or abiotic factors, which might control the productivity of specific genotypes. These techniques will undoubtedly provide the tools for answering more discerning questions concerning their ecology. How the complete genome sequence information is providing insights, and can further facilitate our understanding, of the ecology of these organisms is also discussed.

Closely related Prochlorococcus genotypes show remarkably different depth distributions in two oceanic regions as revealed by in situ hybridization using 16S rRNA-targeted oligonucleotides.

An in situ hybridization method was applied to the identification of marine cyanobacteria assignable to the genus Prochlorococcus using horseradish-peroxidase-labelled 16S rRNA-targeted oligonucleotide probes in combination with tyramide signal amplification (TSA). With this method very bright signals were obtained, in contrast to hybridizations with oligonucleotides monolabelled with fluorochromes, which failed to give positive signals. Genotype-specific oligonucleotides for high light (HL)- and low light (LL)-adapted members of this genus were identified by 16S rRNA sequence analyses and their specificities confirmed in whole-cell hybridizations with cultured strains of Prochlorococcus marinus Chisholm et al., 1992, Prochlorococcus sp. and Synechococcus sp. In situ hybridization of these genotype-specific probes to field samples from stratified water bodies collected in the North Atlantic Ocean and the Red Sea allowed a rapid assessment of the abundance and spatial distribution of HL- and LL-adapted Prochlorococcus. In both oceanic regions the LL-adapted Prochlorococcus populations were localized in deeper water whereas the HL-adapted Prochlorococcus populations were not only distinct in each region but also exhibited strikingly different depth distributions, HLI being confined to shallow water in the North Atlantic, in contrast to HLII, which was present throughout the water column in the Red Sea.