Roseobacter-Like Bacteria in Red and Mediterranean Sea Aerobic Anoxygenic Photosynthetic Populations

Bacteriochlorophyll a-containing aerobic anoxygenic phototrophs (AAnP) have been proposed to account for up to 11% of the total surface water microbial community and to potentially have great ecological importance in the world's oceans. Recently, environmental and genomic data based on analysis of the pufM gene identified the existence of α-proteobacteria as well as possible γ-like proteobacteria among AAnP in the Pacific Ocean. Here we report on analyses of environmental samples from the Red and Mediterranean Seas by using pufM as well as the bchX and bchL genes as molecular markers. The majority of photosynthesis genes retrieved from these seas were related to Roseobacter-like AAnP sequences. Furthermore, the sequence of a novel photosynthetic operon organization from an uncultured Roseobacter-like bacterial artificial chromosome retrieved from the Red Sea is described. The data show the presence of Roseobacter-like bacteria in Red and Mediterranean Sea AAnP populations in the seasons analyzed.     

Abundant presence of the gamma-like Proteobacterial pufM gene in oxic seawater

Known anoxygenic photosynthetic bacteria (APB) affiliated to Gammaproteobacteria usually use anaerobic metabolism and are restricted to oxygen-free habitats. Here, we report abundant (average of 34.5%) presence of diverse APB related to gamma-like Proteobacteria in oxic oceanic surface water as indicated by the pufM gene, that encodes the M subunit of the light reaction centre complex. Thus, our sequences were most likely derived from aerobic anoxygenic phototrophs (AAnP). Two genetically distinct genotypes were revealed: one was from the oligotrophic North Pacific Ocean Gyre and the other, was from the trophic East China Sea and Bering Sea. The discovery of abundant presence of novel gamma-like Proteobacterial pufM gene in the oxic seawater extends the functional ecotypes of AAnP.     

Putative novel photosynthetic reaction centre organizations in marine aerobic anoxygenic photosynthetic bacteria: insights from metagenomics and environmental genomics

Photosynthetic core complexes of anoxygenic bacteria consist of reaction centres (RCs) surrounded by light-harvesting complexes (LHC). The structural proteins of the RC-LHC1 complex are encoded by the puf-operon. We find diverse operon organizations of puf-operons that reflect structural differences of the core complex in marine aerobic anoxygenic photosynthetic bacteria (AAnP). By analysis of environmental DNA records coming from AAnP bacteria we find several unknown proteins downstream to the pufM, which were assigned as novel PufX proteins. As all known pufX genes belong to Rhodobacter strains which carry out anaerobic photosynthesis, this may be the first observation of a PufX-containing RCs in aerobic anoxygenic photosynthetic bacteria. Phylogenetic analyses of PufM proteins from cultured as well as from uncultured bacteria show that PufM from operons containing putative novel pufX genes are grouped with Rhodobacter and not with Roseobacter strains.     

Novel primers reveal wider diversity among marine aerobic anoxygenic phototrophs

Aerobic anoxygenic phototrophic bacteria (AAnPs) were previously proposed to account for up to 11% of marine bacterioplankton and to potentially have great ecological importance in the world's oceans. Our data show that previously used primers based on the M subunit of anoxygenic photosynthetic reaction center genes (pufM) do not comprehensively identify the diversity of AAnPs in the ocean. We have designed and tested a new set of pufM-specific primers and revealed several new AAnP variants in environmental DNA samples and genomic libraries.     

Diversity and distribution of ecotypes of the aerobic anoxygenic phototrophy gene pufM in the Delaware estuary

The diversity of aerobic anoxygenic phototrophic (AAP) bacteria has been examined in marine habitats, but the types of AAP bacteria in estuarine waters and distribution of ecotypes in any environment are not well known. The goal of this study was to determine the diversity of AAP bacteria in the Delaware estuary and to examine the distribution of select ecotypes using quantitative PCR (qPCR) assays for the pufM gene, which encodes a protein in the light reaction center of AAP bacteria. In PCR libraries from the Delaware River, pufM genes similar to those from Beta- (Rhodoferax-like) or Gammaproteobacteria comprised at least 50% of the clones, but the expressed pufM genes from the river were not dominated by these two groups in August 2002 (less than 31% of clones). In four transects, qPCR data indicated that the gammaproteobacterial type of pufM was abundant only near the mouth of the bay whereas Rhodoferax-like AAP bacteria were restricted to waters with a salinity of <5. In contrast, a Rhodobacter-like pufM gene was ubiquitous, but its distribution along the salinity gradient varied with the season. High fractions (12 to 24%) of all three pufM types were associated with particles. The data suggest that different groups of AAP bacteria are controlled by different environmental factors, which may explain current difficulties in predicting the distribution of total AAP bacteria in aquatic environments.     

Microbial community associated with the colonial ascidian Cystodytes dellechiajei

The ascidian Cystodytes dellechiajei (Della Valle, 1877) (phylum Chordata, class Ascidiacea, family Polycitoridae) is a colonial tunicate that inhabits benthic rock environments in the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean Sea. Its life cycle has two phases, the adult sessile colony and the free-living larva. Both adult zooids and larvae are surrounded by a protective tunic that contains several eukaryotic cell lines, is composed mainly of acidic mucopolysacharides associated with collagen and elastin-like proteins, and is covered by a thin cuticle. The microbiota associated with the tunic tissues of adult colonies and larva of C. dellechiajei has been examined by optical, confocal and electron microscopy and by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and 16S rRNA gene clone library analysis. Microscopy analyses indicated the presence inside the tunic, both for the adult and the larva, of a dense community of Bacteria while only the external surface of colony cuticle was colonized by diatoms, rodophyte algae and prokaryotic-like epiphytes. Transmission electron microscopy showed tunic eukaryotic cells that were engulfing and lysing bacteria. 16S rRNA gene analyses (DGGE and clone libraries) and FISH indicated that the community inside the tunic tissues of the adults and larvae was dominated by Alphaproteobacteria. Bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were also detected in the adults. Many of the 16S rRNA gene sequences in the tunic tissues were related to known aerobic anoxygenic phototrophs (AAP), like Roseobacter sp. and Erythrobacter sp. In order to check whether the gene pufM, coding for the M subunit of the reaction centre complex of aerobic anoxygenic photosynthesis, was being expressed inside the ascidian tissues, two libraries, one for an adult colony and one for larva, of cDNA from the expressed pufM gene were also constructed. The sequences most frequently (64% for colony and 67% for larva) retrieved from these libraries presented > 90% aa identity with the pufM gene product of the Roseobacter-like group, a cluster of AAP widely detected in marine planktonic environments.     

Genetic diversity assessment of anoxygenic photosynthetic bacteria by distance-based grouping analysis of pufM sequences

AIM: To assess how completely the diversity of anoxygenic phototrophic bacteria (APB) was sampled in natural environments. METHODS AND RESULTS: All nucleotide sequences of the APB marker gene pufM from cultures and environmental clones were retrieved from the GenBank database. A set of cutoff values (sequence distances 0.06, 0.15 and 0.48 for species, genus, and (sub)phylum levels, respectively) was established using a distance-based grouping program. Analysis of the environmental clones revealed that current efforts on APB isolation and sampling in natural environments are largely inadequate. Analysis of the average distance between each identified genus and an uncultured environmental pufM sequence indicated that the majority of cultured APB genera lack environmental representatives. CONCLUSIONS: The distance-based grouping method is fast and efficient for bulk functional gene sequences analysis. The results clearly show that we are at a relatively early stage in sampling the global richness of APB species. Periodical assessment will undoubtedly facilitate in-depth analysis of potential biogeographical distribution pattern of APB. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first attempt to assess the present understanding of APB diversity in natural environments. The method used is also useful for assessing the diversity of other functional genes.     

Response of Aerobic Anoxygenic Phototrophic Bacterial Diversity to Environment Conditions in Saline Lakes and Daotang River on the Tibetan Plateau,NW China

Variations of the pufM gene [encoding the M subunit of the photosynthetic reaction center in aerobic anoxygenic phototrophic (AAnP) bacteria] diversity in response to environmental changes were investigated in waters of six aqueous regimes (including Daotang River and five saline/hypersaline lakes on the Tibetan Plateau) representing a full salinity gradient from freshwater to NaCl-saturation. AAnP bacterial community structures responded to salinity change: Gamma-like AAnP community was predominant in freshwater Daotang River (0.01% salinity). AAnP community structure shifted from Loktanella-like sequences of the Alphaproteobacteria in saline Qinghai Lake to Roseobacter-like sequences in hypersaline lakes (Gahai, Xiaochaidan and Charhan). In addition to salinity, other environmental variables (e.g. N and P availability, TOC and/or DOC, and HCO^? ₃/CO^2? ₃ ions) were also important in affecting the pufM gene diversity in hypersaline lakes. These data have important implications for our understanding of the response of AAnP bacterial community to environmental variables in high-altitude aquatic ecosystems.     

Abundance, depth distribution, and composition of aerobic bacteriochlorophyll a-producing bacteria in four basins of the central Baltic Sea

The abundance, vertical distribution, and diversity of aerobic anoxygenic phototrophic bacteria (AAP) were studied at four basins of the Baltic Sea. AAP were enumerated by infrared epifluorescence microscopy, and their diversity was analyzed by using pufM gene clone libraries. In addition, numbers of CFU containing the pufM gene were determined, and representative strains were isolated. Both approaches indicated that AAP reached maximal abundance in the euphotic zone. Maximal AAP abundance was 2.5 x 10(5) cells ml(-1) (11% of total prokaryotes) or 1.0 x 10(3) CFU ml(-1) (9 to 10% of total CFU). Environmental pufM clone sequences were grouped into 11 operational taxonomic units phylogenetically related to cultivated members of the Alpha-, Beta-, and Gammaproteobacteria. In spite of varying pufM compositions, five clones were present in all libraries. Of these, Jannaschia-related clones were always found in relative abundances representing 25 to 30% of the total AAP clones. The abundances of the other clones varied. Clones potentially affiliated with typical freshwater Betaproteobacteria sequences were present at three Baltic Sea stations, whereas clones grouping with Loktanella represented 40% of the total cell numbers in the Gotland Basin. For three alphaproteobacterial clones, probable pufM phylogenetic relationships were supported by 16S rRNA gene analyses of Baltic AAP isolates, which showed nearly identical pufM sequences. Our data indicate that the studied AAP assemblages represented a mixture of marine and freshwater taxa, thus characterizing the Baltic Sea as a "melting pot" of abundant, polyphyletic aerobic photoheterotrophic bacteria.     

Source environment feature related phylogenetic distribution pattern of anoxygenic photosynthetic bacteria as revealed by pufM analysis

Anoxygenic photosynthesis, performed primarily by anoxygenic photosynthetic bacteria (APB), has been supposed to arise on Earth more than 3 billion years ago. The long established APB are distributed in almost every corner where light can reach. However, the relationship between APB phylogeny and source environments has been largely unexplored. Here we retrieved the pufM sequences and related source information of 89 pufM containing species from the public database. Phylogenetic analysis revealed that horizontal gene transfer (HGT) most likely occurred within 11 out of a total 21 pufM subgroups, not only among species within the same class but also among species of different phyla or subphyla. A clear source environment feature related phylogenetic distribution pattern was observed, with all species from oxic habitats and those from anoxic habitats clustering into independent subgroups, respectively. HGT among ancient APB and subsequent long term evolution and adaptation to separated niches may have contributed to the coupling of environment and pufM phylogeny.     

Novel Primers Reveal Wider Diversity among Marine Aerobic Anoxygenic Phototrophs

Aerobic anoxygenic phototrophic bacteria (AAnPs) were previously proposed to account for up to 11% of marine bacterioplankton and to potentially have great ecological importance in the world's oceans. Our data show that previously used primers based on the M subunit of anoxygenic photosynthetic reaction center genes (pufM) do not comprehensively identify the diversity of AAnPs in the ocean. We have designed and tested a new set of pufM-specific primers and revealed several new AAnP variants in environmental DNA samples and genomic libraries.     

Ecological traits and environmental affinity explain Red Sea fish introduction into the Mediterranean

Alien species are considered one of the prime threats to biodiversity, driving major changes in ecosystem structure and function. Identifying the traits associated with alien introduction has been largely restricted to comparing indigenous and alien species or comparing alien species that differ in abundance or impact. However, a more complete understanding may emerge when the entire pool of potential alien species is used as a control, information that is rarely available. In the eastern Mediterranean, the marine environment is undergoing an unparalleled species composition transformation, as a flood of aliens have entered from the Red Sea following the opening of the Suez Canal in 1869. In this study, we compile data on species traits, geographical distribution, and environmental affinity of the entire pool of reef‐associated fish species in the Red Sea and more generally across the Indo‐Pacific. We use this extensive data to identify the prime characteristics separating Red Sea species that have become alien in the Mediterranean from those that have not. We find that alien species occupy a larger range of environments in their native ranges, explaining their ability to colonize the seasonal Mediterranean. Red Sea species that naturally experience high maximum temperatures in their native range have a high probability of becoming alien. Thus, contrary to predictions of an accelerating number of aliens following increased water temperatures, hotter summers in this region may prevent the establishment of many alien species. We further find that ecological trait diversity of alien species is substantially more evenly spaced and more divergent than random samples from the pool of Red Sea species, pointing at additional processes, such as competition, promoting ecological diversity among alien species. We use these results to provide a first quantitative ranking of the potential of Red Sea species to become established in the eastern Mediterranean.     

The status of coral reef ecology research in the Red Sea

The Red Sea has long been recognized as a region of high biodiversity and endemism. Despite this diversity and early history of scientific work, our understanding of the ecology of coral reefs in the Red Sea has lagged behind that of other large coral reef systems. We carried out a quantitative assessment of ISI-listed research published from the Red Sea in eight specific topics (apex predators, connectivity, coral bleaching, coral reproductive biology, herbivory, marine protected areas, non-coral invertebrates and reef-associated bacteria) and compared the amount of research conducted in the Red Sea to that from Australia’s Great Barrier Reef (GBR) and the Caribbean. On average, for these eight topics, the Red Sea had 1/6th the amount of research compared to the GBR and about 1/8th the amount of the Caribbean. Further, more than 50 % of the published research from the Red Sea originated from the Gulf of Aqaba, a small area (<2 % of the area of the Red Sea) in the far northern Red Sea. We summarize the general state of knowledge in these eight topics and highlight the areas of future research priorities for the Red Sea region. Notably, data that could inform science-based management approaches are badly lacking in most Red Sea countries. The Red Sea, as a geologically “young” sea located in one of the warmest regions of the world, has the potential to provide insight into pressing topics such as speciation processes as well as the capacity of reef systems and organisms to adapt to global climate change. As one of the world’s most biodiverse coral reef regions, the Red Sea may yet have a significant role to play in our understanding of coral reef ecology at a global scale.     

Rapid microevolution during recent range expansion to harsh environments

Background

Adaptive evolution is one of the crucial mechanisms for organisms to survive and thrive in new environments. Recent studies suggest that adaptive evolution could rapidly occur in species to respond to novel environments or environmental challenges during range expansion. However, for environmental adaptation, many studies successfully detected phenotypic features associated with local environments, but did not provide ample genetic evidence on microevolutionary dynamics. It is therefore crucial to thoroughly investigate the genetic basis of rapid microevolution in response to environmental changes, in particular on what genes and associated variation are responsible for environmental challenges. Here, we genotyped genome-wide gene-associated microsatellites to detect genetic signatures of rapid microevolution of a marine tunicate invader, Ciona robusta, during recent range expansion to the harsh environment in the Red Sea.

Results

The Red Sea population was significantly differentiated from the other global populations. The genome-wide scan, as well as multiple analytical methods, successfully identified a set of adaptive genes. Interestingly, the allele frequency largely varied at several adaptive loci in the Red Sea population, and we found significant correlations between allele frequency and local environmental factors at these adaptive loci. Furthermore, a set of genes were annotated to get involved in local temperature and salinity adaptation, and the identified adaptive genes may largely contribute to the invasion success to harsh environments.

Conclusions

All the evidence obtained in this study clearly showed that environment-driven selection had left detectable signatures in the genome of Ciona robusta within a few generations. Such a rapid microevolutionary process is largely responsible for the harsh environmental adaptation and therefore contributes to invasion success in different aquatic ecosystems with largely varied environmental factors.

     

Different SAR86 subgroups harbour divergent proteorhodopsins

Proteorhodopsins (PRs), bacterial photoactive proton pumps, were originally detected in the uncultured marine gamma-proteobacterial SAR86 group. PRs are now known to occur in both the gamma and alpha marine proteobacterial lineages. Recent environmental shotgun sequence analysis in the Sargasso Sea has added yet more diversity, and a potentially broader taxonomic distribution, to the PR family. Much remains to be learned, however, about within-taxon PR variability and the broader organismal distribution of different PR types. We report here genomic analyses of large genome fragments from different subgroups of the SAR86 lineage, recovered from naturally occurring bacterioplankton populations in coastal Red Sea and open ocean Pacific waters. Sequence comparisons were performed on large bacterial artificial chromosomes (BACs) bearing both rRNA and PR genes, derived from different SAR86 subgroups. Our analyses indicated the presence of different PR sequence types within the same SAR86 rRNA subgroup. The data suggested that the distribution of particular PR types does not necessarily parallel the phylogenetic relationship inferred from highly conserved genes such as rRNA. Further analyses of the genomic regions flanking PR also revealed a potential pathway for the biosynthesis of retinal, the PR chromophore that is required to generate the functionally active photoprotein. Finally, comparison of our results with recently reported Sargasso Sea environmental shotgun sequence assemblies demonstrated the utility of BAC clones for interpreting environmental shotgun sequence data, much of which is represented in short contigs that have an overall low depth of coverage.     

Comparison of cultivation-dependent and molecular methods for studying the diversity of anoxygenic purple phototrophs in sediments of an eutrophic brackish lagoon

Phototrophic anoxygenic purple bacteria play a key role in many aquatic ecosystems by oxidizing sulfur compounds and low-molecular-weight organic compounds using light as energy source. In this study, molecular methods based upon pufM gene (photosynthetic unit forming gene) were compared with culture-dependent methods to investigate anoxygenic purple phototrophic communities in sediments of an eutrophic brackish lagoon. Thirteen strains, belonging to eight different genera of purple phototrophic bacteria were isolated with a large dominance of the metabolically versatile purple non-sulfur bacteria (eight strains), some purple sulfur bacteria (three strains) and two strains belonging to the Roseobacter clade (aerobic phototrophs). The pufM genes amplified from the isolated strains were not detected by the molecular methods [terminal-restriction fragment length polymorphism (T-RFLP)] applied on in situ communities. An environmental clone library of the pufM gene was thus constructed from sediment samples. The results showed that most of the clones probably corresponded to aerobic phototrophic bacteria. Our results demonstrate that the culture-dependent techniques remain the best experimental approach for determining the diversity of phototrophic purple non-sulfur bacteria whereas the molecular approach clearly illustrated the abundance of organisms related to the Roseobacter clade in these eutrophic sediments.     

Thermal refugia against coral bleaching throughout the northern Red Sea

Tropical reefs have been impacted by thermal anomalies caused by global warming that induced coral bleaching and mortality events globally. However, there have only been very few recordings of bleaching within the Red Sea despite covering a latitudinal range of 15° and consequently it has been considered a region that is less sensitive to thermal anomalies. We therefore examined historical patterns of sea surface temperature (SST) and associated anomalies (1982–2012) and compared warming trends with a unique compilation of corresponding coral bleaching records from throughout the region. These data indicated that the northern Red Sea has not experienced mass bleaching despite intensive Degree Heating Weeks (DHW) of >15°C‐weeks. Severe bleaching was restricted to the central and southern Red Sea where DHWs have been more frequent, but far less intense (DHWs <4°C‐weeks). A similar pattern was observed during the 2015–2016 El Niño event during which time corals in the northern Red Sea did not bleach despite high thermal stress (i.e. DHWs >8°C‐weeks), and bleaching was restricted to the central and southern Red Sea despite the lower thermal stress (DHWs < 8°C‐weeks). Heat stress assays carried out in the northern (Hurghada) and central (Thuwal) Red Sea on four key reef‐building species confirmed different regional thermal susceptibility, and that central Red Sea corals are more sensitive to thermal anomalies as compared to those from the north. Together, our data demonstrate that corals in the northern Red Sea have a much higher heat tolerance than their prevailing temperature regime would suggest. In contrast, corals from the central Red Sea are close to their thermal limits, which closely match the maximum annual water temperatures. The northern Red Sea harbours reef‐building corals that live well below their bleaching thresholds and thus we propose that the region represents a thermal refuge of global importance.     

Population genomic response to geographic gradients by widespread and endemic fishes of the Arabian Peninsula

Genetic structure within marine species may be driven by local adaptation to their environment, or alternatively by historical processes, such as geographic isolation. The gulfs and seas bordering the Arabian Peninsula offer an ideal setting to examine connectivity patterns in coral reef fishes with respect to environmental gradients and vicariance. The Red Sea is characterized by a unique marine fauna, historical periods of desiccation and isolation, as well as environmental gradients in salinity, temperature, and primary productivity that vary both by latitude and by season. The adjacent Arabian Sea is characterized by a sharper environmental gradient, ranging from extensive coral cover and warm temperatures in the southwest, to sparse coral cover, cooler temperatures, and seasonal upwelling in the northeast. Reef fish, however, are not confined to these seas, with some Red Sea fishes extending varying distances into the northern Arabian Sea, while their pelagic larvae are presumably capable of much greater dispersal. These species must therefore cope with a diversity of conditions that invoke the possibility of steep clines in natural selection. Here, we test for genetic structure in two widespread reef fish species (a butterflyfish and surgeonfish) and eight range‐restricted butterflyfishes across the Red Sea and Arabian Sea using genome‐wide single nucleotide polymorphisms. We performed multiple matrix regression with randomization analyses on genetic distances for all species, as well as reconstructed scenarios for population subdivision in the species with signatures of isolation. We found that (a) widespread species displayed more genetic subdivision than regional endemics and (b) this genetic structure was not correlated with contemporary environmental parameters but instead may reflect historical events. We propose that the endemic species may be adapted to a diversity of local conditions, but the widespread species are instead subject to ecological filtering where different combinations of genotypes persist under divergent ecological regimes.     

Screening the Sargasso Sea metagenome for data to investigate genome evolution in Ostreococcus (Prasinophyceae, Chlorophyta)

The Sargasso Sea water shotgun sequencing unveiled an unprecedented glimpse of marine prokaryotic diversity and gene content. The sequence data was gathered from 0.8 microm filtered surface water extracts, and revealed picoeukaryotic (cell size<2 microm) sequences alongside the prokaryotic data. We used the available genome sequence of the picoeukaryote Ostreococcus tauri (Prasinophyceae, Chlorophyta) as a benchmark for the eukaryotic sequence content of the Sargasso Sea metagenome. Sequence data from at least two new Ostreococcus strains were identified and analyzed, and showed a bias towards higher coverage of the AT-rich organellar genomes. The Ostreococcus nuclear sequence data retrieved from the Sargasso metagenome is divided onto 731 scaffolds of average size 3917 bp, and covers 23% of the complete nuclear genome and 14% of the total number of protein coding genes in O. tauri. We used this environmental Ostreococcus sequence data to estimate the level of constraint on intronic and intergenic sequences in this compact genome.