Sponge-specific clusters revisited: a comprehensive phylogeny of sponge-associated microorganisms

Marine sponges often contain diverse and abundant communities of microorganisms including bacteria, archaea and eukaryotic microbes. Numerous 16S rRNA-based studies have identified putative 'sponge-specific' microbes that are apparently absent from seawater and other (non-sponge) marine habitats. With more than 7500 sponge-derived rRNA sequences (from clone, isolate and denaturing gradient gel electrophoresis data) now publicly available, we sought to determine whether the current notion of sponge-specific sequence clusters remains valid. Comprehensive phylogenetic analyses were performed on the 7546 sponge-derived 16S and 18S rRNA sequences that were publicly available in early 2010. Overall, 27% of all sequences fell into monophyletic, sponge-specific sequence clusters. Such clusters were particularly well represented among the Chloroflexi, Cyanobacteria, 'Poribacteria', Betaproteobacteria and Acidobacteria, and in total were identified in at least 14 bacterial phyla, as well as the Archaea and fungi. The largest sponge-specific cluster, representing the cyanobacterium 'Synechococcus spongiarum', contained 245 sequences from 40 sponge species. These results strongly support the existence of sponge-specific microbes and provide a suitable framework for future studies of rare and abundant sponge symbionts, both of which can now be studied using next-generation sequencing technologies.     

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.     

Cyanobacterial genomics for ecology and biotechnology

Cyanobacteria are the only prokaryotes that directly convert solar energy and CO(2) into organic matter by oxygenic photosynthesis, explaining their relevance for primary production in many ecosystems and the increasing interest for biotechnology. At present, there are more than 60 cyanobacteria for which a total genome sequence is publicly available. These cyanobacteria belong to different lifestyles and origins, coming from marine and freshwater aquatic environments, as well as terrestrial and symbiotic habitats. Genome sizes vary by a factor of six, from 1.44 Mb to 9.05 Mb, with the number of reported genes ranging from 1241 to 8462. Several studies have demonstrated how these sequences could be used to successfully infer important ecological, physiological and biotechnologically relevant characteristics. However, sequences of cyanobacterial origin also comprise a significant portion of certain metagenomes. Moreover, genome analysis has been employed for culture-independent approaches and for resequencing mutant strains, a very recent tool in cyanobacterial research.     

In silico comparison of bacterial strains using mutual information

Fast-sequencing throughput methods have increased the number of completely sequenced bacterial genomes to about 400 by December 2006, with the number increasing rapidly. These include several strains. In silico methods of comparative genomics are of use in categorizing and phylogenetically sorting these bacteria. Various word-based tools have been used for quantifying the similarities and differences between entire genomes. The simple di-nucleotide frequency comparison, codon specificity and k-mer repeat detection are among some of the well-known methods. In this paper, we show that the Mutual Information function, which is a measure of correlations and a concept from Information Theory, is very effective in determining the similarities and differences among genome sequences of various strains of bacteria such as the plant pathogen Xylella fastidiosa, marine Cyanobacteria Prochlorococcus marinus or animal and human pathogens such as species of Ehrlichia and Legionella. The short-range three-base periodicity, small sequence repeats and long-range correlations taken together constitute a genome signature that can be used as a technique for identifying new bacterial strains with the help of strains already catalogued in the database. There have been several applications of using the Mutual Information function as a measure of correlations in genomics but this is the first whole genome analysis done to detect strain similarities and differences.     

Dark side of cyanobacteria: searching for strategies to control blooms

Cyanobacteria are ecologically one of the most prolific groups of photosynthetic prokaryotes in marine and freshwater habitats. They are primary producer microorganisms and are involved in the production of important secondary metabolites, including toxic compounds such as cyanotoxins. Environmental conditions promote massive growth of these microbes, causing blooms that can have critical ecological and public health implications. In this highlight, we discuss some of the approaches being addressed to prevent these blooms, such as control of nutrient loading, treatments to minimize growth or monitoring interactions with other species.     

Taxonomic and functional microbial signatures of the endemic marine sponge Arenosclera brasiliensis

The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (～150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome.     

The Planctomycetes, Verrucomicrobia, Chlamydiae and sister phyla comprise a superphylum with biotechnological and medical relevance

In the rRNA-based tree of life four bacterial phyla, comprising the Planctomycetes, Verrucomicrobia, Chlamydiae and Lentisphaerae, form together with the candidate phyla Poribacteria and OP3 a monophyletic group referred to as the PVC superphylum. This assemblage contains organisms that possess dramatically different lifestyles and which colonize sharply contrasting habitats. Some members of this group are among the most successful human pathogens, others are abundant soil microbes, and others still are of major importance for the marine nitrogen cycle and hold much promise for sustainable wastewater treatment. Recent comparative genomic and metagenomic analyses of a few representatives of this group revealed many unusual features and generated unexpected hypotheses regarding their physiology, some of which have already been confirmed experimentally. Furthermore, the availability of these genome sequences offered new insights into the evolutionary history of this peculiar group of microbes with major medical, ecological and biotechnological relevance.     

Ma-LMM01 infecting toxic Microcystis aeruginosa illuminates diverse cyanophage genome strategies

Cyanobacteria and their phages are significant microbial components of the freshwater and marine environments. We identified a lytic phage, Ma-LMM01, infecting Microcystis aeruginosa, a cyanobacterium that forms toxic blooms on the surfaces of freshwater lakes. Here, we describe the first sequenced freshwater cyanomyovirus genome of Ma-LMM01. The linear, circularly permuted, and terminally redundant genome has 162,109 bp and contains 184 predicted protein-coding genes and two tRNA genes. The genome exhibits no colinearity with previously sequenced genomes of cyanomyoviruses or other Myoviridae. The majority of the predicted genes have no detectable homologues in the databases. These findings indicate that Ma-LMM01 is a member of a new lineage of the Myoviridae family. The genome lacks homologues for the photosynthetic genes that are prevalent in marine cyanophages. However, it has a homologue of nblA, which is essential for the degradation of the major cyanobacteria light-harvesting complex, the phycobilisomes. The genome codes for a site-specific recombinase and two prophage antirepressors, suggesting that it has the capacity to integrate into the host genome. Ma-LMM01 possesses six genes, including three coding for transposases, that are highly similar to homologues found in cyanobacteria, suggesting that recent gene transfers have occurred between Ma-LMM01 and its host. We propose that the Ma-LMM01 NblA homologue possibly reduces the absorption of excess light energy and confers benefits to the phage living in surface waters. This phage genome study suggests that light is central in the phage-cyanobacterium relationships where the viruses use diverse genetic strategies to control their host's photosynthesis.     

Comparative Genomic Analysis of Archaeal Genotypic Variants in a Single Population and in Two Different Oceanic Provinces

Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated.     

Comparative genomic analysis of archaeal genotypic variants in a single population and in two different oceanic provinces.

Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated.     

<Emphasis Type="Italic">In silico</Emphasis> comparison of bacterial strains using mutual information

Fast-sequencing throughput methods have increased the number of completely sequenced bacterial genomes to about 400 by December 2006, with the number increasing rapidly. These include several strains. In silico methods of comparative genomics are of use in categorizing and phylogenetically sorting these bacteria. Various word-based tools have been used for quantifying the similarities and differences between entire genomes. The simple di-nucleotide frequency comparison, codon specificity and k-mer repeat detection are among some of the well-known methods.In this paper, we show that the Mutual Information function, which is a measure of correlations and a concept from Information Theory, is very effective in determining the similarities and differences among genome sequences of various strains of bacteria such as the plant pathogen Xylella fastidiosa, marine Cyanobacteria Prochlorococcus marinus or animal and human pathogens such as species of Ehrlichia and Legionella. The short-range three-base periodicity, small sequence repeats and long-range correlations taken together constitute a genome signature that can be used as a technique for identifying new bacterial strains with the help of strains already catalogued in the database.There have been several applications of using the Mutual Information function as a measure of correlations in genomics but this is the first whole genome analysis done to detect strain similarities and differences.     

Molecular signatures for the main phyla of photosynthetic bacteria and their subgroups

The bacterial groups corresponding to different photosynthetic prokaryotes are presently identified mainly on the basis of their branching in phylogenetic trees. The availability of genome sequences is enabling identification of many molecular signatures that are specific for different groups of photosynthetic bacteria. Our recent work has identified large numbers of signatures consisting of conserved inserts or deletions (indels) in widely distributed proteins, as well as whole proteins that are specific for various sequenced species/strains from Cyanobacteria, Chlorobi, and Proteobacteria phyla. Based upon these signatures, it is now possible to identify/distinguish bacteria from these phyla of photosynthetic bacteria as well as their major subclades in clear molecular terms. The use of these signatures in conjunction with phylogenomic analyses, summarized here, is leading to a holistic picture concerning the branching order and evolutionary relationships among the above groups of photosynthetic bacteria. Although detailed studies in this regard have not yet been carried on Chloroflexi and Heliobacteriaceae, we have identified some conserved indels that are specific for these groups. Some of the conserved indels for the photosynthetic bacteria are present in photosynthesis-related proteins. These include a 4 aa insert in the pyruvate flavodoxin/ferridoxin oxidoreductase that is specific for the genus Chloroflexus, a 2 aa insert in magnesium chelatase that is uniquely shared by all Cyanobacteria except the deepest branching Clade A (Gloebacterales), a 6 aa insert in an A-type flavoprotein that is specific for various marine unicellular Cyanobacteria, a 2 aa insert in heme oxygenase that is specific for various Prochlorococcus strains/isolates, and 1 aa deletion in the protein protochlorophyllide oxidoreductase that is commonly shared by various Prochlorococcus strains except the deepest branching isolates MIT 9303 and MIT 9313. The identified CSIs are located in the structures of these proteins in surface loops indicating that they may be important in mediating protein–protein interactions. The cellular functions of these conserved indels, or most of the signature proteins are presently unknown, but they provide valuable means for discovering novel properties that are unique to different groups of photosynthetic bacteria.     

Cyanobacterial assimilatory nitrate reductase gene diversity in coastal and oligotrophic marine environments

Cyanobacteria are important primary producers in many marine ecosystems and their abundances and growth rates depend on their ability to assimilate various nitrogen sources. To examine the diversity of nitrate-utilizing marine cyanobacteria, we developed PCR primers specific for cyanobacterial assimilatory nitrate reductase (narB) genes. We obtained amplification products from diverse strains of cultivated cyanobacteria and from several marine environments. Phylogenetic trees constructed with the narB gene are congruent with those based on ribosomal RNA genes and RNA polymerase genes. Analysis of sequence library data from coastal and oligotrophic marine environments shows distinct groups of Synechococcus sp. in each environment; some of which are represented by sequences from cultivated organisms and others that are unrelated to known sequences and likely represent novel phylogenetic groups. We observed spatial differences in the distribution of sequences between two sites in Monterey Bay and differences in the vertical distribution of sequence types at the Hawai'i Ocean Time-series Station ALOHA, suggesting that nitrogen assimilation in Synechococcus living in different ecological niches can be followed with the nitrate reductase gene.     

Impact of genomics on microbial food safety

Genome sequences are now available for many of the microbes that cause food-borne diseases. The information contained in pathogen genome sequences, together with the development of themed and whole-genome DNA microarrays and improved proteomics techniques, might provide tools for the rapid detection and identification of such organisms, for assessing their biological diversity and for understanding their ability to respond to stress. The genomic information also provides insight into the metabolic capacity and versatility of microbes; for example, specific metabolic pathways might contribute to the growth and survival of pathogens in a range of niches, such as food-processing environments and the human host. New concepts are emerging about how pathogens function, both within foods and in interactions with the host. The future should bring the first practical benefits of genome sequencing to the field of microbial food safety, including strategies and tools for the identification and control of emerging pathogens.     

New records of benthic marine algae and Cyanobacteria for Costa Rica,and a comparison with other Central American countries

We present the results of an intensive sampling program carried out from 2000 to 2007 along both coasts of Costa Rica, Central America. The presence of 44 species of benthic marine algae is reported for the first time for Costa Rica. Most of the new records are Rhodophyta (27 spp.), followed by Chlorophyta (15 spp.), and Heterokontophyta, Phaeophycea (2 spp.). Overall, the currently known marine flora of Costa Rica is comprised of 446 benthic marine algae and 24 Cyanobacteria. This species number is an under estimation, and will increase when species of benthic marine algae from taxonomic groups where only limited information is available (e.g., microfilamentous benthic marine algae, Cyanobacteria) are included. The Caribbean coast harbors considerably more benthic marine algae (318 spp.) than the Pacific coast (190 spp.); such a trend has been observed in all neighboring countries. Compared to other Central American countries, Costa Rica has the highest number of reported benthic marine algae; however, Panama may have a similarly high diversity after unpublished results from a Rhodophyta survey (Wysor, unpublished) are included. Sixty-two species have been found along both the Pacific and Caribbean coasts of Costa Rica; we discuss this result in relation to the emergence of the Central American Isthmus.     

Complete Genome Sequences of Two Persicivirga Bacteriophages, P12024S and P12024L

The phylum Bacteroidetes is one of the major bacterial phyla in marine environments, where bacteriophages are highly abundant. Bacteriophages infecting members of the phylum Bacteroidetes, however, have not been well represented in public genome databases. Here we announce the genome sequences of two bacteriophages, P12024S and P12024L, that were isolated from coastal seawater and lytically infect Persicivirga sp. IMCC12024, a marine Bacteroidetes bacterium.     

Synechococcus: 3 billion years of global dominance

Cyanobacteria are among the most important primary producers on the Earth. However, the evolutionary forces driving cyanobacterial species diversity remain largely enigmatic due to both their distinction from macro‐organisms and an undersampling of sequenced genomes. Thus, we present a new genome of a Synechococcus‐like cyanobacterium from a novel evolutionary lineage. Further, we analyse all existing 16S rRNA sequences and genomes of Synechococcus‐like cyanobacteria. Chronograms showed extremely polyphyletic relationships in Synechococcus, which has not been observed in any other cyanobacteria. Moreover, most Synechococcus lineages bifurcated after the Great Oxidation Event, including the most abundant marine picoplankton lineage. Quantification of horizontal gene transfer among 70 cyanobacterial genomes revealed significant differences among studied genomes. Horizontal gene transfer levels were not correlated with ecology, genome size or phenotype, but were correlated with the age of divergence. All findings were synthetized into a novel model of cyanobacterial evolution, characterized by serial convergence of the features, that is multicellularity and ecology.     

Exploring novel bioactive compounds from marine microbes

The historical paradigm of the deep ocean as a biological 'desert' has shifted to one of a 'rainforest' owing to the isolation of many novel microbes and their associated bioactive compounds. Recently, there has been an explosion of information about novel bioactive compounds that have been isolated from marine microbes in an effort to further explore the relatively untapped marine microbes and their secondary metabolites for drug discovery. The microbes are recovered and purified from the ocean by both conventional and innovative isolation methods to obtain those previously thought to be 'uncultivable'. To overcome the difficulties and limitations associated with cultivation techniques, several DNA-based molecular methods have been developed to bypass the culture-dependent bottleneck. Bioactive compounds isolated using the above strategies have not only shown importance in biotechnological and pharmaceutical applications but have also increased our understanding of the diversity of marine microbiota, ecosystem functions and the exploitable biology.     

Marine phage genomics: what have we learned?

Marine phages are the most abundant and diverse form of life on the planet, and their genomes have been described as the largest untapped reservoir of genomic information. To date, however, the complete genome sequences of only 17 marine phage are known. Nevertheless, these genomes have revealed some interesting features, including the presence of photosynthetic genes in cyanophage and common patterns of genomic organization. Intriguing findings are also being made from studies of the uncultivated marine viral community genome ('metavirome'). The greatest challenge in interpreting the biology of these phages, and for making comparisons with their terrestrial counterparts, is the high proportion of unidentifiable open reading frames (approximately 60%). Future studies are likely to focus on sequencing more marine phage genomes from disparate hosts and diverse environments and on further basic studies of the biology of existing marine phages.