Breviolum and Cladocopium Are Dominant Among Symbiodiniaceae of the Coral Holobiont Madracis decactis.

The scleractinian reef building coral Madracis decactis is a cosmopolitan species. Understanding host-symbiont associations is critical for assessing coral's habitat requirements and its response to environmental changes. In this study, we performed a fine grained phylogenetic analyses of Symbiodiniaceae associated with Madracis in two locations in the Southwest Atlantic Ocean (Abrolhos Bank and St. Peter and St. Paul Archipelago). Previous studies have argued that Madracis is a specialist coral, with colonies harboring a single symbiont from the genus Breviolum (formerly clade B). However, these previous studies have not precisely addressed if Madracis is colonized by several types of Symbiodiniaceae simultaneously or whether this coral is a specialist. The hypothesis that Madracis is a generalist coral host was evaluated in the present study. A total of 1.9 million reads of ITS2 nuclear ribosomal DNA were obtained by Illumina MiSeq sequencing. While Symbiodiniaceae ITS2 sequences between two sampling depths were almost entirely (62%) from the genus Breviolum (formerly clade B), shallow (10-15 m) populations in Abrolhos had a greater diversity of ITS2 sequences in comparison to deeper (25-35 m) populations of St. Peter and St. Paul Archipelago. Cladocopium (formerly clade C) and Symbiodinium (formerly clade A) were also found in Abrolhos. A single Madracis colony can host different symbiont types with > 30 Symbiodiniaceae ITS2-type profiles. Abrolhos corals presented a higher photosynthetic potential as a possible result of co-occurrence of multiple Symbiodiniaceae in a single coral colony. Multiple genera/clades of Symbiodiniaceae possibly confer coral hosts with broader environmental tolerance and ability to occupy diverse or changing habitats.

Vibrio tetraodonis sp. nov.: genomic insights on the secondary metabolites repertoire.

Description of a Gram-negative, motile, circular-shaped bacterial strain, designated A511T obtained from the skin of the pufferfish Sphoeroides spengleri (Family Tetraodontidae), collected in Arraial do Cabo, Brazil. Optimum growth occurs at 20-28 °C in the presence of 3% NaCl. The genome sequence of the novel isolate consisted of 4.36 Mb, 3,976 coding genes and G + C content of 42.5%. Genomic taxonomy analyses based on average amino acid (AAI), genome-to-genome-distance (GGDH) and phylogenetic reconstruction placed A511T (= CBAS 712T = CAIM 1939T) into a new species of the genus Vibrio (Vibrio tetraodonis sp. nov.). The genome of the novel species contains eight genes clusters (~ 183.9 Kbp in total) coding for different types of bioactive compounds that hint to several possible ecological roles in the pufferfish host.

A new genomic taxonomy system for the Synechococcus collective.

Cyanobacteria of the genus Synechococcus are major contributors to global primary productivity and are found in a wide range of aquatic ecosystems. This Synechococcus collective (SC) is metabolically diverse, with some lineages thriving in polar and nutrient-rich locations and others in tropical or riverine waters. Although many studies have discussed the ecology and evolution of the SC, there is a paucity of knowledge on its taxonomic structure. Thus, we present a new taxonomic classification framework for the SC based on recent advances in microbial genomic taxonomy. Phylogenomic analyses of 1085 cyanobacterial genomes demonstrate that organisms classified as Synechococcus are polyphyletic at the order rank. The SC is classified into 15 genera, which are placed into five distinct orders within the phylum Cyanobacteria: (i) Synechococcales (Cyanobium, Inmanicoccus, Lacustricoccus gen. Nov., Parasynechococcus, Pseudosynechococcus, Regnicoccus, Synechospongium gen. nov., Synechococcus and Vulcanococcus); (ii) Cyanobacteriales (Limnothrix); (iii) Leptococcales (Brevicoccus and Leptococcus); (iv) Thermosynechococcales (Stenotopis and Thermosynechococcus) and (v) Neosynechococcales (Neosynechococcus). The newly proposed classification is consistent with habitat distribution patterns (seawater, freshwater, brackish and thermal environments) and reflects the ecological and evolutionary relationships of the SC.

Unlocking the Genomic Taxonomy of the Prochlorococcus Collective.

Prochlorococcus is the most abundant photosynthetic prokaryote on our planet. The extensive ecological literature on the Prochlorococcus collective (PC) is based on the assumption that it comprises one single genus comprising the species Prochlorococcus marinus, containing itself a collective of ecotypes. Ecologists adopt the distributed genome hypothesis of an open pan-genome to explain the observed genomic diversity and evolution patterns of the ecotypes within PC. Novel genomic data for the PC prompted us to revisit this group, applying the current methods used in genomic taxonomy. As a result, we were able to distinguish the five genera: Prochlorococcus, Eurycolium, Prolificoccus, Thaumococcus, and Riococcus. The novel genera have distinct genomic and ecological attributes.

Enterovibrio baiacu sp. nov.

We report here the novel species to encompass the isolate A649T (=CBAS 716T = CBRVS P1061T) obtained from viscera of the healthy pufferfish Sphoeroides spengleri (Family Tetraodontidae). Genomic taxonomy analysis demonstrates that the novel strain A649T had < 95% average amino acid identity/average nucleotide identity (AAI/ANI) and < 70% similarity of genome-to-genome distance (GGDH) towards its closest neighbors which places A649T into a new Enterovibrio species (Enterovibrio baiacu sp nov.). In silico phenotyping disclosed several features that may be used to differentiate related Enterovibrio species. The nearly complete genome assembly of strain A649T consisted of 5.4 Mbp and 4826 coding genes.

Halomonas coralii sp. nov. Isolated from Mussismilia braziliensis.

We report here the novel species Halomonas coralii. The nearly complete genome of strain 362.1T consisted of 4.4 Mbp (3989 CDS; 66.3% GC). Genomic taxonomy analysis demonstrates that the novel strain has < 83% AAI and < 29% GGDH towards its closest neighbors.

Description of Endozoicomonas arenosclerae sp. nov. using a genomic taxonomy approach.

The taxonomic position of strains Ab112(T) (CBAS 572(T)) and Ab227_MC (CBAS 573) was evaluated by means of genomic taxonomy. These isolates represent the dominant flora cultured from the healthy marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro. Strains CBAS 572(T) and CBAS 573 shared >98 % 16S rRNA sequence identity with Endozoicomonas numazuensis and Endozoicomonas montiporae. In silico DNA-DNA Hybridization, i.e. genome-to-genome distance (GGD), amino acid identity (AAI) and average nucleotide identity (ANI) further showed that these strains had <70 %, at maximum 71.1 and 78 % of identity, respectively, to their closest neighbours E. numazuensis and E. montiporae. The DNA G+C content of CBAS 572(T) and CBAS 573 were 47.6 and 47.7 mol%, respectively. Phenotypic and chemotaxonomic features also allowed a separation from the type strains of their phylogenetic neighbours. Useful phenotypic features for discriminating CBAS 572(T) and CBAS 573 from E. numazuensis and E. montiporae species include C8 esterase, N-acetyl-ß-glucosaminidase, citric acid, uridine and siderophore. The species Endozoicomonas arenosclerae sp. nov. is proposed to harbour the new isolates. The type strain is CBAS 572(T) (=Ab112(T)).

Microbial taxonomy in the post-genomic era: rebuilding from scratch?

Microbial taxonomy should provide adequate descriptions of bacterial, archaeal, and eukaryotic microbial diversity in ecological, clinical, and industrial environments. Its cornerstone, the prokaryote species has been re-evaluated twice. It is time to revisit polyphasic taxonomy, its principles, and its practice, including its underlying pragmatic species concept. Ultimately, we will be able to realize an old dream of our predecessor taxonomists and build a genomic-based microbial taxonomy, using standardized and automated curation of high-quality complete genome sequences as the new gold standard.

Enterococcus bulliens sp. nov., a novel lactic acid bacterium isolated from camel milk.

Four lactic acid bacteria isolates obtained from fresh dromedary camel milk produced in Dakhla, a city in southern Morocco, were characterised in order to determine their taxonomic position. The four isolates had highly similar MALDI-TOF MS and RAPD fingerprints and identical 16S rRNA gene sequences. Comparative sequence analysis revealed that the 16S rRNA gene sequence of the four isolates was most similar to that of Enterococcus sulfureus ATCC 49903(T) and Enterococcus italicus DSM 15952(T) (99.33 and 98.59% similarity, respectively). However, sequence analysis of the phenylalanyl-tRNA synthase (pheS), RNA polymerase (rpoA) and ATP synthase (atpA) genes revealed that the taxon represented by strain LMG 28766(T) was well separated from E. sulfureus LMG 13084(T) and E. italicus LMG 22039(T), which was further confirmed by DNA-DNA hybridization values that were clearly below the species demarcation threshold. The novel taxon was easily differentiated from its nearest neighbour species through sequence analysis of protein encoding genes, MALDI-TOF mass spectrometry and multiple biochemical tests, but had a similar percentage G+C content of about 39%. We therefore propose to formally classify these isolates as Enterococcus bulliens sp. nov., with LMG 28766(T) (=CCMM B1177(T)) as the type strain.

Finding diagnostic phenotypic features of Photobacterium in the genome sequences.

Photobacterium species are ubiquitous in the aquatic environment and can be found in association with animal hosts including pathogenic and mutualistic associations. The traditional phenotypic characterization of Photobacterium is expensive, time-consuming and restricted to a limited number of features. An alternative is to infer phenotypic information directly from whole genome sequences. The present study evaluates the usefulness of whole genome sequences as a source of phenotypic information and compares diagnostic phenotypes of the Photobacterium species from the literature with the predicted phenotypes obtained from whole genome sequences. All genes coding for the specific proteins involved in metabolic pathways responsible for positive phenotypes of the seventeen diagnostic features were found in the majority of the Photobacterium genomes. In the Photobacterium species that were negative for a given phenotype, at least one or several genes involved in the respective biochemical pathways were absent.

Streptococcus tangierensis sp. nov. and Streptococcus cameli sp. nov., two novel Streptococcus species isolated from raw camel milk in Morocco.

Biochemical and molecular genetic studies were performed on two unidentified Gram-stain positive, catalase and oxidase negative, non-hemolytic Streptococcus-like organisms recovered from raw camel milk in Morocco. Phenotypic characterization and comparative 16S rRNA gene sequencing demonstrated that the two strains were highly different from each other and that they did not correspond to any recognized species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms each formed a hitherto unknown sub-line within the genus Streptococcus, displaying a close affinity with Streptococcus moroccensis, Streptococcus minor and Streptococcus ovis. DNA G+C content determination, MALDI-TOF mass spectrometry and biochemical tests demonstrated the bacterial isolates represent two novel species. Based on the phenotypic distinctiveness of the new bacteria and molecular genetic evidence, it is proposed to classify the two strains as Streptococcus tangierensis sp. nov., with CCMM B832(T) (=LMG 27683(T)) as the type strain, and Streptococcus cameli sp. nov., with CCMM B834(T) (=LMG 27685(T)) as the type strain.

The dispersant Corexit 9500 and (dispersed) oil are lethal to coral endosymbionts.

Endosymbionts (Symbiodiniaceae) play a vital role in the health of corals. Seawater pollution can harm these endosymbionts and dispersants used during oil spill cleanup can be extremely toxic to these organisms. Here, we examined the impact of oil and a specific dispersant, Corexit-9500, on two representative endosymbionts - Symbiodinium and Cladocopium - from the Southwestern endemic coral Mussismilia braziliensis. The survival and photosynthetic potential of the endosymbionts decreased dramatically after exposure to the dispersant and oil by ~25 % after 2 h and ~50 % after 7 days. Low concentrations of dispersant (0.005 ml/l) and dispersed oil (Polycyclic Aromatic Hydrocarbons, 1132 µg/l; Total Petroleum Hydrocarbons, 595 µg/l) proved highly toxic to both Symbiodinium and Cladocopium. These levels triggered a reduction in growth rate, cell size, and cell wall thickness. After a few hours of exposure, cellular organelles were damaged or destroyed. These acute toxic effects underline the fragile nature of coral endosymbionts.

Microbial genomic taxonomy.

A need for a genomic species definition is emerging from several independent studies worldwide. In this commentary paper, we discuss recent studies on the genomic taxonomy of diverse microbial groups and a unified species definition based on genomics. Accordingly, strains from the same microbial species share >95% Average Amino Acid Identity (AAI) and Average Nucleotide Identity (ANI), >95% identity based on multiple alignment genes, <10 in Karlin genomic signature, and > 70% in silico Genome-to-Genome Hybridization similarity (GGDH). Species of the same genus will form monophyletic groups on the basis of 16S rRNA gene sequences, Multilocus Sequence Analysis (MLSA) and supertree analysis. In addition to the established requirements for species descriptions, we propose that new taxa descriptions should also include at least a draft genome sequence of the type strain in order to obtain a clear outlook on the genomic landscape of the novel microbe. The application of the new genomic species definition put forward here will allow researchers to use genome sequences to define simultaneously coherent phenotypic and genomic groups.

Surface microbial consortia from Livarot, a French smear-ripened cheese.

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)(5)-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter , Brevibacterium , Corynebacterium , and Staphylococcus . New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes , Hafnia , Proteus , Pseudomonas , and Psychrobacter . Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.

An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates.

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95% concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).

"Candidatus Colwellia aromaticivorans" sp. nov., "Candidatus Halocyntiibacter alkanivorans" sp. nov., and "Candidatus Ulvibacter alkanivorans" sp. nov. Genome Sequences.

Unplanned oil spills during offshore production are a serious problem for the industry and the marine environment. Here, we present the genome sequence analysis of three novel hydrocarbon-degrading bacteria, namely, "Candidatus Colwellia aromaticivorans" sp. nov., "Candidatus Halocyntiibacter alkanivorans" sp. nov., and "Candidatus Ulvibacter alkanivorans" sp. nov.

Biodiversity of vibrios.

Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years.

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates.

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.

Opinion: Re-evaluating prokaryotic species.

There is no widely accepted concept of species for prokaryotes, and assignment of isolates to species is based on measures of phenotypic or genome similarity. The current methods for defining prokaryotic species are inadequate and incapable of keeping pace with the levels of diversity that are being uncovered in nature. Prokaryotic taxonomy is being influenced by advances in microbial population genetics, ecology and genomics, and by the ease with which sequence data can be obtained. Here, we review the classical approaches to prokaryotic species definition and discuss the current and future impact of multilocus nucleotide-sequence-based approaches to prokaryotic systematics. We also consider the potential, and difficulties, of assigning species status to biologically or ecologically meaningful sequence clusters.