Insights into the genomic repertoire of Aquimarina litoralis CCMR20, a symbiont of coral Mussismilia braziliensis.

Aquimarina litoralis CCMR20 originated from the coral Mussismilia braziliensis (Sebastião Gomes Reef, Brazil, summer 2010). To gain new insights into the genomic repertoire associated with symbioses, we obtained the genome sequence of this strains using Illumina sequencing. CCMR20 has a genome size of 6.3 Mb, 32.6%GC, and 5513 genes (37 tRNA and 4 rRNA). A more fine-grained examination of the gene repertoire of CCMR20 disclosed genes engaged with symbiosis (heterotrophic carbon metabolism, CAZymes, B-vitamins group, carotenoid pigment and antioxidant molecules production). Genomic evidence further expand the possible relevance of this symbiont in the health of Mussismilia holobiont.Whole Genome Shotgun project has been deposited at DDBJ/ENA/GeneBank under the accession number WEKL00000000.

Genetic diversity and connectivity of Flaccisagitta enflata (Chaetognatha: Sagittidae) in the tropical Atlantic ocean (northeastern Brazil).

The phylogeography of the holoplanktonic chaetognath Flaccisagitta enflata was investigated in the Tropical Western Atlantic (TWA). Considering the cosmopolitan range of this species and the fact that its entire life cycle is planktonic, the central hypothesis of this study is that F. enflata exhibits connectivity due to its high dispersal capacity, forming a panmictic population among the study sites. The evaluated areas included neritic (Port of Recife-PR, and Tamandaré - TA) and oceanic (Fernando de Noronha Archipelago-FN, Rocas Atoll-RA, Guará seamount-GS and Saint Peter and Saint Paul's Archipelago-SPSPA) locations of the Brazilian Blue Amazon. We used COI gene sequences as molecular marker. Partial sequences (425 bp) were obtained for 116 specimens and employed to reconstruct the phylogeny, build an haplotype network, evaluate gene flow through a migration model, and estimate diversity indices, population structuring and demographic history. High levels of haplotype diversity (mean: 0.98) and moderate to high levels of nucleotide diversity (mean: 0.023) were observed. The phylogeny and the haplotype network topologies showed some geographic clustering, indicating local structuring in GS and PR. This finding was supported by the AMOVA high global Φst (0.033, significant) and some pairwise Φst comparisons (7 out of 15 were significantly >0). Significant differences suggested lower levels of connectivity when GS population was compared to those of FN and SPSPA; as well as when TA was compared to FN. These results might be related to particularities of the oceanic dynamics which rules the TWA, sustaining such dissimilarities. Structuring was also observed between PR and all oceanic locations. We hypothesize that the topography of the port inlet, enclosured by a reef barrier, may constrain the water turnover ratio and thus migration rates of F. enflata in the TWA. Accordingly, Migrate-N yielded a four metapopulations model (PR ⇌ TA ⇌ SPSPA+FN ⇌ GS+RA) as the best (highest probability; ~0.90) to represent the structuring of F. enflata in the TWA. Therefore, the null hypothesis of one randomly mating population cannot be accepted. The demographic evaluation demonstrated that the neutral hypothesis of stable populations may not be rejected for most of the locations. This work is the start point to broaden the knowledge on the phylogeography and population genetic structure of a numerically dominant species in the Western Atlantic, with key role in the marine trophic web.

New tetrodotoxin analogs in Brazilian pufferfishes tissues and microbiome.

While tetrodotoxin (TTX) is commonly found in pufferfish tissues, it is unclear if bacterial symbionts isolated from pufferfish tissues can produce TTX. In this investigation, UPLC qTOF-MS/MS analysis of tissue extracts obtained from Sphoeroides spengleri and Canthigaster figuereidoi identified TTX in their composition, indicating their consumption is unsafe. UPLC qTOF-MS/MS analysis coupled with Molecular Networking indicated new TTX analogs (methyl-TTX, TTX-acetate, hydroxypropyl-TTX and glycerol-TTX). Bacterial extracts from sixteen strains revealed a compound with a [M+H]+ ion at m/z 320.1088, identical to TTX. However, TTX itself was not detected in these cultures by UPLC-MS/MS. Neurotoxicity of Vibrio A665 purified fraction 2 (with precursor [M+H]+ ion at m/z 320.1088) was significant in human neural stem cells (hNSCs), but the Nav blockage activity was not confirmed by the veratridine/ouabain essays, indicating a possible difference in the mechanism of action between the bacterium A665 purified fraction 2 and TTX. Vibrios symbionts of pufferfish point out involving in the production of TTX precursors.

Genome sequence of Shewanella corallii strain A687 isolated from pufferfish (Sphoeroides spengleri).

We present here the genome sequence of Shewanella corallii strain A687 isolated from pufferfish Sphoeroides spengleri (Family Tetraodontidae). The assembly consists of 5,215,037 bp and contains 284 contigs, with a G+C content of 50.3%.

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.

Environmental and Sanitary Conditions of Guanabara Bay, Rio de Janeiro.

Guanabara Bay is the second largest bay in the coast of Brazil, with an area of 384 km(2). In its surroundings live circa 16 million inhabitants, out of which 6 million live in Rio de Janeiro city, one of the largest cities of the country, and the host of the 2016 Olympic Games. Anthropogenic interference in Guanabara Bay area started early in the XVI century, but environmental impacts escalated from 1930, when this region underwent an industrialization process. Herein we present an overview of the current environmental and sanitary conditions of Guanabara Bay, a consequence of all these decades of impacts. We will focus on microbial communities, how they may affect higher trophic levels of the aquatic community and also human health. The anthropogenic impacts in the bay are flagged by heavy eutrophication and by the emergence of pathogenic microorganisms that are either carried by domestic and/or hospital waste (e.g., virus, KPC-producing bacteria, and fecal coliforms), or that proliferate in such conditions (e.g., vibrios). Antibiotic resistance genes are commonly found in metagenomes of Guanabara Bay planktonic microorganisms. Furthermore, eutrophication results in recurrent algal blooms, with signs of a shift toward flagellated, mixotrophic groups, including several potentially harmful species. A recent large-scale fish kill episode, and a long trend decrease in fish stocks also reflects the bay's degraded water quality. Although pollution of Guanabara Bay is not a recent problem, the hosting of the 2016 Olympic Games propelled the government to launch a series of plans to restore the bay's water quality. If all plans are fully implemented, the restoration of Guanabara Bay and its shores may be one of the best legacies of the Olympic Games in Rio de Janeiro.

Turbulence-driven shifts in holobionts and planktonic microbial assemblages in St. Peter and St. Paul Archipelago, Mid-Atlantic Ridge, Brazil.

The aim of this study was to investigate the planktonic and the holobiont Madracis decactis (Scleractinia) microbial diversity along a turbulence-driven upwelling event, in the world's most isolated tropical island, St Peter and St Paul Archipelago (SPSPA, Brazil). Twenty one metagenomes were obtained for seawater (N = 12), healthy and bleached holobionts (N = 9) before, during and after the episode of high seawater turbulence and upwelling. Microbial assemblages differed between low turbulence-low nutrient (LLR) and high-turbulence-high nutrient (HHR) regimes in seawater. During LLR there was a balance between autotrophy and heterotrophy in the bacterioplankton and the ratio cyanobacteria:heterotrophs ~1 (C:H). Prochlorales, unclassified Alphaproteobacteria and Euryarchaeota were the dominant bacteria and archaea, respectively. Basic metabolisms and cyanobacterial phages characterized the LLR. During HHR C:H < < 0.05 and Gammaproteobacteria approximated 50% of the most abundant organisms in seawater. Alteromonadales, Oceanospirillales, and Thaumarchaeota were the dominant bacteria and archaea. Prevailing metabolisms were related to membrane transport, virulence, disease, and defense. Phages targeting heterotrophs and virulence factor genes characterized HHR. Shifts were also observed in coral microbiomes, according to both annotation-indepent and -dependent methods. HHR bleached corals metagenomes were the most dissimilar and could be distinguished by their di- and tetranucleotides frequencies, Iron Acquision metabolism and virulence genes, such as V. cholerae-related virulence factors. The healthy coral holobiont was shown to be less sensitive to transient seawater-related perturbations than the diseased animals. A conceptual model for the turbulence-induced shifts is put forward.

Diversity and ecological structure of vibrios in benthic and pelagic habitats along a latitudinal gradient in the Southwest Atlantic Ocean.

We analyzed the diversity and population structure of the 775 Vibrio isolates from different locations of the southwestern Atlantic Ocean (SAO), including St. Peter and St. Paul Archipelago (SPSPA), Abrolhos Bank (AB) and the St. Sebastian region (SS), between 2005 and 2010. In this study, 195 novel isolates, obtained from seawater and major benthic organisms (rhodoliths and corals), were compared with a collection of 580 isolates previously characterized (available at www.taxvibrio.lncc.br). The isolates were distributed in 8 major habitat spectra according to AdaptML analysis on the basis of pyrH phylogenetic reconstruction and ecological information, such as isolation source (i.e., corals: Madracis decactis, Mussismilia braziliensis, M. hispida, Phyllogorgia dilatata, Scolymia wellsi; zoanthids: Palythoa caribaeorum, P. variabilis and Zoanthus solanderi; fireworm: Hermodice carunculata; rhodolith; water and sediment) and sampling site regions (SPSPA, AB and SS). Ecologically distinct groups were discerned through AdaptML, which finds phylogenetic groups that are significantly different in their spectra of habitat preferences. Some habitat spectra suggested ecological specialization, with habitat spectra 2, 3, and 4 corresponding to specialization on SPSPA, AB, and SS, respectively. This match between habitat and location may reflect a minor exchange of Vibrio populations between geographically isolated benthic systems. Moreover, we found several widespread Vibrio species predominantly from water column, and different populations of a single Vibrio species from H. carunculata in ecologically distinct groups (H-1 and H-8 respectively). On the other hand, AdaptML detected phylogenetic groups that are found in both the benthos and in open water. The ecological grouping observed suggests dispersal and connectivity between the benthic and pelagic systems in AB. This study is a first attempt to characterize the biogeographic distribution of vibrios in both seawater and several benthic hosts in the SAO. The benthopelagic coupling observed here stands out the importance of vibrios in the global ocean health.

Exploring the Genome of Cheese Starter Lactic Acid Bacterium Lactococcus lactis subsp. lactis CECT 4433.

Here, we present the draft genome sequences of Lactococcus lactis subsp. lactis CECT 4433, a cheese fermentation starter strain. The genome provides further insight into the genomic plasticity, biocomplexity (including gene strain specifics), and evolution of these genera.

Oenococcus alcoholitolerans sp. nov., a lactic acid bacteria isolated from cachaça and ethanol fermentation processes.

Four strains of lactic acid bacteria isolated from cachaça and alcohol fermentation vats in Brazil were characterised in order to determine their taxonomic position. Phylogenetic analysis revealed that they belong to the genus Oenococcus and should be distinguished from their closest neighbours. The 16S rRNA gene sequence similarity against the type strains of the other two species of the genus was below 94.76 % (Oenococcus kitaharae) and 94.62 % (Oenococcus oeni). The phylogeny based on pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparison, Average Amino Acid Identity, Average Nucleotide Identity and Karlin genomic signature confirmed the novelty of the taxon. Distinctive phenotypic characteristics are the ability to metabolise sucrose but not trehalose. The name Oenococcus alcoholitolerans sp. nov. is proposed for this taxon, with the type strain UFRJ-M7.2.18(T) ( = CBAS474(T) = LMG27599(T)). In addition, we have determined a draft genome sequence of the type strain.

Photobacterium sanctipauli sp. nov. isolated from bleached Madracis decactis (Scleractinia) in the St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil.

Five novel strains of Photobacterium (A-394T, A-373, A-379, A-397 and A-398) were isolated from bleached coral Madracis decactis (scleractinian) in the remote St Peter & St Archipelago (SPSPA), Mid-Atlantic Ridge, Brazil. Healthy M. decactis specimens were also surveyed, but no strains were related to them. The novel isolates formed a distinct lineage based on the 16S rRNA, recA, and rpoA gene sequences analysis. Their closest phylogenetic neighbours were Photobacterium rosenbergii, P. gaetbulicola, and P. lutimaris, sharing 96.6 to 95.8% 16S rRNA gene sequence similarity. The novel species can be differentiated from the closest neighbours by several phenotypic and chemotaxonomic markers. It grows at pH 11, produces tryptophane deaminase, presents the fatty acid C18:0, but lacks C16:0 iso. The whole cell protein profile, based in MALDI-TOF MS, distinguished the strains of the novel species among each other and from the closest neighbors. In addition, we are releasing the whole genome sequence of the type strain. The name Photobacterium sanctipauli sp. nov. is proposed for this taxon. The G + C content of the type strain A-394(T) (= LMG27910(T) = CAIM1892(T)) is 48.2 mol%.

Vibrio madracius sp. nov. isolated from Madracis decactis (Scleractinia) in St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil.

Three novel isolates (A-354(T), A-328, and A-384) were retrieved from apparently healthy scleractinian Madracis decactis in the remote St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil. The novel isolates formed a distinct lineage based on the phylogenetic reconstruction using the 16S rRNA and pyrH gene sequences. They fell into the Mediterranei clade and their closest phylogenetic neighbour was V. mediterranei species, sharing upto 98.1 % 16S rRNA gene sequence similarity. Genomic analysis including in silico DDH, MLSA, AAI and genomic signature distinguished A-354(T) from V. mediterranei LMG 19703 (=AK1) with values of 33.3, 94.2, 92 %, and 11.3, respectively. Phenotypically, the novel isolates can be differentiated from V. mediterranei based on the four following features. They do not grow at 8 % NaCl; use D-gluconic acid but not L-galactonic acid lactone as carbon source; and do not have the fatty acid C18:0. Differentiation from both the other Mediterranei clade species (V. maritimus and V. variabilis) is supported by fifteen features. The novel species show lysine decarboxylase and tryptophan deaminase, but not gelatinase and arginine dihydrolase activity; produce acetoin; use α-D-lactose, N-acetyl-D-galactosamine, myo-Inositol, D-gluconic acid, and ß-hydroxy-D,L-butyric acid; and present the fatty acids C14:0 iso, C15:0 anteiso, C16:0 iso, C17:0 anteiso, and C17:1x8c . Whole-cell protein profiles, based on MALDI-TOF, showed that the isolates are not clonal and also distinguished them from the closes phylogenetic neighbors. The name Vibrio madracius sp. nov. is proposed to encompass these novel isolates. The G+C content of the type strain A-354(T) (=LMG 28124(T)=CBAS 482(T)) is 44.5 mol%.

Culturable heterotrophic bacteria associated with healthy and bleached scleractinian Madracis decactis and the fireworm Hermodice carunculata from the remote St. Peter and St. Paul Archipelago, Brazil.

We report on the first characterization of the culturable heterotrophic bacteria of the scleractinian Madracis decactis. In addition, we characterized the culturable bacteria associated with the fireworm Hermodice carunculata, observed predating partially bleached coral colonies. Our study was carried out in the remote St. Peter and St. Paul Archipelago (SPSPA), Mid-Atlantic Ridge, Brazil. We constituted a 403 isolates collection and subsequently characterized it by means of pyrH and 16S rRNA partial sequences. We identified Photobacterium, Bacillus, and Vibrio species as members of the culturable microbiota of healthy M. decactis. V. campbellii, V. harveyi, V. communis, and V. maritimus were the most commonly found Vibrio species in healthy corals, representing more than 60 % of all vibrio isolates. Most of the vibrios isolated from the fireworm's tissues (n = 143; >90 %) were identified as V. shiloi. However, we did not recover V. shiloi from bleached M. decactis. Instead, we isolated V. communis, a novel Photobacterium species, Bacillus, Kocuria, and Pseudovibrio, suggesting a possible role of other facultative anaerobic bacteria and/or environmental features (such as water quality) in the onset of bleaching in SPSPA's M. decactis.

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.

Genome sequences of the ethanol-tolerant Lactobacillus vini strains LMG 23202T and JP7.8.9.

We report on the genome sequences of Lactobacillus vini type strain LMG 23202(T) (DSM 20605) (isolated from fermenting grape musts in Spain) and the industrial strain L. vini JP7.8.9 (isolated from a bioethanol plant in northeast Brazil). All contigs were assembled using gsAssembler, and genes were predicted and annotated using Rapid Annotation using Subsystem Technology (RAST). The identified genome sequence of LMG 23202(T) had 2.201.333 bp, 37.6% G+C, and 1,833 genes, whereas the identified genome sequence of JP7.8.9 had 2.301.037 bp, 37.8% G+C, and 1,739 genes. The gene repertoire of the species L. vini offers promising opportunities for biotechnological applications.

Vibrio alfacsensis sp. nov., isolated from marine organisms.

Five strains (CAIM 1831(T), CAIM 1832, CAIM 1833, CAIM 1834 and CAIM 1836) were isolated from cultured sole (Solea senegalensis) in two regions of Spain, two strains (CAIM 404 and CAIM 1294) from wild-caught spotted rose snapper (Lutjanus guttatus) in Mexico, and one strain (CAIM 1835) from corals in Brazil. The 16S rRNA gene sequences of the novel isolates showed similarity to Vibrio ponticus (98.2-98.3%, GenBank accession no. AJ630103) and to a lesser degree to Vibrio furnissii (97.2-97.3%, X76336) and to Vibrio fluvialis (96.9-97.1%, X74703). Multilocus sequence analysis clustered these strains closely together and clearly separated them from phylogenetically related species of the genus Vibrio. Genomic fingerprinting by rep-PCR clustered the novel strains according to their geographical origin. Phenotypic analyses showed a large variation among the new strains, but many tests enabled them to be differentiated from other species of the genus Vibrio. The mean ΔT(m) values between the strains analysed here and closely related type strains were above 6.79 °C. The values between the novel isolates were below 2.35 °C, well outside the limit suggested for the delineation of a bacterial species. The phenotypic and genotypic data presented here clearly place these new strains as a coherent group within the genus Vibrio, for which we propose the name Vibrio alfacsensis sp. nov. with CAIM 1831(T) ( = DSM 24595(T) = S277(T)) as the type strain.

Usefulness of a real-time PCR platform for G+C content and DNA-DNA hybridization estimations in vibrios.

The aim of this study was to evaluate the utility of a real-time PCR platform to estimate the DNA G+C content (mol%) and DNA-DNA hybridization (DDH) values in the genus Vibrio. In total, nine vibrio strains were used to determine the relationship between genomic DNA G+C content and T(m) (°C). The T(m) and HPLC datasets fit a linear regression curve with a significant correlation coefficient, corroborating that this methodology has a high correlation with the standard methodology based on HPLC (R(2) = 0.94). Analysis of 31 pairs of vibrios provided a wide range of ΔT(m) values, varying between 0.72 and 12.5 °C. Pairs corresponding to strains of the same species or strains from sister species showed the lowest ΔT(m) values. For instance, the ΔT(m) of the sister species Vibrio harveyi LMG 4044(T) and Vibrio campbellii LMG 11216(T) was 5.2 °C, whereas the ΔT(m) of Vibrio coralliilyticus LMG 20984(T) and Vibrio neptunius LMG 20536(T) was 8.75 °C. The mean ΔT(m) values corresponding to pairs of strains with DDH values lower than 60 % or higher than 80 % were, respectively, 8.29 and 2.21 °C (significant difference, P<0.01). The high correlation between DDH values obtained in previous studies and the ΔT(m) values (R(2) = 0.7344) indicates that the fluorimetric methodology is a reliable alternative for the estimation of both DNA G+C content and ΔT(m) in vibrios. We suggest that strains of the same Vibrio species will have less than 4 °C ΔT(m). The use of a real-time PCR platform represents a valuable alternative for the development of the taxonomy of vibrios.

Diversity of lactic acid bacteria of the bioethanol process.

BACKGROUND: Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil. RESULTS: A total of 489 LAB isolates were obtained from four distilleries in 2007 and 2008. The abundance of LAB in the fermentation tanks varied between 6.0 × 105 and 8.9 × 108 CFUs/mL. Crude sugar cane juice contained 7.4 × 107 to 6.0 × 108 LAB CFUs. Most of the LAB isolates belonged to the genus Lactobacillus according to rRNA operon enzyme restriction profiles. A variety of Lactobacillus species occurred throughout the bioethanol process, but the most frequently found species towards the end of the harvest season were L. fermentum and L. vini. The different rep-PCR patterns indicate the co-occurrence of distinct populations of the species L. fermentum and L. vini, suggesting a great intraspecific diversity. Representative isolates of both species had the ability to grow in medium containing up to 10% ethanol, suggesting selection of ethanol tolerant bacteria throughout the process. CONCLUSIONS: This study served as a first survey of the LAB diversity in the bioethanol process in Brazil. The abundance and diversity of LAB suggest that they have a significant impact in the bioethanol process.