Microarray tools to unveil viral-microbe interactions in nature

The interactions between viruses and their microbial hosts play a central role in the control of microbial communities in nature. However, the study of such interactions within the uncultured majority is technically very challenging. Here, we review how microarray tools can be used to analyze the interactions between viruses and their microbial hosts in nature, away from laboratory pure culture-based models. We show examples of how DNA arrays have been used to study the expression of viral assemblages in natural samples, and to assign viruses to hosts within uncultured communities. Finally, we briefly discuss the possibilities of protein and glycan arrays to gain insight into the ways microbes interact with their viruses.Our current studies with viral microarrays are supported by projects CGL2012-39627-C03-01 (to Josefa Antón) and AYA2011-24803 (to Víctor Parro) of the Spanish Ministry of Science and Innovation, which are co-financed with FEDER support from the European Union

Shifts in marine invertebrate bacterial assemblages associated with tissue necrosis during a heat wave

Marine heat waves (MHWs) are periods of extremely high seawater temperature that affect marine ecosystems in several ways. Anthozoans (corals and gorgonians) and Porifera (sponges) are usually among the taxa most affected by MHWs. Both are holobiont entities that form complex interactions with a wide range of microbes, which are an essential part of these organisms and play key roles in their health status. Here, we determine microbial community changes suffered in two corals (Cladocora caespitosa and Oculina patagonica), one gorgonian (Leptogorgia sarmentosa) and one sponge (Sarcotragus fasciculatus) during the 2015 MHW. The microbial communities were different among hosts and displayed shifts related to host health status, with a higher abundance in necrosed tissues of Ruegeria species or of potential pathogens like Vibrio. We also carry out a meta-analysis using 93 publicly accessible 16S rRNA gene libraries from O. patagonica, C. caespitosa and L. sarmentosa to establish a Mediterranean core microbiome in these species. We have identified one Ruegeria OTU that maintained a stable and consistent association with these species, which was also related to tissue necrosis in their hosts. Therefore, Ruegeria sp. could play an important and still underexplored role in the health status of its hosts.This work has been carried out within the CIESM project “Tropical Signals” and it was funded by the European Union’s framework program Horizon 2020 (LEIT-BIO-2015-685474, Metafluidics, to JA)

Exploring changes in bacterial communities to assess the influence of fish farming on marine sediments

Changes in bacterial assemblages along an environmental gradient determined by the distance to aquaculture installations were analysed, using denaturing gradient gel electrophoresis to assess the influence of fish farming on marine sediments. Our findings show that changes in the structure of the bacterial community are a useful indicator for determining the environmental impact of aquaculture farms, due to the rapid response to changes in nutrient load, and could be an alternative strategy for monitoring programmes. Delta and Epsilonproteobacteria linked to the sulphur cycle were detected in the sediments beneath the cages. Since these groups were not found in the sediments at control stations, they serve as indicators for assessing the impact of the organic load from fish farming on marine sediments.This study forms part of the ‘Selección de indicadores, determinación de valores de referencia, diseño de programas y protocolos de métodos y medidas para estudios ambientales en acuicultura marina’ project funded by the ‘Spanish National Plans of Aquaculture’ (JACUMAR). The research was supported by Spanish Ministry of Economy and Competitiveness project CLG2015_66686-C3-3 (JA), which was also supported by financing from the European Regional Development Fund (FEDER). ERP thanks the Generalitat Valenciana for a postdoctoral grant (APOSTD-2016-091)

Retinal-binding proteins mirror prokaryotic dynamics in multipond solar salterns

Microbial opsin (i.e. retinal-binding protein) dynamics has been studied along a salinity gradient in Santa Pola solar salterns (Alicante, Spain) by using culture-independent approaches and statistical analyses. Five ponds of salinities ranging from 18% to above 40% were sampled nine times along a year. Forty-three opsin-like sequences were retrieved by denaturing gradient gel electrophoresis and clustered into 18 different phylogroups, indicating that their diversity was higher than expected according to previous data. Moreover, the statistical correlation between environmental factors controlling microbial community structure and dynamics of environmental rhodopsin proteins indicated almost identical temporal fluctuations between the opsin-related sequences and their corresponding putative ‘producers’ in nature. Although most sequences were related to others previously detected in hypersaline environments, some pond-specific opsins putatively belonged to previously uncharacterized hosts. Furthermore, we propose that subtle changes in the bacteriorhodopsin ‘retinal proton binding pocket’, which is key in the photocycle function, could be the molecular basis behind a fine ‘photocycle-tuning’ mechanism to avoid inter/intraspecies light-competition in hypersaline environments.This work was supported by project CGL2012-39627-C03-01 of the Spanish Ministry of Economy and Competitiveness, which was also co-funded with FEDER support from the European Union

Interactions between Closely Related Bacterial Strains Are Revealed by Deep Transcriptome Sequencing

Comparative genomics, metagenomics, and single-cell technologies have shown that populations of microbial species encompass assemblages of closely related strains. This raises the question of whether individual bacterial lineages respond to the presence of their close relatives by modifying their gene expression or, instead, whether assemblages simply act as the arithmetic addition of their individual components. Here, we took advantage of transcriptome sequencing to address this question. For this, we analyzed the transcriptomes of two closely related strains of the extremely halophilic bacterium Salinibacter ruber grown axenically and in coculture. These organisms dominate bacterial assemblages in hypersaline environments worldwide. The strains used here cooccurred in the natural environment and are 100% identical in their 16S rRNA genes, and each strain harbors an accessory genome representing 10% of its complete genome. Overall, transcriptomic patterns from pure cultures were very similar for both strains. Expression was detected along practically the whole genome albeit with some genes at low levels. A subset of genes was very highly expressed in both strains, including genes coding for the light-driven proton pump xanthorhodopsin, genes involved in the stress response, and genes coding for transcriptional regulators. Expression differences between pure cultures affected mainly genes involved in environmental sensing. When the strains were grown in coculture, there was a modest but significant change in their individual transcription patterns compared to those in pure culture. Each strain sensed the presence of the other and responded in a specific manner, which points to fine intraspecific transcriptomic modulation.The group of J.A. is funded by grant CGL2012-39627-C03-01 from the Spanish Ministry of Economy and Competitiveness (MINECO), which is cofinanced with FEDER support from the European Union. P.G.-T. was an FPI-MINECO fellow. Research by the group of T.G. is funded in part by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund (NPRP 5-298-3-086), and a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement no. ERC-2012-StG-310325). L.P.P. was funded through the La Caixa-CRG international fellowship program

Diversity of extremely halophilic cultivable prokaryotes in Mediterranean, Atlantic and Pacific solar salterns: Evidence that unexplored sites constitute sources of cultivable novelty

The culturable fraction of aerobic, heterotrophic and extremely halophilic microbiota retrieved from sediment and brine samples of eight sampling sites in the Mediterranean, Canary Islands and Chile was studied by means of a tandem approach combining large-scale cultivation, MALDI-TOF MS targeting whole cell biomass, and phylogenetic reconstruction based on 16S rRNA gene analysis. The approach allowed the identification of more than 4200 strains and a comparison between different sampling sites. The results indicated that the method constituted an excellent tool for the discovery of taxonomic novelty. Four new genera and nine new species could be identified within the archaeal family Halobacteriaceae, as well as one new bacterial species, and a representative of Salinibacter ruber phylotype II, a group that had been refractory to isolation for the last fifteen years. Altogether, the results indicated that in order to provide better yields for the retrieval of novel taxa from the environment, performance of non-redundant environment sampling is recommended together with the screening of large sets of strains.The current study was funded with the scientific support given by the Spanish Ministry of Economy through the projectsCGL2012-39627-C03-01 and CGL2012-39627-C03-03, which were also supported with European Regional Development Fund (FEDER) funds, and the preparatory phase of the Microbial Resource Research Infrastructure (MIRRI) funded by the EU (grant number 312251). In addition, the funding from competitive research groups (Microbiology) of the Government of the Balearic Islands (also co-supported with FEDER funds), is also acknowledged. TVP acknowledges the predoctoral fellowship of the Ministerio de Economía y Competitividad of the Spanish Government for the FPI fellowship (Nr BES-2013-064420) supporting his research activities

Virulence as a Side Effect of Interspecies Interaction in Vibrio Coral Pathogens

The increase in prevalence and severity of coral disease outbreaks produced by Vibrio pathogens, and related to global warming, has seriously impacted reef-building corals throughout the oceans. The coral Oculina patagonica has been used as a model system to study coral bleaching produced by Vibrio infection. Previous data demonstrated that when two coral pathogens (Vibrio coralliilyticus and Vibrio mediterranei) simultaneously infected the coral O. patagonica, their pathogenicity was greater than when each bacterium was infected separately. Here, to understand the mechanisms underlying this synergistic effect, transcriptomic analyses of monocultures and cocultures as well as experimental infection experiments were performed. Our results revealed that the interaction between the two vibrios under culture conditions overexpressed virulence factor genes (e.g., those encoding siderophores, the type VI secretion system, and toxins, among others). Moreover, under these conditions, vibrios were also more likely to form biofilms or become motile through induction of lateral flagella. All these changes that occur as a physiological response to the presence of a competing species could favor the colonization of the host when they are present in a mixed population. Additionally, during coral experimental infections, we showed that exposure of corals to molecules released during V. coralliilyticus and V. mediterranei coculture induced changes in the coral microbiome that favored damage to coral tissue and increased the production of lyso-platelet activating factor. Therefore, we propose that competition sensing, defined as the physiological response to detection of harm or to the presence of a competing Vibrio species, enhances the ability of Vibrio coral pathogens to invade their host and cause tissue necrosis.This research was supported in part by the EU-H2020 MetaFluidics project with grant agreement number 685474 (to J.A.) and NSF-PIRE grant number OISE1243541 (to F.R.). E.R.-P. was funded by the postdoctoral program Vali+d (GVA) (grant number APOSTD-2016-091). A.M.C.-R. and P.C.D. were supported by the National Sciences Foundation grant IOS-1656481

Elucidating Viral Communities During a Phytoplankton Bloom on the West Antarctic Peninsula

In Antarctic coastal waters where nutrient limitations are low, viruses are expected to play a major role in the regulation of bloom events. Despite this, research in viral identification and dynamics is scarce, with limited information available for the Southern Ocean (SO). This study presents an integrative-omics approach, comparing variation in the viral and microbial active communities on two contrasting sample conditions from a diatom-dominated phytoplankton bloom occurring in Chile Bay in the West Antarctic Peninsula (WAP) in the summer of 2014. The known viral community, initially dominated by Myoviridae family (∼82% of the total assigned reads), changed to become dominated by Phycodnaviridae (∼90%), while viral activity was predominantly driven by dsDNA members of the Phycodnaviridae (∼50%) and diatom infecting ssRNA viruses (∼38%), becoming more significant as chlorophyll a increased. A genomic and phylogenetic characterization allowed the identification of a new viral lineage within the Myoviridae family. This new lineage of viruses infects Pseudoalteromonas and was dominant in the phage community. In addition, a new Phycodnavirus (PaV) was described, which is predicted to infect Phaeocystis antarctica, the main blooming haptophyte in the SO. This work was able to identify the changes in the main viral players during a bloom development and suggests that the changes observed in the virioplankton could be used as a model to understand the development and decay of blooms that occur throughout the WAP.This work was financially supported by a Ph.D. scholarship CONICYT N° 21130667; INACH15-10, INACH RG_09-17, CONICYT for international cooperation DPI20140044, FONDAP N° 15110009, and FONDECYT N° 1190998

High-throughput functional metagenomics for the discovery of glycan metabolizing pathways

Glycans are widely distributed in nature. Produced by almost all organisms, they are involved in numerous cellular processes, such as energy supply and storage, cell structuration, protein maturation and signalling, and cell recognition. Glycans are thus key elements mediating the interactions between mammals, plants, bacteria, fungi and even viruses. They also represent a reliable source of carbon for microbes, which have developed complex strategies to face their structural diversity and to harvest them. However between 70 and 99% of these microorganisms are still uncultured, while they represent a goldmine for the discovery of new enzymes. In order to boost their identification and characterization, a functional metagenomic approach was developed, based on the design of various high-throughput, robust and sensitive screening strategies. The functional potential of Gbp of metagenomic DNA from various origins was explored, revealing dozens of novel enzyme families and functions. Integration of biochemical, structural, meta-omic and omic data allowed us to decipher, from the molecular to the ecosystemic scale, novel mechanisms of plant, microbial and mammal glycan metabolization. These new metabolic pathways involve batteries of glycoside-hydrolases, glycoside-phosphorylases and sugar transporters. These fascinating proteins appear as new targets to control host-microbe interactions. They also constitute very efficient biotechnological tools for biorefineries and synthetic biology

Seasonal dynamics of extremely halophilic microbial communities in three Argentinian salterns

Seasonal sampling was carried out at three Argentinian salterns, Salitral Negro (SN), Colorada Grande (CG) and Guatraché (G), to analyze abiotic parameters and microbial diversity and dynamics. Microbial assemblages were correlated to environmental factors by statistical analyses. Principal component analysis of the environmental data grouped SN and CG samples separately from G samples owing to G's higher pH values and sulfate concentration. Differences in microbial assemblages were also found. Many archaeal sequences belonged to uncultured members of Haloquadratum and Haloquadratum-related genera, with different environmental optima. Notably, nearly half of the archaeal sequences were affiliated to the recently described ‘Candidatus Haloredividus’ (phylum Nanohaloarchaeota), not previously detected in salt-saturated environments. Most bacterial sequences belonged to Salinibacter representatives, while sequences affiliated to the recently described genus Spiribacter were also found. Seasonal analysis showed at least 40% of the microbiota from the three salterns was prevalent through the year, indicating they are well adapted to environmental fluctuations. On the other hand, a minority of archaeal and bacterial sequences were found to be seasonally distributed. Five viral morphotypes and also eukaryal predators were detected, suggesting different mechanisms for controlling prokaryotic numbers. Notably, Guatraché was the saltern that harbored the highest virus-to-cell ratios reported to date for hypersaline environments.This work was supported by Agencia Española de Cooperación Internacional (AECI) [Grant number AECIA/023820/09], the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Mar del Plata (UNMdP), Argentina and by the Ministerio Español de Economía y Competitividad (MINECO) [Grant number CGL2015-66686-C3-3-P]