Coral mucus as a reservoir of bacteriophages targeting Vibrio pathogens.

The increasing trend in sea surface temperature promotes the spread of Vibrio species, which are known to cause diseases in a wide range of marine organisms. Among these pathogens, Vibrio mediterranei has emerged as a significant threat, leading to bleaching in the coral species Oculina patagonica. Bacteriophages, or phages, are viruses that infect bacteria, thereby regulating microbial communities and playing a crucial role in the coral's defense against pathogens. However, our understanding of phages that infect V. mediterranei is limited. In this study, we identified two phage species capable of infecting V. mediterranei by utilizing a combination of cultivation and metagenomic approaches. These phages are low-abundance specialists within the coral mucus layer that exhibit rapid proliferation in the presence of their hosts, suggesting a potential role in coral defense. Additionally, one of these phages possesses a conserved domain of a leucine-rich repeat protein, similar to those harbored in the coral genome, that plays a key role in pathogen recognition, hinting at potential coral-phage coevolution. Furthermore, our research suggests that lytic Vibrio infections could trigger prophage induction, which may disseminate genetic elements, including virulence factors, in the coral mucus layer. Overall, our findings underscore the importance of historical coral-phage interactions as a form of coral immunity against invasive Vibrio pathogens.

Deepening the knowledge of universal stress proteins in Haloferax mediterranei.

Haloarchaea, like many other microorganisms, have developed defense mechanisms such as universal stress proteins (USPs) to cope with environmental stresses affecting microbial growth. Despite the wide distribution of these proteins in Archaea, their biochemical characteristics still need to be discovered, and there needs to be more knowledge about them focusing on halophilic Archaea. Therefore, elucidating the role of USPs would provide valuable information to improve future biotechnological applications. Accordingly, transcriptional expression of the 37 annotated USPs in the Haloferax mediterranei genome has been examined under different stress conditions. From a global perspective, finding a clear tendency between particular USPs and specific stress conditions was not possible. Contrary, data analysis indicates that there is a recruitment mechanism of proteins with a similar sequence able to modulate the H. mediterranei growth, accelerating or slowing it, depending on their number. In fact, only three of these USPs were expressed in all the tested conditions, pointing to the cell needing a set of USPs to cope with stress conditions. After analysis of the RNA-Seq data, three differentially expressed USPs were selected and homologously overexpressed. According to the growth data, the overexpression of USPs induces a gain of tolerance in response to stress, as a rule. Therefore, this is the only work that studies all the USPs in an archaeon. It represents a significant first base to continue advancing, not only in this important family of stress proteins but also in the field of biotechnology and, at an industrial level, to improve applications such as designing microorganisms resistant to stress situations. KEY POINTS: • Expression of Haloferax mediterranei USPs has been analyzed in stress conditions. • RNA-seq analysis reveals that most of the USPs in H. mediterranei are downregulated. • Homologous overexpression of USPs results in more stress-tolerant strains.

Factors structuring microbial communities in highly impacted coastal marine sediments (Mar Menor lagoon, SE Spain).

Coastal marine lagoons are environments highly vulnerable to anthropogenic pressures such as agriculture nutrient loading or runoff from metalliferous mining. Sediment microorganisms, which are key components in the biogeochemical cycles, can help attenuate these impacts by accumulating nutrients and pollutants. The Mar Menor, located in the southeast of Spain, is an example of a coastal lagoon strongly altered by anthropic pressures, but the microbial community inhabiting its sediments remains unknown. Here, we describe the sediment prokaryotic communities along a wide range of environmental conditions in the lagoon, revealing that microbial communities were highly heterogeneous among stations, although a core microbiome was detected. The microbiota was dominated by Delta- and Gammaproteobacteria and members of the Bacteroidia class. Additionally, several uncultured groups such as Asgardarchaeota were detected in relatively high proportions. Sediment texture, the presence of Caulerpa or Cymodocea, depth, and geographic location were among the most important factors structuring microbial assemblages. Furthermore, microbial communities in the stations with the highest concentrations of potentially toxic elements (Fe, Pb, As, Zn, and Cd) were less stable than those in the non-contaminated stations. This finding suggests that bacteria colonizing heavily contaminated stations are specialists sensitive to change.

Marine heatwaves drive recurrent mass mortalities in the Mediterranean Sea.

Climate change is causing an increase in the frequency and intensity of marine heatwaves (MHWs) and mass mortality events (MMEs) of marine organisms are one of their main ecological impacts. Here, we show that during the 2015-2019 period, the Mediterranean Sea has experienced exceptional thermal conditions resulting in the onset of five consecutive years of widespread MMEs across the basin. These MMEs affected thousands of kilometers of coastline from the surface to 45 m, across a range of marine habitats and taxa (50 taxa across 8 phyla). Significant relationships were found between the incidence of MMEs and the heat exposure associated with MHWs observed both at the surface and across depths. Our findings reveal that the Mediterranean Sea is experiencing an acceleration of the ecological impacts of MHWs which poses an unprecedented threat to its ecosystems' health and functioning. Overall, we show that increasing the resolution of empirical observation is critical to enhancing our ability to more effectively understand and manage the consequences of climate change.

Metagenomics Unveils Posidonia oceanica "Banquettes" as a Potential Source of Novel Bioactive Compounds and Carbohydrate Active Enzymes (CAZymes).

Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea. It produces large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." In recent years, interest in the potential uses of these P. oceanica banquettes has increased, and it was demonstrated that biomass extracts showed antioxidant, antifungal, and antiviral activities. The discovery of new compounds through the culture of microorganisms is limited, and to overcome this limitation, we performed a metagenomic study to investigate the microbial community associated with P. oceanica banquettes. Our results showed that the microbial community associated with P. oceanica banquettes was dominated by Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria. Pseudoalteromonas was the dominant genus, followed by Alteromonas, Labrenzia, and Aquimarina. The metagenome reads were binned and assembled into 23 nearly complete metagenome-assembled genomes (MAGs), which belonged to new families of Cyanobacteria, Myxococcota, and Granulosicoccaceae and also to the novel genus recently described as Gammaproteobacteria family UBA10353. A comparative analysis with 60 published metagenomes from different environments, including seawater, marine biofilms, soils, corals, sponges, and hydrothermal vents, indicated that banquettes have numbers of natural products and carbohydrate active enzymes (CAZymes) similar to those found for soils and were only surpassed by marine biofilms. New proteins assigned to cellulosome modules and lignocellulose-degrading enzymes were also found. These results unveiled the diverse microbial composition of P. oceanica banquettes and determined that banquettes are a potential source of bioactive compounds and novel enzymes. IMPORTANCE Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea that forms large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." These banquettes accumulate on the shore, where they can prevent erosion, although they also cause social concern due to their impact on beach use. Furthermore, Posidonia dry material has been considered a source of traditional remedies in several areas of the Mediterranean, and a few studies have been carried out to explore pharmacological activities of Posidonia extracts. The work presented here provides the first characterization of the microbiome associated with Posidonia banquettes. We carried out a metagenomic analysis together with an in-depth comparison of the banquette metagenome with 60 published metagenomes from different environments. This comparative analysis has unveiled the potential that Posidonia banquettes have for the synthesis of natural products, both in abundance (only surpassed by marine biofilms) and novelty. These products include mainly nonribosomal peptides and carbohydrate active enzymes. Thus, the interest of our work lies in the interest of Posidonia "waste" material as a source of new bioactive compounds and CAZymes.

The Domestic Environment and the Lung Mycobiome.

This study analyzes the relationship between the mycobiome of the Lower Respiratory Tract (LRT) and the fungi in the domestic environment. Samples studied consisted of Broncho-Alveolar Lavage (BAL) from 45 patients who underwent bronchoscopy for different diagnostic purposes, and dust and air from the houses (ENV) of 20 of them (44.4%). Additionally, five bronchoscopes (BS) were also analyzed and negative controls were included for every procedure. All samples were processed for DNA extraction and cultures, which were performed in Sabouraud Dextrose and Potato Dextrose Agar. The fungal Internal Transcribed Spacer (ITS2) was sequenced by the Solexa/Illumina system and sequences were analyzed by QIIME 1.8.0 and compared with the UNITE Database for identification. The similarity between the two fungal communities (BAL and ENV) for a specific patient was assessed via the percentage of coincidence in the detection of specific operational taxonomic units (OTUs), and about 75% of co-occurrence was detected between the mycobiome of the LRT and the houses. Cultures confirmed the presence of the core mycobiome species. However, the low rate of isolation from BAL suggests that most of its mycobiome corresponds to non-culturable cells. This likely depends on the patient's immune system activity and inflammatory status.

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.IMPORTANCEVibrio coralliilyticus and Vibrio mediterranei are important coral pathogens capable of inducing serious coral damage, which increases severely when they infect the host simultaneously. This has consequences related to the dispersion of these pathogens among different locations that could enhance deleterious effects on coral reefs. However, the mechanisms underlying this synergistic interaction are unknown. The work described here provides a new perspective on the complex interactions among these two Vibrio coral pathogens, suggesting that coral infection could be a collateral effect of interspecific competition. Major implications of this work are that (i) Vibrio virulence mechanisms are activated in the absence of the host as a response to interspecific competition and (ii) release of molecules by Vibrio coral pathogens produces changes in the coral microbiome that favor the pathogenic potential of the entire Vibrio community. Thus, our results highlight that social cues and competition sensing are crucial determinants of development of coral diseases.

Biophysical and physiological processes causing oxygen loss from coral reefs.

The microbialization of coral reefs predicts that microbial oxygen consumption will cause reef deoxygenation. Here we tested this hypothesis by analyzing reef microbial and primary producer oxygen metabolisms. Metagenomic data and in vitro incubations of bacteria with primary producer exudates showed that fleshy algae stimulate incomplete carbon oxidation metabolisms in heterotrophic bacteria. These metabolisms lead to increased cell sizes and abundances, resulting in bacteria consuming 10 times more oxygen than in coral incubations. Experiments probing the dissolved and gaseous oxygen with primary producers and bacteria together indicated the loss of oxygen through ebullition caused by heterogenous nucleation on algae surfaces. A model incorporating experimental production and loss rates predicted that microbes and ebullition can cause the loss of up to 67% of gross benthic oxygen production. This study indicates that microbial respiration and ebullition are increasingly relevant to reef deoxygenation as reefs become dominated by fleshy algae.

Biogeographic Differences in the Microbiome and Pathobiome of the Coral Cladocora caespitosa in the Western Mediterranean Sea.

The endemic Mediterranean zooxanthellate scleractinian reef-builder Cladocora caespitosa is among the organisms most affected by warming-related mass mortality events in the Mediterranean Sea. Corals are known to contain a diverse microbiota that plays a key role in their physiology and health. Here we report the first study that examines the microbiome and pathobiome associated with C. caespitosa in three different Mediterranean locations (i.e., Genova, Columbretes Islands, and Tabarca Island). The microbial communities associated with this species showed biogeographical differences, but shared a common core microbiome that probably plays a key role in the coral holobiont. The putatively pathogenic microbial assemblage (i.e., pathobiome) of C. caespitosa also seemed to depend on geographic location and the human footprint. In locations near the coast and with higher human influence, the pathobiome was entirely constituted by Vibrio species, including the well-known coral pathogens Vibrio coralliilyticus and V. mediterranei. However, in the Columbretes Islands, located off the coast and the most pristine of the analyzed locations, no changes among microbial communities associated to healthy and necrosed samples were detected. Hence, our results provide new insights into the microbiome of the temperate corals and its role in coral health status, highlighting its dependence on the local environmental conditions and the human footprint.

Vibrio communities in scleractinian corals differ according to health status and geographic location in the Mediterranean Sea.

The increase in seawater temperature associated with global warming is a significant threat to coral health and is linked to increasing mass mortality events and Vibrio-related coral diseases. In the Mediterranean Sea, the endemic Cladocora caespitosa and the invasive species Oculina patagonica are the main scleractinian corals affected by mass mortalities. In this study, culturable Vibrio spp. assemblages associated with healthy and unhealthy colonies of these two shallow coral species were characterized to assess the presence of Vibrio pathogens in tissue necrosis. Vibrio communities associated with O. patagonica and C. caespitosa showed geographical differences, although these became more homogeneous in unhealthy specimens of both species. Furthermore, the number of recovered Vibrio specimens was more than five times higher in unhealthy than in healthy corals. Within these culturable vibrios, the known pathogens Vibrio mediterranei and Vibrio coralliilyticus were present in unhealthy colonies of both coral species in the two localities, suggesting that they could play a role in the health status of C. caespitosa and thus act as generalist pathogens in Mediterranean corals. Nonetheless, a clonal type of V. coralliilyticus detected in C. caespitosa was not associated with disease signs, suggesting that this species could encompass assemblages with different levels of virulence.

Effects of the 2015 heat wave on benthic invertebrates in the Tabarca Marine Protected Area (southeast Spain).

In the late summer of 2015, extensive mortality of scleratinian corals, gorgonians, and sponges was observed in the Marine Protected Area of Tabarca (southeast Spain). Quantitative data indicated that at 25 m depth the sea fan Eunicella singularis was the most affected species (50% of colonies affected by partial mortality); while in shallow waters more than 40% of the endemic scleractinian coral Cladocora caespitosa population showed tissue lesions that affected more than 10% of their surfaces. Other affected species were the scleractinian corals Oculina patagonica and Phyllangia mouchezii, the sea fan Leptogorgia sarmentosa and the sponge Sarcotragus fasciculatus. This mortality event coincided with an abnormal rise in seawater temperature in this region. Microbiological analysis showed a higher abundance of culturable Vibrio species in invertebrates exhibiting tissue lesions, which indicated that these opportunistic pathogens could be a key factor in the process.

Structure and temporal dynamics of the bacterial communities associated to microhabitats of the coral Oculina patagonica.

Corals are known to contain a diverse microbiota that plays a paramount role in the physiology and health of holobiont. However, few studies have addressed the variability of bacterial communities within the coral host. In this study, bacterial community composition from the mucus, tissue and skeleton of the scleractinian coral Oculina patagonica were investigated seasonally at two locations in the Western Mediterranean Sea, to further understand how environmental conditions and the coral microbiome structure are related. We used denaturing gradient gel electrophoresis in combination with next-generation sequencing and electron microscopy to characterize the bacterial community. The bacterial communities were significantly different among coral compartments, and coral tissue displayed the greatest changes related to environmental conditions and coral health status. Species belonging to the Rhodobacteraceae and Vibrionaceae families form part of O. patagonica tissues core microbiome and may play significant roles in the nitrogen cycle. Furthermore, sequences related to the coral pathogens, Vibrio mediterranei and Vibrio coralliilyticus, were detected not only in bleached corals but also in healthy ones, even during cold months. This fact opens a new view onto unveiling the role of pathogens in the development of coral diseases in the future.

Selection of the N-Acylhomoserine Lactone-Degrading Bacterium Alteromonas stellipolaris PQQ-42 and of Its Potential for Biocontrol in Aquaculture.

The production of virulence factors by many pathogenic microorganisms depends on the intercellular communication system called quorum sensing, which involves the production and release of signal molecules known as autoinducers. Based on this, new-therapeutic strategies have emerged for the treatment of a variety of infections, such as the enzymatic degradation of signaling molecules, known as quorum quenching (QQ). In this study, we present the screening of QQ activity amongst 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of the strain PQQ-42, which degrades a wide range of N-acylhomoserine lactones (AHLs). The selected strain, identified as Alteromonas stellipolaris, degraded the accumulation of AHLs and reduced the production of protease and chitinase and swimming motility of a Vibrio species in co-cultivation experiments in vitro. In the bio-control experiment, strain PQQ-42 significantly reduced the pathogenicity of Vibrio mediterranei VibC-Oc-097 upon the coral Oculina patagonica showing a lower degree of tissue damage (29.25 ± 14.63%) in its presence, compared to when the coral was infected with V. mediterranei VibC-Oc-097 alone (77.53 ± 13.22%). Our results suggest that this AHL-degrading bacterium may have biotechnological applications in aquaculture.

Eukarya associated with the stony coral Oculina patagonica from the Mediterranean Sea.

Oculina patagonica is a putative alien scleractinian coral from the Southwest Atlantic that inhabits across the Mediterranean Sea. Here, we have addressed the diversity of Eukarya associated with this coral and its changes related to the environmental conditions and coral status. A total of 46 colonies of O. patagonica were taken from Alicante coast (Spain) and Pietra Ligure coast (Italy) and analyzed using denaturing gradient gel electrophoresis (DGGE) of the small-subunit 18S rRNA and 16S plastid rRNA genes, internal transcribed spacer region 2 (ITS 2) analyses, and electron microscopy. Our results show that Eukarya and plastid community associated to O. patagonica change with environmental conditions and coral status. Cryptic species, which can be difficult to identify by optical methods, were distinguished by 18S rRNA gene DGGE: the barnacle Megatrema anglicum, which was detected at two locations, and two boring sponges related to Cliona sp. and Siphonodictyon coralliphagum detected in samples from Tabarca and Alicante Harbour, respectively. Eukaryotic phototrophic community from the skeletal matrix of healthy corals was dominated by Ochrosphaera sp. while bleached corals from the Harbour and Tabarca were associated to different uncultured phototrophic organism. Differences in ultrastructural morphologies of the zooxanthellae between healthy and bleached corals were observed. Nevertheless, no differences were found in Symbiodinium community among time, environments, coral status and location, showing that O. patagonica hosted only one genotype of Symbiodinium belonging to clade B2. The fact that this clade has not been previously detected in other Mediterranean corals and is more frequent in the tropical Western Atlantic, is a new evidence that O. patagonica is an alien species in the Mediterranean Sea.

New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities.

Bleaching of Oculina patagonica has been extensively studied in the Eastern Mediterranean Sea, although no studies have been carried out in the Western basin. In 1996 Vibrio mediterranei was reported as the causative agent of bleaching in O. patagonica but it has not been related to bleached or healthy corals since 2003, suggesting that it was no longer involved in bleaching of O. patagonica. In an attempt to clarify the relationship between Vibrio spp., seawater temperature and coral diseases, as well as to investigate the putative differences between Eastern and Western Mediterranean basins, we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. Two sampling points located in the Spanish Mediterranean coast were chosen for this study: Alicante Harbour and the Marine Reserve of Tabarca. A complex and dynamic assemblage of Vibrio spp. was present in O. patagonica along the whole year and under different environmental conditions and coral health status. While some Vibrio spp. were detected all year around in corals, the known pathogens V. mediteranei and V. coralliilyticus were only present in diseased specimens. The pathogenic potential of these bacteria was studied by experimental infection under laboratory conditions. Both vibrios caused diseased signs from 24 °C, being higher and faster at 28 °C. Unexpectedly, the co-inoculation of these two Vibrio species seemed to have a synergistic pathogenic effect over O. patagonica, as disease signs were readily observed at temperatures at which bleaching is not normally observed.