Potential of the quorum-quenching and plant-growth promoting halotolerant Bacillus toyonensis AA1EC1 as biocontrol agent.

The use of fertilizers and pesticides to control plant diseases is widespread in intensive farming causing adverse effects together with the development of antimicrobial resistance pathogens. As the virulence of many Gram-negative phytopathogens is controlled by N-acyl-homoserine lactones (AHLs), the enzymatic disruption of this type of quorum-sensing (QS) signal molecules, mechanism known as quorum quenching (QQ), has been proposed as a promising alternative antivirulence therapy. In this study, a novel strain of Bacillus toyonensis isolated from the halophyte plant Arthrocaulon sp. exhibited numerous traits associated with plant growth promotion (PGP) and degraded a broad range of AHLs. Three lactonases and an acylase enzymes were identified in the bacterial genome and verified in vitro. The AHL-degrading activity of strain AA1EC1 significantly attenuated the virulence of relevant phytopathogens causing reduction of soft rot symptoms on potato and carrots. In vivo assays showed that strain AA1EC1 significantly increased plant length, stem width, root and aerial dry weights and total weight of tomato and protected plants against Pseudomonas syringae pv. tomato. To our knowledge, this is the first report to demonstrate PGP and QQ activities in the species B. toyonensis that make this strain as a promising phytostimulant and biocontrol agent.

The synergy of halotolerant PGPB and mauran mitigates salt stress in tomato (Solanum lycopersicum) via osmoprotectants accumulation.

Salinity stress is one of the major abiotic factors limiting sustainable agriculture. Halotolerant plant growth-promoting bacteria (PGPB) increased salt stress tolerance in plants, but the mechanisms underlying the tolerance are poorly understood. This study investigated the PGP activity of four halotolerant bacteria under salinity stress and the tomato salt-tolerance mechanisms induced by the synergy of these bacteria with the exopolysaccharide (EPS) mauran. All PGPB tested in this study were able to offer a significant improvement of tomato plant biomass under salinity stress; Peribacillus castrilensis N3 being the most efficient one. Tomato plants treated with N3 and the EPS mauran showed greater tolerance to NaCl than the treatment in the absence of EPS and PGPB. The synergy of N3 with mauran confers salt stress tolerance in tomato plants by increasing sodium transporter genes' expression and osmoprotectant content, including soluble sugars, polyols, proline, GABA, phenols and the polyamine putrescine. These osmolytes together with the induction of sodium transporter genes increase the osmotic adjustment capacity to resist water loss and maintain ionic homeostasis. These findings suggest that the synergy of the halotolerant bacterium N3 and the EPS mauran could enhance tomato plant growth by mitigating salt stress and could have great potential as an inductor of salinity tolerance in the agriculture sector.

Corrigendum: Identification of volatile organic compounds in extremophilic bacteria and their effective use in biocontrol of postharvest fungal phytopathogens.

[This corrects the article DOI: 10.3389/fmicb.2021.773092.].

Peribacillus castrilensis sp. nov.: A Plant-Growth-Promoting and Biocontrol Species Isolated From a River Otter in Castril, Granada, Southern Spain.

A strictly aerobic, chemoheterotrophic, endospore-forming, Gram-positive, rod-shaped bacterial strain N3T was isolated from the feces of a river otter in Castril (Granada, southern Spain). It is halotolerant, motile, and catalase-, oxidase-, ACC deaminase-, and C4- and C8-lipase-positive. It promotes tomato plant growth and can reduce virulence in Erwinia amylovora CECT 222T and Dickeya solani LMG 25993T through interference in their quorum-sensing systems, although other antagonistic mechanisms could also occur. A phylogenetic analysis of the 16S rRNA gene sequence as well as the phenotypic and phylogenomic analyses indicated that the strain N3T is a novel species of the genus Peribacillus, with the highest 16S rRNA sequence similar to that of Bacillus frigoritolerans DSM 8801T (99.93%) and Peribacillus simplex DSM 1321T (99.80%). Genomic digital DNA-DNA hybridization (dDDH) between the strain N3T and Bacillus frigoritolerans DSM 8801T and Peribacillus simplex was 12.8 and 69.1%, respectively, and the average nucleotide identity (ANIb) of strain N3T and Bacillus frigoritolerans DSM 8801T and Peribacillus simplex was 67.84 and 93.21%, respectively. The genomic G + C content was 40.3 mol%. Its main cellular fatty acids were anteiso-C15:0 and iso-C15:0. Using 16S rRNA phylogenetic and in silico phylogenomic analyses, together with the chemotaxonomic and phenotypic data, we demonstrated that the type strain N3T (=CECT 30509T = LMG 32505T) is a novel species of the genus Peribacillus and the name Peribacillus castrilensis sp. nov. is proposed.

Quorum Quenching Strains Isolated from the Microbiota of Sea Anemones and Holothurians Attenuate Vibriocorallilyticus Virulence Factors and Reduce Mortality in Artemiasalina.

Interference with quorum-sensing (QS) intercellular communication systems by the enzymatic disruption of N-acylhomoserine lactones (AHLs) in Gram-negative bacteria has become a promising strategy to fight bacterial infections. In this study, seven strains previously isolated from marine invertebrates and selected for their ability to degrade C6 and C10-HSL, were identified as Acinetobacter junii, Ruegeria atlantica, Microbulbifer echini, Reinheimera aquimaris, and Pseudomonas sihuiensis. AHL-degrading activity against a wide range of synthetic AHLs were identified by using an agar well diffusion assay and Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 and VIR07 as biosensors. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis indicated that this activity was not due to an AHL lactonase. All the strains degraded Vibrio coralliilyticus AHLs in coculture experiments, while some strains reduced or abolished the production of virulence factors. In vivo assays showed that strains M3-111 and M3-127 reduced this pathogen's virulence and increased the survival rate of Artemia salina up to 3-fold, indicating its potential use for biotechnological purposes. To our knowledge, this is the first study to describe AHL-degrading activities in some of these marine species. These findings highlight that the microbiota associated with marine invertebrates constitute an important underexplored source of biological valuable compounds.

Identification of Volatile Organic Compounds in Extremophilic Bacteria and Their Effective Use in Biocontrol of Postharvest Fungal Phytopathogens.

Phytopathogenic fungal growth in postharvest fruits and vegetables is responsible for 20-25% of production losses. Volatile organic compounds (VOCs) have been gaining importance in the food industry as a safe and ecofriendly alternative to pesticides for combating these phytopathogenic fungi. In this study, we analysed the ability of some VOCs produced by strains of the genera Bacillus, Peribacillus, Pseudomonas, Psychrobacillus and Staphylococcus to inhibit the growth of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Monilinia fructicola, Monilinia laxa and Sclerotinia sclerotiorum, in vitro and in vivo. We analysed bacterial VOCs by using GC/MS and 87 volatile compounds were identified, in particular acetoin, acetic acid, 2,3-butanediol, isopentanol, dimethyl disulphide and isopentyl isobutanoate. In vitro growth inhibition assays and in vivo experiments using cherry fruits showed that the best producers of VOCs, Bacillus atrophaeus L193, Bacillus velezensis XT1 and Psychrobacillus vulpis Z8, exhibited the highest antifungal activity against B. cinerea, M. fructicola and M. laxa, which highlights the potential of these strains to control postharvest diseases. Transmission electron microscopy micrographs of bacterial VOC-treated fungi clearly showed antifungal activity which led to an intense degeneration of cellular components of mycelium and cell death.

AhaP, A Quorum Quenching Acylase from Psychrobacter sp. M9-54-1 That Attenuates Pseudomonas aeruginosa and Vibrio coralliilyticus Virulence.

Although Psychrobacter strain M9-54-1 had been previously isolated from the microbiota of holothurians and shown to degrade quorum sensing (QS) signal molecules C6 and C10-homoserine lactone (HSL), little was known about the gene responsible for this activity. In this study, we determined the whole genome sequence of this strain and found that the full 16S rRNA sequence shares 99.78-99.66% identity with Psychrobacter pulmonis CECT 5989T and P. faecalis ISO-46T. M9-54-1, evaluated using the agar well diffusion assay method, showed high quorum quenching (QQ) activity against a wide range of synthetic N-acylhomoserine lactone (AHLs) at 4, 15, and 28 °C. High-performance liquid chromatography-mass-spectrometry (HPLC-MS) confirmed that QQ activity was due to an AHL-acylase. The gene encoding for QQ activity in strain M9-54-1 was identified from its genome sequence whose gene product was named AhaP. Purified AhaP degraded substituted and unsubstituted AHLs from C4- to C14-HSL. Furthermore, heterologous expression of ahaP in the opportunistic pathogen Pseudomonas aeruginosa PAO1 reduced the expression of the QS-controlled gene lecA, encoding for a cytotoxic galactophilic lectin and swarming motility protein. Strain M9-54-1 also reduced brine shrimp mortality caused by Vibrio coralliilyticus VibC-Oc-193, showing potential as a biocontrol agent in aquaculture.

Roseovarius bejariae sp. nov., a moderately halophilic bacterium isolated from a hypersaline steep-sided river bed.

An aerobic, Gram-stain-negative ovoid, designated as strain A21T, was isolated using the dilution-to-extinction method from a soil sample taken from Rambla Salada, an athalassohaline habitat located in Murcia (south-eastern Spain). Strain A21T is non-motile, has a respiratory metabolism and grows at NaCl concentrations within the range 0.5-15 % (w/v) [optimum, 5 % (w/v)], at 5-35 °C (optimum, 28 °C) and at pH 6-8 (optimum, pH 7.0). This strain is positive for catalase activity, oxidase activity and nitrate reduction. The 16S rRNA gene sequence indicates that it belongs to the genus Roseovarius in the class Alphaproteobacteria. The most closely related species are Roseovarius pacificus and Roseovarius halotolerans to which the strain A21T shows 16S rRNA gene sequence similarity values of 98.06 and 97.7 %, respectively. The average nucleotide identity in blast and digital DNA-DNA hybridization values between strain A21T and R. pacificus LMG 24575T are 76.8 and 21 %, respectively. The DNA G+C content based on the genome is 61.28 mol%. The major fatty acids (>5 % of the total fatty acids) of strain A21T are C18 : 1 ω7c/C18 : 1 ω6c and C16 : 0. The only detected isoprenoid quinone in strain A21T is ubiquinone 10 (Q-10). The polar lipid profile contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and three unidentified polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Roseovarius, for which the name Roseovarius bejariae sp. nov. is proposed. Strain A21T (=CECT 9817T=LMG 31311T) is the type strain.

Effects of Halophyte Root Exudates and Their Components on Chemotaxis, Biofilm Formation and Colonization of the Halophilic Bacterium Halomonas Anticariensis FP35T.

Increase in soil salinity poses an enormous problem for agriculture and highlights the need for sustainable crop production solutions. Plant growth-promoting bacteria can be used to boost the growth of halophytes in saline soils. Salicornia is considered to be a promising salt-accumulating halophyte for capturing large amounts of carbon from the atmosphere. In addition, colonization and chemotaxis could play an important role in Salicornia-microbe interactions. In this study, the role of chemotaxis in the colonization of the halophilic siredophore-producing bacteria, Halomonas anticariensis FP35T, on Salicornia hispanica plants was investigated. The chemotactic response of FP35T to Salicornia root exudates showed optimum dependence at a salt concentration of 5 % NaCl (w/v). Oleanolic acid, the predominant compound in the exudates detected by HPLC and identified by UPLC-HRMS Q-TOF, acts as a chemoattractant. In vitro experiments demonstrated the enhanced positive effects of wild-type H. anticariensis strain FP35T on root length, shoot length, germination and the vigour index of S. hispanica. Furthermore, these positive effects partially depend on an active chemotaxis system, as the chemotaxis mutant H. anticariensis FP35 ΔcheA showed reduced plant growth promotion for all the parameters tested. Overall, our results suggest that chemotaxis responses to root exudates play an important role in interactions between Salicornia and halophilic bacteria, enhance their colonization and boost plant growth promotion. Preliminary results also indicate that root exudates have a positive impact on H. anticariensis FP35T biofilm formation under saline conditions, an effect which totally depends on the presence of the cheA gene.

Plant growth-promoting activity and quorum quenching-mediated biocontrol of bacterial phytopathogens by Pseudomonas segetis strain P6.

Given the major threat of phytopathogenic bacteria to food production and ecosystem stability worldwide, novel alternatives to conventional chemicals-based agricultural practices are needed to combat these bacteria. The objective of this study is to evaluate the ability of Pseudomonas segetis strain P6, which was isolated from the Salicornia europaea rhizosphere, to act as a potential biocontrol agent given its plant growth-promoting (PGP) and quorum quenching (QQ) activities. Seed biopriming and in vivo assays of tomato plants inoculated with strain P6 resulted in an increase in seedling height and weight. We detected QQ activity, involving enzymatic degradation of signal molecules in quorum sensing communication systems, against a broad range of N-acylhomoserine lactones (AHLs). HPLC-MRM data and phylogenetic analysis indicated that the QQ enzyme was an acylase. The QQ activity of strain P6 reduced soft rot symptoms caused by Dickeya solani, Pectobacterium atrosepticum and P. carotovorum on potato and carrot. In vivo assays showed that the PGP and QQ activities of strain P6 protect tomato plants against Pseudomonas syringae pv. tomato, indicating that strain P6 could have biotechnological applications. To our knowledge, this is the first report to show PGP and QQ activities in an indigenous Pseudomonas strain from Salicornia plants.

Characterization of HLA-A*01:302 in two Spanish individuals by sequencing-based typing.

HLA-A*01:302 was likely generated by intralocus conversion between an A*01:01:01 allele and an A*29 allele.

Psychrobacillus vulpis sp. nov., a new species isolated from faeces of a red fox in Spain.

A facultative anaerobic, chemoheterotrophic, endospore-forming, Gram-stain-positive rod, designated as strain Z8T, was isolated from red fox (Vulpes vulpes) faeces sampled at Tablas de Daimiel National Park, Ciudad Real, Spain. Strain Z8T grew at 0-37 °C (optimum, 28 °C), in the presence of 0-5.5 % (w/v) NaCl (2.5 %, w/v) and at pH 6-10 (pH 7). The strain was motile and positive for catalase, oxidase, H2S and siderophore production, acid and alkaline phosphatases, and N-acetylglucosamine, adipic acid and malate assimilation. It hydrolysed starch, DNA, l-tyrosine, Tween 20, Tween 80 and lecithovitellin. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Z8T is a member of the genus Psychrobacillus, showing high sequence similarity to Psychrobacillus lasiicapitis NEAU-3TGS17T (99.2 %) and Psychrobacillus soli NHI-2TT (99.1 %), and around 98 % to other known species of the genus Psychrobacillus. Digital DNA-DNA hybridization and average nucleotide identity values were lower than 24 and 79 %, respectively, with the most related species. In silico G+C content was 35.9 mol%. The major cellular fatty acids of strain Z8T were iso-C14 : 0, iso-C15 : 0 and anteiso-C15 : 0. The novel strain contained diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol as predominant polar lipids, and the main respiratory isoprenoid quinone was MK-8. Based on the 16S rRNA phylogenetic analysis, together with MLSA (recA, rpoB and gyrB), phylogenomic, chemotaxonomic and phenotypic results, we demonstrate that strain Z8T represents a novel species of the genus Psychrobacillus, for which the name Psychrobacillus vulpis sp. nov., is proposed. The type strain is Z8T (=CECT 9721T=LMG 31001T).

Paenibacillus lutrae sp. nov., A Chitinolytic Species Isolated from A River Otter in Castril Natural Park, Granada, Spain.

A highly chitinolytic facultative anaerobic, chemoheterotrophic, endospore-forming, Gram-stain-positive, rod-shaped bacterial strain N10T was isolated from the feces of a river otter in the Castril Natural Park (Granada, Spain). It is a slightly halophilic, motile, catalase-, oxidase-, ACC deaminase- and C4 and C8 lipase-positive strain. It is aerobic, respiratory and has a fermentative metabolism using oxygen as an electron acceptor, produces acids from glucose and can fix nitrogen. Phylogenetic analysis of the 16S rRNA gene sequence, multilocus sequence analysis (MLSA) of 16S rRNA, gyrB, recA and rpoB, as well as phylogenomic analyses indicate that strain N10T is a novel species of the genus Paenibacillus, with the highest 16S rRNA sequence similarity (95.4%) to P. chitinolyticus LMG 18047T and <95% similarity to other species of the genus Paenibacillus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANIb) were 21.1% and <75%, respectively. Its major cellular fatty acids were anteiso-C15:0, C16:0, and iso-C15:0. G + C content ranged between 45%-50%. Using 16S rRNA phylogenetic and in silico phylogenomic analyses, together with chemotaxonomic and phenotypic data, we demonstrate that type strain N10T (= CECT 9541T =LMG 30535T) is a novel species of genus Paenibacillus and the name Paenibacillus lutrae sp. nov. is proposed.

Silencing of Phytopathogen Communication by the Halotolerant PGPR Staphylococcus equorum Strain EN21.

Increasing world food demand together with soil erosion and indiscriminate use of chemical fertilization highlight the need to adopt sustainable crop production strategies. In this context, a combination of plant growth-promoting rhizobacteria (PGPR) and pathogen management represents a sustainable and efficient alternative. Though little studied, halophilic and halotolerant PGPR could be a beneficial plant growth promotion strategy for saline and non-saline soils. The virulence of many bacterial phytopathogens is regulated by quorum sensing (QS) systems. Quorum quenching (QQ) involves the enzymatic degradation of phytopathogen-generated signal molecules, mainly N-acyl homoserine lactones (AHLs). In this study, we investigate plant growth-promoting (PGP) activity and the capacity of the halotolerant bacterium Staphylococcus equorum strain EN21 to attenuate phytopathogens virulence through QQ. We used biopriming and in vivo tomato plant experiments to analyse the PGP activity of strain EN21. AHL inactivation was observed to reduce Pseudomonas syringae pv. tomato infections in tomato and Arabidopsis plants. Our study of Dickeya solani, Pectobacterium carotovorum subsp. carotovorum and Erwinia amylovora bacteria in potato tubers, carrots and pears, respectively, also demonstrated the effectiveness of QS interruption by EN21. Overall, this study highlights the potential of strain S. equorum EN21 in plant growth promotion and QQ-driven bacterial phytopathogen biocontrol.

A Quorum-Sensing Inhibitor Strain of Vibrio alginolyticus Blocks Qs-Controlled Phenotypes in Chromobacterium violaceum and Pseudomonas aeruginosa.

The cell density-dependent mechanism, quorum sensing (QS), regulates the expression of virulence factors. Its inhibition has been proposed as a promising new strategy to prevent bacterial pathogenicity. In this study, 827 strains from the microbiota of sea anemones and holothurians were screened for their ability to produce quorum-sensing inhibitor (QSI) compounds. The strain M3-10, identified as Vibrio alginolyticus by 16S rRNA gene sequencing, as well as ANIb and dDDH analyses, was selected for its high QSI activity. Bioassay-guided fractionation of the cell pellet extract from a fermentation broth of strain M3-10, followed by LC-MS and NMR analyses, revealed tyramine and N-acetyltyramine as the active compounds. The QS inhibitory activity of these molecules, which was confirmed using pure commercially available standards, was found to significantly inhibit Chromobacterium violaceum ATCC 12472 violacein production and virulence factors, such as pyoverdine production, as well as swarming and twitching motilities, produced by Pseudomonas aeruginosa PAO1. This constitutes the first study to screen QSI-producing strains in the microbiota of anemones and holothurians and provides an insight into the use of naturally produced QSI as a possible strategy to combat bacterial infections.

Saline Environments as a Source of Potential Quorum Sensing Disruptors to Control Bacterial Infections: A Review.

Saline environments, such as marine and hypersaline habitats, are widely distributed around the world. They include sea waters, saline lakes, solar salterns, or hypersaline soils. The bacteria that live in these habitats produce and develop unique bioactive molecules and physiological pathways to cope with the stress conditions generated by these environments. They have been described to produce compounds with properties that differ from those found in non-saline habitats. In the last decades, the ability to disrupt quorum-sensing (QS) intercellular communication systems has been identified in many marine organisms, including bacteria. The two main mechanisms of QS interference, i.e., quorum sensing inhibition (QSI) and quorum quenching (QQ), appear to be a more frequent phenomenon in marine aquatic environments than in soils. However, data concerning bacteria from hypersaline habitats is scarce. Salt-tolerant QSI compounds and QQ enzymes may be of interest to interfere with QS-regulated bacterial functions, including virulence, in sectors such as aquaculture or agriculture where salinity is a serious environmental issue. This review provides a global overview of the main works related to QS interruption in saline environments as well as the derived biotechnological applications.

Genomic analyses of two Alteromonas stellipolaris strains reveal traits with potential biotechnological applications.

The Alteromonas stellipolaris strains PQQ-42 and PQQ-44, previously isolated from a fish hatchery, have been selected on the basis of their strong quorum quenching (QQ) activity, as well as their ability to reduce Vibrio-induced mortality on the coral Oculina patagonica. In this study, the genome sequences of both strains were determined and analyzed in order to identify the mechanism responsible for QQ activity. Both PQQ-42 and PQQ-44 were found to degrade a wide range of N-acylhomoserine lactone (AHL) QS signals, possibly due to the presence of an aac gene which encodes an AHL amidohydrolase. In addition, the different colony morphologies exhibited by the strains could be related to the differences observed in genes encoding cell wall biosynthesis and exopolysaccharide (EPS) production. The PQQ-42 strain produces more EPS (0.36 g l-1) than the PQQ-44 strain (0.15 g l-1), whose chemical compositions also differ. Remarkably, PQQ-44 EPS contains large amounts of fucose, a sugar used in high-value biotechnological applications. Furthermore, the genome of strain PQQ-42 contained a large non-ribosomal peptide synthase (NRPS) cluster with a previously unknown genetic structure. The synthesis of enzymes and other bioactive compounds were also identified, indicating that PQQ-42 and PQQ-44 could have biotechnological applications.

Stenotrophomonas maltophilia AHL-Degrading Strains Isolated from Marine Invertebrate Microbiota Attenuate the Virulence of Pectobacterium carotovorum and Vibrio coralliilyticus.

Many Gram-negative aquacultural and agricultural pathogens control virulence factor expression through a quorum-sensing (QS) mechanism involving the production of N-acylhomoserine (AHL) signalling molecules. Thus, the interruption of QS systems by the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), has been proposed as a novel strategy to combat these infections. Given that the symbiotic bacteria of marine invertebrates are considered to be an important source of new bioactive molecules, this study explores the presence of AHL-degrading bacteria among 827 strains previously isolated from the microbiota of anemones and holothurians. Four of these strains (M3-1, M1-14, M3-13 and M9-54-2), belonging to the species Stenotrophomonas maltophilia, were selected on the basis of their ability to degrade a broad range of AHLs, and the enzymes involved in their activity were identified. Strain M9-54-2, which showed the strongest AHL-degrading activity, was selected for further study. High-performance liquid chromatography-mass-spectrometry confirmed that the QQ enzyme is not a lactonase. Strain M9-54-2 degraded AHL accumulation and reduced the production of enzymatic activity in Pectobacterium carotovorum CECT 225T and Vibrio coralliilyticus VibC-Oc-193 in in vitro co-cultivation experiments. The effect of AHL inactivation was confirmed by a reduction in potato tuber maceration and brine shrimp (Artemia salina) mortality caused by P. carotovorum and Vibrio coralliilyticus, respectively. This study strengthens the evidence of marine organisms as an underexplored and promising source of QQ enzymes, useful to prevent infections in aquaculture and agriculture. To our knowledge, this is the first time that anemones and holothurians have been studied for this purpose.

Study of Bacterial Community Composition and Correlation of Environmental Variables in Rambla Salada, a Hypersaline Environment in South-Eastern Spain.

We studied the bacterial community in Rambla Salada in three different sampling sites and in three different seasons and the effect of salinity, oxygen, and pH. All sites samples had high diversity and richness (Rr > 30). The diversity indexes and the analysis of dendrograms obtained by DGGE fingerprint after applying Pearson's and Dice's coefficient showed a strong influence of sampling season. The Pareto-Lorenz (PL) curves and Fo analysis indicated that the microbial communities were balanced and despite the changing environmental conditions, they can preserve their functionality. The main phyla detected by DGGE were Bacteroidetes (39.73%), Proteobacteria (28.43%), Firmicutes (8.23%), and Cyanobacteria (5.14%). The majority of the sequences corresponding to uncultured bacteria belonged to Bacteroidetes phylum. Within Proteobacteria, the main genera detected were Halothiobacillus and Roseovarius. The environmental factors which influenced the community in a higher degree were the salinity and oxygen. The bacteria belonging to Bacteroidetes and Proteobacteria were positively influenced by salinity. Nevertheless, bacteria related to Alpha- and Betaproteobacteria classes and phylum Firmicutes showed a positive correlation with oxygen and pH but negative with salinity. The phylum Cyanobacteria were less influenced by the environmental variables. The bacterial community composition of Rambla Salada was also studied by dilution-to-extinction technique. Using this method, 354 microorganisms were isolated. The 16S sequences of 61 isolates showed that the diversity was very different to those obtained by DGGE and with those obtained previously by using classic culture techniques. The taxa identified by dilution-to-extinction were Proteobacteria (81.92%), Firmicutes (11.30%), Actinobacteria (4.52%), and Bacteroidetes (2.26%) phyla with Gammaproteobacteria as predominant class (65.7%). The main genera were: Marinobacter (38.85%), Halomonas (20.2%), and Bacillus (11.2%). Nine of the 61 identified bacteria showed less than 97% sequence identity with validly described species and may well represent new taxa. The number of bacteria in different samples, locations, and seasons were calculated by CARD-FISH, ranging from 54.3 to 78.9% of the total prokaryotic population. In conclusion, the dilution-to-extinction technique could be a complementary method to classical culture based method, but neither gets to cultivate the major taxa detected by DGGE. The bacterial community was influenced significantly by the physico-chemical parameters (specially the salinity and oxygen), the location and the season of sampling.

Diversity and antimicrobial potential in sea anemone and holothurian microbiomes.

Marine invertebrates, as holobionts, contain symbiotic bacteria that coevolve and develop antimicrobial substances. These symbiotic bacteria are an underexplored source of new bioactive molecules to face the emerging antibiotic resistance in pathogens. Here, we explored the antimicrobial activity of bacteria retrieved from the microbiota of two sea anemones (Anemonia sulcata, Actinia equina) and two holothurians (Holothuria tubulosa, Holothuria forskali). We tested the antimicrobial activity of the isolated bacteria against pathogens with interest for human health, agriculture and aquaculture. We isolated 27 strains with antibacterial activity and 12 of these isolates also showed antifungal activity. We taxonomically identified these strains being Bacillus and Vibrio species the most representative producers of antimicrobial substances. Microbiome species composition of the two sea anemones was similar between them but differed substantially of seawater bacteria. In contrast, microbiome species composition of the two holothurian species was different between them and in comparison with the bacteria in holothurian feces and seawater. In all the holobiont microbiomes Bacteroidetes was the predominant phylum. For each microbiome, we determined diversity and the rank-abundance dominance using five fitted models (null, pre-emption, log-Normal, Zipf and Zipf-Mandelbrot). The models with less evenness (i.e. Zipf and Zipf-Mandelblot) showed the best fits in all the microbiomes. Finally, we tracked (using the V4 hypervariable region of 16S rRNA gene) the relative abundance of these 27 isolates with antibacterial activity in the total pool of sequences obtained for the microbiome of each holobiont. Coincidences, although with extremely low frequencies, were detected only in the microbiome of H. forskali. This fact suggests that these isolated bacteria belong to the long tail of rare symbiotic bacteria. Therefore, more and more sophisticated culture techniques are necessary to explore this apparently vast pool of rare symbiontic bacteria and to determine their biotechnological potentiality.