Interference of AHL signal production in the phytophatogen Pantoea agglomerans as a sustainable biological strategy to reduce its virulence.

Pantoea agglomerans is considered one of the most ubiquitous and versatile organisms that include strains that induce diseases in various crops and occasionally cause opportunistic infections in humans. To develop effective strategies to mitigate its impact on plant health and agricultural productivity, a comprehensive investigation is crucial for better understanding its pathogenicity. One proposed eco-friendly approach involves the enzymatic degradation of quorum sensing (QS) signal molecules like N-acylhomoserine lactones (AHLs), known as quorum quenching (QQ), offering potential treatment for such bacterial diseases. In this study the production of C4 and 3-oxo-C6HSL was identified in the plant pathogenic P. agglomerans CFBP 11141 and correlated to enzymatic activities such as amylase and acid phosphatase. Moreover, the heterologous expression of a QQ enzyme in the pathogen resulted in lack of AHLs production and the attenuation of the virulence by mean of drastically reduction of soft rot disease in carrots and cherry tomatoes. Additionally, the interference with the QS systems of P. agglomerans CFBP 11141 by two the plant growth-promoting and AHL-degrading bacteria (PGP-QQ) Pseudomonas segetis P6 and Bacillus toyonensis AA1EC1 was evaluated as a potential biocontrol approach for the first time. P. segetis P6 and B. toyonensis AA1EC1 demonstrated effectiveness in diminishing soft rot symptoms induced by P. agglomerans CFBP 11141 in both carrots and cherry tomatoes. Furthermore, the virulence of pathogen notably decreased when co-cultured with strain AA1EC1 on tomato plants.

Metagenomic insights into the impact of litter from poultry Concentrated Animal Feeding Operations (CAFOs) to adjacent soil and water microbial communities.

In recent decades, human food consumption has led to an increased demand for animal-based foods, particularly chicken meat production. The state of Georgia, USA is one of the top broiler chicken producers in the United States, where animals are raised in Concentrated Animal Feeding Operations (CAFOs). Without proper management, CAFOs could negatively impact the environment and become a public health risk as a source of water and air pollution and/or by spreading antimicrobial resistance genes. In this study, we used metagenome sequencing to investigate the impact of the application of the CAFO's litter on adjacent soils and downstream creek waters in terms of microbial diversity and antimicrobial resistance profile changes. Our data indicate that while a few microbial groups increased in abundance within a short period of time after litter application, these populations subsequently decreased to levels similar to those found prior to the litter application or to below the detection limit of our metagenome sequencing effort. Microbial taxonomic composition analyses, relative abundance of Metagenome-Assembled Genomes (MAGs) and detection of Antimicrobial Resistance Genes (ARGs) allow us to conclude that this practice of litter application had a negligible effect on the microbiome or resistome profile of these soils and nearby waterways, likely due to its dilution in the field and/or outcompetition by indigenous microbes, revealing a minimal impact of these poultry facilities on the natural microbial communities.

Discovery of the Streamlined Haloarchaeon Halorutilus salinus, Comprising a New Order Widespread in Hypersaline Environments across the World.

The class Halobacteria is one of the most diverse groups within the Euryarchaeota phylum, whose members are ubiquitously distributed in hypersaline environments, where they often constitute the major population. Here, we report the discovery and isolation of a new halophilic archaeon, strain F3-133T exhibiting ≤86.3% 16S rRNA gene identity to any previously cultivated archaeon, and, thus, representing a new order. Analysis of available 16S rRNA gene amplicon and metagenomic data sets showed that the new isolate represents an abundant group in intermediate-to-high salinity ecosystems and is widely distributed across the world. The isolate presents a streamlined genome, which probably accounts for its ecological success in nature and its fastidious growth in culture. The predominant osmoprotection mechanism appears to be the typical salt-in strategy used by other haloarchaea. Furthermore, the genome contains the complete gene set for nucleotide monophosphate degradation pathway through archaeal RuBisCO, being within the first halophilic archaea representatives reported to code this enzyme. Genomic comparisons with previously described representatives of the phylum Euryarchaeota were consistent with the 16S rRNA gene data in supporting that our isolate represents a novel order within the class Halobacteria for which we propose the names Halorutilales ord. nov., Halorutilaceae fam. nov., Halorutilus gen. nov. and Halorutilus salinus sp. nov. IMPORTANCE The discovery of the new halophilic archaeon, Halorutilus salinus, representing a novel order, family, genus, and species within the class Halobacteria and phylum Euryarchaeota clearly enables insights into the microbial dark matter, expanding the current taxonomical knowledge of this group of archaea. The in-depth comparative genomic analysis performed on this new taxon revealed one of the first known examples of an Halobacteria representative coding the archaeal RuBisCO gene and with a streamlined genome, being ecologically successful in nature and explaining its previous non-isolation. Altogether, this research brings light into the understanding of the physiology of the Halobacteria class members, their ecological distribution, and capacity to thrive in hypersaline environments.

Genomic-based phylogenetic and metabolic analyses of the genus Natronomonas, and description of Natronomonas aquatica sp. nov.

The genus Natronomonas is classified on the family Haloarculaceae, within the class Halobacteria and currently includes six species isolated from salterns, saline or soda lakes, and salt mines. All are extremely halophilic (optimal growth at 20-25% [w/v] NaCl) and neutrophilic, except Natronomonas pharaonis, the type species of the genus, that is haloalkaliphilic (showing optimal growth at pH 9.0) and possesses distinct phenotypic features, such as a different polar lipid profile than the rest of species of the genus. We have carried out a genome-based study in order to determine the phylogenetic structure of the genus Natronomonas and elucidate its current taxonomic status. Overall genomic relatedness indexes, i.e., OrthoANI (Average Nucleotide Identity), dDDH (digital DNA-DNA hybridization), and AAI (Average Amino acid Identity), were determined with respect to the species of Natronomonas and other representative taxa of the class Halobacteria. Our data show that the six species of Natronomonas constitute a coherent cluster at the genus level. Besides, we have characterized a new haloarchaeon, strain F2-12T, isolated from the brine of a pond of a saltern in Isla Cristina, Huelva, Spain, and we determined that it constitutes a new species of Natronomonas, for which we propose the name Natronomonas aquatica sp. nov. Besides, the metabolic analysis revealed a heterotrophic lifestyle and a versatile nitrogen metabolism for members of this genus. Finally, metagenomic fragment recruitments from a subset of hypersaline habitats, indicated that the species of Natronomonas are widely distributed in saline lakes and salterns as well as on saline soils. Species of this haloarchaeal genus can be considered as ubiquitous in intermediate to high salinity habitats.

Genomic Insights Into New Species of the Genus Halomicroarcula Reveals Potential for New Osmoadaptative Strategies in Halophilic Archaea.

Metagenomic studies on prokaryotic diversity of hypersaline soils from the Odiel saltmarshes, South-west Spain, revealed a high proportion of genomic sequences not related to previously cultivated taxa, that might be related to haloarchaea with a high environmental and nutritional flexibility. In this study, we used a culturomics approach in order to isolate new haloarchaeal microorganisms from these hypersaline soils. Four haloarchaeal strains, designated strains F24AT, F28, F27T, and F13T, phylogenetically related to the genus Halomicroarcula, were isolated and characterized in detail. The phylogenomic tree based on the 100 orthologous single-copy genes present in the genomes of these four strains as well as those of the type strains of the species Halomicroarcula pellucida CECT 7537T, Halomicroarcula salina JCM 18369T and Halomicroarcula limicola JCM 18640T, that were determined in this study, revealed that these four new isolates clustered on three groups, with strains F24AT and F28 within a single cluster, and altogether with the species of Halomicroarcula. Additionally, Orthologous Average Nucleotide Identity (OrthoANI), digital DNA-DNA hybridization (dDDH) and Average Amino-acid Identity (AAI) values, likewise phenotypic characteristics, including their polar lipids profiles, permitted to determine that they represent three new species, for which we propose the names Halomicroarcula rubra sp. nov. (type strain F13T), Halomicroarcula nitratireducens sp. nov. (type strain F27T) and Halomicroarcula salinisoli sp. nov. (type strain F24AT). An in deep comparative genomic analysis of species of the genus Halomicroarcula, including their metabolism, their capability to biosynthesize secondary metabolites and their osmoregulatory adaptation mechanisms was carried out. Although they use a salt-in strategy, the identification of the complete pathways for the biosynthesis of the compatible solutes trehalose and glycine betaine, not identified before in any other haloarchaea, might suggest alternative osmoadaptation strategies for this group. This alternative osmoregulatory mechanism would allow this group of haloarchaea to be versatile and eco-physiologically successful in hypersaline environments and would justify the capability of the species of this genus to grow not only on environments with high salt concentrations [up to 30% (w/v) salts], but also under intermediate to low salinities.

Taxogenomic and Metabolic Insights into Marinobacterium ramblicola sp. nov., a New Slightly Halophilic Bacterium Isolated from Rambla Salada, Murcia.

A Gram-negative, motile, rod-shaped bacteria, designated D7T, was isolated by using the dilution-to-extinction method, from a soil sample taken from Rambla Salada (Murcia, Spain). Growth of strain D7T was observed at 15-40 °C (optimum, 37 °C), pH 5-9 (optimum, 7) and 0-7.5% (w/v) NaCl (optimum, 3%). It is facultatively anaerobic. Phylogenetic analysis based on 16S rRNA gene sequence showed it belongs to the genus Marinobacterium. The in silico DDH and ANI against closest Marinobacterium relatives support its placement as a new species within this genus. The major fatty acids of strain D7T were C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The polar lipid profile consists of phosphatidylethanolamine, phosphatidylglycerol and two uncharacterized lipids. Ubiquinone 8 was the unique isoprenoid quinone detected. The DNA G + C content was 59.2 mol%. On the basis of the phylogenetic, phenotypic, chemotaxonomic and genomic characterization, strain D7T (= CECT 9818T = LMG 31312T) represents a novel species of the genus Marinobacterium for which the name Marinobacterium ramblicola sp. nov. is proposed. Genome-based metabolic reconstructions of strain D7T suggested a heterotrophic and chemolitotrophic lifestyle, as well as the capacity to biosynthetize and catabolize compatible solutes, and to degrade hydrocarbon aromatic compounds.

Culturomics-based genomics sheds light on the ecology of the new haloarchaeal genus Halosegnis.

The development of culture-independent techniques has revolutionized our understanding of microbial ecology, especially through the illustration of the vast gap between the environmentally abundant microbial diversity and that accessible through cultivation. However, culture-based approaches are not only crucial for understanding the evolutionary, metabolic and ecological milieu of microbial diversity but also for the development of novel biotechnological applications. In this study, we used a culturomics-based approach in order to isolate novel microbial taxa from hypersaline environments (i.e. Isla Cristina and Isla Bacuta salterns in Huelva, Spain). We managed to obtain axenic cultures of four haloarchaeal strains that belong to a new haloarchaeal genus and to obtain their genomic sequences. The phylogenomic and phylogenetic analyses (together with AAI, ANI and digital DDH indices) showed that the isolates constitute two new species, for which we propose the names Halosegnis longus sp. nov. and Halosegnis rubeus sp. nov. The genomic-based metabolic reconstructions indicated that members of this new haloarchaeal genus have photoheterotrophic aerobic lifestyle with a typical salt-in signature. 16S rRNA gene sequence reads abundance profiles and genomic recruitment analyses revealed that the Halosegnis genus has a worldwide geographical distribution, reaching high abundance (up to 8%) in habitats with intermediate salinities.

Natronomonas salsuginis sp. nov., a New Inhabitant of a Marine Solar Saltern.

A halophilic archaeon, strain F20-122T, was isolated from a marine saltern of Isla Bacuta (Huelva, Spain). Cells were Gram-stain-negative, aerobic, and coccoid in morphology. It grew at 25-50 °C (optimum 37 °C), pH 6.5-9.0 (optimum pH 8.0), and 10-30% (w/v) total salts (optimum 25% salts). The phylogenetic analyses based on the 16S rRNA and rpoB' genes showed its affiliation with the genus Natronomonas and suggested its placement as a new species within this genus. The in silico DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) analyses of this strain against closely related species supported its placement in a new taxon. The DNA G + C content of this isolate was 63.0 mol%. The polar lipids of strain F20-122T were phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol (PG), and phosphatidylglycerol sulfate (PGS). Traces of biphosphatidylglycerol (BPG) and other minor phospholipids and unidentified glycolipids were also present. Based on the phylogenetic, genomic, phenotypic, and chemotaxonomic characterization, we propose strain F20-122T (= CCM 8891T = CECT 9564T = JCM 33320T) as the type strain of a new species within the genus Natronomonas, with the name Natronomonas salsuginis sp. nov. Rhodopsin-like sequence analysis of strain F20-122T revealed the presence of haloarchaeal proton pumps, suggesting a lightmediated ATP synthesis for this strain and a maximum wavelength absorption in the green spectrum.

Haloglomus irregulare gen. nov., sp. nov., a New Halophilic Archaeon Isolated from a Marine Saltern.

A halophilic archaeal strain, designated F16-60T, was isolated from Isla Cristina marine saltern in Huelva, Spain. Cells were pleomorphic, irregular, non-motile, and Gram-stain-negative. It produced red-pigmented colonies on agar plates. Strain F16-60T was extremely halophilic (optimum at 30% (w/v) NaCl) and neutrophilic (optimum pH 7.5). Phylogenetic tree reconstructions based on 16S rRNA and rpoB´ gene sequences revealed that strain F16-60T was distinct from species of the related genera Natronomonas, Halomarina, and Halomicrobium, of the order Halobacteriales. The polar lipids are phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS), and one glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1). The DNA G+C content is 68.0 mol%. The taxonomic study, based on a combination of phylogenetic, genomic, chemotaxonomic, and phenotypic analyses, suggest that strain F16-60T (= CECT 9635T = JCM 33318T), represents a novel species of a new genus within the family Haloarculaceae and the order Halobacteriales, for which the name Haloglomus irregulare gen. nov., sp. nov. is proposed. Metagenomic fragment recruitment analysis revealed the worldwide distribution of members of this genus and suggested the existence of other closely related species to be isolated.

New Halonotius Species Provide Genomics-Based Insights Into Cobalamin Synthesis in Haloarchaea.

Hypersaline aquatic and terrestrial ecosystems display a cosmopolitan distribution. These environments teem with microbes and harbor a plethora of prokaryotic lineages that evaded ecological characterization due to the prior inability to cultivate them or to access their genomic information. In order to close the current knowledge gap, we performed two sampling and isolation campaigns in the saline soils of the Odiel Saltmarshes and the salterns of Isla Cristina (Huelva, Spain). From the isolated haloarchaeal strains subjected to high-throughput phylogenetic screening, two were chosen (F15BT and F9-27T) for physiological and genomic characterization due of their relatedness to the genus Halonotius. Comparative genomic analyses were carried out between the isolated strains and the genomes of previously described species Halonotius pteroides CECT 7525T, Halonotius aquaticus F13-13T and environmentaly recovered metagenome-assembled representatives of the genus Halonotius. The topology of the phylogenomic tree showed agreement with the phylogenetic ones based on 16S rRNA and rpoB' genes, and together with average amino acid and nucleotide identities suggested the two strains as novel species within the genus. We propose the names Halonotius terrestris sp. nov. (type strain F15BT = CECT 9688T = CCM 8954T) and Halonotius roseus sp. nov. (type strain F9-27T = CECT 9745T = CCM 8956T) for these strains. Comparative genomic analyses within the genus highlighted a typical salt-in signature, characterized by acidic proteomes with low isoelectric points, and indicated heterotrophic aerobic lifestyles. Genome-scale metabolic reconstructions revealed that the newly proposed species encode all the necessary enzymatic reactions involved in cobalamin (vitamin B12) biosynthesis. Based on the worldwide distribution of the genus and its abundance in hypersaline habitats we postulate that its members perform a critical function by being able to provide "expensive" commodities (i.e., vitamin B12) to the halophilic microbial communities at large.

Halorientalis pallida sp. nov., an extremely halophilic archaeon isolated from a marine saltern.

An extremely halophilic archaeon, strain F13-25T, was isolated from a marine saltern located in Isla Cristina, Huelva, on the south-west coast of Spain. The novel strain had pink-pigmented, non-motile, coccoid cells. Optimal growth was achieved at 25 % (w/v) NaCl, pH 7.5 and 37 °C. Strain F13-25T possessed two heterogeneous 16S rRNA genes (rrnA and rrnB) most closely related to Halorientalis persicus D108T (97.6-99.2 % sequence similarity) and Halorientalis regularis TNN28T (95.9-98.8 %). On the basis of the results of rpoB' gene sequence analysis, strain F13-25T was also closely related to Halorientalis persicus IBRC-M 10043T (89.9 %) and Halorientalis regularis TNN28T (92.3 %). Relatedness values, computed using the Genome-to-Genome Distance Calculator, between strain F13-25T and Halorientalis persicus IBRC-M 10043T and Halorientalis regularis IBRC-M 10760T were 34.6 and 36.2 %, respectively. Average nucleotide identity values based on orthoANI, ANIb and ANIm of strain F13-25T and Halorientalis persicus IBRC-M 10043T and Halorientalisregularis IBRC-M 10760T were 88.0 and 88.8, 87.1 and 87.6 %, and 89.2 and 89.6 %, respectively. All values were far below the threshold accepted for prokaryotic species delineation. The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and one glycolipid chromatographically identical to sulfated diglycosyl diether. The DNA G+C content was 65.7 mol% (genome). The results of phylogenetic, phenotypic and chemotaxonomic analyses indicated that strain F13-25T represents a novel species of the genus Halorientalis, for which the name Halorientalis pallida sp. nov., with type strain F13-25T (=CECT 9384T=IBRC-M 11176T), is proposed.

Halonotius aquaticus sp. nov., a new haloarchaeon isolated from a marine saltern.

A halophilic archaeon, strain F13-13T, was isolated from a marine saltern located in Isla Cristina, Huelva, on the south-west coast of Spain. It was a Gram-stain-negative, motile and aerobic haloarchaeon. It grew at 28-50 °C (optimum, 37 °C), pH 6.0-8.5 (pH 7.5) and in 15-30 % (w/v) total salts (25 %). Phylogenetic analyses based on the 16S rRNA gene sequences showed that strain F13-13T is a member of the genus Halonotius, the most closely related species being Halonotius pteroides 1.15.5T (96.7 % sequence similarity). The 16S rRNA gene sequence similarity to species of other genera is lower than 93.4 %. Strain F13-13T was also found to be closely related to Halonotius pteroides 1.15.5T (91.4 %) on the basis of rpoB' gene sequence analysis. The Genome-to-Genome Distance Calculator relatedness result between strain F13-13T and Halonotius pteroides CECT 7525T was 35 %, a value lower than the 70 % threshold accepted for species delineation. The average nucleotide identity values based on OrthoANI, ANIb and ANIm of strain F13-13T and Halonotius pteroides CECT 7525T were 88.7, 87.8 and 89.4 %, respectively, these values are also lower than the threshold accepted for species delineation. The DNA G+C content of this isolate was 61.2 mol%. The major lipids of strain F13-13T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulphated diglycosyl diether. Based on the phylogenetic, phenotypic, genotypic and chemotaxonomic characterization, we propose the placement of strain F13-13T as a new species within the genus Halonotius, with the name Halonotius aquaticus sp. nov. The type strain is F13-13T (=CECT 9386T=IBRC-M 11204T).

Spatial distribution of prokaryotic communities in hypersaline soils.

Increasing salinization in wetland systems is a major threat to ecosystem services carried out by microbial communities. Thus, it is paramount to understand how salinity drives both microbial community structures and their diversity. Here we evaluated the structure and diversity of the prokaryotic communities from a range of highly saline soils (EC1:5 from 5.96 to 61.02 dS/m) from the Odiel Saltmarshes and determined their association with salinity and other soil physicochemical features by analyzing 16S rRNA gene amplicon data through minimum entropy decomposition (MED). We found that these soils harbored unique communities mainly composed of halophilic and halotolerant taxa from the phyla Euryarchaeota, Proteobacteria, Balneolaeota, Bacteroidetes and Rhodothermaeota. In the studied soils, several site-specific properties were correlated with community structure and individual abundances of particular sequence variants. Salinity had a secondary role in shaping prokaryotic communities in these highly saline samples since the dominant organisms residing in them were already well-adapted to a wide range of salinities. We also compared ESV-based results with OTU-clustering derived ones, showing that, in this dataset, no major differences in ecological outcomes were obtained by the employment of one or the other method.