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Journal Article; Gluten content from barley, rye, wheat and in certain oat varieties, must be avoid in individuals with celiac disease. In most of the Western countries, the level of gluten content in food to be considered as gluten-free products is below 20 parts per million measured by ELISA based on specific anti-gluten peptide antibody. However, in beverages or food suffering complex hydrolytic processes as beers, the relative proportion of reactive peptides for celiac patients and the analytical techniques may differ, because of the diversity of the resulting peptide populations after fermentations. A beer below 20 parts per million of gluten but yet detectable levels of gluten peptides by anti-gliadin 33-mer antibodies (G12 and A1) was analyzed. We identified and characterized the relevant peptides for either antibody recognition or immunoactivity in celiac patients. The beer was fractionated by HPLC. The relative reactivity of the different HPLC fractions to the G12/A1 antibodies correlated to the reactivity of peripheral blood mononuclear cells isolated from 14 celiac individuals. Peptides from representative fractions classified according to the relative reactivity to G12/A1 antibodies were identified by mass spectrometry. The beer peptides containing sequences with similarity to those of previously described G12 and A1 epitopes were synthesized and confirmed significant reactivity for the antibodies. The most reactive peptides for G12/A1 also confirmed the highest immunogenicity by peripheral blood mononuclear cell activation and interferon γ production from celiac patients. We concluded that preparative HPLC combined with anti-gliadin 33-mer G12/A1 antibodies were very sensitive and specific methods to analyze the relevant immunogenic peptides in hydrolyzed gluten. Yes

Real, Ana et al., A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences. © 2012 Real et al., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO,Programa INNPACTO, IPT-2011-1321-010000) and the European Regional Development Fund (FEDER). FP is supported by a Ramón y Cajal research contract from the MINECO (RYC-2010-07345).

Human trichuriasis is a neglected tropical disease which affects millions of people worldwide, mostly living in low socio-economic conditions. Numerous studies have been conducted over the past 10 years to compare the different techniques for T. trichiura eggs detection. Our study provides the first geometric morphometric analysis for the specific detection of eggs of Trichuris sp. isolated from stools of macaque (M. sylvanus), colobus (C. g. kikuyensis), grivets (C. aethiops) and the Brazza's monkey (C. neglectus) from zoos in Spain. Principal Component Analysis (PCA) arises as an efficient method to determine Trichuris spp. eggs. The selected measurements to be included in the PCA were proposed for the first time in the present work, as far as we know, as we could not find previous studies reporting standardized parameters. Ministry of Economy, Industry and Competitiveness (CGL2017-83057), which included FEDER funds Junta de Andalucía (BIO-338) V Plan Propio de Investigacion of the University of Seville

Celiac disease (CeD) is a common autoimmune disorder induced by consuming gluten proteins found in wheat, barley, and rye. Glutens resist breakdown by gastrointestinal proteases, ultimately resulting in peptides with chemical structures that elicit inflammation in patients with CeD. Despite well-established connections between digestion-resistant glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, which have impeded our knowledge of potentially diverse peptide species that encounter the immune system. Here, we developed a novel liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. Using this protocol, we detected 679 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and 5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differed from control subjects. Our urinary peptidomic workflow provides new foundations for understanding gluten immunogenicity and enhancing CeD management. Additionally, it should promote new understandings of the uncharacterized dietary and urinary peptidomes.

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.

The haloarchaeal genera Natrinema and Haloterrigena were described almost simultaneously by two different research groups and some strains studied separately were described as different species of these genera. Furthermore, the description of additional species were assigned to either Natrinema or Haloterrigena, mainly on the basis of the phylogenetic comparative analysis of single genes (16S rRNA gene and more recently rpoB’ gene), but these species were not adequately separated or assigned to the corresponding genus. Some studies suggested that the species of these two genera should be unified into a single genus, while other studies indicated that the genera should remain but some of the species should be reassigned. In this study, we have sequenced or collected the genomes of the type strains of species of Natrinema and Haloterrigena and we have carried out a comparative genomic analysis in order to clarify the controversy related to these two genera. The phylogenomic analysis based on the comparison of 525 translated single-copy orthologous genes and the Overall Genome Relatedness Indexes (i.e., AAI, POCP, ANI, and dDDH) clearly indicate that the species Haloterrigena hispanica, Haloterrigena limicola, Haloterrigena longa, Haloterrigena mahii, Haloterrigena saccharevitans, Haloterrigena thermotolerans, and Halopiger salifodinae should be transferred to the genus Natrinema, as Natrinema hispanicum, Natrinema limicola, Natrinema longum, Natrinema mahii, Natrinema saccharevitans, Natrinema thermotolerans, and Natrinema salifodinae, respectively. On the contrary, the species Haloterrigena turkmenica, Haloterrigena salifodinae, and Haloterrigena salina will remain as the only representative species of the genus Haloterrigena. Besides, the species Haloterrigena daqingensis should be reclassified as a member of the genus Natronorubrum, as Natronorubrum daqingense. At the species level, Haloterrigena jeotgali and Natrinema ejinorense should be considered as a later heterotypic synonyms of the species Haloterrigena (Natrinema) thermotolerans and Haloterrigena (Natrinema) longa, respectively. Synteny analysis and phenotypic features also supported those proposals.

Pantoea agglomerans RSO7, a rhizobacterium previously isolated from Spartina maritima grown on metal polluted saltmarshes, had demonstrated good plant growth promoting activity for its host halophyte, but was never tested in crops. The aims of this study were: (1) testing PGP activity on a model plant (alfalfa) in vitro; (2) testing a bacterial consortium including RSO7 as biofertilizer in a pilot experiment in urban orchard; and (3) identifying the traits related to PGP activities. RSO7 was able to enhance alfalfa growth in vitro, particularly the root system, besides improving plant survival and protecting plants against fungal contamination. In addition, in a pilot experiment in urban orchard, a consortium of three bacteria including RSO7 was able to foster the growth and yield of several winter crops between 1.5 and 10 fold, depending on species. Moreover, the analysis of chlorophyll fluorescence revealed that photosynthesis was highly ameliorated. Genome analysis of RSO7 depicted the robustness of this bacterial strain which showed resilience to multiple stresses (heat, cold, UV radiation, several xenobiotics). Together with wide metabolic versatility, genes conferring resistance to oxidative stress were identified. Many genes involved in metal resistance (As, Cu, Ni, Co, Zn, Se, Te) and in tolerance toward high osmolality (production of a battery of osmoprotectans) were also found. Regarding plant growth promoting properties, traits for phosphate solubilization, synthesis of a battery of siderophores and production of IAA were detected. In addition, the bacterium has genes related to key processes in the rhizosphere including flagellar motility, chemotaxis, quorum sensing, biofilm formation, plant-bacteria dialog, and high competitiveness in the rhizosphere. Our results suggest the high potential of this bacterium as bioinoculant for an array of crops. However, the classification in biosecurity group 2 prevents its use according to current European regulation. Alternative formulations for the application of the bioinoculant are discussed.

Rhinonyssidae (Mesostigmata) is a family of nasal mites only found in birds. All species are hematophagous endoparasites, which may damage the nasal cavities of birds, and also could be potential reservoirs or vectors of other infections. However, the role of members of Rhinonyssidae as disease vectors in wild bird populations remains uninvestigated, with studies of the microbiomes of Rhinonyssidae being almost non-existent. In the nasal mite (Tinaminyssus melloi) from rock doves (Columba livia), a previous study found evidence of a highly abundant putatively endosymbiotic bacteria from Class Alphaproteobacteria. Here, we expanded the sample size of this species (two different hosts- ten nasal mites from two independent samples per host), incorporated contamination controls, and increased sequencing depth in shotgun sequencing and genome-resolved metagenomic analyses. Our goal was to increase the information regarding this mite species and its putative endosymbiont. We obtained a metagenome assembled genome (MAG) that was estimated to be 98.1% complete and containing only 0.9% possible contamination. Moreover, the MAG has characteristics typical of endosymbionts (namely, small genome size an AT bias). Overall, our results support the presence of a potential endosymbiont, which is the first described for avian nasal mites to date, and improve the overall understanding of the microbiota inhabiting these mites. US National Science Foundation DEB-1926919, DEB-1925487

Trichuris trichiura is a nematode considered as the whipworm present in humans and primates. The systematics of the genus Trichuris is complex. Morphological studies of Trichuris isolated from primates and humans conclude that the species infecting these hosts is the same. Furthermore, numerous molecular studies have been carried out so far to discriminate parasite species from humans and Non-Human Primates using molecular techniques, but these studies were not performed in combination with a parallel morphological study. The hypothesised existence of more species of Trichuris in primates opens the possibility to revise the zoonotic potential and host specificity of T. trichiura and other putative new species of whipworms. In the present work, a study of Trichuris Roederer, 1761 (Nematoda:Trichuridae) parasitizing C. g. kikuyensis, P. ursinus, Macaca sylvanus, Pan troglodytes, and Sus scrofa domestica has been carried out using modern morphometric techniques in order to differentiate populations of Trichuris isolated from four species of captive NHP from different geographical regions, and swine, respectively. The results obtained revealed strong support for geometrical morphometrics as a useful tool to differentiate male Trichuris populations. Therefore, morphometrics in combination with other techniques, such as molecular biology analyses, ought to be applied to further the differentiation of male populations. On the other hand, morphometrics applied to female Trichuris species does not seem to contribute new information as all the measurements combinations of obtained from females always showed similar results Ministry of Economy,Industry and Competitiveness(CGL2017-83057) FEDER funds, the Junta de Andalucía (BIO-338) V Plan Propio de Investigación of the University of Seville

Background Treatment for coeliac disease is a lifelong strict gluten‐free diet. Although guidelines recommend regular follow‐up with dietary interviews and coeliac serology, these methods may be inaccurate. Aim To evaluate the usefulness of faecal gluten immunogenic peptides to support the diagnosis and to determine the adherence to the gluten‐free diet in coeliac children. Methods Multicentre prospective observational study including 64 coeliac children. Faecal gluten peptides, and tissue transglutaminase and deamidated gliadin peptide antibodies were analyzed at diagnosis, and 6, 12 and 24 months thereafter. Gluten consumption was estimated from gluten peptide levels. Results Most children (97%) had detectable gluten peptides at diagnosis. On a gluten‐free diet, the rate of gluten peptides increased from 13% at 6 months to 25% at 24 months. Mean estimated gluten exposure dropped from 5543 mg/d at diagnosis to 144 mg/d at 6 months, then increased to 606 mg/d by 24 months. In contrast, deamidated gliadin peptide antibodies normalised and only 20% had elevated tissue transglutaminase antibody by 24 months. The elevation of tissue transglutaminase antibody was more prolonged in patients with detectable gluten peptides (P < 0.05). Nevertheless, absolute levels of tissue transglutaminase antibody had low sensitivity to identify patients with detectable gluten peptides (P > 0.1). Dietitian assessment was only moderately correlated with gluten peptide detection (κ = 0.5). Conclusions Faecal gluten peptides testing may guide treatment of coeliac disease prior to diagnosis and during the assessment diet adherence. Further studies could determine if early identification of gluten exposure reduces the need for expensive/invasive investigations for non‐responsive coeliac disease. ClinicalTrials.gov Number: NCT02711397. España, Ministerio de Ciencia e Innovación and FEDER funds DELIAC, IPT‐2011‐0952‐900000 España, Corporación Tecnológica de Andalucía SINGLUCHECK, 1737/0118

To date, the only treatment for celiac disease (CD) consists of a strict lifelong gluten-free diet (GFD), which has numerous limitations in patients with CD. For this reason, dietary transgressions are frequent, implying intestinal damage and possible long-term complications. There is an unquestionable need for non-dietary alternatives to avoid damage by involuntary contamination or voluntary dietary transgressions. In recent years, different therapies and treatments for CD have been developed and studied based on the degradation of gluten in the intestinal lumen, regulation of the immune response, modulation of intestinal permeability, and induction of immunological tolerance. In this review, therapeutic lines for CD are evaluated with special emphasis on phase III and II clinical trials, some of which have promising results. Federación de Asociaciones de Celíacos de España (FACE) SUBN/2019/005

Human trichuriasis is a Neglected Tropical Disease, which affects hundreds of millions of persons worldwide. Several studies have reported that non-human primates (NHP) represent important reservoirs for several known zoonotic infectious diseases. In this context,Trichurisinfections have been found in a range of NHP species living in natural habitats, including colobus monkeys, macaques, baboons, and chimpanzees. To date, the systematics of the genusTrichurisparasitizing humans and NHP is unclear. During many years,Trichuris trichiurawas considered as the whipworm present in humans and primates. Subsequently, molecular studies suggested thatTrichurisspp. in humans and NHP represent several species that differ in host specificity. This work examines the current knowledge ofT. trichiuraand its relationship to whipworm parasites in other primate host species. A phylogenetic hypothesis, based on three mitochondrial genes (cytochrome c oxidasesubunit 1,cytochromeb, and large subunit rRNA-encoding gene) and two fragments of ribosomal DNA (Internal Transcribed Spacer 1 and 2), allowed us to define a complex of populations ofT. trichiurahosting in a large variety of NHP species, in addition to humans. These populations were divided into four phylogenetic groups with a different degree of host specificity. From these data, we carry out a new morphological and biometrical description of the populations ofTrichurisbased on data cited by other authors as well as those provided in this study. The presence ofT. trichiurais analyzed in several NHP species in captivity from different garden zoos as possible reservoir of trichuriasis for humans. This study contributes to clarify questions that lead to identification of new taxa and will determine parasite transmission routes between these primates, allowing the implementation of appropriate control and prevention measures.

Trichuriasis is among the most prevalent worldwide parasitism caused by helminths. For many years, Trichuris spp. have been described with a relatively narrow range of both morphological and biometrical features. The use of the complete mitochondrial genome (mitogenome) is an alternative and powerful molecular method for inferring phylogenies. Here, we present an overview of the contributions of mitogenome for Trichuris spp. from human and non-human primates. In addition, we carry out structural and phylogenetic comparative analyses with genomes of Trichuris species available in public datasets. The complete mt genomes of Trichuris trichiura and Trichuris sp. from Macaca sylvanus and T. trichiura from Papio papio are 14,091 bp, 14,047 bp and 14,089 bp in length, respectively. The three mt genomes are circular and consist of 37 genes—13 PCGs (cox1–3, nad1–6, nad4L, atp6, atp8 and cob), 22 transfer RNA genes (tRNAs), and two rRNAs (rrnL and rrnS). The molecular evidence presented here supports the hypothesis that T. trichiura de M. sylvanus (TMF31) and T. trichiura de P. papio (TPM1) were similar but genetically different with respect to Trichuris sp. from macaques (TMM5). The phylogenetic study also supported the evolution of the different Trichuris species. In conclusion, we suggest the existence of two cryptic species parasitizing M. sylvanus.

The genome of Pseudomonas fluorescens F113, a model rhizobacterium and a plant growth-promoting agent, encodes three putative type VI secretion systems (T6SSs); F1-, F2- and F3-T6SS. Bioinformatic analysis of the F113 T6SSs has revealed that they belong to group 3, group 1.1, and group 4a, respectively, similar to those previously described in Pseudomonas aeruginosa. In addition, in silico analyses allowed us to identify genes encoding a total of five orphan VgrG proteins and eight putative effectors (Tfe), some with their cognate immunity protein (Tfi) pairs. Genes encoding Tfe and Tfi are found in the proximity of P. fluorescens F113 vgrG, hcp, eagR and tap genes. RNA-Seq analyses in liquid culture and rhizosphere have revealed that F1- and F3-T6SS are expressed under all conditions, indicating that they are active systems, while F2-T6SS did not show any relevant expression under the tested conditions. The analysis of structural mutants in the three T6SSs has shown that the active F1- and F3-T6SSs are involved in interbacterial killing while F2 is not active in these conditions and its role is still unknown.. A rhizosphere colonization analysis of the double mutant affected in the F1- and F3-T6SS clusters showed that the double mutant was severely impaired in persistence in the rhizosphere microbiome, revealing the importance of these two systems for rhizosphere adaption.

Soil salinization is an environmental problem that adversely affects plant growth and crop productivity worldwide. As an alternative to the conventional approach of breeding salt-tolerant plant cultivars, we explored the use of plant-growth-promoting rhizobacteria (PGPR) from halophytic plants to enhance crop growth under saline conditions. Here, we report the effect of five PGPR consortia from halophytes on the growth of eight (alfalfa, flax, maize, millet, rice, strawberry, sunflower, and wheat) of the crops most commonly produced on salinized soils worldwide. To test the efficiency of halotolerant consortia, we designed a complex environmental matrix simulating future climate-change scenarios, including increased CO2 levels and temperature. Overall, biofertilizers enhanced growth of most crops with respect to non-inoculated control plants under different CO2 concentrations (400/700 ppm), temperatures (25/+4 °C), and salinity conditions (0 and 85 mM NaCl). Biofertilizers counteracted the detrimental effect of salinity on crop growth. Specifically, strawberry and rice showed the greatest positive additive response to inoculation in the presence of salt; above-ground biomasses were 35% and 3% greater, respectively, than their respective control grown without salt. Furthermore, depending on the interaction of environmental factors (salinity × CO2 × temperature) analyzed, the results varied—influencing the most effective biofertilizer determined for each crop now, or in the future. Our findings highlight the importance of conducting studies that consider stress interaction for realistic assessments of the potential of biofertilizers in a climate-changed world

[Backgound] The ingestion of wheat and other cereals are related to several gut disorders. The specific components responsible for non-celiac wheat-sensitivity (NCWS) may include gluten and other compounds. Tritordeum is a new cereal derived from crossing durum wheat with a wild barley species, which differs from bread wheat in its gluten composition. In the present work, we examined the response of NCWS patients to tritordeum bread Gastrointestinal symptoms as well as tritordeum acceptability, gluten immunogenic peptides excretion, and the composition and structure of the intestinal microbiota were evaluated., [Results] Gastrointestinal symptoms of the subjects showed no significant change between the gluten-free bread and the tritordeum bread. Participating subjects rated tritordeum bread higher than the gluten-free bread. Analysis of the bacterial gut microbiota indicated that tritordeum consumption does not alter the global structure and composition of the intestinal microbiota, and only a few changes in some butyrate-producing bacteria were observed. [Conclusions] All the results derived from acceptability, biochemical and microbiological tests suggest that tritordeum may be tolerated by a sub-set of NCWS sufferers who do not require strict exclusion of gluten from their diet. © 2020 Society of Chemical Industry., This research was funded by Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidad, project AT-5985 and the European Regional Development Fund (FEDER).

Summary Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS‐dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC‐GlxA‐like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post‐transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyectoine production., Besides the main ectoine hydroxylase EctD, the halophilic bacterium Chromohalobacter salexigens possess a secondary ectoine hydroxylases, EctE, which contributes to C. salexigens thermoprotection. Expression of ectD and ectE is dependent on the growth phase and the general stress factor RpoS. In addition, the EctZ transcriptional regulator plays a dual function under exponential growth as both an activator of osmorregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD.

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coli ∆arsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli ∆arsC and ∆arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli ∆arsC and ∆arsRBC, possibly by chelation of arsenic or by increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome., Terribacillus harbors an unusual ars operon that confers resistance to arsenite in Escherichia coli.

Bistable expression of the Salmonella enterica pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1OFF FlagOFF, SPI-1OFF FlagON, SPI-1ON FlagOFF, and SPI-1ON FlagON. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that FlagOFF cells might contribute to optimal invasion as previously proposed for SPI-1OFF cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1ON cells. As a consequence, growth resumption in SPI-1ON FlagON cells requires switching to both SPI-1OFF and FlagOFF states. Ministerio de Economía, Industria y Competitividad. Gobierno de España-BIO2016-75235-P Fondo Europeo de Desarrollo Regional. Unión Europea VI Plan Propio de Investigación y Transferencia. Universidad de Sevilla. España

Prokaryotic communities and physico-chemical characteristics of 30 brine samples from the thalassohaline Tuz Lake (Salt Lake), Deep Zone, Kayacik, Kaldirim, and Yavsan salterns (Turkey) were analyzed using 16S rRNA amplicon sequencing and standard methods, respectively. Archaea (98.41% of reads) was found to dominate in these habitats in contrast to the domain Bacteria (1.38% of reads). Representatives of the phylum Euryarchaeota were detected as the most predominant, while 59.48% and 1.32% of reads, respectively, were assigned to 18 archaeal genera, 19 bacterial genera, 10 archaeal genera, and one bacterial genus that were determined to be present, with more than 1% sequences in the samples. They were the archaeal genera Haloquadratum, Haloarcula, Halorhabdus, Natronomonas, Halosimplex, Halomicrobium, Halorubrum, Halonotius, Halolamina, Halobacterium, and Salinibacter within the domain Bacteria. The genera Haloquadratum and Halorhabdus were found in all sampling sites. While Haloquadratum, Haloarcula, and Halorhabdus were the most abundant genera, two uncultured Tuz Lake Halobacteria (TLHs) 1 and 2 were detected in high abundance, and an additional uncultured haloarchaeal TLH-3 was found as a minor abundant uncultured taxon. Their future isolation in pure culture would permit us to expand our knowledge on hypersaline thalassohaline habitats, as well as their ecological role and biomedical and biotechnological potential applications.

Microorganisms can potentially colonise volcanic rocks using the chemical energy in reduced gases such as methane, hydrogen (H2) and carbon monoxide (CO). In this study, we analysed soil metagenomes from Chilean volcanic soils, representing three different successional stages with ages of 380, 269 and 63 years, respectively. A total of 19 metagenome-assembled genomes (MAGs) were retrieved from all stages with a higher number observed in the youngest soil (1640: 2 MAGs, 1751: 1 MAG, 1957: 16 MAGs). Genomic similarity indices showed that several MAGs had amino-acid identity (AAI) values &gt, 50% to the phyla Actinobacteria, Acidobacteria, Gemmatimonadetes, Proteobacteria and Chloroflexi. Three MAGs from the youngest site (1957) belonged to the class Ktedonobacteria (Chloroflexi). Complete cellular functions of all the MAGs were characterised, including carbon fixation, terpenoid backbone biosynthesis, formate oxidation and CO oxidation. All 19 environmental genomes contained at least one gene encoding a putative carbon monoxide dehydrogenase (CODH). Three MAGs had form I coxL operon (encoding the large subunit CO-dehydrogenase). One of these MAGs (MAG-1957-2.1, Ktedonobacterales) was highly abundant in the youngest soil. MAG-1957-2.1 also contained genes encoding a [NiFe]-hydrogenase and hyp genes encoding accessory enzymes and proteins. Little is known about the Ktedonobacterales through cultivated isolates, but some species can utilise H2 and CO for growth. Our results strongly suggest that the remote volcanic sites in Chile represent a natural habitat for Ktedonobacteria and they may use reduced gases for growth.

National Science Foundation NSF DEB-1239788, DEB-1342604, DEB-1855812

The draft genome sequence of Saccharomonospora piscinae KCTC 19743T, with a size of 4,897,614 bp, was assembled into 11 scaffolds containing 4,561 open reading frames and a G+C content of 71.0 mol%. Polyketide synthase and nonribosomal peptide synthetase gene clusters, which are responsible for the biosynthesis of several biomolecules, were identified and located in different regions in the genome.

Background: Rhinonyssid mites are permanent parasites of birds that inhabit their respiratory tract. There are around 600 species described worldwide and almost all species of birds are found to have embedded rhinonyssid mites. Despite their presumed relevance, these mites are largely unstudied due to the difficulty in sampling them and, therefore, the majority of mite-host associations and species-prevalence data are unknown. New information: In this study, 179 mite specimens belonging to 27 species and eight genera were identified. Notably, 18 new mite-bird associations were documented for the first time, thus increasing the known host range for these mite species. In addition, mite-host associations found in this study were compared with known associations from these species of birds in the European part of Russia and in Europe. Overall, this study represents the largest survey to date carried out on rhinonyssid mites in Russia and one of the most comprehensive datasets on rhinonyssid host-range

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&acute, 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.

Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the α‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in α‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low‐gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties., The Spanish Ministry of Economy and Competitiveness (Projects AGL2013‐48946‐C3‐1‐R and AGL2016‐80566‐P) and the European Regional Development Fund (FEDER) supported this work. Javier Gil‐Humanes acknowledges the Fundacion Alfonso Martín Escudero for its funding support.

A comparative taxonomic study of Halorubrum distributum , Halorubrum terrestre , Halorubrum arcis and Halorubrum litoreum was carried out using different approaches, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), phylogenomic analysis based on the comparison of the core genome, orthologous average nucleotide identity (OrthoANI), Genome-to-Genome Distance Calculator (GGDC), synteny plots and polar lipid profile (PLP). The MLSA study, using the five concatenated housekeeping genes atpB, EF-2, glnA, ppsA and rpoB′, and the phylogenomic analysis based on 1347 core translated gene sequences obtained from their genomes showed that Halorubrum distributum JCM 9100T, Halorubrum terrestre JCM 10247T, Halorubrum arcis JCM 13916T and Halorubrum litoreum JCM 13561T formed a robust cluster, clearly separated from the rest of species of the genus Halorubrum . The OrthoANI and digital DDH values, calculated by the GGDC, showed percentages among Hrr. distributum JCM 9100T, Hrr. terrestre JCM 10247T, Hrr. arcis JCM 13916T and Hrr. litoreum JCM 13561T that ranged from 98.1 to 97.5 %, and 84.0 to 78.0 %, respectively, while these values among those strains and the type strains of their most related species of Halorubrum were equal or lower than 90.8 and 41.2 %, respectively. Moreover, degree of synteny across the four genomes was very high, especially between the genomes of Halorubrum litoreum JCM 13561T and Halorubrum arcis JCM 13916T. In addition, the PLP is quite similar among the four strains studied, showing a common pattern typical of the neutrophilic species of the genus Halorubrum . Overall, these data show that Hrr. distributum, Hrr. terrestre, Hrr. arcis and Hrr. litoreum constitute a single species. Thus, the latter three should be considered as later, heterotypic synonyms of Hrr. distributum based on the rules for priority of names. We propose an emended description of Hrr. distributum, including the features of Hrr. terrestre, Hrr. arcis and Hrr. litoreum.

The organisms thriving under extreme conditions better than any other organism living on Earth, fascinate by their hostile growing parameters, physiological features, and their production of valuable bioactive metabolites. This is the case of microorganisms (bacteria, archaea, and fungi) that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As along as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions on which the compounds are produced in the microbial community without more stress than sharing the same substratum with their peers, the salt. In this review are reported the molecules described and produced by halophilic microorganisms with a spectrum of action in vitro: antimicrobial and anticancer. The action mechanisms of these molecules, the urgent need to introduce alternative lead compounds and the current aspects on the exploitation and its limitations are discussed. España, MINECO CGL2017-83385-P

Calicotome villosa is a spontaneous Mediterranean legume that can be a good candidate as pioneer plants to limit regression of vegetation cover and loss of biodiversity in Tunisian arid soils. In order to grow legumes in such soils, pairing rhizobia and nodule associated bacteria (NAB) might provide numerous advantages. In this work, cultivable biodiversity of rhizobial symbionts and NAB in nodules of C. villosa plants growing in five arid regions of south Tunisia was characterized. Phylogenetic analysis using 16S rDNA gene, dnak, recA and nodD sequences separated nodule-forming bacteria in six clades associated to genera Ensifer, Neorhizobium, Phyllobacterium and Rhizobium. Among NAB, the strain Variovorax sp. CT7.15 was selected due to its capacity to solubilise phosphate and, more interestingly, its high level of aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity. C. villosa plants were inoculated with representative rhizobia of each phylogenetic group and co-inoculated with the same rhizobia and strain CT7.15. Compared with single rhizobia inoculation, co-inoculation significantly improved plant growth and nodulation, ameliorated plant physiological state and increased nitrogen content in the plants, independently of the rhizobia used. These results support the benefits of pairing rhizobia and selected NAB to promote legume growth in arid or degraded soils España, Ministerio de Economía y Competitividad (MINECO Project CGL2016-75550-R cofounded by FEDER) Junta de Andalucía (Project 2017/391)

17 páginas, 2 figuras, 3 tablas, Strain RA15T was isolated from the rhizosphere of the halophyte plant Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). RA15T cells were Gram stain-negative, non-spore-forming, aerobic rods and formed cream-coloured, opaque, mucoid, viscous, convex, irregular colonies with an undulate margin. Optimal growth conditions were observed on tryptic soy agar (TSA) plates supplemented with 2.5 % NaCl (w/v) at pH 7.0 and 28 °C, although it was able to grow at 4–32 °C and at pH values of 5.0–9.0. The NaCl tolerance range was from 0 to 15 %. The major respiratory quinone was Q8 but Q9 was also present. The most abundant fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C17 : 1 ω8c and C16 : 0. The polar lipids profile comprised phosphatidylglycerol and phosphatidylethanolamine as the most abundant representatives. Phylogenetic analyses confirmed the well-supported affiliation of strain RA15T within the genus Pseudoalteromonas , close to the type strains of Pseudoalteromonas neustonica , Pseudoalteromonas prydzensis and Pseudoalteromonas mariniglutinosa . Results of comparative phylogenetic and phenotypic studies between strain RA15T and its closest related species suggest that RA15T could be a new representative of the genus Pseudoalteromonas , for which the name Pseudoalteromonas rhizosphaerae sp. nov. is proposed. The type strain is RA15T (=CECT 9079T=LMG 29860T). The whole genome has 5.3 Mb and the G+C content is 40.4 mol%., This work has been possible thanks to Junta de Andalucía (P11-RNM-7274MO project) and INIA (RTA 2012–0006 C03-03 project).

The genus Cyclobacterium belongs to the phylum Bacteroidetes and includes eight species. Our study, based on the genomic parameters in silico DNA&ndash, DNA hybridization (GGDC), average nucleotide identity (OrthoANI), and average amino acid identity (AAI), confirmed that all current species of Cyclobacterium belong to this genus and constitute a coherent phylogenomic group, but with species forming two separate branches. In addition, the genome-based analyses revealed that Cyclobacterium xiamenense and Cyclobacterium halophilum are members of the same species. Besides, we carried out a taxonomic characterization of the new strain GBPx2T, isolated from the halophytic plant Salicornia sp. Analysis of its 16S rRNA gene sequence showed the highest sequence similarity (97.5%) to Cyclobacterium lianum HY9T. Percentages of GGDC and OrthoANI between strain GBPx2T and species of the genus Cyclobacterium were lower than the threshold value for species delineation. The DNA G+C content was 43.0 mol%. The polar lipids included phosphatidylethanolamine as well as one unidentified phospholipid and four unidentified lipids, and its major cellular fatty acids were iso-C15:0 and summed feature 3 (C16:1&omega, 7c and/or iso-C15:0 2-OH). The only quinone present was menaquinone 7. Based on a combination of phenotypic, chemotaxonomic, and phylogenomic features, the GBPx2T strain represents a novel species of the genus Cyclobacterium, for which the name Cyclobacterium plantarum sp. nov. is proposed. The type strain of Cyclobacterium plantarum is GBPx2T (= IBRC-M 10634T = LMG 28551T).

Minimally processed or fresh fruits and vegetables are unfortunately linked to an increasing number of food-borne diseases, such as salmonellosis. One of the relevant virulence factors during the initial phases of the infection process is the bacterial flagellum. Although its function is well studied in animal systems, contradictory results have been published regarding its role during plant colonization. In this study, we tested the hypothesis that Salmonella’s flagellin plays a versatile function during the colonization of tomato plants. We have assessed the persistence in plant tissues of a Salmonella enterica wild type strain, and of a strain lacking the two flagellins, FljB and FliC., The work carried out in the C.R.B. and J.R. laboratory has been granted by Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain, RTI2018-095069-B-100) awarded to C.R.B. and J.R., and Proyectos de Excelencia (Junta de Andalucía; PY18-2398) awarded to C.B. This work was co-funded by Fondos Europeos de Desarrollo Regional (FEDER). N.L. received funding for a short training mission (STSM) in the A.S. laboratory from CA 16110 HUPLANT from the EU Cost Action Program. We would like to thank DAAD for scholarship funding of A.A.Z.

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&ndash, 50 &deg, C (optimum 37 &deg, C), pH 6.5&ndash, 9.0 (optimum pH 8.0), and 10&ndash, 30% (w/v) total salts (optimum 25% salts). The phylogenetic analyses based on the 16S rRNA and rpoB&rsquo, genes showed its affiliation with the genus Natronomonas and suggested its placement as a new species within this genus. The in silico DNA&ndash, 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.

© 2020 by the authors., Rhynonyssidae is a family of endoparasitic hematophagous mites, which are still largely unknown even though they could act as vector or reservoir of different pathogens like dermanyssids. Sampling requirements have prevented deeper analysis. Rhinonyssids have been explored in a few host specimens per species, leading to undetailed morphological descriptions and inaccurate epidemiology. We explore the relationships established between these parasites in two Columbiformes urban birds (domestic pigeon (Columba livia domestica) and Eurasian collared dove (Streptopelia decaocto)), assesing 250 individuals of each type in Seville (Spain). As expected, Mesonyssus melloi (Castro, 1948) and Mesonyssus columbae (Crossley, 1950) were found in domestic pigeons, and Mesonyssus streptopeliae (Fain, 1962) in Eurasian collared doves. However, M. columbae was found for the first time in Eurasian collared doves. This relationship could be common in nature, but sampling methodology or host switching could also account for this result. An additional unknown specimen was found in a Eurasian collared dove, which could be a new species or an aberrant individual. We also provide an epidemiological survey of the three mite species, with M. melloi being the most common one followed by M. streptopeliae and M. columbae. High variation between previous epidemiological measurements and ours highlights the importance of developing deeper studies to uncover the factors regulating mite prevalence and intensities of infection., This research was funded by “V Plan Propio de Investigación of the University of Seville, Spain” and by “Ministero de Ciencia, Innovación y Universidades (MCIU/AEI/FEDER, UE)” project number PGC2018-097426-B-C22.

Wheat gluten proteins are responsible for the bread-making properties of the dough but also for triggering important gastrointestinal disorders. Celiac disease (CD) affects approximately 1% of the population in Western countries. The only treatment available is the strict avoidance of gluten in the diet. Interference RNA (RNAi) is an excellent approach for the down-regulation of genes coding for immunogenic proteins related to celiac disease, providing an alternative for the development of cereals suitable for CD patients. In the present work, we report a comparative study of the stimulatory capacity of seven low-gluten RNAi lines differing in grain gluten and non-gluten protein composition, relevant for CD and other gluten pathologies. Peripheral blood mononuclear cells (PBMCs) of 35 patients with active CD were included in this study to assess the stimulatory response induced by protein extracts from the RNAi lines. Analysis of the proliferative response and interferon-gamma (INF-&gamma, ) release of PBMCs demonstrated impaired stimulation in response to all RNAi lines. The lower response was provided by lines with a very low content of &alpha, and &gamma, gliadins, and low or almost devoid of DQ2.5 and p31&ndash, 43 &alpha, gliadin epitopes. The non-gluten protein seems not to play a key role in PBMC stimulation.

In a scenario of climate change and growing population, halophyte root microbiota interactions may be a sustainable solution to improve alternative crop production while combating abiotic stress. In this work, seeds of the cash crop halophyte Salicornia ramosissima were inoculated with five different plant growth-promoting rhizobacteria consortia, isolated from the rhizosphere of five halophytes in southwestern Spain salt marshes. For the first time, we recorded seed germination response to three interactive abiotic stressors, CO2 (400 and 700 ppm), temperature (25 and 29 ℃) and salinity (171, 510 and 1030 mM NaCl), all of them related to climate change. Salinity played a decisive role, as no significant differences were registered between treatments at 171 mM NaCl and no germination took place at 1030 mM NaCl. At 510 mM NaCl, one rhizobacterial consortium improved seed parameters notably, increasing up to 114% germination percentage and 65% seedlings biomass. These first findings encourage us to think that cash crop halophytes like S. ramosissima and halophyte root microbiota may be valuable resources for human or animal feeding in a future climate reality. Ministerio de Economía y Competitividad (CGL2016-75550-R) Ministerio de Educación, Cultura y Deporte (FPU014/03987)

In the genomic era phylogenetic relationship among prokaryotes can be inferred from the core orthologous genes (OGs) or proteins in order to elucidate their evolutionary history and current taxonomy should benefits of that. The genus Salinivibrio belongs to the family Vibrionaceae and currently includes only five halophilic species, in spite the fact that new strains are very frequently isolated from hypersaline environments. Species belonging to this genus have undergone several reclassifications and, moreover, there are many strains of Salinivibrio with available genomes which have not been affiliated to the existing species or have been wrongly designated. Therefore, a phylogenetic study using the available genomic information is necessary to clarify the relationships of existing strains within this genus and to review their taxonomic affiliation. For that purpose, we have also sequenced the first complete genome of a Salinivibrio species, Salinivibrio kushneri AL184T, which was employed as a reference to order the contigs of the draft genomes of the type strains of the current species of this genus, as well as to perform a comparative analysis with all the other available Salinivibrio sp. genomes. The genome of S. kushneri AL184T was assembled in two circular chromosomes (with sizes of 2.84 Mb and 0.60 Mb, respectively), as typically occurs in members of the family Vibrionaceae, with nine complete ribosomal operons, which might explain the fast growing rate of salinivibrios cultured under laboratory conditions. Synteny analysis among the type strains of the genus revealed a high level of genomic conservation in both chromosomes, which allow us to hypothesize a slow speciation process or homogenization events taking place in this group of microorganisms to be tested experimentally in the future. Phylogenomic and orthologous average nucleotide identity (OrthoANI)/average amino acid identity (AAI) analyses also evidenced the elevated level of genetic relatedness within members of this genus and allowed to group all the Salinivibrio strains with available genomes in seven separated species. Genome-scale attribute study of the salinivibrios identified traits related to polar flagellum, facultatively anaerobic growth and osmotic response, in accordance to the phenotypic features described for species of this genus. Spanish Ministry of Economy and Competitiveness Project CGL2017- 83385-P España Junta de Andalucía BIO-213 Spanish, University of Seville VIPPIT-US-2018

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. Conclusions Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism. University of Murcia and University of Seville was supported by projects: BIO2015-63949-R, BIO2014-54411-C2-1-R Europa MINECO/FEDER RTI2018-094393-B-C21 Fundación Séneca (Grant no. 19236/PI/14)

Soil contamination with heavy metals is a constraint for plant establishment and development for which phytoremediation may be a solution, since rhizobacteria may alleviate plant stress under these conditions. A greenhouse experiment was conducted to elucidate the effect of toxic metals on growth, the activities of ROS (reactive oxygen species)-scavenging enzymes, and gene expression of Medicago sativa grown under different metal and/or inoculation treatments. The results showed that, besides reducing biomass, heavy metals negatively affected physiological parameters such as chlorophyll fluorescence and gas exchange, while increasing ROS-scavenging enzyme activities. Inoculation of M. sativa with a bacterial consortium of heat- and metallo-resistant bacteria alleviated metal stress, as deduced from the improvement of growth, lower levels of antioxidant enzymes, and increased physiological parameters. The bacteria were able to effectively colonize and form biofilms onto the roots of plants cultivated in the presence of metals, as observed by scanning electron microscopy. Results also evidenced the important role of glutathione reductase (GR), phytochelatin synthase (PCS), and metal transporter NRAMP1 genes as pathways for metal stress management, whereas the gene coding for cytochrome P450 (CP450) seemed to be regulated by the presence of the bacteria. These outcomes showed that the interaction of metal-resistant rhizobacteria/legumes can be used as an instrument to remediate metal-contaminated soils, while cultivation of inoculated legumes on these soils is still safe for animal grazing, since inoculation with bacteria diminished the concentrations of heavy metals accumulated in the aboveground parts of the plants to below toxic levels Marruecos. Centre National pour la Recherche Scientifique et Technique (CNRST)-España, Ministerio de Economía y Competitividad (MINECO)-PPR2 /2016/42 Unión Europea (FEDER)-CGL2016-75550-R

Technical ceramics are being widely employed in the electric power, medical and engineering industries because of their thermal and mechanical properties, as well as their high resistance qualities. The manufacture of technical ceramic components involves complex processes, including milling and stirring of raw materials in aqueous solutions, spray drying and dry pressing. In general, the spray-dried powders exhibit an important degree of variability in their performance when subjected to dry-pressing, which affects the efficiency of the manufacturing process. Commercial additives, such as deflocculants, biocides, antifoam agents, binders, lubricants and plasticizers are thus applied to ceramic slips. Several bacterial and fungal species naturally occurring in ceramic raw materials, such as Sphingomonas, Aspergillus and Aureobasidium, are known to produce exopolysaccharides. These extracellular polymeric substances (EPS) may confer unique and potentially interesting properties on ceramic slips, including viscosity control, gelation, and flocculation. In this study, the microbial communities present in clay raw materials were identified by both culture methods and DNA-based analyses to select potential EPS producers based on the scientific literature for further assays based on the use of EPS for enhancing the performance of technical ceramics. Potential exopolysaccharide producers were identified in all samples, such as Sphingomonas sp., Pseudomonas xanthomarina, P. stutzeri, P. koreensis, Acinetobacter lwoffi, Bacillus altitudinis and Micrococcus luteus, among bacteria. Five fungi (Penicillium citrinum, Aspergillus niger, Fusarium oxysporum, Acremonium persicinum and Rhodotorula mucilaginosa) were also identified as potential EPS producers., This research was funded by Fundação para a Ciência e a Tecnologia, project EXPL/CTM-CER/0637/2012.

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.

Gluten is a complex of proteins present in barley, wheat, rye and several varieties of oats that triggers celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and therefore, proline-rich digestion-resistant peptides contain multiple immunogenic epitopes. Prolyl endopeptidases (PEP) hydrolyse internal proline residues on the carboxyl side of peptides and have been proposed for food gluten detoxification and as oral enzyme supplementation for celiacs. The aim of this study was to identify new gluten-degrading microbial enzymes with the potential to reduce gluten immunogenicity by neutralizing its antigenic epitopes. Using a gluten-degrading colony screening approach, a bacterial isolate (2RA3) displaying the highest glutenase activity was selected, characterized and its genome completely sequenced. The identification through 16S rDNA gene sequencing showed a 99,1% similarity to Chryseobacterium taeanense. Hydrolysis of gluten immunogenic peptides (GIP) was further monitored, over a 48-hour period, by colony encapsulation in gliadin-containing microspheres, followed by detection with the G12 anti-GIP monoclonal antibody. Glutenase activity was detected in the extracellular medium of 2RA3 cultures, where gel electrophoresis and gliadin zymography revealed the presence of a ~50 kDa gluten-degrading enzyme. Nano-ESI-Q-TOF of the excised active band identified 7 peptides contained in the protein product predicted for an open reading frame (ORF) in the 2RA3 genome. Based on sequence similarity to the PEP family, the new enzyme was named PEP 2RA3. The PEP 2RA3 coding sequence was PCR-amplified from C. taeanense 2RA3, cloned and expressed in Escherichia coli as a C-terminally His-tagged recombinant protein and purified by Ni-NTA affinity chromatography. The recombinant protein, with predicted molecular mass and isoelectric point of 78.95 kDa and 6.8, respectively, shows PEP activity with standard chromogenic substrates, works optimally at pH 8.0 and 30°C and remains stable at pH 6.0 and 50°C, indicating a potential use in gluten-containing food process applications. The ability of the recombinant enzyme to degrade GIP in beer into smaller peptides was confirmed. Ministry of Science and Innovation AGL2009-09069

The high global demand of wheat and its subsequent consumption arise from the physicochemical properties of bread dough and its contribution to the protein intake in the human diet. Gluten is the main structural complex of wheat proteins and subjects affected by celiac disease (CD) cannot tolerate gluten protein. Within gluten proteins, &alpha, gliadins constitute the most immunogenic fraction since they contain the main T-cell stimulating epitopes (DQ2.5-glia-&alpha, 1, DQ2.5-glia-&alpha, 2, and DQ2.5-glia-&alpha, 3). In this work, the celiac immunotoxic potential of &alpha, gliadins was studied within Triticeae: diploid, tetraploid, and hexaploid species. The abundance and immunostimulatory capacity of CD canonical epitopes and variants (with one or two mismatches) in all &alpha, gliadin sequences were determined. The results showed that the canonical epitopes DQ2.5-glia-&alpha, 1 and DQ2.5-glia-&alpha, 3 were more frequent than DQ2.5-glia-&alpha, 2. A higher abundance of canonical DQ2.5-glia-&alpha, 1 epitope was found to be associated with genomes of the BBAADD, AA, and DD types, however, the abundance of DQ2.5-glia-&alpha, 3 epitope variants was very high in BBAADD and BBAA wheat despite their low abundance in the canonical epitope. The most abundant substitution was that of proline to serine, which was disposed mainly on the three canonical DQ2.5 domains on position 8. Interestingly, our results demonstrated that the natural introduction of Q to H at any position eliminates the toxicity of the three T-cell epitopes in the &alpha, gliadins. The results provided a rational approach for the introduction of natural amino acid substitutions to eliminate the toxicity of three T-cell epitopes, while maintaining the technological properties of commercial wheats.

Gluten is a complex mixture of storage proteins in cereals like wheat, barley, and rye. Prolamins are the main components of gluten. Their high content in proline and glutamine makes them water-insoluble and difficult to digest in the gastrointestinal tract. Partial digestion generates peptide sequences which trigger immune responses in celiac and gluten-sensitive patients. Gluten detection in food is challenging because of the diversity, in various food matrices, of protein proportions or modifications and the huge number of immunogenic sequences with differential potential immunoactivity. Attempts to develop standard reference materials have been unsuccessful. Recent studies have reported the detection of a limited number of dominant Gluten Immunogenic Peptides (GIP) that share similarities to epitopes presented in the α-gliadin 33-mer, which showed to be highly proteolytic resistant and is considered to be the most immunodominant peptide within gluten in celiac disease (CD). GIP were detectable and quantifiable in very different kind of difficult to analyze food, revealing the potential immunogenicity by detecting T-cell activity of celiac patients. But GIP were also found in stool and urine of celiac patients on a supposedly gluten-free diet (GFD), showing the capacity to resist and be absorbed and excreted from the body, providing the first simple and objective means to assess adherence to the GFD. Methods to specifically and sensitively detect the most active GIP in food and biological fluids are rational candidates may use similar analytical standard references for determination of the immunopathological risk of gluten exposure in gluten-related diseases. España, MINECO AGL2013-48946-C España, Ministerio de Ciencia, Innovación y Universidades y Corporación Tecnológica de Andalucía (CTA)

The study evaluated the symptoms, acceptance, and digestibility of bread made from transgenic low-gliadin wheat, in comparison with gluten free bread, in Non-coeliac gluten sensitivity (NCGS) patients, considering clinical/sensory parameters and gut microbiota composition. This study was performed in two phases of seven days each, comprising a basal phase with gluten free bread and an E82 phase with low-gliadin bread. Gastrointestinal clinical symptoms were evaluated using the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire, and stool samples were collected for gluten immunogenic peptides (GIP) determination and the extraction of gut microbial DNA. For the basal and E82 phases, seven and five patients, respectively, showed undetectable GIPs content. The bacterial 16S rRNA gene V1-V2 hypervariable regions were sequenced using the Illumina MiSeq platform and downstream analysis was done using a Quantitative Insights into Microbial Ecology (QIIME) pipeline. No significant differences in the GSRS questionnaires were observed between the two phases. However, we observed a significantly lower abundance of some gut genera Oscillospira, Dorea, Blautia, Bacteroides, Coprococcus, and Collinsella, and a significantly higher abundance of Roseburia and Faecalibacterium genera during the E82 phase compared with the basal phase. The consumption of low-gliadin bread E82 by NCGS subjects induced potentially positive changes in the gut microbiota composition, increasing the butyrate-producing bacteria and favoring a microbial profile that is suggested to have a key role in the maintenance or improvement of gut permeability., The Spanish Ministry of Economy and Competitiveness (Projects AGL2013-48946-C3-1-R, AGL2013-48946-C and AGL2016-80566-P) and the European Regional Development Fund (FEDER) supported this research.

Polycyclic aromatic hydrocarbon (PAHs) are common soil contaminants of concern due to their toxicity toward plants, animals and microorganisms. The use of indigenous or added microbes (bioaugmentation) is commonly used for bioremediation of PAHs. In this work, the biodegradation rates and changes in the bacterial community structure were evaluated. The enrichment culture was useful for unambiguously identifying members of the soil bacterial community associated with PAH degradation and yielded a low diversity community. No significant difference in the rate of PAH degradation was observed between the microcosm receiving only PAHs or PAHs and bioaugmentation. Moreover, identical matches to the bioaugmentation inoculum were only observed at the initial stages of PAH degradation on day 8. After 22 days of incubation, the substantial degradation of all PAHs had occurred in both microcosms and the PAH contaminated soil had statistically significant increases in Alphaproteobacteria. There were also increases in Betaproteobacteria. In contrast, the PAH contaminated and bioaugmented soil was not enriched in PAH degrading Proteobacteria genera and, instead, an increase from 1.6% to 8% of the population occurred in the phylum Bacteroidetes class Flavobacteria, with Flavobacterium being the only identified genus. In addition, the newly discovered genus Ohtaekwangia increased from 0% to 3.2% of the total clones. These results indicate that the same soil microbial community can give rise to different PAH degrading consortia that are equally effective in PAH degradation efficiency. Moreover, these results suggest that the lack of efficacy of bioaugmentation in soils can be attributed to a lack of persistence of the introduced microbes, yet nonetheless may alter the microbial community that arises in response to PAH contamination in unexpected ways.

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related Halorubrum strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge. España, MINECO Projects CGL2013-46941-P and CGL2017-83385-P

Hypersaline environments encompass aquatic and terrestrial habitats. While only a limited number of studies on the microbial diversity of saline soils have been carried out, hypersaline lakes and marine salterns have been thoroughly investigated, resulting in an aquatic-biased knowledge about life in hypersaline environments. To improve our understanding of the assemblage of microbes thriving in saline soils, we assessed the phylogenetic diversity and metabolic potential of the prokaryotic community of two hypersaline soils (with electrical conductivities of ~24 and 55 dS/m) from the Odiel saltmarshes (Spain) by metagenomics. Comparative analysis of these soil databases with available datasets from salterns ponds allowed further identification of unique and shared traits of microbial communities dwelling in these habitats. Saline soils harbored a more diverse prokaryotic community and, in contrast to their aquatic counterparts, contained sequences related to both known halophiles and groups without known halophilic or halotolerant representatives, which reflects the physical heterogeneity of the soil matrix. Our results suggest that Haloquadratum and certain Balneolaeota members may preferentially thrive in aquatic or terrestrial habitats, respectively, while haloarchaea, nanohaloarchaea and Salinibacter may be similarly adapted to both environments. We reconstructed 4 draft genomes related to Bacteroidetes, Balneolaeota and Halobacteria and appraised their metabolism, osmoadaptation strategies and ecology. This study greatly improves the current understanding of saline soils microbiota. España, GL2013-46941-P and CGL2017-83385-P

Two 454 shotgun metagenomes were sequenced from hypersaline soil samples collected in the Odiel salt marsh area in Huelva, southwestern Spain. Analysis of contigs and 16S rRNA-related sequences showed that Halobacteria , Balneolaeota , and Bacteroidetes were the dominant groups. Rhodothermaeota and Nanohaloarchaeota were also abundant.

Objective Gluten-free diet (GFD) is the only management for coeliac disease (CD). Available methods to assess GFD compliance are insufficiently sensitive to detect occasional dietary transgressions that may cause gut mucosal damage. We aimed to develop a method to determine gluten intake and monitor GFD compliance in patients with CD and to evaluate its correlation with mucosal damage. Design Urine samples of 76 healthy subjects and 58 patients with CD subjected to different gluten dietary conditions were collected. A lateral flow test (LFT) with the highly sensitive and specific G12 monoclonal antibody for the most dominant gluten immunogenic peptides (GIP) and a LFT reader were used to quantify GIP in solid-phase extracted urines. Results GIP were detectable in concentrated urines from healthy individuals previously subjected to GFD as early as 4–6 h after single gluten intake, and remained detectable for 1–2 days. The urine assay revealed infringement of the GFD in about 50% of the patients. Analysis of duodenal biopsies revealed that most of patients with CD (89%) with no villous atrophy had no detectable GIP in urine, while all patients with quantifiable GIP in urine showed incomplete intestinal mucosa recovery. Conclusion GIP are detected in urine after gluten consumption, enabling a new and non-invasive method to monitor GFD compliance and transgressions. The method was sensitive, specific and simple enough to be convenient for clinical monitoring of patients with CD as well as for basic and clinical research applications including drug development. Trial registration number NCT02344758.