Search

Resumen del trabajo presentado en el International Symposium Microbe-assisted crop production – opportunities, challenges and needs (MICROPE 2022), celebrado en Viena (Austria), del 11 al 14 de julio de 2022, Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants’ responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota., This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2013-49125-C2-1-P, BIO2016-78747-P and PID2019-104685GB-100), the Government of Navarra (refs. P1044 AGROESTI, P1004 PROMEBIO and P1046 MICROBIOME), and the project “Plants as a tool for sustainable global development” (registration number: CZ.02.1.01/0.0/0.0/16_019/0000827) within the program Research, Development and Education (OP RDE)

Biofilm development and quorum sensing (QS) are closely interconnected processes. Biofilm formation is a cooperative group behaviour that involves bacterial populations living embedded in a self-produced extracellular matrix. QS is a cell-cell communication mechanism that synchronizes gene expression in response to population cell density. Intuitively, it would appear that QS might coordinate the switch to a biofilm lifestyle when the population density reaches a threshold level. However, compelling evidence obtained in different bacterial species coincides in that activation of QS occurs in the formed biofilm and activates the maturation and disassembly of the biofilm in a coordinate manner. The aim of this review is to illustrate, using four bacterial pathogens as examples, the emergent concept that QS activates the biofilm dispersion process, Work in the Laboratory of Microbial Biofilms is funded by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, as well as ERA-NET Pathogenomics (PIM2010EPA-00606) and grant from the Departamento de Innovación (IIM13329.RI1), Gobierno de Navarra.

In this work we compiled information on current and emerging microbial-based fertilization practices, especially the use of cell-free microbial culture filtrates (CFs), to promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identified limitations to bring microbial CFs to the market as biostimulants. In nature, plants act as metaorganisms, hosting microorganisms that communicate with the plants by exchanging semiochemicals through the phytosphere. Such symbiotic interactions are of high importance not only for plant yield and quality, but also for functioning of the soil microbiota. One environmentally sustainable practice to increasing crop productivity and/or protecting plants from (a)biotic stresses while reducing the excessive and inappropriate application of agrochemicals is based on the use of inoculants of beneficial microorganisms. However, this technology has a number of limitations, including inconsistencies in the field, specific growth requirements and host compatibility. Beneficial microorganisms release diffusible substances that promote plant growth and enhance yield and stress tolerance. Recently, evidence has been provided that this capacity also extends to phytopathogens. Consistently, soil application of microbial cell-free culture filtrates (CFs) has been found to promote growth and enhance the yield of horticultural crops. Recent studies have shown that the response of plants to soil application of microbial CFs is associated with strong proliferation of the resident beneficial soil microbiota. Therefore, the use of microbial CFs to enhance both crop yield and stress tolerance, and to activate beneficial soil microbiota could be a safe, efficient and environmentally friendly approach to minimize shortfalls related to the technology of microbial inoculation. In this review, we compile information on microbial CFs and the main constituents (especially volatile compounds) that promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identify challenges and limitations for their use as biostimulants to bring them to the market and we propose remedial actions and give suggestions for future work, This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grant PID2019-104685GB-100).

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains., This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant No. 646869 to A.T.-A.) and by the Spanish Ministry of Science and Innovation grants (BIO2017-83035-R to I.L. and PID2019-105216GBI00 to A.T.-A.). Funding for open access charge was provided by the CSIC Open Access Publication Support Initiative, Unit of Information Resources for Research (URICI).

[Background] MicroRNAs (miRNAs) are short endogenous, single-stranded, noncoding small RNA molecules of approximately 22 nucleotides in length. They regulate gene expression posttranscriptionally by silencing mRNA expression, thus orchestrating many physiological processes. The Small Ruminant Lentiviruses (SRLV) group includes the Visna Maedi Virus (VMV) and Caprine Arthritis Encephalitis (CAEV) viruses, which cause a disease in sheep and goats characterized by pneumonia, mastitis, arthritis and encephalitis. Their main target cells are from the monocyte/macrophage lineage. To date, there are no studies on the role of miRNAs in this viral disease., [Results] Using RNA-seq technology and bioinformatics analysis, the expression levels of miRNAs during different clinical stages of infection were studied. A total of 212 miRNAs were identified, of which 46 were conserved sequences in other species but found for the first time in sheep, and 12 were completely novel. Differential expression analysis comparing the uninfected and seropositive groups showed changes in several miRNAs; however, no significant differences were detected between seropositive asymptomatic and diseased sheep. The robust increase in the expression level of oar-miR-21 is consistent with its increased expression in other viral diseases. Furthermore, the target prediction of the dysregulated miRNAs revealed that they control genes involved in proliferation-related signalling pathways, such as the PI3K-Akt, AMPK and ErbB pathways., [Conclusions] To the best of our knowledge, this is the first study reporting miRNA profiling in sheep in response to SRLV infection. The known functions of oar-miR-21 as a regulator of inflammation and proliferation appear to be a possible cause of the lesions caused in the sheep’s lungs. This miRNA could be an indicator for the severity of the lung lesions, or a putative target for therapeutic intervention., This work was supported by a UPV/EHU grant (GIU14/23) provided to B.M. Jugo, two predoctoral fellowships from the UPV/EHU to M. Bilbao-Arribas (PIF17/306) and E. Varela-Martínez (PIF15/361) and a postdoctoral fellowship from the UPV/EHU to Dr. N. Abendaño (ESPDOC16/43).

Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii., The results of this work have been funded by the Projects PI11/01034 to MP and PI14/00059 to MP and AB and PI12/00552 and PI15/00860 to GB, integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the ISCIII- General Subdirection of Assessment and Promotion of the Research – European Regional Development Fund (FEDER) “A way of making Europe” and Miami University (Ohio, USA) Research Funds. We also thank Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to GB), cofinanced by the European Development Regional Fund (EDRF) “A Way to Achieve Europe, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad. A.P. was financially supported by the Galician Plan for Research, Innovation and Growth (I2C Plan 2012-2016). J.A. Vallejo was financially supported by the Sara Borrell Program (ISCIII, Spain CD13/00373). We wish to thank grant GEIH/REIPI-Ab2010 from Instituto de Salud Carlos III - Ministerio Economía y Competitividad for providing MAR002 strain and M.I. Voskuil for pMo130.

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression., This work was supported by the Spanish Ministry of Economy and Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union). A.T.-A. is supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme Grant Agreement 646869.

ADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identified during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specific RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions., This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) (grant numbers BIO2013-49125-C2-1-P and BIO2016-78747-P). A.M.V. is a Career Researcher of the Consejo Nacional de Investigaciones Cientificas y Técnicas de Argentina (CONICET) and Professor of Micribiology at the National University of Rosario (U.N.R., Argentina). A.M.V. expresses his gratitude to the Ministerio de Educación y Cultura, the Consejo Superior de Investigaciones Científicas, and the Public University of Navarra for financial support.

23 p.-9 fig.-4 tab., Tracking bacterial evolution during chronic infection provides insights into how host selection pressures shape bacterial genomes. The human-restricted opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) infects the lower airways of patients suffering chronic obstructive pulmonary disease (COPD) and contributes to disease progression. To identify bacterial genetic variation associated with bacterial adaptation to the COPD lung, we sequenced the genomes of 92 isolates collected from the sputum of 13 COPD patients over 1 to 9years. Individuals were colonized by distinct clonal types (CTs) over time, but the same CT was often reisolated at a later time or found in different patients. Although genomes from the same CT were nearly identical, intra-CT variation due to mutation and recombination occurred. Recurrent mutations in several genes were likely involved in COPD lung adaptation. Notably, nearly a third of CTs were polymorphic for null alleles of ompP1 (also called fadL), which encodes a bifunctional membrane protein that both binds the human carcinoembryonic antigen-related cell adhesion molecule 1 (hCEACAM1) receptor and imports long-chain fatty acids (LCFAs). Our computational studies provide plausible three-dimensional models for FadL's interaction with hCEACAM1 and LCFA binding. We show that recurrent fadL mutations are likely a case of antagonistic pleiotropy, since loss of FadL reduces NTHi's ability to infect epithelia but also increases its resistance to bactericidal LCFAs enriched within the COPD lung. Supporting this interpretation, truncated fadL alleles are common in publicly available NTHi genomes isolated from the lower airway tract but rare in others. These results shed light on molecular mechanisms of bacterial pathoadaptation and guide future research toward developing novel COPD therapeutics.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidate the bacterial pathways undergoing in vivo evolutionary adaptation, we compared bacterial genomes collected over time from 13 COPD patients and identified recurrent genetic changes arising in independent bacterial lineages colonizing different patients. Besides finding changes in phase-variable genes, we found recurrent loss-of-function mutations in the ompP1 (fadL) gene. We show that loss of OmpP1/FadL function reduces this bacterium's ability to infect cells via the hCEACAM1 epithelial receptor but also increases its resistance to bactericidal fatty acids enriched within the COPD lung, suggesting a case of antagonistic pleiotropy that restricts DeltafadL strains' niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways., This work has been funded by MINECO grants SAF2012-31166 and SAF2015-66520-R to J.G. and grants CTQ2014-57141-R and CTQ2017-88353-R to S.M.-S.; grant 03/2016 from the Health Department, Regional Government of Navarra, Spain, and SEPAR grant 31/2015 to J.G.; and NIDCD grant 5R01 DC 02148 and NIDDK grant 1U01 DK082316 from the U.S. National Institutes of Health to G.D.E. CIBERES is an initiative from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. J.M. and L.P.-R. were funded by Ph.D.studentships BES-2013-062644 and BES-2012-053653 from MINECO, Spain.

Previous studies on Arabidopsis under long-term exposure to elevated CO2 have been conducted using starch synthesis and breakdown mutants cultured under short day conditions. These studies showed that starch synthesis can ameliorate the photosynthetic reduction caused by soluble sugar-mediated feedback regulation. In this work we characterized the effect of long-term exposure to elevated CO2 (800 ppm) on growth, photosynthesis and content of primary photosynthates in long-day grown wild type plants as well as the near starch-less (aps1) and the starch-excess (gwd) mutants. Notably, elevated CO2 promoted growth of both wild type and aps1 plants but had no effect on gwd plants. Growth promotion by elevated CO2 was accompanied by an increased net photosynthesis in WT and aps1 plants. However, the plants with the highest starch content (wild type at elevated CO2, gwd at ambient CO2, and gwd at elevated CO2) were the ones that suffered decreased in in vivo maximum carboxylation rate of Rubisco, and therefore, photosynthetic down-regulation. Further, the photosynthetic rates of wild type at elevated CO2 and gwd at elevated CO2 were acclimated to elevated CO2. Notably, elevated CO2 promoted the accumulation of stress-responsive and senescence-associated amino acid markers in gwd plants. The results presented in this work provide evidence that under long-day conditions, temporary storage of overflow photosynthate as starch negatively affect Rubisco performance. These data are consistent with earlier hypothesis that photosynthetic acclimation can be caused by accelerated senescence and hindrance of CO2 diffusion to the stroma due to accumulation of large starch granules., This work was partially supported by the Spanish National Research and Development Programme (AGL2016-79868-R), by Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) (grant number BIO2016-78747-P) and the Basque Government (IT-932-16).

12 Pags.- 2 Tabls.- 6 Figs. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited., The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin–thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI–LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way., This work was supported by the Spanish Ministry of Science and Innovation (AGL2016-79868) and from the Basque Government (UPV/EHU-GV IT-932-16). MA is a holder of a PhD fellowship from the Spanish Ministry of Education (FPU13/01675).

The major challenges in diagnosing small ruminant lentivirus (SRLV) infection include early detection and genotyping of strains of epidemiological interest. A longitudinal study was carried out in Rasa Aragonesa sheep experimentally infected with viral strains of genotypes A or B from Spanish neurological and arthritic SRLV outbreaks, respectively. Sera were tested with two commercial ELISAs, three based on specific peptides and a novel combined peptide ELISA. Three different PCR assays were used to further assess infection status.The kinetics of anti-viral antibody responses were variable, with early diagnosis dependent on the type of ELISA used. Peptide epitopes of SRLV genotypes A and B combined in the same ELISA well enhanced the overall detection rate, whereas single peptides were useful for genotyping the infecting strain (A vs. B). The results of the study suggest that a combined peptide ELISA can be used for serological diagnosis of SRLV infection, with single peptide ELISAs useful for subsequent serotyping., Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1R) and Navarra's Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI-fellow of the Spanish MINECO and R. Reina had a contract of the Public University of Navarra.

Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis. Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status., This work was co-funded by the Italian Ministry of Instruction, University and Research PRIN 2008 (no. 20084CSFLT), by Piedmont Region, “Ricerca Sanitaria Finalizzata” 2008 and 2009, and by University of Turin, “Fondi ricerca locale (ex-60%)” 2009. The Authors acknowledge Mr. R. Maritano, CISRA for his valuable contribution in animal management, and Mr. D. Arnulfo and R. Vanni for their competent work and assistance during animal autopsies. R. Reina was supported by Spanish Ministry of Science and Innovation ‘Ramón y Cajal’ contract (AGL2013-49137-C3-1R).

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life., This research was supported by the Institute for Tropical Health funders (Obra Social la CAIXA, Fundaciones Caja Navarra and Roviralta, PROFAND, Ubesol, ACUNSA, and Artai) and grants MINECO (AGL2014-58795-C4-1-R, Bru-Epidia 291815- FP7/ERANET/ANIHWA), Aragón Government (Consolidated Group A14), andMarie Curie Career Integration Grant U-KARE (PCIG13-GA-2013-618162). TLB is the recipient of a Ph.D. Fellowship funded by the Department for Employment and Learning (Northern Ireland, United Kingdom).

Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules., A.F.C was funded by a contract from Ministerio Economía y Competitividad-MINECO, reference 20132RC947, Spain; I.R.A. is funded by a PhD studentship from Universidad Pública de Navarra, Spain; J.M. was funded by PhD studentship BES-2013-062644 from MINECO; S.M. is funded by a postdoctoral contract from CIBERES; L.C. was funded by a contract from MINECO, reference CS_NAV_IDAB_005, Spain; T.L.B. is the recipient of a PhD fellowship funded by the Department for Employment and Learning (Northern Ireland, UK). This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520-R, from Health Department, Regional Govern from Navarra, Spain, reference 03/2016, and from SEPAR 31/2015 to J.G.; and by grant from MINECO DPI2015-64221 to COdS. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

12 Páginas, 7 Figuras. Contiene información suplementaria en: http://dx.doi.org/10.1038/s41467-018-02949-y, Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches., This work was supported by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, BIO2014-53530-R, SAF2014-56716-REDT , and RTC-2015-3184-1. J.V. was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness.

Rice nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides including ADP-glucose and ATP. Under high temperature and elevated CO2 conditions (HT + ECO2), the npp1 knockout rice mutant displayed rapid growth and high starch content phenotypes, indicating that NPP1 exerts a negative effect on starch accumulation and growth. To gain further insight into the mechanisms involved in the NPP1 downregulation induced starch overaccumulation, in this study we conducted photosynthesis, leaf proteomic, and chloroplast phosphoproteomic analyses of wild-type (WT) and npp1 plants cultured under HT + ECO2. Photosynthesis in npp1 leaves was significantly higher than in WT. Additionally, npp1 leaves accumulated higher levels of sucrose than WT. The proteomic analyses revealed upregulation of proteins related to carbohydrate metabolism and the protein synthesis system in npp1 plants. Further, our data indicate the induction of 14-3-3 proteins in npp1 plants. Our finding demonstrates a higher level of protein phosphorylation in npp1 chloroplasts, which may play an important role in carbohydrate accumulation. Together, these results offer novel targets and provide additional insights into carbohydrate metabolism regulation under ambient and adverse conditions., This research was supported by Grants-in-Aid for Scientific Research (A) (15H02486) and Strategic International Collaborative Research Program by the Japan Science and Technology Agency (JST SICORP).

There have been few in vivo studies on the effect of aluminum hydroxide adjuvant and its influence on the immune response to vaccination. In this study, lambs received a parallel subcutaneous treatment with either commercial vaccines containing aluminum hydroxide or an equivalent dose of this compound only with the aim of identifying the activated molecular signature. Blood samples were taken from each animal at the beginning and at the end of the experiment and PBMCs isolated. Total RNA and miRNA libraries were prepared and sequenced. After alignment to the Oar3.1 reference genome and differential expression with 3 programs, gene enrichment modeling was performed. For miRNAs, miRBase and RNAcentral databases were used for detection and characterization. Three expression comparisons were made: vaccinated animals at the beginning and at the end of the treatment, adjuvanted animals at the same times, and animals of both treatments at the end of the experiment. After exposure to both treatments, a total of 2,473; 2,980 and 429 differentially expressed genes were identified in vaccinated animals, adjuvanted animals and animals at the end of both treatments, respectively. In both adjuvant and vaccine treated animals the NF-κB signaling pathway was enriched. On the other hand, it can be observed a downregulation of cytokines and cytokine receptors in the adjuvanted group compared to the vaccinated group at the final time, suggesting a milder induction of the immune response when the adjuvant is alone. As for the miRNA analysis, 95 miRNAs were detected: 64 previously annotated in Ovis aries, 11 annotated in Bos taurus and 20 newly described. Interestingly, 6 miRNAs were differentially expressed in adjuvant treated animals, and 3 and 1 in the other two comparisons. Lastly, an integrated miRNA-mRNA expression profile was developed, in which a miRNA-mediated regulation of genes related to DNA damage stimulus was observed. In brief, it seems that aluminum-containing adjuvants are not simple delivery vehicles for antigens, but also induce endogenous danger signals that can stimulate the immune system. Whether this contributes to long-lasting immune activation or to the overstimulation of the immune system remains to be elucidated., This work was supported by a MINECO project grant (AGL2013-49137-C3-3-R to BJ and AGL2013-49137-C3-2-R to LL and AGL2013-49137-C3-1 to DdA), a predoctoral fellowship from the UPV/EHU to EV-M (PIF15/361) and a postdoctoral fellowship from the UPV/EHU to NA (ESPDOC16/43).

Los métodos tradicionales utilizados en la conservación del patrimonio histórico incluyen principalmente el uso de insecticidas sintéticos, que presentan las desventajas de su toxicidad y de su falta de inocuidad sobre el sustrato a tratar. En este artículo se presenta una alternativa a estos métodos basada en el empleo de sustancias biocidas extraídas o provenientes de hongos entomopatógenos, como son Beauveria bassiana y Metarhizium anisopliae. La presente propuesta incluye dos variantes no excluyentes como son el uso de conidios y la aplicación de geles con enzimas y toxinas provenientes de dichas especies. Esta metodología aporta ventajas con respecto a los métodos tradicionales, como la no toxicidad para el manipulador, la aplicabilidad a diferentes sustratos independientemente de su tamaño y localización, y su prácticamente nula reactividad frente al sustrato.

Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g., water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success. All work was funded by the University of Reading.

The plastid-localized phosphoglucose isomerase isoform PGI1 is an important determinant of growth in Arabidopsis thaliana, likely due to its involvement in the biosynthesis of plastidial isoprenoid-derived hormones. Here, we investigated whether PGI1 also influences seed yields. PGI1 is strongly expressed in maturing seed embryos and vascular tissues. PGI1-null pgi1-2 plants had ∼60% lower seed yields than wild-type plants, with reduced numbers of inflorescences and thus fewer siliques and seeds per plant. These traits were associated with low bioactive gibberellin (GA) contents. Accordingly, wild-type phenotypes were restored by exogenous GA application. pgi1-2 seeds were lighter and accumulated ∼50% less fatty acids (FAs) and ∼35% less protein than wild-type seeds. Seeds of cytokinin-deficient plants overexpressing CYTOKININ OXIDASE/DEHYDROGENASE1 (35S:AtCKX1) and GA-deficient ga20ox1 ga20ox2 mutants did not accumulate low levels of FAs, and exogenous application of the cytokinin 6-benzylaminopurine and GAs did not rescue the reduced weight and FA content of pgi1-2 seeds. Seeds from reciprocal crosses between pgi1-2 and wild-type plants accumulated wild-type levels of FAs and proteins. Therefore, PGI1 is an important determinant of Arabidopsis seed yield due to its involvement in two processes: GA-mediated reproductive development and the metabolic conversion of plastidial glucose-6-phosphate to storage reserves in the embryo., This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) (Grants BIO2013-49125-C2-1-P and BIO2016-78747-P), the Ministry of Education, Youth, and Sports of the Czech Republic (Grant LO1204 from the National Program of Sustainability I), the Government of Navarra (ref. P1004 PROMEBIO), the Università degli Studi di Milano (UNIMI-RTD-A, Linea2-DBS 2017-2018), and the H2020-MSCA-RISE Project (ExpoSeed GA-691109).

β-glucans exhibited in cell walls of several pathogens as bacteria or fungi are sensed by pathogen recognition receptors such as scavenger receptors present in antigen presenting cells, i.e., macrophages. β-glucans obtained from Shiitake mushrooms were chemically characterized. A β-glucan supplemented diet was assayed for 30 days in rabbits aiming to characterize the immune response elicited in blood-derived macrophages. M1 and M2 profiles of macrophage differentiation were confirmed in rabbits by in vitro stimulation with IFN-γ and IL-4 and marker quantification of each differentiation pathway. Blood derived macrophages from rabbits administered in vivo with the β-glucan supplemented diet showed higher IL-4, IFN-γ and RAGE together with lower IL-10 relative expression, indicative of an ongoing immune response. Differences in IL-1β, IL-13 and IL-4 expression were also found in rabbit sera by ELISA suggesting further stimulation of the adaptive response. Recent challenges in the rabbit industry include the search of diet supplements able to elicit an immune stimulation with particular interest in facing pathogens such as viruses or bacteria. β–glucans from fungi may contribute to maintain an immune steady state favouring protection and thus reducing antibiotic treatment., This work was funded by MAGRAMA, IDi 2014 (REF.:20140020001815), CDTI(Ref.: IDI 20140852) and ADER (Ref.:2013-l-lDD-00074). Ramsés Reina was supported by Spanish Ministryof Science and Innovation‘Ramón y Cajal’contract.

Chrysodeixis chalcites, an important pest of banana crops on the Canary Islands, is usually controlled by chemical insecticides. The present study aimed to evaluate the efficacy of the most prevalent isolate of the Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV, Baculoviridae) as a biological insecticide. Overall the prevalence of ChchNPV infection in C. chalcites populations was 2.3% (103 infected larvae out of 4,438 sampled), but varied from 0–4.8% on Tenerife and was usually low (0–2%) on the other islands. On Tenerife, infected larvae were present at 11 out of 17 plantations sampled. The prevalence of infection in larvae on bananas grown under greenhouse structures was significantly higher (3%) than in open-field sites (1.4%). The ChchNPV-TF1 isolate was the most abundant and widespread of four genetic variants of the virus. Application of 1.0x109 viral occlusion bodies (OBs)/l of ChchNPV-TF1 significantly reduced C. chalcites foliar damage in young banana plants as did commonly used pesticides, both in greenhouse and open-field sites. The insecticidal efficacy of ChchNPV-TF1 was similar to that of indoxacarb and a Bacillus thuringiensis (Bt)-based insecticide in one year of trials and similar to Bt in the following year of trails in greenhouse and field crops. However, larvae collected at different time intervals following virus treatments and reared in the laboratory experienced 2–7 fold more mortality than insects from conventional insecticide treatments. This suggests that the acquisition of lethal dose occurred over an extended period (up to 7 days) compared to a brief peak in larvae on plants treated with conventional insecticides. These results should prove useful for the registration of a ChchNPV-based insecticide for integrated management of this pest in banana crops on the Canary Islands., This study received financial support from the National Institute of the Agricultural Research from the Spanish Ministry of Economy, Industry and Competitiveness project numbers RTA2010-00016-C02 and RTA2013-00114-C02 and from the Spanish Ministry for Science and Technology AGL2014-57752-C02-1R). E. G. F. received a student scholarship from National Institute of the Agricultural Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Biological insecticides based on Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) can efficiently control S. exigua larvae on field and greenhouse crops in many parts of the world. Spanish wild populations and laboratory colonies of S. exigua are infected by two iflaviruses (SeIV-1 and SeIV-2). Here we evaluated the effect of iflavirus co-infection on the insecticidal characteristics of SeMNPV occlusion bodies (OBs). Overall, iflavirus co-inoculation consistently reduced median lethal concentrations (LC50) for SeMNPV OBs compared to larvae infected with SeMNPV alone. However, the speed of kill of SeMNPV was similar in the presence or absence of the iflaviruses. A reduction of the weight gain (27%) associated with iflavirus infection resulted in a 30% reduction in total OB production per larva. Adult survivors of SeMNPV OB inoculation were examined for covert infection. SeMNPV DNA was found to be present at a high prevalence in all SeIV-1 and SeIV-2 co-infection treatments. Interestingly, co-inoculation of SeMNPV with SeIV-2 alone or in mixtures with SeIV-1 resulted in a significant increase in the SeMNPV load of sublethally infected adults, suggesting a role for SeIV-2 in vertical transmission or reactivation of sublethal SeMNPV infections. In conclusion, iflaviruses are not desirable in insect colonies used for large scale baculovirus production, as they may result in diminished larval growth, reduced OB production and, depending on their host-range, potential risks to non-target Lepidoptera., This study was funded by the Spanish Ministerio de Ciencia e Innovacio´n, project number AGL2011-30352-C02-01. AC received a student scholarship from the Ministerio de Economı´a y Competitividad.

The ISO 6579:2002/Amd 1:2007 (ISO) standard has been the bacteriological standard method used in the European Union for the detection of Salmonella spp. in pig mesenteric lymph nodes (MLN), but there are no published estimates of the diagnostic sensitivity (Se) of the method in this matrix. Here, the Se of the ISO (SeISO) was estimated on 675 samples selected from two populations with different Salmonella prevalences (14 farms with a ≥20% prevalence and 13 farms with a, This study was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) of Spain (research grants no. RTA2007-65 and FAU2008-16) and the Gobierno de Navarra (project reference IIQ14064.RI1).

13 p.-5 fig.3 tab. Martínez, Isidoro et al., Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here., Research activities in the participating laboratories received further funding from the following sources: Centro Nacional de Microbiología, ISCIII, PI15CIII/00024 and MINECO (SAF2015- 67033-R); Centro Nacional de Biotecnología, MINECO (BFU2014-57797-R); Hospital Universitari Germans Trias I Pujol, Spanish Society of Pneumology and Thoracic Surgery (SEPAR 054/2011); Departamento de Bioquímica y Biología Molecular I, MINECO (SAF2015-65307-R); Centro de Investigaciones Biológicas, MINECO (SAF2012-39444-C01/02); Fundación de Investigación Sanitaria de las Islas Baleares, MINECO (SAF2012-39841); Instituto de Agrobiotecnología, MINECO (SAF2015-66520-R); Instituto de Química Física Rocasolano, MINECO (BFU2015-70052-R) and the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA- 2012-317297). Subprograma Estatal de Formación (BES-2013- 065355).

Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities., This research was supported by the Spanish Ministry of Science and Innovation (grant reference AGL2009-13340-C02) and by the Universidad Pública de Navarra (PhD contract awarded to Leopoldo Palma).

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 μg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein's target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins. © 2014 by the authors; licensee MDPI, Basel, Switzerland., This research was supported by the Spanish Ministry of Science and Innovation (grant ref. AGL2009-13340-C02) and by the Universidad Pública de Navarra (P hD contract awarded to L.P.).

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. © 2014, American Society for Microbiology., J. Valle was supported by Spanish Ministry of Science and Innovation “Ramón y Cajal” contract. This research was supported by grants ERANET Pathogenomic (GEN2006-27792-C2-1-E/PAT), BIO2011-30503- C02-02, and AGL2011-23954 from the Spanish Ministry of Economy and Competitivity and IIQ14066.RI1 from Innovation Department of the Government of Navarra. We thank Enrique Calvo from the Proteomic Service of CNIC for his expert help in protein identification and Victor Segura from the Genomics, Proteomics and Bioinformatics Unit at the Center for Applied Medical Research, University of Navarra, for microarray data analysis.

The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased., The authors would like to thank the Government of Navarra, Department of Innovation, Business and Employment for financial support within the project SABioD

A longitudinal study was conducted to investigate the presence of multidrug antimicrobial resistance (multi-AR) in Salmonella enterica in pigs reared under conventional preventative medicine programmes in Spain and the possible association of multi-AR with ceftiofur or tulathromycin treatment during the pre-weaning period. Groups of 7-day-old piglets were treated by intramuscular injection with ceftiofur on four farms (n = 40 piglets per farm) and with tulathromycin on another four farms (n = 40 piglets per farm). A control group of untreated piglets (n = 30 per farm) was present on each farm. Faecal swabs were collected for S. enterica culture prior to treatment, at 2, 7 and 180 days post-treatment, and at slaughter. Minimal inhibitory concentrations of 14 antimicrobial agents, pulsed-field gel electrophoresis and detection of resistance genes representing five families of antimicrobial agents were performed. Plasmids carrying cephalosporin resistant (CR) genes were characterised. Sixty-six S. enterica isolates were recovered from five of eight farms. Forty-seven isolates were multi-AR and four contained blaCTX-M genes harboured in conjugative plasmids of the IncI1 family; three of these isolates were recovered before treatment with ceftiofur. The most frequent AR genes detected were tet(A) (51/66, 77%), sul1 (17/66, 26%); tet(B) (15/66, 23%) and qnrB (10/66, 15%). A direct relation between the use of ceftiofur in these conditions and the occurrence of CR S. enterica was not established. However, multi-AR was common, especially for ampicillin, streptomycin, sulphonamides and tetracycline. These antibiotics are used frequently in veterinary medicine in Spain and, therefore, should be used sparingly to minimise the spread of multi-AR., This work was supported by project AGL2011-28836 from the Ministerio de Economia y Competitividad of Spain, the Departamento de Innovación, Empresa y Empleo del Gobierno de Navarra (project reference IIQ14064.RI1) and Fundación Caja Navarra, Spain (project reference VATC 2014-0411 y VATC 2015-70628). Contract of LMG was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the European Social Fund. Contract of VG was supported by Fundación Caja Navarra, Spain (project reference VATC 2015-70628). We acknowledge the Centres de Recerca de Catalunya programme for financial support, and to John G. Wild for review of English.

Antibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010–2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration., IR is funded by a Ph.D. studentship from Universidad Pública de Navarra, Spain; JM is funded by Ph.D. studentship BES-2013-062644 from Ministerio Economía y Competitividad-MINECO, Spain; SM is funded by a postdoctoral contract from CIBER Enfermedades Respiratorias (CIBERES); NL is funded by a contract from Department of Economy, Regional Govern from Navarra, Spain, reference 0011-1307-2015-000037. This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520-R, Health Department, Regional Govern from Navarra, Spain, reference 03/2016, and SEPAR 31/2015 to JG. CIBERES is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Composting in bins is one of the most practical home composting methods. There is currently a need for greater information to improve the management of the composting process and to create home composting programs, which ensure sustainable production of high quality compost. This study investigates how two aspects of the bin feeding regime—the feeding frequency and the amount of waste applied at each feed—influence the process's evolution and the quality of the compost. Compost bins were assayed after introducing the same amount of kitchen and garden waste according to three different frequencies: in a single batch, weekly, or every 3 weeks. A fourth treatment was applied to calculate the potential waste reduction achieved by the composting process, filling the bins to the brim on a weekly basis. Temperature, mass, and volume changes; the microbial diversity (by Biolog); and gas emissions (CO2, CH4, N2O, and NH3) were all determined during the process. At the end of the experiment, all of the composts were weighed and characterized. Results show that the main differences were very dependent on the quantity of waste provided. Large amounts of waste were added increasing the compost's temperature and maturity during the process, while slightly affecting the salinity and phytotoxicity of the final compost but without any clear effects on microbial diversity and gas emission. Therefore, from a technical point of view, the shared use of compost bins among several households (community composting) is preferable to individual use.

During an insect sampling program in alfalfa crops near Montpellier, France in 2011, Lacanobia oleracea larvae were collected that died due to nucleopolyhedrovirus infection (LaolNPV). This virus was subjected to molecular and biological characterization. The virus was a multiple nucleocapsid NPV that showed similar restriction profiles to Mamestra configurata NPV-A (MacoNPV-A) but with significant differences. Polypeptide analysis demonstrated similar proteins in occlusion bodies and occlusion derived virions, to those observed in NPVs from Mamestra spp. Terminal sequencing revealed that the genome organization shared similarity with that of MacoNPV-A. The most homologous virus was MacoNPV-A 90/2 isolate (95.63% identity and 96.47% similarity), followed by MacoNPV-A 90/4 strain (95.37% and 96.26%), MacoNPV-B (89.21% and 93.53%) and M. brassicae MNPV (89.42% and 93.74%). Phylogenetic analysis performed with lef-8, lef-9, polh and a concatenated set of genes showed that LaolNPV and the Mamestra spp. NPVs clustered together with HaMNPV, but with a closer genetic distance to MacoNPV-A strains. The Kimura 2-parameter (K-2-P) distances of the complete genes were greater than 0.05 between LaolNPV and the MbMNPV/MacoNPV-B/HaMNPV complex, which indicates that LaolNPV is a distinct species. K-2-P distances were in the range 0.015–0.050 for comparisons of LaolNPV with MacoNPV-A strains, such that additional biological characteristics should be evaluated to determine species status. While MacoNPV-A was pathogenic to seven lepidopteran species tested, LaolNPV was only pathogenic to Chrysodeixis chalcites. Given these findings, Lacanobia oleracea nucleopolyhedrovirus should be considered as a new species in the Alphabaculovirus genus., This study received financial support from the National Institute of Agricultural Research from the Spanish Ministry project RTA2013-00114-C02-02 (Dr. Oihane Simón) and from the Department of Industry of the Government of Navarre project IIQ1406-RI1 (Dr. Primitivo Caballero). This study was supported in part by funding from Agriculture and Agri-Food Canada Research Project J000197 (Dr. Martin A. Erlandson). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Although there is a great wealth of data supporting the occurrence of simultaneous synthesis and breakdown of storage carbohydrate in many organisms, previous 13CO2 pulse-chase based studies indicated that starch degradation does not operate in illuminated Arabidopsis leaves. Here we show that leaves of gwd, sex4, bam4, bam1/bam3 and amy3/isa3/lda starch breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured under continuous light (CL) conditions. We also show that leaves of CL grown dpe1 plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose than WT leaves, the overall data providing evidence for the occurrence of extensive starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown mex1/pglct plants impaired in the chloroplastic maltose and glucose transporters display a severe dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1 and pGlcT transporters are involved in the export of starch breakdown products to the cytosol to support growth during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition (δ13C) in starch of leaves of 13CO2 pulsed-chased WT and AGP lacking aps1 plants. Notably, the rate of increase of δ13C in starch of aps1 leaves during the pulse was exceedingly higher than that of WT leaves. Furthermore, δ13C decline in starch of aps1 leaves during the chase was much faster than that of WT leaves, which provides strong evidence for the occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves., Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) Grant numbers: BIO2010-18239 and BIO2013-49125-C2-1-P, http://www.mineco.gob.es/portal/site/mineco/.

Impairments in leaf nitrogen (N) assimilation in C3 plants have been identified as processes conditioning photosynthesis under elevated [CO2], especially when N is supplied as nitrate. Leaf N status is usually improved under ammonium nutrition and elevated [CO2]. However, ammonium fertilization is usually accompanied by the appearance of oxidative stress symptoms, which constrains plant development. To understand how the limitations of direct fertilization with ammonium (growth reduction attributed to ammonium toxicity) can be overcome, the effects of elevated [CO2] (800 ppm) exposure were studied in the Arabidopsis thaliana double nitrate reductase defective mutant, nia1-1/chl3-5 (which preferentially assimilates ammonium as its nitrogen source). Analysis of the physiology, metabolites and gene expression was carried out in roots and shoot organs. Our study clearly showed that elevated [CO2] improved the inhibited phenotype of the nitrate reductase double mutant. Both the photosynthetic rates and the leaf N content of the NR mutant under elevated CO2 were similar to wild type plants. The growth of the nitrate reductase mutant was linked to its ability to overcome ammonium-associated photoinhibition processes at 800 ppm [CO2]. More specifically: (i) the capacity of NR mutants to equilibrate energy availability, as reflected by the electron transport equilibrium reached (photosynthesis, photorespiration and respiration), (ii) as well as by the upregulation of genes involved in stress tolerance were identified as the processes involved in the improved performance of NR mutants., This work has been funded by the Spanish National Research and Development Programme (AGL2009-13339-C02-02, AGL2011-30386-C02-02 and AGL2012-37815-C05-05). Ivan Jauregui was the holder of a FPI fellowship from the Spanish Ministry of Economy and Competitiveness.

The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is an important cause of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) that requires efficient treatments. A previous screening for host genes differentially expressed upon NTHi infection identified sirtuin-1, which encodes a NAD-dependent deacetylase protective against emphysema and is activated by resveratrol. This polyphenol concomitantly reduces NTHi viability, therefore highlighting its therapeutic potential against NTHi infection at the COPD airway. In this study, resveratrol antimicrobial effect on NTHi was shown to be bacteriostatic and did not induce resistance development in vitro. Analysis of modulatory properties on the NTHi-host airway epithelial interplay showed that resveratrol modulates bacterial invasion but not subcellular location, reduces inflammation without targeting phosphodiesterase 4B gene expression, and dampens β defensin-2 gene expression in infected cells. Moreover, resveratrol therapeutics against NTHi was evaluated in vivo on mouse respiratory and zebrafish septicemia infection model systems, showing to decrease NTHi viability in a dose-dependent manner and reduce airway inflammation upon infection, and to have a significant bacterial clearing effect without signs of host toxicity, respectively. This study presents resveratrol as a therapeutic of particular translational significance due to the attractiveness of targeting both infection and overactive inflammation at the COPD airway., We thank Javier Moleres for his technical support. N.L.L. was funded by a contract from Department of Economy, Regional Govern from Navarra, Spain, reference 0011–1307; I.R.A. is funded by a PhD studentship from Universidad Pública de Navarra, Spain. This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520-R, Health Department, Regional Govern from Navarra, Spain, reference 3/2016, and SEPAR 31/2015 to J.G. CIBERES is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

Sánchez-López, Ángela María et al., It is known that volatile emissions from some beneficial rhizosphere microorganisms promote plant growth. Here we show that volatile compounds (VCs) emitted by phylogenetically diverse rhizosphere and non-rhizhosphere bacteria and fungi (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote growth and flowering of various plant species, including crops. In Arabidopsis plants exposed to VCs emitted by the phytopathogen Alternaria alternata, changes included enhancement of photosynthesis and accumulation of high levels of cytokinins (CKs) and sugars. Evidence obtained using transgenic Arabidopsis plants with altered CK status show that CKs play essential roles in this phenomenon, because growth and flowering responses to the VCs were reduced in mutants with CK-deficiency (35S:AtCKX1) or low receptor sensitivity (ahk2/3). Further, we demonstrate that the plant responses to fungal VCs are light-dependent. Transcriptomic analyses of Arabidopsis leaves exposed to A. alternata VCs revealed changes in the expression of light- and CK-responsive genes involved in photosynthesis, growth and flowering. Notably, many genes differentially expressed in plants treated with fungal VCs were also differentially expressed in plants exposed to VCs emitted by the plant growth promoting rhizobacterium Bacillus subtilis GB03, suggesting that plants react to microbial VCs through highly conserved regulatory mechanisms., This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) (grant numbers BIO2010-18239 and BIO2013- 49125-C2-1-P), the Government of Navarra (grant number IIM010491.RI1), the I-Link0939 project from the Ministerio de Economía y Competitividad, the Ministry of Education, Youth and Sports of the Czech Republic (Grant L01204 from the National Program of Sustainability) and Palacky University institutional support. AM S-L and P G-G gratefully acknowledge predoctoral fellowships from the Spanish Ministry of Science and Innovation. M B and G A acknowledge post-doctoral fellowships awarded by the Public University of Navarra.

10 p., il., tablas y bibliografía. Junkal Garmedia [et al.], Non-typable Haemophilus influenzae (NTHi) is a Gram negative pathogen that causes acute respiratory infections and is associated with the progression of chronic respiratory diseases. Previous studies have established the existence of a remarkable genetic variability among NTHi strains. In this study we show that, in spite of a high level of genetic heterogeneity, NTHi clinical isolates display a prevalent molecular feature, which could confer fitness during infectious processes. A total of 111 non-isogenic NTHi strains from an identical number of patients, isolated in two distinct geographical locations in the same period of time, were used to analyse nine genes encoding bacterial surface molecules, and revealed the existence of one highly prevalent molecular pattern (lgtF+, lic2A+, lic1D+, lic3A+, lic3B+, siaA-, lic2C+, ompP5+, oapA+) displayed by 94.6% of isolates. Such a genetic profile was associated with a higher bacterial resistance to serum mediated killing and enhanced adherence to human respiratory epithelial cells., This work was supported by grants from Instituto de Salud Carlos III (ISCIII, Spain) with references CP05-0027, PI06-1251 and PS09-0130, and from Fundación Mutua Madrileña a 2008 to JG, and by a grant from Instituto de Salud Carlos III (ISCIII, Spain) with reference PS09-1904 to JL.

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria., This research was supported by the Spanish Ministry of Economy and Competitiveness grants AGL2011-23954, BIO2014-53530-R and BFU2013-44763-P. JV was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness.

[Background] Detailed descriptions of the early development of parasitic nematodes are seldom available. The embryonic development of the plant-parasitic nematode Meloidogyne incognita was studied, focusing on the early events., [Results] A fixed pattern of repeated cell cleavages was observed, resulting in the appearance of the six founder cells 3 days after the first cell division. Gastrulation, characterized by the translocation of cells from the ventral side to the center of the embryo, was seen 1 day later. Approximately 10 days after the first cell division a rapidly elongating two-fold stage was reached. The fully developed second stage juvenile hatched approximately 21 days after the first cell division., [Conclusions] When compared to the development of the free-living nematode Caenorhabditis elegans, the development of M. incognita occurs approximately 35 times more slowly. Furthermore, M. incognita differs from C. elegans in the order of cell divisions, and the early cleavage patterns of the germ line cells. However, cytoplasmic ruffling and nuclear migration prior to the first cell division as well as the localization of microtubules are similar between C. elegans and M. incognita., This work was funded by grants from the California Agricultural Research Initiative (grant #: ARI/CATI/Calderón-Urrea/Cell Death/03-2-006-31), the College of Science and Mathematics at Fresno State, the California State University Program for Education and Research in Biotechnology (CSUPERB), and the RIMI Facility at Fresno State (development of this facility was funded by NIH-NIMHD grant “Research Infrastructure for Minority Institutions P20MD002732”).

Background: The use of reactive rock phosphate (RP) in acidic soils as a phosphate (P) source for pastures and crops presents attractive economic advantages with respect to soluble phosphate. However, some studies have demonstrated that the short-term (1-year) efficiency of RP, compared with that of water-soluble P, is relatively poor. This fact penalizes not only the yield and quality of the earlier harvests, but even the whole final yield when the crop is affected by some abiotic or biotic stress at the beginning of the cycle. In the present study, we investigated the ability of new edaphic biostimulants to increase the short-term efficiency of RP-based fertilizer as a P source for pastures cultivated in acid soils. To this end, we have granulated rock phosphate with two edaphic biostimulants: tryptophan and a heteromolecular organic complex formed by humic acid and tryptophan through iron bridges, and compared their short-term P fertilizer efficacy with that of single superphosphate and rock phosphate. Results: Soil incubation studies showed that the heteromolecular complex humic acid–tryptophan and Tryptophan were able to increase soil CO2 production compared with native soil, rock phosphate, and superphosphate. Likewise, the presence of humic acid–tryptophan complex and Trp significantly increases plant-available phosphate compared with rock phosphate, up to levels similar to those of superphosphate. Plant (ray grass)–soil–pot studies showed that rock phosphate/(humic acid–tryptophan) formulation yielded values for both ray grass dry matter production and shoot P concentration, clearly higher than those of rock phosphate and rock phosphate/tryptophan. In addition, the results associated with rock phosphate/(humic acid–tryptophan) were similar to those for superphosphate, after 3 months of cultivation. Conclusions: Taken together, these results showed the suitability of the use of specific humic acid-based edaphic biostimulants to improve the short-term effect of rock phosphate fertilizers as a phosphate source for pastures cultivated in acid soils. This Research Project has been supported by a Grant from CDTI and Government of Navarra, as well the Roullier Group.

6 p., 4 tables, 1 figure and bibliography, Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis., This work was supported in part by MICINN-CICYT, Spain (AGL2008-04514-C03-01 and AGL2010-20247), and by the joint Costa Rica-Spain Bilateral Cooperation (CRUSA-CSIC) (2008CR0006). Contract support for P.M.M. from the Instituto Nacional de Investigaciones Agrarias (INIA) of Spain (DR08-0064) and the Grupo Consolidado de Brucelosis (A-14) of the Aragón Government is also gratefully acknowledged.

The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda . Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda . All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died ∼24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N - glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB.

Staphylococcus aureus pathogenicity islands (SaPIs) have an intimate relationship with temperate staphylococcal phages. During phage growth, SaPIs are induced to replicate and are efficiently encapsidated into special small phage heads commensurate with their size. We have analyzed by amino acid sequencing and mass spectrometry the protein composition of the specific SaPI particles. This has enabled identification of major capsid and tail proteins and a putative portal protein. As expected, all these proteins were phage encoded. Additionally, these analyses suggested the existence of a protein required for the formation of functional phage but not SaPI particles. Mutational analysis demonstrated that the phage proteins identified were involved only in the formation and possibly the function of SaPI or phage particles, having no role in other SaPI or phage functions. This work was supported by grant BIO2005-08399-C02-02 from the Comisión Interministerial de Ciencia y Tecnología (C.I.C.Y.T.) and grants from the Cardenal Herrera-CEU University and from the Generalitat Valenciana (ACOMP07/258) to J.R.P. Fellowship support for María Desamparados Ferrer and for Elisa Maiques from the Cardenal Herrera-CEU University is gratefully acknowledged.

Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats., Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1-R) and Navarra’s Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI fellow of the Spanish MINECO and R. Reina had contracts from the Public University of Navarra and CSIC. We acknowledge Marta Gil Antona for sampling in hunting expeditions and Hunting Associations (FEDEMCA, Tragsa, INTIA, etc.) for their collaboration in the obtention of samples. We acknowledge support in the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

[Abstract] Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2Aby hmw1Aresulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1Awas confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1Amight be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1Abacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation., [Author Summary] Many bacteria are naturally competent, actively taking up DNA from their surroundings and incorporating it into their genomes by homologous recombination. This cellular process has had a large impact on the evolution of these species, for example by enabling pathogens to acquire virulence factors and antibiotic resistances from their relatives. But natural competence can also be exploited by researchers to identify the underlying genetic variation responsible for naturally varying phenotypic traits, similar to how eukaryotic geneticists use meiotic recombination during sexual reproduction to create genetically admixed populations. Here we exploited natural competence, phenotypic selection, and deep sequencing to rapidly identify the hmw1 locus as a major contributor to intracellular invasion of airway epithelial cells by the human pathogen Haemophilus influenzae, a trait that likely allows bacterial cells to evade the immune system and therapeutic interventions during chronic infections. Genetic variation in this locus can strongly modulate bacterial intracellular invasion rates, and possession of a certain allele favors adhesion and self-aggregation, which appear to prompt bacteria to invade airway cells as groups, rather than as individuals. Overall, our findings indicate that targeting HMW1 could block the ability of H. influenzae to invade airway cells, which would make antibiotic therapy to treat chronic lung infections more effective. Furthermore, our new approach to identifying the genetic basis of natural phenotypic variation is applicable to a wide-range of phenotypically selectable traits within the widely distributed naturally competent bacterial species, including pathogenesis traits in many human pathogens., This work was supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship F32AI084427 (to JCM); a Canadian Institute of Health Research operating grant (to RJR); Genome British Columbia grant SOF122 (to RJR and JCM); the Faculty of Pharmaceutical Sciences, Canadian Foundation for Innovation (to CN); National Institutes of Heath R01 grant DC002873 (to JWSG); and (to JG) grants from Ministerio Economía y Competitividad-MINECO SAF2012-31166 and SAF2015-66520-R, Dpto. Salud Gobierno Navarra 359/2012 and Ministerio de Educación PRX12/00191.

British Ecological Society Special Feature: Demography Beyond the Population., Red deer populations in the Iberian glacial refugium were the main source for postglacial recolonization and subspecific radiation in north-western Europe. However, the phylogenetic history of Iberian red deer (Cervus elaphus hispanicus) and its relationships with northern European populations remain uncertain. Here, we study DNA sequences at the mitochondrial control region along with STR markers for over 680 specimens from all the main red deer populations in Spain and other west European areas. Our results from mitochondrial and genomic DNA show contrasting patterns, likely related to the nature of these types of DNA markers and their specific processes of change over time. The results, taken together, bring support to two distinct, cryptic maternal lineages for Iberian red deer that predated the last glacial maximum and that have maintained geographically well differentiated until present. Haplotype relationships show that only one of them contributed to the northern postglacial recolonization. However, allele frequencies of nuclear markers evidenced one main differentiation between Iberian and northern European subspecies although also supported the structure of both matrilines within Iberia. Thus, our findings reveal a paraphyletic nature for Iberian red deer but also its genetic identity and differentiation with respect to northern subspecies. Finally, we suggest that maintaining the singularity of Iberian red deer requires preventing not only restocking practices with red deer specimens belonging to other European populations but also translocations between both Iberian lineages., This work was partly supported by project CGL2010-17163/BOS from the Spanish MInistry of Science and by Extremadura and Andalusian Regional Goverments.

Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus–virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides., This study received financial support from the Spanish Ministry for Science and Technology (AGL2011-30352-C02 and AGL2014-57752-C2). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Incluye material complementario Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin- Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms. This work was supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional, Spain (grant nos. BIO2010–18239 and BIO2013–49125–C2–1–P), by the Government of Navarra (grant no. IIM010491.RI1), by the I-Link0939 project from the Ministerio de Economía y Competitividad, by the Ministry of Education, Youth, and Sports of the Czech Republic (grant no. LO1204 from the National Program of Sustainability), by Palacky University institutional support, by predoctoral fellowships from the Spanish Ministry of Science and Innovation (to A.M.S.-L. and P.G.-G.), and by postdoctoral fellowships from the Public University of Navarra (to M.B. and G.A.).