Mutualistic interaction between Salmonella enterica and Aspergillus niger and its effects on Zea mays colonization

Salmonella Typhimurium inhabits a variety of environments and is able to infect a broad range of hosts. Throughout its life cycle, some hosts can act as intermediates in the path to the infection of others. Aspergillus niger is a ubiquitous fungus that can often be found in soil or associated to plants and microbial consortia. Recently, S. Typhimurium was shown to establish biofilms on the hyphae of A. niger. In this work, we have found that this interaction is stable for weeks without a noticeable negative effect on either organism. Indeed, bacterial growth is promoted upon the establishment of the interaction. Moreover, bacterial biofilms protect the fungus from external insults such as the effects of the anti-fungal agent cycloheximide. Thus, the Salmonella–Aspergillus interaction can be defined as mutualistic. A tripartite gnotobiotic system involving the bacterium, the fungus and a plant revealed that co-colonization has a greater negative effect on plant growth than colonization by either organism in dividually. Strikingly, co-colonization also causes a reduction in plant invasion by S. Typhimurium. This work demonstrates that S. Typhimurium and A. niger establish a mutualistic interaction that alters bacterial colonization of plants and affects plant physiology

Maintenance of Microbial Cooperation Mediated by Public Goods in Single- and Multiple-Trait Scenarios

Microbes often form densely populated communities, which favor competitive and cooperative interactions. Cooperation among bacteria often occurs through the production of metabolically costly molecules produced by certain individuals that become available to other neighboring individuals; such molecules are called public goods. This type of cooperation is susceptible to exploitation, since nonproducers of a public good can benefit from it while saving the cost of its production (cheating), gaining a fitness advantage over producers (cooperators). Thus, in mixed cultures, cheaters can increase in frequency in the population, relative to cooperators. Sometimes, and as predicted by simple game-theoretic arguments, such increases in the frequency of cheaters cause loss of the cooperative traits by exhaustion of the public goods, eventually leading to a collapse of the entire population. In other cases, however, both cooperators and cheaters remain in coexistence. This raises the question of how cooperation is maintained in microbial populations. Several strategies to prevent cheating have been studied in the context of a single trait and a unique environmental constraint. In this review, we describe current knowledge on the evolutionary stability of microbial cooperation and discuss recent discoveries describing the mechanisms operating in multiple-trait and multiple-constraint settings. We conclude with a consideration of the consequences of these complex interactions, and we briefly discuss the potential role of social interactions involving multiple traits and multiple environmental constraints in the evolution of specialization and division of labor in microbes.info:eu-repo/semantics/publishedVersio

Evolutionary Mechanisms Shaping the Maintenance of Antibiotic Resistance

The deposited review is a post-print version and has been submitted to peer review.This publication hasn't any creative commons license associated.This deposit is composed by the review, and it hasn't any supplementary materials associated.The publication deposited was in a state of Epub Ahead of Print, at the time when the upload was made.Antibiotics target essential cellular functions but bacteria can become resistant by acquiring either exogenous resistance genes or chromosomal mutations. Resistance mutations typically occur in genes encoding essential functions; these mutations are therefore generally detrimental in the absence of drugs. However, bacteria can reduce this handicap by acquiring additional mutations, known as compensatory mutations. Genetic interactions (epistasis) either with the background or between resistances (in multiresistant bacteria) dramatically affect the fitness cost of antibiotic resistance and its compensation, therefore shaping dissemination of antibiotic resistance mutations. This Review summarizes current knowledge on the evolutionary mechanisms influencing maintenance of resistance mediated by chromosomal mutations, focusing on their fitness cost, compensatory evolution, epistasis, and the effect of the environment on these processes.PD and RB were supported by Fundação para a Ciência e Tecnologia (FCT), fellowships SFRH/BPD/118474/2016 and SFRH/BPD/109517/2015, respectively. Current research is supported by project JPIAMR/0001/2016-ERA NET and ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by FEEI – ‘Fundos Europeus Estruturais e de Investimento’ from ‘Programa Operacional Regional Lisboa 2020’, and by national funds from FCT – ‘Fundação para a Ciência e a Tecnologia’.info:eu-repo/semantics/acceptedVersio

Insertion hot spot for horizontally acquired DNA within a bidirectional small-RNA locus in Salmonella enterica

In Escherichia coli and Salmonella enterica, RyeA and RyeB RNAs are encoded on opposite DNA strands at the same locus. We present evidence indicating that the last 23 bp of the ryeB gene, corresponding to an internal portion of the ryeA gene, served repeatedly as the integration site for exogenous DNA during Salmonella evolution and still act as an attachment site for present-day bacteriophages. Interestingly, ryeA sequence and expression are modified upon lysogenization.Ministerio de Educación y Ciencia BIO2004-3455-CO2-0

Multidrug-resistant bacteria compensate for the epistasis between resistances

Mutations conferring resistance to antibiotics are typically costly in the absence of the drug, but bacteria can reduce this cost by acquiring compensatory mutations. Thus, the rate of acquisition of compensatory mutations and their effects are key for the maintenance and dissemination of antibiotic resistances. While compensation for single resistances has been extensively studied, compensatory evolution of multiresistant bacteria remains unexplored. Importantly, since resistance mutations often interact epistatically, compensation of multiresistant bacteria may significantly differ from that of single-resistant strains. We used experimental evolution, next-generation sequencing, in silico simulations, and genome editing to compare the compensatory process of a streptomycin and rifampicin double-resistant Escherichia coli with those of single-resistant clones. We demonstrate that low-fitness double-resistant bacteria compensate faster than single-resistant strains due to the acquisition of compensatory mutations with larger effects. Strikingly, we identified mutations that only compensate for double resistance, being neutral or deleterious in sensitive or single-resistant backgrounds. Moreover, we show that their beneficial effects strongly decrease or disappear in conditions where the epistatic interaction between resistance alleles is absent, demonstrating that these mutations compensate for the epistasis. In summary, our data indicate that epistatic interactions between antibiotic resistances, leading to large fitness costs, possibly open alternative paths for rapid compensatory evolution, thereby potentially stabilizing costly multiple resistances in bacterial populations.Fundação para a Ciência e a Tecnologia, European Research Council.info:eu-repo/semantics/publishedVersio

The Mitogenome Relationships and Phylogeography of Barn Swallows (Hirundo rustica)

The barn swallow (Hirundo rustica) poses a number of fascinating scientific questions, including the taxonomic status of postulated subspecies. Here, we obtained and assessed the sequence variation of 411 complete mitogenomes, mainly from the European H. r. rustica, but other subspecies as well. In almost every case, we observed subspecies-specific haplogroups, which we employed together with estimated radiation times to postulate a model for the geographical and temporal worldwide spread of the species. The female barn swallow carrying the Hirundo rustica ancestral mitogenome left Africa (or its vicinity) around 280 thousand years ago (kya), and her descendants expanded first into Eurasia and then, at least 51 kya, into the Americas, from where a relatively recent (<20 kya) back migration to Asia took place. The exception to the haplogroup subspecies specificity is represented by the sedentary Levantine H. r. transitiva that extensively shares haplogroup A with the migratory European H. r. rustica and, to a lesser extent, haplogroup B with the Egyptian H. r. savignii. Our data indicate that rustica and transitiva most likely derive from a sedentary Levantine population source that split at the end of the Younger Dryas (YD) (11.7 kya). Since then, however, transitiva received genetic inputs from and admixed with both the closely related rustica and the adjacent savignii. Demographic analyses confirm this species' strong link with climate fluctuations and human activities making it an excellent indicator for monitoring and assessing the impact of current global changes on wildlife

Análisis Genético y Molecular de ARNs pequeños reguladores en Salmonella enterica

En bacterias, los ARN no codificantes reguladores o "ARNs pequeños" (sRNA) regulan la expresión de genes-diana interaccionando con la región 5’ no traducida del ARN mensajero y facilitando o dificultando el acceso del ribosoma y/o la degrad ... En bacterias gram-negativas, condiciones que afectan al plegamiento de proteínas de la membrana externa (OMPs) activan el factor sigma alternativo σE, responsable de la transcripción de un conjunto de genes cuyos productos confieren tolerancia a dichas condiciones. En los estudios realizados durante esta tesis, se describió la activación constitutiva de la respuesta mediada por σE en ausencia de Hfq. La proteína Hfq es una “chaperona de ARNs” esencial para la estabilidad y función de la inmensa mayoría de sRNAs. Los experimentos realizados durante esta tesis definieron que la inducción de la respuesta σE se debe a la estimulación de la degradación de un factor anti-σE llamado RseA, que en condiciones normales secuestra a σE, impidiendo su acción. Se describió que la degradación de RseA está relacionada con la pérdida de la represión de varias OMPs por parte de dos sRNAs pertenecientes al regulón σE: MicA y RybB.MicA reprime la traducción de las proteínas de la membrana externa OmpA y LamB. Por su parte, RybB inhibe la síntesis de varias proteínas de la membrana externa, reprimiendo la traducción y/o estimulando la degradación de los correspondientes ARNs mensajeros. Dos de los ARNm-diana son el de ompC y el de ompD. Con el objeto de identificar las secuencias determinantes en la interacción RybB:ompC, obtuvimos mutantes alterados en dicha interacción gracias a un procedimiento que combina la PCR mutagénica con la recombinación mediada por el sistema lambda Red. Fragmentos de ADN que contenían el gen rybB o la región situada corriente abajo del promotor de ompC fueron amplificados en condiciones propensas a error y transferidos al cromosoma de una estirpe que porta una fusión ompC::lacZ y expresa constitutivamente σE. Los mutantes se detectaron por cambios de color en placas indicadoras. De este modo se obtuvo una colección de mutantes, posteriormente ratificados por secuenciación y caracterizados por Northern. Esta estrategia ha permitido identificar las secuencias de ARN implicadas en la interacción RybB:ompC, así como las relacionadas con la estabilidad de RybB y la interacción con la proteína Hfq. Analizando los efectos de los mutantes en RybB sobre ompD, se ha descrito el primer ejemplo de interacción pequeño ARN:ARNm en dos localizaciones diferentes, con la particularidad adicional de que dichas interacciones tienen lugar dentro de la secuencia codificante del gen. Además, se ha descrito un nuevo gen perteneciente al regulón RybB: la quitoporina ChiP. Durante esta tesis también se estudió la posible participación de los sRNA en procesos biológicos más allá de la regulación de la expresión génica. Se definió la existencia de un locus, en el que están codificados dos sRNAs (ryeA y ryeB), que ha resultado un punto preferente de inserción de profagos bacterianos. Además, se describió que dicha inserción tenía efecto sobre los niveles de expresión relativa de ambos sRNAs, invirtiéndola, y sugiriendo unas consecuencias biológicas aún por estudiar. En cualquier caso, la clara implicación de los profagos, en tanto que elementos genómicos móviles, en procesos de transferencia genética horizontal, convierte a los sRNAs en principales participantes del proceso de evolución bacteriana. El conjunto de estudios realizados ha subrayado la enorme utilidad de la genética clásica en el estudio de la regulación mediada por pequeños ARN no codificantes, y ha supuesto el esclarecimiento de eventos clave en dichos procesos

Recombineering 101: Making an in-Frame Deletion Mutant

DNA recombineering uses phage λ Red recombination functions to promote integration of DNA fragments generated by polymerase chain reaction (PCR) into the bacterial chromosome. The PCR primers are designed to have the last 18-22 nt anneal on either side of the donor DNA and to carry 40- to 50-nt 5' extensions homologous to the sequences flanking the chosen insertion site. The simplest application of the method results in knockout mutants of nonessential genes. Deletions can be constructed by replacing a portion or the entirety of a target gene with an antibiotic-resistance cassette. In some commonly used template plasmids, the antibiotic-resistance gene can be coamplified with a pair of flanking FRT (Flp recombinase recognition target) sites that, following insertion of the fragment into the chromosome, allow excision of the antibiotic-resistance cassette via the activity of the site-specific Flp recombinase. The excision step leaves behind a "scar" sequence comprising an FRT site and flanking primer annealing sequences. Removal of the cassette minimizes undesired perturbations on the expression of neighboring genes. Even so, polarity effects can result from the occurrence of stop codons within, or downstream of, the scar sequence. These problems can be avoided by the appropriate choice of the template and by designing primers so that the reading frame of the target gene is maintained past the deletion end point. This protocol is optimized for use with Salmonella enterica and Escherichia coli.Agence Nationale de la Recherche ANR-15-CE11-0024-0

Juvenile dispersal behaviour and conspecific attraction: An alternative approach with translocated Spanish imperial eagles

The transient stage prior to definitive recruitment, known as juvenile dispersal, is thought to be under great evolutionary pressure and subject to a trade-off between associated costs and long-term benefits for fitness. Conspecific attraction has been shown to be an adaptive mechanism driving dispersal behaviours that may lead to negative density-dependent dispersal patterns. However, conspecific attraction can be scarcely discernible from imprinting to the natal area in wild populations. Reintroductions in the absence of settled individuals can be used as alternative colonization-like contexts to investigate the relative role of conspecific attraction in juvenile dispersal behaviours. We examined the spatiotemporal development of dispersal movements in reintroduced juveniles of a long-lived species with deferred maturity, the Spanish imperial eagle, Aquila adalberti, in comparison with nonmanipulated juveniles from a nearby population. We found that reintroduced birds started dispersal earlier and were initially more philopatric, probably encouraged by the advantageous competitive environment in the release area. Conversely, they revealed a more expansive strategy as they matured and approached the time when settlement decisions would be made, especially in females. They returned less frequently, increased exploratory movements and dispersal ranges, and visited breeding areas, probably as a consequence of the relatively lower reproductive prospects in the release area than in nearby populations. Therefore, the singular social cueing in reintroductions may eventually lead to juvenile wandering behaviours characteristic of colonization contexts in this territorial long-lived species. Such dispersive strategies relying on conspecifics may have important consequences for population dynamics and management. They may hinder the initial settlement phase in reintroductions, although behaviours such as longer returns may enhance recruitment prospects. Translocation programmes should consider specific dispersal scenarios, as well as postrelease monitoring to increase philopatry and success probabilities.The reintroduction programme was supported by G.I.A.S.A., E.G.M.A.S.A. and Junta de Andalucía under the scientific supervision of the Biological Station of Doñana (CSIC). R.M. was FPU fellow (grant ref. 2003-3252, Ministry of Education, Spain). Part of the work carried out in the Doñana Natural Area was possible thanks to the equipment and facilities of the Doñana Biological Reserve (RBD) cofunded by the FEDER program (2007–2013) through the Ministry of Economy and Competitiveness of Spain

Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica

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