First description of a new worm bait fishery in the NW Mediterranean Sea

Most commercial clam stocks in the NW Mediterranean Sea have collapsed over the last few decades and, as a result, most clam dredge fishermen have been forced to leave the fishing sector. Recently, in order to sustain their economic activity, some fishermen have modified dredges to target sea worms to sell as bait for recreational fisheries. This study provides the first information about this new worm bait fishery on the Catalan Maresme coast (NE Spain). The local administration has regulated only a few aspects of the fishery: users (2 boats), geographical limits (40 km; between 0-7 m depth), fishing time (6:00-14:00) and dredge design (the same as those used for smooth clams but with interior structures to retain worms and an open back). Fishing activity takes place throughout the year. Fishermen target three worm bait species: Sigalion squamosus, Ophelia neglecta and Halla parthenopeia. A mean of 233.37 individuals of S. squamosus, 167.93 of O. neglecta and 2.17 of H. parthenopeia are gathered per boat and day. Worm baits are sold directly to local recreational fishing shops as a quality product at the highest prices on the market. This fishery has a high social and economic value for the Maresme coast, helping to maintain small-scale fishermen jobs with an economic benefit similar to clam fishing

FleN and FlhF as new regulatory elements in the lifestyle switch in Pseudomonas putida

Motivation:Pseudomonas putida is a soil bacterium that can be found in nature as individual motile cells or as part of sessile communities called biofilms (1). Biofilm formation could be considered an adaptive strategy as it provide higher resistance against adverse agents, antimicrobial treatments, ultraviolet radiation or dehydration, increasing bacteria survival (2). The switch from a planktonic lifestyle to biofilm formation in P.putida is regulated by FleQ and the intracellular levels of c-di-GMP (3). Isolation of insertion mutants in flhF gene, defective in biofilm formation, suggests the involvement of additional elements in the regulation of this process. In this work, we have characterized the transcriptional organization of flhA, flhF, fleN and fliA genes and the role of FlhF and FleN in biofilm development in P.putida. Methods: Planktonic growth and biofilm formation curves, adhesion and swimming-motility assays. Gene expression analysis: β-galactosidase assays and RT-PCR. Electrophoretic mobility shift assays (EMSA).Results and Conclusions: To test the role of FlhF and FleN in biofilm development, we carried out a phenotypic characterization of ΔflhF and ΔfleN mutants. Experiments shown that ΔfleN mutant is not able to form biofilm whereas ΔflhF mutant exhibits a wild-type phenotype. Adhesion assays indicate that ΔfleN mutant has a reduced adhesion, whereas ΔflhF can properly adhere to the surface. On the other hand, both mutant show reduced swimming motility. These resuts suggests that both, FleN and FlhF, are involved in swimming motility, but only FleN is necessary for biofilm formation, probably by altering adhesion capacity of the bacteria. Bioinformatic tools predict that flhF and fleN form an operon with the upstream gene flhA and the downstream gene fliA. In order to corroborate this hypothesis, we have done RT-PCR using RNA from the mutants. Results shown that these genes are structured in a single operon: flhAFfleNfliA.To determine a possible regulatory role of these elements in biofilm formation and motility, β-galactosidase assays were performed to analyse the expression of biofilm and flagellum related promoters. Results shown that FleN downregulates both types of promoters whereas FlhF is only involved in the regulation of one flagellar gene. The regulatory role of FleN has been further studied in vitro by EMSA with the regulator FleQ. Results suggest that FleN is required for FleQ to bind to its target promoters

Prospective individual patient data meta-analysis of two randomized trials on convalescent plasma for COVID-19 outpatients

Data on convalescent plasma (CP) treatment in COVID-19 outpatients are scarce. We aimed to assess whether CP administered during the first week of symptoms reduced the disease progression or risk of hospitalization of outpatients. Two multicenter, double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when = 50 years and symptomatic for <= 7days were included. The intervention consisted of 200-300mL of CP with a predefined minimum level of antibodies. Primary endpoints were a 5-point disease severity scale and a composite of hospitalization or death by 28 days. Amongst the 797 patients included, 390 received CP and 392 placebo; they had a median age of 58 years, 1 comorbidity, 5 days symptoms and 93% had negative IgG antibody-test. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The odds ratio (OR) of CP for improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311); OR for hospitalization or death was 0.919 (CI 0.592-1.416). CP effect on hospital admission or death was largest in patients with <= 5 days of symptoms (OR 0.658, 95%CI 0.394-1.085). CP did not decrease the time to full symptom resolution

Nuevos elementos reguladores de la movilidad y la adhesión en Pseudomonas putida

Resumen del trabajo presentado a la XI Reunión del Grupo de Microbiología Molecular, celebrada en Sevilla del 6 al 8 de septiembre de 2016.Pseudomonas putida es una bacteria de suelo que puede encontrarse en la naturaleza como células móviles individuales o como parte de comunidades sésiles adheridas a sustratos denominadas biofilm. La transición entre ambos estilos de vida está regulado por la proteína FleQ y los niveles intracelulares de di-GMPc. El aislamiento de mutantes de inserción en el gen flhF, defectivos en la formación de biofilm, sugieren la implicación de elementos adicionales en la regulación de este proceso. flhF codifica una GTPasa necesaria para la correcta localización del flagelo, y se encuentra localizado aguas arriba de fleN, que regula el número de flagelos, y fliA, que codifica un factor sigma específico. Con el fin de estudiar el posible papel regulador de estos elementos, se han caracterizado mutantes de deleción en flhF, fleN y un doble mutante flhFfleN. Para ello se han llevado a cabo curvas de crecimiento planctónico y formación de biofilm, ensayos de adhesión y movilidad tipo swimming y swarming. También se han elaborado tinciones de flagelo de todos los mutantes para su posterior visualización al microscopio óptico. Además, se han realizado RT-PCR para el estudio de la organización transcripcional de los genes flhF y fleN, Para estudiar la implicación de estos elementos en regulación de la movilidad y el desarrollo del biofilm, se han realizado ensayos β-galactosidasa de fusiones transcripcionales al gen lacZ de promotores relacionados tanto con la síntesis de flagelo como la formación de biofilm. Los resutados indican que flhA, flhF, fleN y fliA están organizados en un único operón. FleN y FlhF están involucrados en movilidad tipo swimming, pero solo FleN es necesario para la adhesión y formación de biofilm. Por otro lado, ambos genes son necesarios para la movilidad tipo swarming. El análisis de la expresión génica indica que FleN está implicado en la regulación de genes relacionados con movilidad y formación de biofilm. Además, flhF parece estar involucrado en la regulación de, al menos, uno de los promotores flagelares estudiados. Todo ello, sugiere que FleN es un elemento importante en la regulación de la movilidad flagelar y de la adhesión en P. putida.Este trabajo ha sido financiado por los proyectos BIO2010-17853 (MICINN) y BIO2013-42073-P (MINECO).Peer Reviewe

First description of a new worm bait fishery in the NW Mediterranean Sea

Most commercial clam stocks in the NW Mediterranean Sea have collapsed over the last few decades and, as a result, most clam dredge fishermen have been forced to leave the fishing sector. Recently, in order to sustain their economic activity, some fishermen have modified dredges to target sea worms to sell as bait for recreational fisheries. This study provides the first information about this new worm bait fishery on the Catalan Maresme coast (NE Spain). The local administration has regulated only a few aspects of the fishery: users (2 boats), geographical limits (40 km; between 0-7 m depth), fishing time (6:00-14:00) and dredge design (the same as those used for smooth clams but with interior structures to retain worms and an open back). Fishing activity takes place throughout the year. Fishermen target three worm bait species: Sigalion squamosus, Ophelia neglecta and Halla parthenopeia. A mean of 233.37 individuals of S. squamosus, 167.93 of O. neglecta and 2.17 of H. parthenopeia are gathered per boat and day. Worm baits are sold directly to local recreational fishing shops as a quality product at the highest prices on the market. This fishery has a high social and economic value for the Maresme coast, helping to maintain small-scale fishermen jobs with an economic benefit similar to clam fishing

La interacción entre FleQ, FleN y el di-GMPc regula la expresión de genes implicados en la movilidad y la formación de biofilm en P. putida

Resumen del trabajo presentado a la XI Reunión del Grupo de Microbiología Molecular, celebrada en Sevilla del 6 al 8 de septiembre de 2016.La alternancia entre un estilo de vida planctónico y la formación de estructuras complejas llamadas biofilm es un fenómeno habitual en el ciclo de vida de muchas bacterias ambientales. Trabajos previos llevados a cabo en nuestro grupo en la bacteria Gram-negativa de suelo Pseudomonas putida mostraron que la proteína FleQ, un activador de promotores dependientes de σ54, junto con el mensajero secundario 3',5'-diguanilato cíclico (di-GMPc) participan coordinadamente en la regulación de funciones relacionadas con movilidad y colonización de superficies. Recientemente hemos comprobado que FleN, la proteína encargada de regular el número de flagelos en la bacteria, es también necesaria para el reconocimiento de ciertos promotores por parte de FleQ. Con el objetivo de aumentar nuestro conocimiento acerca de la red de regulación que controla esta alternancia entre estilos de vida en P. putida, se llevó a cabo un screening de promotores potencialmente involucrados en movilidad y desarrollo de biofilm fusionados a gfp y lacZ que permitió medir la expresión en diferentes fondos mutantes, incluyendo ΔfleQ y ΔfleN. Además, llevamos a cabo ensayos in vitro tales como ensayos de retardos en gel o footprint para describir la posible interacción entre los componentes en las regiones promotoras. Nuestros resultados muestran que la capacidad de unión de FleQ a sus promotores diana se encuentra modulada por la interacción con FleN, la cual actúa como proteína auxiliar a FleQ. A su vez, esta interacción se encuentra modulada de manera diferencial por los niveles de diGMPc en cada promotor. El mecanismo mediante el cual FleN interacciona con FleQ y cómo el diGMPc modifica la afinidad de esta interacción serán objeto de discusión.Este trabajo ha sido financiado por los proyectos BIO2010-17853 (MICINN) y BIO2013-42073-P (MINECO).Peer Reviewe

Complex interplay between FleQ, cyclic diguanylate and multiple σ factors coordinately regulates flagellar motility and biofilm development in Pseudomonas putida

Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-Associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in Pseudomonas putida. Disruption of fleQ caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to gfp and lacZ, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σN and FliA in the transcription of FleQregulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σN-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σN-and FliA-independent PlapA and PbcsD promoters revealed two disparate regulatory mechanisms leading to a similar outcome: The synthesis of biofilm matrix components in response to increased cyclic diguanylate levels.This work was supported by Ministerio de Ciencia e Innovación, Spain (http://www.idi.mineco.gob.es/) and European Regional Development fund (http://ec.europa.eu/regional_policy/en/funding/erdf/), Grant BIO2010-17853, awarded to FG; Ministerio de Economía y Competitividad, Spain (http://www.idi.mineco.gob.es/) and European Regional Development fund (http://ec.europa.eu/regional_policy/en/funding/erdf/), Grant BIO2013-420173-P, awarded to FG; and Consejo Superior de Investigaciones Científicas, Spain (http://www.csic.es/), JAE-Predoc 2010 scholarship, awarded to AJ-F.Peer Reviewe

Transcriptional organization, regulation and functional analysis of flhF and fleN in Pseudomonas putida

© 2019 Navarrete et al.The Pseudomonas putida flhA-flhF-fleN-fliA cluster encodes a component of the flagellar export gate and three regulatory elements potentially involved in flagellar biogenesis and other functions. Here we show that these four genes form an operon, whose transcription is driven from the upstream PflhA promoter. A second promoter, PflhF, provides additional transcription of the three distal genes. PflhA and PflhF are σN-dependent, activated by the flagellar regulator FleQ, and negatively regulated by FleN. Motility, surface adhesion and colonization defects of a transposon insertion mutant in flhF revealed transcriptional polarity on fleN and fliA, as the former was required for strong surface adhesion and biofilm formation, and the latter was required for flagellar synthesis. On the other hand, FlhF and FleN were necessary to attain proper flagellar location and number for a fully functional flagellar complement. FleN, along with FleQ and the second messenger c-di-GMP differentially regulated transcription of lapA and the bcs operon, encoding a large adhesion protein and cellulose synthase. FleQ positively regulated the PlapA promoter and activation was antagonized by FleN and c-di-GMP. PbcsD was negatively regulated by FleQ and FleN, and repression was antagonized by c-di-GMP. FleN promoted FleQ binding to both PlapA and PbcsD in vitro, while c-di-GMP antagonized interaction with PbcsD and stimulated interaction with PlapA. A single FleQ binding site in PlapA was critical to activation in vivo. Our results suggest that FleQ, FleN and c-di-GMP cooperate to coordinate the regulation of flagellar motility and biofilm development.This work was supported by Ministerio de Ciencia e Innovación, Spain (http://www.idi.mineco. gob.es/) and European Regional Development fund (http://ec.europa.eu/regional_policy/en/funding/erdf/), Grant BIO2010-17853, awarded to FG; Ministerio de Economía y Competitividad, Spain (http://www.idi.mineco.gob.es/) and European Regional Development fund (http://ec.europa.eu/regional_policy/en/funding/erdf/), Grant BIO2013-420173-P, awarded to FG; and Consejo Superior de Investigaciones Científicas, Spain (http://www.csic.es/), JAE-Predoc 2010 scholarship, awarded to AJ-F. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Complex Interplay between FleQ, Cyclic Diguanylate and Multiple σ Factors Coordinately Regulates Flagellar Motility and Biofilm Development in <i>Pseudomonas putida</i>

<div><p>Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in <i>Pseudomonas putida</i>. Disruption of <i>fleQ</i> caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to <i>gfp</i> and <i>lacZ</i>, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σ^N and FliA in the transcription of FleQ-regulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σ^N-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σ^N- and FliA-independent P<i>lapA</i> and P<i>bcsD</i> promoters revealed two disparate regulatory mechanisms leading to a similar outcome: the synthesis of biofilm matrix components in response to increased cyclic diguanylate levels.</p></div

Gel mobility shift assays of FleQ on the P<i>lapA</i> and P<i>bcsD</i> promoters.

<p><b>Top:</b> Cartoon of the probes used for each of the promoter regions, indicating the restriction enzymes used for cleavage, the sizes (in bp) of the resulting fragments, and the location of the predicted FleQ binding motifs (open boxes). Drawn to scale. <b>Bottom:</b> Ethidium bromide-stained PAGE showing the results of a typical gel mobility shift assay. Each promoter region was challenged with 0, 0.45 and 4.5 μM FleQ.</p