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CRUE-CSIC (Acuerdos Transformativos 2022), The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built-on the surface of magnetic microbeads functionalized with nucleocapsid (N) and inhouse expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8) screen-printed electrodes using the HQ/HRP/H2O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency and even identification of the variant responsible for the infection., Ministerio de Ciencia e Innovación (MICINN), Comunidad de Madrid, Instituto de Salud Carlos III (ISCIII), Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

Many bacteria can move chemotactically to a variety of compounds and the recognition of chemoeffectors by the chemoreceptor ligand binding domain (LBD) defines the specificity of response. Many chemoreceptors were found to recognize different amino and organic acids, but the McpU chemoreceptor from Pseudomonas putida was identified as the first chemoreceptor that bound specifically polyamines. We report here the three-dimensional structure of McpU-LBD in complex with putrescine at a resolution of 2.4 Å which fitted well a solution structure generated by small-angle X-ray scattering. Putrescine bound to a negatively charged pocket in the membrane distal module of McpU-LBD. Similarities exist in the binding of putrescine to McpU-LBD and taurine to the LBD of the Mlp37 chemoreceptor of Vibrio cholerae. In both structures, the primary amino group of the respective ligand is recognized by hydrogen bonds established by two aspartate and a tyrosine side chain. This feature may be used to predict the ligands of chemoreceptors with unknown function. Analytical ultracentrifugation revealed that McpU-LBD is monomeric in solution and that ligand binding does not alter this oligomeric state. This sensing mode thus differs from that of the well-characterised four-helix bundle domains where ligands bind to two sites at the LBD dimer interface. Although there appear to be different sensing modes, results are discussed in the context of data, indicating that chemoreceptors employ the same mechanism of transmembrane signaling. This work enhances our understanding of CACHE domains, which are the most abundant sensor domains in bacterial chemoreceptors and sensor kinases.

The interference of plant compounds with bacterial quorum sensing (QS) is a major mechanism through which plants and bacteria communicate. However, little is known about the modes of action and effects on signal integrity during this type of communication. We have recently shown that the plant compound rosmarinic acid (RA) specifically binds to the Pseudomonas aeruginosa RhlR QS receptor. To determine the effect of RA on expression patterns, we carried out global RNA-seq analysis. The results show that RA induces the expression of 128 genes, amongst which many virulence factor genes. RA triggers a broad QS response because 88% of the induced genes are known to be controlled by QS, and because RA stimulated genes were found to be involved in all four QS signalling systems within P. aeruginosa. This finding was confirmed through the analysis of transcriptional fusions transferred to wt and a rhlI/lasI double mutant. RA did not induce gene expression in the rhlI/lasI/rhlR triple mutant indicating that the effects observed are due to the RA-RhlR interaction. Furthermore, RA induced seven sRNAs that were all encoded in regions close to QS and/or RA induced genes. This work significantly enhances our understanding of plant bacteria interaction.

Título en ingles: Quantification of cultivable bacteria by the “Massive Stamping Drop Plate” method Resumen: Cuando se desea cuantificar el número de bacterias presentes en múltiples muestras, los procedimientos de rutina suelen consumir mucho tiempo. En ese periodo las muestras podrían sufrir modificaciones en su población. En el presente trabajo se evaluó una metodología alternativa para cuantificar bacterias cultivables de forma masiva, rápida y económica en la que se implica el sellado, estampado o impresión de diluciones seriadas de muestras de diversa procedencia. El tiempo requerido para preparar 22 muestras para su sellado en placa es de 15 minutos. El método se basó en realizar diluciones seriadas (base 10) de las muestras líquidas originales contenidas en una placa multipozos con la ayuda de una pipeta multicanal. Después, con un replicador se tomó un volumen (aproximadamente 1,65 µl) de muestra de cada pozo, que se inoculó por sellado en un medio de crecimiento gelificado de interés. Las placas se incubaron el tiempo necesario, se contó el número de colonias presentes en la dilución contable y se calculó el número de Unidades Formadoras de Colonia por mililitro (UFC/ml) para cada muestra. La metodología se denominó “Goteo por Sellado en Placa Masivo” (GSPM) y ha sido aplicada para cuantificar exitosamente bacterias provenientes de diferentes muestras de laboratorio, por ejemplo, de cultivos líquidos, muestras clínicas (como exudados y secreciones) y bacterias presentes en la rizósfera de plantas de maíz. Sin embargo, la metodología GSPM podría aplicarse para contabilizar masivamente a bacterias de cualquier otra procedencia. Palabras clave: UFC; goteo en placa; plaqueo; vaciado en placa; GSPM. Abstract: In an attempt to quantify the number of bacteria present in a high number of samples, routine procedures are usually very time-consuming. During this period of time, bacterial population could be modified. In this work, an alternative for a massive, quick

In an attempt to quantify the number of bacteria present in a high number of samples, routine procedures are usually very time-consuming. During this period of time, bacterial population could be modified. In this work, an alternative for a massive, quick and economic method was evaluated in order to count viable bacteria, consisting in the sealing or stamping of serial dilutions performed in samples from different origins. The time required to prepare 22 samples for plate stamping is only 15 minutes. The quantification was based in performing serial dilutions (10-fold) of the original liquid samples contained in a multiwell plate using a multichannel micropipette. Afterwards, using a replicator, the same volume of each sample (approximately 1,65 ìl) was recovered from each well, and then it was inoculated and sealed in a solid growth media of interest. Plates were incubated as needed, colonies were counted in the quantifiable dilution and Colony Forming Units per milliliter (CFU/ml) was calculated for each sample. We called this method "Massive Stamping Drop Plate" (MSDP) and it has been successfully applied to count bacteria from different lab samples, including liquid cultures, clinical samples (exudates and secretions) and bacteria recovered from the rhizosphere of corn plants. However, MSDP could also be applied to massively count bacteria from any other source., Cuando se desea cuantificar el numero de bacterias presentes en multiples muestras, los procedimientos de rutina suelen consumir mucho tiempo. En ese periodo las muestras podrian sufrir modificaciones en su poblacion. En el presente trabajo se evaluo una metodologia alternativa para cuantificar bacterias cultivables de forma masiva, rapida y economica en la que se implica el sellado, estampado o impresion de diluciones seriadas de muestras de diversa procedencia. El tiempo requerido para preparar 22 muestras para su sellado en placa es de 15 minutos. El metodo se baso en realizar diluciones seriadas (base 10) de las muestras liquidas originales contenidas en una placa multipozos con la ayuda de una pipeta multicanal. Despues, con un replicador se tomo un volumen (aproximadamente 1,65 Êl) de muestra de cada pozo, que se inoculo por sellado en un medio de crecimiento gelificado de interes. Las placas se incubaron el tiempo necesario, se conto el numero de colonias presentes en la dilucion contable y se calculo el numero de Unidades Formadoras de Colonia por mililitro (UFC/ml) para cada muestra. La metodologia se denomino "Goteo por Sellado en Placa Masivo" (GSPM) y ha sido aplicada para cuantificar exitosamente bacterias provenientes de diferentes muestras de laboratorio, por ejemplo, de cultivos liquidos, muestras clinicas (como exudados y secreciones) y bacterias presentes en la rizosfera de plantas de maiz. Sin embargo, la metodologia GSPM podria aplicarse para contabilizar masivamente a bacterias de cualquier otra procedencia.