BUSCADOR RECOLECTA

Two-component systems (TCSs) are a prominent sensory system in bacteria. A prototypical TCS comprises a membrane-bound sensor histidine kinase (HK) responsible for sensing the signal and a cytoplasmic response regulator (RR) that controls target gene expression. Signal binding activates a phosphotransfer cascade from the HK to the RR. As a result, the phosphorylated RR undergoes a conformational change that leads to activation of the response. Growing experimental evidence indicates that unphosphorylated RRs may also have regulatory functions, and thus, the classical view that the RR is only active when it is phosphorylated needs to be revisited. In this review, we highlight the most recent findings showing that RRs in the non-phosphorylated state control critical bacterial processes that range from secretion of factors to the host, antibiotic resistance, iron transport, stress response, and cell-wall metabolism to biofilm development., This work was financially supported by the Spanish Ministry of Science, Innovation and Universities grant BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L. and C.S.

In this thesis, we have focused on studying variants found in IGRs adjacent to the most important genes involved in S. aureus biofilm formation; the icaADBCR locus, and the genes encoding the family of surface adhesins. For this purpose, we sequenced the whole genome of a collection of 71 S. aureus isolates from periprosthetic joint infections (PJI) and wound infections stored at the Clinical Bacteriological Laboratory of the Sahlgrenska University Hospital and at the Culture Collection University of Gothenburg (CCUG), respectively.
In the first chapter, we explored the regulatory regions of the icaADBCR locus to identify patterns that might be associated with an increased capacity of the isolates to produce PIA/PNAG and form a biofilm. This study compared the regulatory regions of the icaADBCR locus in the genomes of PJI and wound isolates with those in the genome of the reference strain MW2. From these analyses, strains were grouped based on the SNPs found in the IGRs of the operon and also within the coding region of the transcriptional regulator IcaR.
These regions showed high conservation rates, and no pattern associated with the origin of the isolates, either PJI or wounds, was detected. On the other hand, using transcriptional fusions between the regulatory region of the icaADBCR locus and the green fluorescent protein gene (gfp), we demonstrated that the expression of icaADBC genes was not affected by the presence of variations in IGRs. Notably, a SNP within the coding region of icaR, which results in an amino acid change in the transcriptional repressor IcaR V176E, led to a significant increase in the transcription of the icaADBC operon and the production of PIA/PNAG. Using a Galleria mellonella infection model, we were able to demonstrate a significant reduction in S. aureus virulence associated with the
increase in PIA/PNAG production.
In the second chapter, we focused on analyzing the association between SNPs in the promoter regions of genes encoding adhesion-related proteins with adhesins expression levels and therefore, the ability of the strain to adhere to medical devices. Genome analyses of PJI and wound isolates showed different profiles in the content of adhesin-encoding genes. Some of these, such as sasG and cna, were lineage-associated, and fifteen genes were present in the whole collection of strains. When the variability in the SNPs contained in regulatory regions that control the expression of each adhesin was investigated, different variation rates were found among the isolates. Following the same approach as in chapter I, based on transcriptional fusions between regulatory regions and the gfp gene, results showed that each genetic lineage contained a specific profile of adhesins expression under the same environmental condition.
Moreover, we developed a biomaterial-associated murine infection model together with a metagenomic analysis to simultaneously compare the capacity of different S. aureus isolates to colonize medical implants.
In summary, our results evidenced that SNPs in the IGRs flanking the genes encoding factors important for biofilm development may contribute to the generation of variability in the capacity of S. aureus to colonize medical implants.
In particular, our results revealed that IGRs controlling the expression of the icaADBC locus and production of the PIA/PNAG exopolysaccharide are highly conserved and that very few silent SNPs can be detected between strains. On the
contrary, SNPs in the IGRs of genes encoding surface adhesins provide a profile of proteins expression that is specific for each S. aureus clonal complex (CC).
Altogether, these studies emphasize the importance of investigating the potential impact of SNPs inside IGRs on gene expression and specific bacterial traits, such as pathogen colonization success., European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586; Spanish Ministry of Economy, Industry and Competitiveness grant BIO2017-83035-R; Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization., JV was supported by SAF2015-74267-JIN. This research was supported by grants BIO2014-53530-R, BIO2017-83035-R and RTC-2015-3184-1 from the Spanish Ministry of Economy and Competitivity.

Staphylococcus aureus es una bacteria versátil que se puede encontrar estableciendo una relación comensal en la piel de, aproximadamente, el 30% de la población sin causar ningún problema. Sin embargo, cuando ésta atraviesa la barrera epitelial y dependiendo del estado inmunológico del portador, puede causar patologías leves, como los abscesos, o severas, como la endocarditis u osteomielitis. Para el tratamiento de estas patologías, existe un amplio abanico de fármacos, no obstante, S. aureus ha desarrollado mecanismos que le permiten incrementar su resistencia y evadir el efecto de estos antibióticos.
En el capítulo I de esta tesis doctoral, hemos analizado la implicación de los TCSs de S. aureus en el posible desarrollo de resistencia a dos antibióticos glicopéptidos de reciente descubrimiento, la complestatina (Cm) y la corbomicina (Cb). Para ello, hemos estudiado la susceptibilidad frente a ambos antibióticos de una colección compuesta por mutantes simples en cada TCS en la cepa MW2 y otra compuesta por derivados de una cepa mutante múltiple en todos los TCSs no esenciales (ΔXV) complementados con un plásmido portador de un único TCS. Con ayuda de estas colecciones, observamos que el sistema VraSR es el único que controla la susceptibilidad de S. aureus a la Cm y Cb sugiriendo que uno o varios componentes específicos del regulón de VraSR podrían ser los responsables de este control. Estudiando el regulón de VraSR en profundidad, determinamos que la regulación directa de SpdC e indirecta de SagB por parte de este TCS podrían estar relacionadas con la menor susceptibilidad que presentan las cepas portadoras del sistema VraSR. Al estar SpdC y SagB implicados en la longitud de las cadenas de azúcar del peptidoglicano, analizamos la posible presencia de cambios en la composición del mismo en los mutantes objeto de estudio. De entre las cepas estudiadas, se apreció un incremento en la cantidad de muropéptido compuesto por dímeros M5-5Gly-M4-1Ala en la cepa mutante en vraSR, pero no se pudo demostrar si este cambio está relacionado con la mayor susceptibilidad de esta cepa a la Cm y Cb.
Por otra parte, el capítulo II está dedicado a revisar las evidencias que demuestran la regulación ejercida por los TCSs en su estado no fosforilado, lo que supone un cambio de paradigma en la regulación de los TCSs.
Finalmente, en el capítulo III, hemos analizado la regulación de la expresión del operón icaADBC y de otros factores de virulencia por parte del TCS ArlRS. En concreto, analizamos como la forma fosforilada y no fosforilada de ArlR regulan la expresión de icaADBC y del represor icaR, responsables de la producción del exopolisacárido PIA/PNAG, elemento mayoritario del biofilm de S. aureus. Los resultados indicaron que la forma no fosforilada de ArlR actúa como represor de icaR causando un aumento de la producción de las proteínas IcaADBC y un aumento de la producción de PIA/PNAG., Staphylococcus aureus is a versatile bacterium that can be found establishing a commensal relationship on the skin of approximately 30% of the population without causing any harm. However, when it crosses the epithelial barrier and depending on the immune status of the carrier, it can cause mild pathologies such as abscesses, or severe ones, such as endocarditis or osteomyelitis. For the treatment of these pathologies, there is a wide range of available drugs. Nevertheless, S. aureus has developed mechanisms that allow it to increase its resistance and evade the effect of these antibiotics.
In chapter I, we have analyzed the implication of the TCSs of S. aureus in the possible development of resistance against two recently discovered glycopeptide antibiotics, complestatin (Cm) and corbomycin (Cb). For this purpose, we tested the susceptibility to both antibiotics of a collection of single mutants in each TCS in the MW2 strain and another collection composed of derivatives of a multiple mutant in each non essential TCS (ΔXV strain) complemented with a plasmid carrying a single TCS. Using these collections, we observed that the VraSR system is the only TCS that controls the susceptibility of S. aureus to Cm and Cb, suggesting that one or several specific components of the VraSR regulon might be responsible for this control. By futher studying the VraSR regulon, we determined that the direct regulation of SpdC and the indirect regulation of SagB by this TCS might be related to the lower susceptibility phenotype shown by the strains carrying the VraSR system. Since SpdC and SagB are involved in the length of the peptidoglycan chains, we analysed the presence of changes in the peptidoglycan in the mutants under study. Among the strains analysed, the vraSR mutant showed an increase in the amount of muropeptide composed of M5-5Gly-M4-1Ala dimers, however, it could not be demonstrated whether this change is related to the increased susceptibility to Cm and Cb.
On the other hand, chapter II is aimed at reviewing the literature related to the regulation exerted by TCSs in their non-phosphorylated state, which represents a paradigm shift in TCS regulation.
Finally, in chapter III, we have analysed the regulation of the expression of the icaADBC operon and other virulence factors. Specifically, we analysed how the phosphorylated and non-phosphorylated forms of ArlR regulate the expression of the icaR repressor and icaADBC operon, responsible for the production of the exopolysaccharide PIA/PNAG, the main component of the S. aureus biofilm. Results indicated that the non-phosphorylated form of ArlR acts as a repressor of icaR causing an increased production of IcaADBC and PIA/PNAG., Este trabajo ha sido realizado dentro de los proyectos de investigación:
• Título del proyecto: ‘Caracterización funcional de los determinantes moleculares para la adaptación de Staphylococcus aureus a la virulencia’. BIO2017-83035-R. Ministerio de Economía, Industria y Competitividad.
• Título del proyecto: ‘Operones no-contiguos: un nuevo nivel de regulación génica en bacterias’. PID2020-113494RB-100. Ministerio de Ciencia e Innovación., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Incluye material complementario, Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels., This study was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014‐53530‐R and BIO2017‐83035‐R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I. Lasa and C. Solano and grants BIO2016‐79736‐R, PCIN‐2014‐113 and PCI2019‐111833‐2 to E. Díaz. P. Dorado‐Morales was supported by a F.P.I. (BES‐2015‐072859) contract from the Spanish Ministry of Science, Innovation and Universities.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer, Parkinson or Type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity towards that diversity of amyloid fibrils, or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here, we show the feasibility of the approach by designing, synthesizing and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The more soluble derivative, compound 1D, stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein or amylin, provides a sensitivity only slightly lower than that of Thioflavin T, displays a large Stokes shift, allows an efficient excitation in the UV spectral region,and it is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by Staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining,S. aureus biofilms containing amyloid forming phenol soluble modulins from those lacking them., IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SVis supported by grant BIO2016-783-78310-R and by ICREA (ICREA Academia 2015). MDD is supported by the Government of Aragon (GA E-102). JS is supported by grants BFU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). JS and IL acknowledge financial support from grant CI-2017/001-3 (Campus Iberus, Spain). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government.

Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed., This research was supported by grants RTI2018-096011-B-I00 and BIO2017-83035-R from the Spanish Ministry of Science, Innovation and Universities, and Proyecto Intramural Incorporación-2018 CSIC.

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA)., SB was supported by a predoctoral contract from the Public University of Navarra. CG, ME and BG were recipients of postdoctoral contracts under Grant BIO2014-53530-R. This research was supported by grant IIM 13329.RI1 from the Departamento de Innovación, Empresa y Empleo, Government of Navarra and grants BIO2014-53530-R and BIO2017-83035-R from the Spanish Ministry of Economy and Competitiveness (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union).

In bacteria, adaptation to changes in the environment is mainly controlled through two-component signal transduction systems (TCSs). Most bacteria contain dozens of TCSs, each of them responsible for sensing a different range of signals and controlling the expression of a repertoire of target genes (regulon). Over the years, identification of the regulon controlled by each individual TCS in different bacteria has been a recurrent question. However, limitations associated with the classical approaches used have left our knowledge far from complete. In this report, using a pioneering approach in which a strain devoid of the complete nonessential TCS network was systematically complemented with the constitutively active form of each response regulator, we have reconstituted the regulon of each TCS of S. aureus in the absence of interference between members of the family. Transcriptome sequencing (RNA-Seq) and proteomics allowed us to determine the size, complexity, and insulation of each regulon and to identify the genes regulated exclusively by one or many TCSs. This gain-of-function strategy provides the first description of the complete TCS regulon in a living cell, which we expect will be useful to understand the pathobiology of this important pathogen. IMPORTANCE Bacteria are able to sense environmental conditions and respond accordingly. Their sensorial system relies on pairs of sensory and regulatory proteins, known as two-component systems (TCSs). The majority of bacteria contain dozens of TCSs, each of them responsible for sensing and responding to a different range of signals. Traditionally, the function of each TCS has been determined by analyzing the changes in gene expression caused by the absence of individual TCSs. Here, we used a bacterial strain deprived of the complete TC sensorial system to introduce, one by one, the active form of every TCS. This gain-of-function strategy allowed us to identify the changes in gene expression conferred by each TCS without interference of other members of the family., This work was supported by the Spanish Ministry of Economy and Competitiveness grant BIO2017-83035-R (AEI/FEDER, EU) awarded to I.L. and a Tenovus Scotland project grant S16/12 awarded to A.F.H. A.F.H. is supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program awarded to J.R.P. (grant agreement ERC-ADG-2014 proposal no. 670932 Dut-signal from EU). V.D.C. was supported by the Medical Research Council (MRC grant MC_UU_12016) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer Ingelheim, GlaxoSmithKline, and Merck KGaA).

En este trabajo de tesis hemos evaluado dos aproximaciones diferentes dedicadas a la búsqueda de fármacos que tengan como diana de acción los TCSs. Por un lado, dentro del proyecto 'Nuevas estrategias para el control de infecciones nosocomiales (RTC-2015-3184-1) hemos participado en la evaluación de una colección de fármacos aprobados por la FDA (colección Prestwick) para distintas patologías como posibles agentes antimicrobianos frente a S. aureus. El objetivo era reconvertir alguno de estos fármacos (repurposing) y que además de utilizarse para la finalidad para la que fue descrito, también actuara como antimicrobiano. En el caso concreto de nuestro estudio, el escrutinio se hizo frente al TCS GraRS. La segunda estrategia, recogida en el cuarto capítulo de esta tesis, ha consistido en el desarrollo de dos terapias alternativas frente a S. aureus basadas en inmunoterapias: anticuerpos monoclonales y nanoanticuerpos. Para ello, hemos elegido como diana de acción la HK del TCS esencial WalKR, en particular los anticuerpos se han generado frente al dominio de unión a ligando (ligand-bindig domain, LBD) de la HK. La elección de esta diana se debe a dos razones, por un lado, se trata del único TCS esencial de S. aureus y, por otro lado, ensayos realizados con sueros de pacientes infectados por S. aureus mostraron la presencia de anticuerpos frente a este dominio. A lo largo de la tesis hemos producido anticuerpos monoclonales y nanoanticuerpos frente a diferentes epítopos del LBD. Hemos realizado diferentes ensayos para probar el reconocimiento de la proteína tanto in vitro como in vivo y para analizar el efecto de los anticuerpos en el crecimiento de S. aureus. Los ensayos preliminares indican que varios de los nanoanticuerpos desarrollados activan el crecimiento de la bacteria en lugar de reducir su crecimiento. Este resultado, aunque inesperado, indica que es posible desarrollar nanoanticuerpos capaces de afectar la funcionalidad de la HK y por tanto de bloquear su actividad., During the course of the thesis we have participated in the development of two potential complementary therapies against S. aureus. The first strategy was carried out within the framework of the project ‘New strategies to control nosocomial infections’ (RTC-2015-3184-1) and we have participated in the screening of a collection of FDA’s approved drugs (Prestwick collection) as new antimicrobial agents. The objective was to repurpose one the compounds as a new drug against S. aureus, in this case the target of the screening was the TCS GraRS. The second strategy is presented in the fourth chapter of the thesis. The goal was to develop a potential complementary therapy against S. aureus based on two different immunetherapy strategies: monoclonal antibodies and nanobodies. For that, we have focused on the HK of S. aureus and in particular the antibodies were developed against the ligand-binding domain (LBD) of the HK from WalRK. We have selected this target because WalRK is the only essential TCS of S. aureus and besides, assays performed with serum from patients with an S. aureus infection showed an immunogenic response against the LBD. We have been able to obtain both, the monoclonal antibody and also different nanobodies against the LBD region of WalK able to recognize the peptide in vitro and in vivo. Unexpectedly, incubation of S. aureus with various nanobodies promoted instead of inhibited bacterial growth. These surprising results indicate that nanobodies can modify the activity of HKs and as a consequence, interfere with the signal transduction system., Este trabajo ha sido realizado dentro de los siguientes proyectos de investigación: ‘Descifrando las singularidades del exopolisacárido universal del biofilm (PNAG) y evaluación de su potencial biotecnológico’. BIO2014-53530-R MINECO. ‘Nuevas estrategias para el control de infecciones nosocomiales’. RTC-2015-3184-1, MINECO. ‘Caracterización funcional de los determinantes moleculares para la adaptación de Staphylococcus aureus a la virulencia’. BIO2017-83035-R. MINECO, Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Infections caused by Staphylococcus aureus pose a serious and sometimes fatal health issue. With the
aim of exploring a novel therapeutic approach, we chose GraXRS, a Two-Component System (TCS)
that determines bacterial resilience against host innate immune barriers, as an alternative target
to disarm S. aureus. Following a drug repurposing methodology, and taking advantage of a singular
staphylococcal strain that lacks the whole TCS machinery but the target one, we screened 1.280 offpatent
FDA-approved drug for GraXRS inhibition. Reinforcing the connection between this signaling
pathway and redox sensing, we found that antioxidant and redox-active molecules were capable of
reducing the expression of the GraXRS regulon. Among all the compounds, verteporfin (VER) was
really efficient in enhancing PMN-mediated bacterial killing, while topical administration of such drug
in a murine model of surgical wound infection significantly reduced the bacterial load. Experiments
relying on the chemical mimicry existing between VER and heme group suggest that redox active
residue C227 of GraS participates in the inhibition exerted by this FDA-approved drug. Based on these
results, we propose VER as a promising candidate for sensitizing S. aureus that could be helpful to
combat persistent or antibiotic-resistant infections., This study was supported by Centre for the Development of Industrial Technology (CDTI), (NEO16RECOMBINA; EXP 00112635/SNEO-20161233). Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities grant BIO2017-83035-R (AEI/FEDER, EU).

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 Investigacion/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.

Bacteria use two-component systems (TCSs) to sense and respond to their environments. Free-living bacteria usually contain dozens of TCSs, each of them responsible for sensing and responding to a different range of signals. Differences in the content of two-component systems are related with the capacity of the bacteria to colonize different niches or improve the efficiency to grow under the conditions of the existing niche. This review highlights differences in the TCS content between Staphylococcus aureus and Staphylococcus saprophyticus as a case study to exemplify how the ability to sense and respond to the environment is relevant for bacterial capacity to colonize and survive in/on different body surfaces., B.R is recipient of a PhD grant from Universidad Pública de Navarra. Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities grant BIO2017-83035-R Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union. A.F.H. is supported by the European Research Council ERC under the European Union's Horizon 2020 research and innovation program Grant Agreement ERC-ADG-2014 Proposal no 670932 Dut-signal from EU awarded to José R. Penadés and was the recipient of a Tenovus Project Grant (S16-12)., B.R is recipient of a PhD grant from Universidad Publica de Navarra. Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities grant BIO2017-83035-R Agencia Espanola de Investigacio'n/Fondo Europeo de Desarrollo Regional, European Union. A.F.H. is supported by the European Research Council ERC under the European Union's Horizon 2020 research and innovation program Grant Agreement ERC-ADG-2014 Proposal no 670932 Dut-signal from EU awarded to Jose R. Penades) and was the recipient of a Tenovus Project Grant (S16-12).

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 PID2019-105216GB-I00 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).

In this article, we propose a change of paradigm where ammonium nutrition may be considered not exclusively as an undesirable situation for plant performance, but as a way to provoke changes in plant metabolism that can be beneficial for crop quality and plant physiology. While some of the positive effects of ammonium referred here still require further evaluation, the cross-tolerance induction of NH+4 to certain subsequent stresses, notably salinity, is clear. However, the molecular actors governing these interactions are almost completely unknown, and future works will be essential in order to fully exploit the benefits of ammonium-based fertilizers., This work was funded by the Basque Government (IT-932-16) and the Spanish Ministry of Economy and Competitiveness (Grants BIO2017-84035-R and AGL2017-86293-P, both co-funded by FEDER).

Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids., This work was financially supported by the Spanish Ministry of Science, Innovationand Universities grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L. and C.S.
P.D.-M. was supported by an F.P.I. (BES-2015-072859) contract from the Spanish Ministry of Science, Innovation and Universities.

Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). σB is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that σB is required for polysaccharide-independent biofilms, controversy persists over the role of σB in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus σB-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the σB mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active σB, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity., Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities (grants SAF2015-74267-JIN and BIO2017-83035-R [Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union])., Peer reviewed

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., Peer reviewed

In the last decade, the implementation of high-throughput methods for RNA profiling has uncovered that a large part of the bacterial genome is transcribed well beyond the boundaries of known genes. Therefore, the transcriptional space of a gene very often invades the space of a neighbouring gene, creating large regions of overlapping transcription. The biological significance of these findings was initially regarded with scepticism. However, mounting evidence suggests that overlapping transcription between neighbouring genes conforms to regulatory purposes and provides new strategies for coordinating bacterial gene expression. In this MicroReview, considering the discoveries made in a pioneering transcriptome analysis performed on Listeria monocytogenes as a starting point, we discuss the progress in understanding the biological meaning of overlapping transcription that has given rise to the excludon concept. We also discuss new conditional transcriptional termination events that create antisense RNAs depending on the metabolite concentrations and new genomic arrangements, known as noncontiguous operons, which contain an interspersed gene that is transcribed in the opposite direction to the rest of the operon., This review is a tribute to part of the research legacy of P. Cossart's lab, to whom the authors have had the honour of belonging. This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC Consolidator Grant Agreement No. 646869) to A.T‐A. and Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union (BIO2017‐83035‐R) to I.L.

Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed., This research was supported by grants RTI2018-096011-B-I00 and BIO2017-83035-R from the Spanish Ministry of Science, Innovation and Universities, and Proyecto Intramural Incorporación-2018 CSIC.

15 p.-4 fig.-2 tab., Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di‐GMP (c‐di‐GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels., This study was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014‐53530‐R and BIO2017‐83035‐R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I. Lasa and C. Solano and grants BIO2016‐79736‐R, PCIN‐2014‐113 and PCI2019‐111833‐2 to E. Díaz. P. Dorado‐Morales was supported by a F.P.I. (BES‐2015‐072859) contract from the Spanish Ministry of Science, Innovation and Universities., Peer reviewed

One of the major components of the staphylococcal biofilm is surface proteins that assemble as scaffold components of the biofilm matrix. Among the different surface proteins able to contribute to biofilm formation, this review is dedicated to the Biofilm Associated Protein (Bap). Bap is part of the accessory genome of Staphylococcus aureus but orthologs of Bap in other staphylococcal species belong to the core genome. When present, Bap promotes adhesion to abiotic surfaces and induces strong intercellular adhesion by self-assembling into amyloid like aggregates in response to the levels of calcium and the pH in the environment. During infection, Bap enhances the adhesion to epithelial cells where it binds directly to the host receptor Gp96 and inhibits the entry of the bacteria into the cells. To perform such diverse range of functions, Bap comprises several domains, and some of them include several motifs associated to distinct functions. Based on the knowledge accumulated with the Bap protein of S. aureus, this review aims to summarize the current knowledge of the structure and properties of each domain of Bap and their contribution to Bap functionality., Work in the laboratories of JV and IL is supported by grants RTI2018-096011-B-I00 and BIO2017-83035-R from Spanish Ministry of Science, Innovation and Universities. Work in the laboratory of XF is supported by grants from the National Natural Science Foundation of China (No. 31872712) and the Beijing Advanced Innovation Center for Structural Biology., Peer reviewed

Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization., JV was supported by SAF2015-74267-JIN. This research was supported by grants BIO2014-53530-R, BIO2017-83035-R and RTC-2015-3184-1 from the Spanish Ministry of Economy and Competitivity.

© 2021 Dorado-Morales et al., Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids., This work was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L. and C.S. P.D.-M. was supported by an F.P.I. (BES-2015-072859) contract from the Spanish Ministry of Science, Innovation and Universities.

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).