Estudio de los regulones de las proteínas Hfq y Crc de Pseudomonas putida,y de la expresión del ARN pequeño CrcZ que controla la actividad de estas proteínas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-05-2018Esta tesis tiene embargado el acceso al texto completo hasta el 21-11-2019Pseudomonas putida KT2440 es una bacteria Gram-negativa de gran importancia biotecnológica debido, entre otras cosas, a su enorme versatilidad metabólica que le permite utilizar multitud de compuestos como fuentes de carbono y energía. El uso de estos compuestos está jerárquicamente regulado por las proteínas Hfq y Crc. Cuando en el medio hay fuentes de carbono preferentes, estas proteínas inhiben la traducción de los ARN mensajeros de genes implicados en el transporte o asimilación de fuentes de carbono no preferentes. Hfq y Crc se unen a secuencias ricas en adeninas, denominadas motivos CA, presentes en las inmediaciones del sitio de inicio de traducción de los ARN mensajeros susceptibles de regulación, impidiendo así que sean traducidos. Cuando los sustratos preferentes se han consumido, Hfq y Crc son secuestradas por los ARN pequeños CrcZ y CrcY, que contienen varios motivos CA en su secuencia, y la traducción de los ARNm de genes implicados en la asimilación de sustratos no preferidos puede tener lugar. Además de ser sintetizados desde sus propios promotores PcrcZ y PcrcY, CrcZ y CrcY también pueden originarse desde los promotores de los genes localizados por encima de ellos. En la primera parte de esta tesis doctoral hemos estudiado cómo tiene lugar este proceso en el caso de CrcZ. Hemos comprobado que desde el promotor de cbrB, el gen anterior a crcZ, se origina un tránscrito que contiene cbrB y crcZ y que, tras ser procesado en un punto cercano al inicio de transcripción de PcrcZ, se genera un tránscrito de CrcZ procesado, que hemos denominado CrcZ*, con un tamaño muy similar al tránscrito CrcZ primario. En la segunda parte de esta tesis doctoral hemos estudiado qué otras funciones ejercen Hfq y Crc, a fin de entender mejor cómo funciona su colaboración. Mediante ensayos de transcriptómica y proteómica se ha comparado el efecto que la inactivación de los genes crc y/o hfq tiene sobre la bacteria cuando ésta crece en un medio mínimo con succinato como única fuente de carbono. Los resultados obtenidos indican que el efecto de ausencia de Crc, en estas condiciones, es pequeño e indirecto, mientras que la falta de Hfq provoca un efecto más amplio. En las condiciones analizadas, el mutante hfq presenta menor cantidad de los ARNs pequeños PrrF1 y PrrF2 implicados en la regulación del hierro. Además, tiene afectada la expresión de numerosos genes, entre ellos los relacionados con la producción y captación del sideróforo pioverdina. Así pues, la ausencia de Hfq desregula la homeostasis del hierro, un microelemento esencial para la bacteria

Novel regulatory mechanism of establishment genes of conjugative plasmids

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg. Reg also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg. Each operator is bound with extreme cooperativity by two Regdimers. The X-ray structure revealed that Reg has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition

Pseudomonas aeruginosa mutants defective in glucose uptake have pleiotropic phenotype and altered virulence in non-mammal infection models

Pseudomonas spp. are endowed with a complex pathway for glucose uptake that relies on multiple transporters. In this work we report the construction and characterization of Pseudomonas aeruginosa single and multiple mutants with unmarked deletions of genes encoding outer membrane (OM) and inner membrane (IM) proteins involved in glucose uptake. We found that a triple \u394gltKGF \u394gntP \u394kguT mutant lacking all known IM transporters (named GUN for Glucose Uptake Null) is unable to grow on glucose as unique carbon source. More than 500 genes controlling both metabolic functions and virulence traits show differential expression in GUN relative to the parental strain. Consistent with transcriptomic data, the GUN mutant displays a pleiotropic phenotype. Notably, the genome-wide transcriptional profile and most phenotypic traits differ between the GUN mutant and the wild type strain irrespective of the presence of glucose, suggesting that the investigated genes may have additional roles besides glucose transport. Finally, mutants carrying single or multiple deletions in the glucose uptake genes showed attenuated virulence relative to the wild type strain in Galleria mellonella, but not in Caenorhabditis elegans infection model, supporting the notion that metabolic functions may deeply impact P. aeruginosa adaptation to specific environments found inside the host

Influence of the Hfq and Crc global regulators on the control of iron homeostasis in Pseudomonas putida

Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low‐level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1‐F2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1‐F2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes.We are grateful to V. de Lorenzo and E. Duque for providing the P. putida Hfq‐null and PvdD‐null strains, respectively, and to J.C. Oliveros for help in the design and interpretation of RNA‐seq assays. DS‐H received a predoctoral fellowship from the Spanish Ministry of Science and Competitiveness (MINECO). This work was funded by grant BIO2015–66203‐P (AEI/FEDER, UE)

Novel regulatory mechanism of establishment genes of conjugative plasmids

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.Economy and Competitiveness of the Spanish Government [BFU2016-75471-C2-1-P (AEI/FEDER, EU) to C.A., BIO2013-41489-P (AEI/FEDER, EU) and BIO2016-77883-C2-1-P (AEI/FEDER, EU) to W.M., BIO2016-77883-C2-2-P (AEI/FEDER, EU) to R.B., BIO-2015-66203-P (AEI/FEDER, EU) to F.R., FIS2016-78313-P (AEI/FEDER, EU) to S.A.]; BIO2013-41489-P (AEI/FEDER, EU) and BIO2016-77883-C2-1-P (AEI/FEDER, EU) also supported J.V., A.M., and C.G.; Wellcome Investigator Award [209500 to Jeff Errington] supported L.W; ‘Ramón y Cajal’ Contract Supported S.A.; ‘Agencia Estatal de Investigaci ´on’ (AEI); ‘Fondo Europeo de Desarrollo Regional (FEDER); European Union (EU

Novel regulatory mechanism of establishment genes of conjugative plasmids

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg. Reg also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg. Each operator is bound with extreme cooperativity by two Regdimers. The X-ray structure revealed that Reg has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit