The role of the Crc/Hfq/CrcZ-CrcY global regulatory system on the regulation of metabolic and cellular processes in Pseudomonas putida

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-09-2015Metabolically versatile bacteria have evolved diverse strategies to adapt and to colonize different environments and niches. This ensures high fitness and the ability to withstand changing surrounding conditions and diverse kinds of stresses. These abilities rely on a varied genetic repertoire and on complex regulatory networks that integrate external and internal signals, providing efficient responses. Pseudomonas putida possesses a large genome and a remarkable metabolic and physiologic versatility, with several regulatory networks that assure metabolic responses for surviving in a wide variety of environments. One of the main regulatory networks that serve to optimize metabolism, growth speed and cellular fitness is the so-called Carbon Catabolite Repression (CCR). This regulatory mechanism ensures that, when mixtures of carbon sources are available at sufficient concentrations, those with higher energetic yield are preferentially assimilated, limiting the use of those that are less preferred. This process allows a hierarchical and sequential assimilation of the carbon sources available, optimizing metabolism and the overall fitness. In P. putida, the Crc and Hfq proteins and the CrcZ and CrcY small RNAs are key components of CCR. The Crc and Hfq proteins work together to inhibit the translation of some mRNAs that present an A-rich sequence, named CA motif, located close to the ribosome binding site. In turn, Crc/Hfq action is antagonised by the CrcZ and CrcY sRNAs. The levels of these two sRNAs are low in media that generate high CCR conditions and increase significantly when CCR decreases; it is believed that the function of these sRNAs is to bind the Hfq protein, perhaps together with Crc, sequestering them when their effects are not necessary. The work described in this thesis expands our knowledge on the influence of CCR, and in particular of the Crc/Hfq/CrcZ-CrcY system, in shaping P. putida metabolism according to environmental conditions. The results presented illustrate the role of the Crc regulator in controlling several metabolic processes. We have determined the hierarchy of assimilation of organic acids and amino acids when cells grow in a complex medium, where CCR is strong, and the role of the Crc protein in regulating this process. We have also clarified the influence of the Crc regulator in balancing the carbon fluxes through central metabolic pathways under mild CCR conditions, optimizing metabolism. Moreover, we have shown that the Crc/Hfq/CrcZCrcY regulatory network can modulate the synthesis of polyhydroxyalkanoates, which are important storage compounds, thus balancing carbon flow. The influence of the Crc protein in the synthesis of polyhydroxyalkanoates extends the role of this regulator beyond that of repressing the uptake and assimilation of non-preferred carbon sourcesLas bacterias que tienen un metabolismo versátil han desarrollado diversas estrategias para adaptarse a diferentes ambientes, y colonizarlos. Entre ellas destaca la capacidad de resistir condiciones ambientales cambiantes y diversos tipos de estrés, características que confieren una elevada eficacia biológica. Pseudomonas putida tiene una gran versatilidad metabólica y fisiológica, características que derivan de un genoma grande cuya expresión está controlada por diversas redes de regulación global que aseguran respuestas adecuadas frente a señales fisiológicas y del medio ambiente. Esto facilita su supervivencia en condiciones y ecosistemas muy variados. Una de las redes de regulación más importantes para optimizar el metabolismo, la velocidad de crecimiento y la eficacia biológica es la llamada represión catabólica. Este mecanismo regulador asegura que, cuando las células disponen de diversas fuentes de carbono en concentraciones suficientes, se metabolicen preferentemente aquellas que facilitan un mejor rendimiento energético, limitando el uso de otros compuestos menos preferidos. Este proceso regulador permite una asimilación secuencial y jerárquica de las fuentes de carbono disponibles, optimizando el metabolismo y la capacidad competitiva de la bacteria. En P. putida, las proteínas Crc y Hfq, y los RNAs pequeños CrcZ y CrcY, son componentes clave de la represión catabólica. Crc y Hfq trabajan juntas para inhibir la traducción de determinados mRNAs que tienen una secuencia rica en adeninas (llamada motivo CA) en la región de inicio de la traducción. Los RNAs pequeños CrcZ y CrcY regulan la acción de Crc y Hfq, actuando como antagonistas. Los niveles de estos dos RNAs son bajos en situaciones que generan una fuerte represión catabólica, pero aumentan significativamente cuando la represión disminuye. Se piensa que la función de CrcZ y CrcY es unir la proteína Hfq, quizá junto con Crc, secuestrándolas cuando su efecto no es necesario. El trabajo que se describe en esta tesis aporta información nueva sobre la influencia de la represión catabólica, y en particular del sistema Crc/Hfq/CrcZ-CrcY, en la adaptación del metabolismo de P. putida a condiciones ambientales cambiantes. Los resultados obtenidos ilustran la función de la proteína reguladora Crc en el control de varios procesos metabólicos. Hemos analizado la jerarquía de asimilación de diversos ácidos orgánicos y aminoácidos cuando la célula crece en un medio rico, en el que la represión catabólica es muy fuerte, y la función de Crc en este proceso. También hemos clarificado la influencia del regulador Crc en el balance de los flujos de metabolitos a través de las rutas metabólicas centrales, optimizando el metabolismo cuando las células crecen en condiciones en las que la represión catabólica es moderada. Además, mostramos que el sistema regulador Crc/Hfq/CrcZ-CrcY puede modular la síntesis de polihidroxialcanoatos, que son compuestos de reserva importantes. En conjunto, los resultados implican que este sistema regulador no solo controla el transporte y asimilación de fuentes de carbono, sino que influye en todo el proceso del flujo de compuestos carbonadosEste trabajo se ha realizado gracias a una beca predoctoral de la Fundación “La Caix

Convergent Metabolic Specialization through Distinct Evolutionary Paths in <i>Pseudomonas aeruginosa</i>

ABSTRACT Evolution by natural selection under complex and dynamic environmental conditions occurs through intricate and often counterintuitive trajectories affecting many genes and metabolic solutions. To study short- and long-term evolution of bacteria in vivo, we used the natural model system of cystic fibrosis (CF) infection. In this work, we investigated how and through which trajectories evolution of Pseudomonas aeruginosa occurs when migrating from the environment to the airways of CF patients, and specifically, we determined reduction of growth rate and metabolic specialization as signatures of adaptive evolution. We show that central metabolic pathways of three distinct Pseudomonas aeruginosa lineages coevolving within the same environment become restructured at the cost of versatility during long-term colonization. Cell physiology changes from naive to adapted phenotypes resulted in (i) alteration of growth potential that particularly converged to a slow-growth phenotype, (ii) alteration of nutritional requirements due to auxotrophy, (iii) tailored preference for carbon source assimilation from CF sputum, (iv) reduced arginine and pyruvate fermentation processes, and (v) increased oxygen requirements. Interestingly, although convergence was evidenced at the phenotypic level of metabolic specialization, comparative genomics disclosed diverse mutational patterns underlying the different evolutionary trajectories. Therefore, distinct combinations of genetic and regulatory changes converge to common metabolic adaptive trajectories leading to within-host metabolic specialization. This study gives new insight into bacterial metabolic evolution during long-term colonization of a new environmental niche. IMPORTANCE Only a few examples of real-time evolutionary investigations in environments outside the laboratory are described in the scientific literature. Remembering that biological evolution, as it has progressed in nature, has not taken place in test tubes, it is not surprising that conclusions from our investigations of bacterial evolution in the CF model system are different from what has been concluded from laboratory experiments. The analysis presented here of the metabolic and regulatory driving forces leading to successful adaptation to a new environment provides an important insight into the role of metabolism and its regulatory mechanisms for successful adaptation of microorganisms in dynamic and complex environments. Understanding the trajectories of adaptation, as well as the mechanisms behind slow growth and rewiring of regulatory and metabolic networks, is a key element to understand the adaptive robustness and evolvability of bacteria in the process of increasing their in vivo fitness when conquering new territories

Notulae to the Italian alien vascular flora: 7

In this contribution, new data concerning the distribution of vascular flora alien to Italy are presented. It includes new records, confirmations, and status changes for Italy or for Italian administrative regions of taxa in the genera Abies, Actinidia, Alooe, Amaryllis, Anredera, Arctotheca, Bidens, Cardiospermum, Celosia, Commelina, Cotoneaster, Cyclamen, Eclipta, Euphorbia, Grevillea, Hedera, Hibiscus, Impatiens, Juglans, Kalanchoe, Koelreuteria, Lindernia, Melinis, Myriophyllum, Nandina, Nicotiana, Oenothera, Oxalis, Parthenocissus, Phoenix, Phyllanthus, Physalis, Plumbago, Pteris, Quercus, Setaria, Symphytum, Tagetes, and Washingtonia. Nomenclatural and distribution updates, published elsewhere are provided as Suppl. material 1

A Cell Cycle Role for the Epigenetic Factor CTCF-L/BORIS

CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, downregulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements