Biosynthesis and physiology of coenzyme Q in bacteria.

International audienceUbiquinone, also called coenzyme Q, is a lipid subject to oxido-reduction cycles. It functions in the respiratory electron transport chain and plays a pivotal role in energy generating processes. In this review, we focus on the biosynthetic pathway and physiological role of ubiquinone in bacteria. We present the studies which, within a period of five decades, led to the identification and characterization of the genes named ubi and involved in ubiquinone production in Escherichia coli. When available, the structures of the corresponding enzymes are shown and their biological function is detailed. The phenotypes observed in mutants deficient in ubiquinone biosynthesis are presented, either in model bacteria or in pathogens. A particular attention is given to the role of ubiquinone in respiration, modulation of two-component activity and bacterial virulence. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference

The ubiK protein is an accessory factor necessary for bacterial Ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK. Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli. Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.Agence Nationale de la Recherche ANR-15-CE11-0001-02Centre National de la Recherche Scientifique PICS07279French State Program "Investissements d'Avenir" ANR-11-LABX-001

ubiJ, a New Gene Required for Aerobic Growth and Proliferation in Macrophage, Is Involved in Coenzyme Q Biosynthesis in Escherichia coli and Salmonella enterica Serovar Typhimurium.

International audienceUbiquinone (coenzyme Q or Q8) is a redox active lipid which functions in the respiratory electron transport chain and plays a crucial role in energy-generating processes. In both Escherichia coli and Salmonella enterica serovar Typhimurium, the yigP gene is located between ubiE and ubiB, all three being likely to constitute an operon. In this work, we showed that the uncharacterized yigP gene was involved in Q8 biosynthesis in both strains, and we have renamed it ubiJ. Under aerobic conditions, an ubiJ mutant was found to be impaired for Q8 biosynthesis and for growth in rich medium but did not present any defect anaerobically. Surprisingly, the C-terminal 50 amino acids, predicted to interact with lipids, were sufficient to restore Q8 biosynthesis and growth of the ubiJ mutant. Salmonella ubiE and ubiB mutants were impaired in Q8 biosynthesis and in respiration using different electron acceptors. Moreover, ubiE, ubiJ, and ubiB mutants were all impaired for Salmonella intracellular proliferation in macrophages. Taken together, our data establish an important role for UbiJ in Q8 biosynthesis and reveal an unexpected link between Q8 and virulence. They also emphasize that Salmonella organisms in an intracellular lifestyle rely on aerobic respiration to survive and proliferate within macrophages

Phenotypic heterogeneity in Pseudomonas syringae

The notion of isogenic bacterial populations displaying phenotypic differences is widely accepted today. A particular case of phenotypic heterogeneity is bistability. Bistability occurs when bacterial population splits into two subpopulations showing distinct phenotypes. Phenotypic heterogeneity can allow some individuals to survive sudden environmental changes (risk-spreading) and can also lead to the cooperation (division of labour) between individuals. The relevance of this process has been highlighted in some animal pathogens, nevertheless, little is known about the occurrence or impact of these processes in the adaptation of bacteria to non-animal hosts. Pseudomonas syringae is a plant-pathogenic bacterium whose virulence depend of the T3SS expression. We have reported that T3SS expression is bistable in hrp-induction medium. This bistability generates two subpopulations, that show differences in virulence. Flagella is also an important virulence determinant for Pseudomonas syringae colonization. Here, we show how flagella expression also displays markedly phenotypic heterogeneity during growth within the plant. Although subpopulations displaying flagONT3SSON and flagOFFT3SSOFF can be identified within the plant, we provide evidence of cross regulation between T3SS and flagella expression at the individual cell level and propose phenotypic heterogeneity as an adaptative value for Pseudomonas adaptation to the plant host.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

First mid-infrared spectrum of a faint high-z galaxy: Observations of CFRS 14.1157 with the Infrared Spectrograph on the Spitzer Space Telescope

The unprecedented sensitivity of the Infrared Spectrograph on the Spitzer Space Telescope allows for the first time the measurement of mid-infrared spectra from 14 to 38 microns of faint high-z galaxies. This unique capability is demonstrated with observations of sources having 16 micron fluxes of 3.6 mJy (CFRS 14.1157) and 0.35 mJy (CFRS 14.9025). A spectral-fitting technique is illustrated which determines the redshift by fitting emission and absorption features characteristic of nearby galaxies to the spectrum of an unknown source. For CFRS 14.1157, the measured redshift is z = 1.00+/-0.20 in agreement with the published result of z = 1.15. The spectrum is dominated by emission from an AGN, similar to the nucleus of NGC 1068, rather than a typical starburst with strong PAH emission like M82. Such spectra will be crucial in characterizing the nature of newly discovered distant galaxies, which are too faint for optical follow-up.Comment: Accepted in ApJ Sup. Spitzer Special Issue, 4 pages, 5 figure

Heterogeneidad de la expresión del T3SS y del flagelo en Pseudomonas syringae

La heterogeneidad o variación fenotípica en bacterias es un fenómeno descrito en poblaciones clonales desde hace décadas. Puede estar determinada por mecanismos epigenéticos, y dar lugar a linajes bacterianos con perfiles de expresión génica diferentes. Bajo el control de ciertos circuitos regulatorios, la heterogeneidad en la expresión génica puede dar lugar a un perfil de expresión bimodal en ambientes homogéneos, proceso conocido como biestabilidad. La relevancia de este proceso se ha demostrado en Salmonella entérica y en otros patógenos humanos durante el establecimiento de la resistencia a antibióticos, y se ha observado la implicación de este proceso en la expresión de genes de virulencia. Pseudomonas syringae es una bacteria fitopatógena cuya virulencia depende del Sistema de Secreción Tipo III (T3SS). Nuestro equipo ha descrito que genes de diferentes elementos del T3SS en P. syringae pv. phaseolicola muestran biestabilidad en condiciones de inducción en el laboratorio. Además, las subpoblaciones bacterianas separadas según niveles de expresión muestran diferencias en virulencia, y dicha expresión es marcadamente heterogénea durante la colonización de los tejidos de la planta. Por otro lado, el flagelo es otro elemento importante tanto en el estilo de vida de P. syringae como en su interacción con el huésped, donde dispara inmunidad basal. Hemos observado que fliC el gen que codifica para la flagelina presenta expresión heterogénea, en este caso tanto en cultivos de laboratorio, como durante la proliferación en los espacios intercelulares de la hoja huésped. Dado que resultados previos de nuestro y otros laboratorios indican la existencia de un cierto grado de contra-regulación entre el flagelo y el T3SS, se pretende profundizar en la relación entre la motilidad flagelar y la regulación de la expresión génica del T3SS, así como en el impacto potencial que la expresión biestable del T3SS puede tener sobre la motilidad

Expresión heterogénea de genes relevantes para la virulencia de Pseudomonas syringae

La heterogeneidad o variación fenotípica ha sido descrita en poblaciones clonales microbianas desde hace décadas. Bajo el control de ciertos circuitos regulatorios, la heterogeneidad en la expresión génica puede dar lugar a un perfil de expresión bimodal en ambientes homogéneos, proceso conocido como biestabilidad. La relevancia de este proceso se ha demostrado en Salmonella entérica y en otros patógenos humanos durante el establecimiento de la resistencia a antibióticos, y se ha observado la implicación de este proceso en la expresión de genes de virulencia. Pseudomonas syringae es una bacteria fitopatógena de gran importancia económica que requiere del Sistema de Secreción Tipo III (T3SS) para suprimir las respuestas de defensa de la planta. Nuestro equipo ha descrito que genes de diferentes elementos del T3SS muestran biestabilidad en su expresión en condiciones de inducción en el laboratorio, asociada a diferencias fenotípicas en virulencia, y que dicha expresión es marcadamente heterogénea durante la colonización de la planta. Por otro lado, el flagelo es otro elemento importante tanto en el estilo de vida de P. syringae como en su interacción con el huésped, donde dispara inmunidad basal, que asimismo presenta expresión heterogénea, tanto en cultivos, como durante la proliferación en los espacios intercelulares de la hoja huésped. Dado que resultados previos de nuestro y otros laboratorios indican la existencia de un cierto grado de contra-regulación entre el flagelo y el T3SS, este trabajo pretende profundizar en la relación entre la motilidad flagelar y la regulación de la expresión génica del T3SS, así como en el impacto potencial que la expresión biestable del T3SS puede tener sobre la motilidad a nivel de una sola célula.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Expresión heterogénea y colonización de la planta en Pseudomonas syringae

La heterogeneidad fenotípica se ha descrito en poblaciones clonales durante décadas. La heterogeneidad o ruido en la expresión génica puede dar lugar a un patrón de expresión bimodal. Este proceso donde la población se divide en una subpoblación ON y una subpoblación OFF para la expresión de un determinado gen es conocido como biestabilidad. La relevancia de este proceso se ha demostrado en patógenos de animales durante la persistencia a antibióticos. Un fenómeno similar se ha observado en proteínas asociadas a virulencia. En P. syringae, una bacteria fitopatógena Gram negativa, hemos observado que genes importantes para su virulencia muestran expresión heterogénea durante la colonización de la planta. Para analizar la expresión a nivel de célula individual se han generado fusiones transcripcionales en el cromosoma de Ps. syringae pv. phaseolicola a genes codificantes de proteínas fluorescentes. Los genes seleccionados codifican diferentes elementos del sistema de secreción tipo III (T3SS) y la flagelina. Su análisis ha usado técnicas de microscopía confocal y de citometría. La expresión de genes del T3SS y del flagelo es heterogénea en los espacios intercelulares de la hoja huésped. En medio de inducción en el laboratorio, los genes del T3SS, pero no del flagelo, muestran expresión biestable que requiere división celular activa. Resultados previos de nuestro laboratorio indican un cierto grado de contra-regulación entre la expresión del T3SS y la motilidad flagelar. Mediante análisis de expresión de estos genes a nivel de célula individual, hemos confirmado que esta contra-regulación existe, pero no es todo o nada pudiéndose observar subpoblaciones que además de expresar uno u otro sistema, expresan ambos o ninguna.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A new gene involved in coenzyme Q biosynthesis in Escherichia coli: UbiI functions in aerobic C5-hydroxylation

International audienceCoenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known

Phenotypic heterogeneity during plant colonization

Isogenic bacterial populations can display phenotypic differences. Cell-to-cell phenotypic differences can be a consequence of noisy gene expression, or a programmed event under genetic or epigenetic control. Bistability occurs when populations splits into subpopulations showing distinct phenotypes. This heterogeneity can allow individuals to survive environmental changes or can lead to cooperation between individuals. This is highly relevant for some animal pathogens, but little is known about it for plant colonizing bacteria. We have reported that T3SS expression in Pseudomonas syringae is bistable in hrp-induction medium and displays phenotypic heterogeneity during plant colonization. This bistability generates two subpopulations that show differences in virulence. Flagella is also an important virulence determinant for Pseudomonas syringae colonization that displays a degree of counter-regulation with the T3SS. Salmonella enterica serovar typhimurium SPI-1 T3SS is also expressed bistably and counter-regulates with flagella. SPI-1 bistable expression is a important asset during mouse infection as it leads to cooperative virulence. But although S. enterica colonize plants as alternative hosts and requires SPI-1 to do so efficiently, little is know about the expression of this system during plant colonization We present our newest findings regarding phenotypic heterogeneity of virulence relevant traits of P. syringae and S. enterica during plant colonization.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech