RESUMEN
As a ubiquitous environmental organism and an important human pathogen, Pseudomonas aeruginosa readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow P. aeruginosa to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp. P. aeruginosa produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (ΔSR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated pqsA and pqsR expression and reduced repression by the rhl system. We also found that (p)ppGpp is required for full expression of both rhl and las AHL (acyl-homoserine lactone) quorum-sensing systems, since the ΔSR mutant has reduced rhlI, rhlR, lasI, and lasR expression, butanoyl-homoserine lactone (C4-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the las system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.
Asunto(s)
Pseudomonas aeruginosa/fisiología , Quinolinas/metabolismo , Percepción de Quorum , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Nucleótidos de Guanina/biosíntesis , Nucleótidos de Guanina/metabolismo , Pseudomonas aeruginosa/genéticaRESUMEN
The function of a putative galacturonosyltransferase from Arabidopsis (Arabidopsis thaliana; At1g02720; GALACTURONOSYLTRANSFERASE-LIKE5 [AtGATL5]) was studied using a combination of molecular genetic, chemical, and immunological approaches. AtGATL5 is expressed in all plant tissues, with highest expression levels in siliques 7 DPA. Furthermore, its expression is positively regulated by several transcription factors that are known to regulate seed coat mucilage production. AtGATL5 is localized in both endoplasmic reticulum and Golgi, in comparison with marker proteins resident to these subcellular compartments. A transfer DNA insertion in the AtGATL5 gene generates seed coat epidermal cell defects both in mucilage synthesis and cell adhesion. Transformation of atgatl5-1 mutants with the wild-type AtGATL5 gene results in the complementation of all morphological phenotypes. Compositional analyses of the mucilage isolated from the atgatl5-1 mutant demonstrated that galacturonic acid and rhamnose contents are decreased significantly in atgatl5-1 compared with wild-type mucilage. No changes in structure were observed between soluble mucilage isolated from wild-type and mutant seeds, except that the molecular weight of the mutant mucilage increased 63% compared with that of the wild type. These data provide evidence that AtGATL5 might function in the regulation of the final size of the mucilage rhamnogalacturonan I.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mucílago de Planta/metabolismo , Semillas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ácidos Hexurónicos/metabolismo , Hibridación in Situ , Microscopía Confocal , Microscopía Electrónica de Rastreo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Pectinas/metabolismo , Mucílago de Planta/análisis , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ramnosa/metabolismo , Semillas/genética , Semillas/ultraestructura , TranscriptomaRESUMEN
Bacteria become highly tolerant to antibiotics when nutrients are limited. The inactivity of antibiotic targets caused by starvation-induced growth arrest is thought to be a key mechanism producing tolerance. Here we show that the antibiotic tolerance of nutrient-limited and biofilm Pseudomonas aeruginosa is mediated by active responses to starvation, rather than by the passive effects of growth arrest. The protective mechanism is controlled by the starvation-signaling stringent response (SR), and our experiments link SR-mediated tolerance to reduced levels of oxidant stress in bacterial cells. Furthermore, inactivating this protective mechanism sensitized biofilms by several orders of magnitude to four different classes of antibiotics and markedly enhanced the efficacy of antibiotic treatment in experimental infections.