Initial approaches towards the understanding of catabolite repression in halophilic Archaea and the characterization of an alpha-glucosidase (maltase) from Haloquadratum walsbyi

Abstract

The work of Carl Woese based on ribosomal RNA sequence analysis from organisms have led scientist to classify life as we know it into three domains. One such domain is the Archaea. There is a lot of interest in studying regulation of gene expression on these microorganisms. There are few reports about gene regulation in halophilic Archaea, especially with genes related to carbohydrate metabolism. Also there are no reports describing the properties of an extremely halophilic alpha glucosidase (maltase) from the Archaea. The purpose of this research project was to study the putative catabolic repression-like system occurring in the extreme halophilic archaea using Halogeometricum borinquense and Haloquadratum walsbyi as models. To achieve this goal, a set of enzymes involved in carbohydrate metabolism were selected (αglucosidase, β-glucosidase, and β-galactosidase) and their specific activity levels were determined on different carbon sources supplementing a minimal medium. The experiments revealed that H. borinquense prefers glucose as a carbon source, but the three enzymes seem to be fully repressed at this growth condition. These might indicate that H. borinquense uses the system of catabolite repression to regulate the genes involved in carbohydrate metabolism. In addition the activity of the three enzymes studied in this work, were markedly dependent on the salt (NaCl or KCl) present in the enzymatic buffer. Also, this project focused on the cloning, expression, purification, and partial characterization of an alpha glucosidase from the extremely halophilic archaeon H. walsbyi. The recombinant enzyme showed optimum conditions at 40 0C, 15% NaCl (w/v), and pH of 6.0. This study constitutes the first attempt to study gene regulation by catabolite repression in extreme halophilic archaea.

El trabajo de Carl Woese basado en el análisis de secuencias del ARN ribosomal en organismos, ha llevado a clasificar la vida tal como la conocemos en tres dominios, siendo Archaea uno de estos dominios. Hay pocos informes acerca de la regulación génica en arqueas halofílicas, especialmente con los genes relacionados con el metabolismo de carbohidratos. El propósito de este trabajo se ha centrado en el estudio del posible sistema de represión catabólica en la haloarchaea Halogeometricum borinquense. Para lograr este objetivo, un conjunto de enzimas que intervienen en el metabolismo de carbohidratos fueron seleccionados (α-glucosidasa, β-glucosidasa, y βgalactosidasa). Sus niveles de actividades específicas se determinaron utilizando diferentes fuentes de carbono en un medio mínimo de sales. Además, la actividad de las tres enzimas estudiadas en este trabajo depende de la sal (NaCl o KCl) utilizada en el amortiguador de ensayos enzimáticos. Por otra parte, todas las alfa glucosidasas caracterizadas hasta el momento corresponden a arqueas hipertermofílicas. No hay reportes en la literatura donde se describa un alfa glucosidasa halofílica (maltasa). La segunda parte de este trabajo se centró en la purificación, expresión y caracterización parcial de la enzima alfa glucosidasa en la arquea halofilica H. walsbyi. Esta enzima presentó su nivel óptimo de expresión a 400C, 15% NaCl y pH 6.0. Este estudio representa el primer intento en estudiar el fenómeno de represión catabólica en las arqueas halófilas.