RESUMEN
The inoculum size effect in the dimorphic fungus Candida albicans results from production of an extracellular quorum-sensing molecule (QSM). This molecule prevents mycelial development in both a growth morphology assay and a differentiation assay using three chemically distinct triggers for germ tube formation (GTF): L-proline, N-acetylglucosamine, and serum (either pig or fetal bovine). In all cases, the presence of QSM prevents the yeast-to-mycelium conversion, resulting in actively budding yeasts without influencing cellular growth rates. QSM exhibits general cross-reactivity within C. albicans in that supernatants from strain A72 are active on five other strains of C. albicans and vice versa. The QSM excreted by C. albicans is farnesol (C(15)H(26)O; molecular weight, 222.37). QSM is extracellular, and is produced continuously during growth and over a temperature range from 23 to 43 degrees C, in amounts roughly proportional to the CFU/milliliter. Production is not dependent on the type of carbon source nor nitrogen source or on the chemical nature of the growth medium. Both commercial mixed isomer and (E,E)-farnesol exhibited QSM activity (the ability to prevent GTF) at a level sufficient to account for all the QSM activity present in C. albicans supernatants, i.e., 50% GTF at ca. 30 to 35 microM. Nerolidol was ca. two times less active than farnesol. Neither geraniol (C(10)), geranylgeraniol (C(20)), nor farnesyl pyrophosphate had any QSM activity.
Asunto(s)
Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Farnesol/metabolismo , Farnesol/farmacología , Candida albicans/genética , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Medios de Cultivo , Farnesol/química , Farnesol/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Regulación Fúngica de la Expresión Génica , CalorRESUMEN
We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.