RESUMEN
Bacteria communicate at high cell density through quorum sensing, however, there are no reports about this mechanism in archaea. The archaeon Natronococcus occultus produces an extracellular protease at the end of growth. Early production of protease activity was observed when a low density culture was incubated with late exponential conditioned medium suggesting the presence of factor(s) inducing this activity. Conditioned medium and ethyl acetate extracts corresponding to the transition from exponential to stationary phase showed a positive signal in Agrobacterium biosensor. We report the detection of potential autoinducer molecules of the acylated homoserine lactone type in the archaeon N. occultus. These molecules may be responsible for the production/activation of extracellular protease.
Asunto(s)
Endopeptidasas/metabolismo , Regulación de la Expresión Génica Arqueal , Natronococcus/crecimiento & desarrollo , Transducción de Señal , Medios de Cultivo , Medios de Cultivo Condicionados/farmacología , Natronococcus/enzimología , Cloruro de SodioRESUMEN
The haloalkaliphilic archaeon Natronococcus occultus produces an extracellular serine protease in the stationary growth phase and upon starvation. Two proteins immunologically related to the extracellular protease were detected into the cells: P200 and P190. P200 was detected at early stages of growth and its relative amount decreased as the culture reached the stationary growth phase, concomitantly with the appearance of P190 and proteolytic activity, suggesting that P200 may be the precursor of the secreted protease and P190 the mature enzyme. Both proteins were also detected in the culture medium. Conversion of inactive P200 into active P190 was attained in cell-free culture medium from stationary phase but not from exponential phase. This process was prevented in the presence of PMSF and could be attained by addition of purified mature extracellular protease to P200. Altogether these results indicate that activation of Natronococcus occultus extracellular protease may be autoproteolytic and that factor/s present in stationary phase culture medium may be required for this process.
Asunto(s)
Natronococcus/enzimología , Serina Endopeptidasas/metabolismo , Activación Enzimática , Serina Endopeptidasas/análisisRESUMEN
A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests that Natronococcus occultus extracellular protease may be a novel enzyme.
Asunto(s)
Natronococcus/enzimología , Serina Endopeptidasas/aislamiento & purificación , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Peso Molecular , Serina Endopeptidasas/química , Serina Endopeptidasas/inmunologíaRESUMEN
A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.