Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus

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.     

Purification and characterization of a membrane-associated ATPase from Natronococcus occultus, a haloalkaliphilic archaeon

Isolated membranes of the extreme haloalkaliphilic archaeon Natronococcus occultus were able to hydrolyze ATP via an ATPase, which required the presence of Mg(2+), high concentrations of NaCl, and a pH value of 9. The native molecular mass of the purified ATPase was 130 kDa and was composed of 74- and 61-kDa subunits. Enzyme activity was specific for the hydrolysis of ATP with slight activity towards GTP, CTP, and ITP. The enzyme required NaCl for maximal activity but Na(2)SO(4) and (NH(4))(2)SO(4) could substitute. The enzyme showed no activity if Na(2)SO(3) or sodium citrate was substituted for NaCl. The ATPase from N. occultus was inhibited by NBD-Cl, NaN(3), and ouabain, and was sensitive to nitrate, vanadate, DCCD, and bafilomycin A(1). It was not inhibited by NEM in contrast to other previously characterized halophile ATPases. The ATPase had a K(M) of 0.5 mM and appeared to be non-competitively inhibited by NaN(3) with a K(I) of 3.1 mM.     

High level, intrinsic resistance of Natronococcus occultus to potassium tellurite

Natronococcus occultus, a haloalkaliphilic archaeon, was examined for its resistance to potassium tellurite. Cells grown in the presence of 1 mM potassium tellurite reduced it to metallic tellurium resulting in the deposition of intracellular crystals in the cytoplasm. The minimal inhibitory concentration for potassium tellurite was 10 mM. N. occultus had an inducible tellurite reductase activity. Cell-free extracts catalyzed the enzymatic reduction of potassium tellurite in a reaction which was dependent on NADH oxidation and a reduced environment.     

Neue sesquiterpene und norditerpene aus vertretern der gattung Libanothamnus

The aerial parts of L. granatesianus afforded in addition to known compounds two nor- ent-kaurenes, while those of L. occultus yielded in addition to several diterpenes, two new dialdehydes derived from silinene. The chemotaxonomic situation is discussed briefly.     

Extracellular protease of Natrialba magadii: purification and biochemical characterization

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°C in the presence of 1.5 M 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. Received: October 1, 1999 / Accepted: February 1, 2000     

Ultrastructural study of the proboscis endothelium of Riseriellus occultus (Nemertea, Heteronemertea)

We have examined with transmission electron microscopythe epithelial layer exposed to the rhynchocoel fluidof the proboscis in the heteronemertine Riseriellus occultus. This epithelium is organized asa monociliated, pseudostratified myoepitheliumconsisting of two cell types: apically situatedmonociliated supportive cells and subapical myocyteslacking cilia. The low supportive cells form acontinuous adluminal sheet and reach with numerouscytoplasmic processes into the extracellular matrix;these cells are characterized by numerous, irregularlyshaped, apical folds projecting into the rhynchocoelfluid, delimiting broad extracellular spaces. Theauthors suppose that both apical and basal folds couldaccommodate stretching of the endothelium when theproboscis is everted. The apical folds of thesupportive cells increase the interface of these withthe rhynchocoel fluid; this feature, together with thepresence of pinocytotic vesicles in such cells,suggest that they could be involved in the exchange ofsubstances between the rhynchocoel fluid and theproboscis. The myocytes are scattered singly withinthe monociliated pseudostratified myoepithelium. Theyare situated between the supportive cells and thesubjacent extracellular matrix. Basement membraneseparating both cells types is lacking. Myofibrillarparts protrude basally from the myocyte somata. Themyofibrillar parts lie in direct apposition to theextracellular matrix, and are oriented circular to thelongitudinal axis of the proboscis. We consider themyocytes to be intra-epithelial, myoepithelial cells.     

Detection and preliminary characterization of extracellular proteolytic activities of the haloalkaliphilic archaeon Natronococcus occultus

Extracellular proteolytic activity was detected in the haloalkaliphilic archaeon Natronococcus occultus as the culture reached the stationary growth phase. Proteolytic activity was precipitated with ethanol and subjected to a preliminary characterization. Optimal conditions for activity were attained at 60° C and 1–2 M NaCl or KCl. Gelatin zymography in the presence of 4 M betaine revealed a complex pattern of active species with apparent molecular masses ranging from 50 to 120 kDa. Experiments performed with inhibitors of the various groups of proteases indicated that the extracellular proteolytic enzymes of N. occultus are of the serine type. Individual protein species showed some differences in salt and thermal stability. Received: 10 January 1997 / Accepted: 27 June 1997