No abstract

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-R) is the major ratelimiting enzyme of the mevalonate pathway in many organisms, including yeasts. In the yeast Saccharomyces cerevisiae, there are two isoenzymes of HMG-R (Hmg1p and Hmg2p). Both consist of an anchoring transmembrane domain and a catalytic domain. We have removed the known controlling features of HMG-R by overproducing the catalytic domain of Hmg1p. This overproduction leads to an enhancement of squalene production, implying that HMG-R has been deregulated. The enhancement is apparent under semianaerobic and aerobic conditions. Despite the increase in squalene production, the amount of ergosterol produced by the HMG-R-overproducing yeast was not increased. This result suggests the presence of another regulatory step between squalene and ergosterol formation. Squalene levels generated by cells overproducing the catalytic domain of HMG-R were estimated to be up to 10 times those produced by wild-type cells. The enhancement in squalene production coincided with a reduction in growth rate. This reduction may be a direct consequence of the buildup of high concentrations of squalene and presqualene intermediates of the pathway.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P i) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P i concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P i typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.

No abstract

No abstract