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.

The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp. PCC 7942. A new high-CO2-requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5'-flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco). The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli. The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO2 for Rubisco. Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr O221, Rubisco was markedly associated with the carboxysomes. Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP ؉ and, to a lesser extent, NAD ؉ , suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [ P]NAD؉ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.In cyanobacteria, as in most prokaryotes, glutamine synthetase (GS; EC 6.3.1.2) plays a key role in nitrogen assimilation. The enzyme catalyzes the ATP-dependent biosynthesis of glutamine from NH 4 ϩ and glutamate and thus represents a key point for the interaction of carbon and nitrogen metabolism. Given this importance, one would expect that both the synthesis and the activity of the enzyme would be tightly regulated. Three types of GS, termed GSI to GSIII, are found in prokaryotes; GSI is the typical form found in many organisms (31). In the enterobacteriaceae, GS activity is regulated by an adenylylation/deadenylylation system, the activity of the adenyltransferase itself being regulated by the uridylylation state of the protein P II , which also exterts an effect on the transcription of the glnALG operon through its interaction with the NR II protein (14, 23). In gram-positive organisms, GS is not subject to adenylation and activity appears to be regulated by allosteric mechanisms (2).GSs have been purified from a number of cyanobacteria (18) and exhibit the typical size and subunit composition of GSI, although recently the presence of a GSIII has been reported in Synechocystis sp. strain PCC 6803 (24). The regulation of GS activity in cyanobacteria, however, is not well understood. Fisher et al. (4) demonstrated that a cloned cyanobacterial GS was functionally expressed in Escherichia coli but was not subject to adenylation, although cyanobacteria have been shown to possess a P II homolog (28), the activity of which in Synechococcus sp. strain PCC 7942 is regulated by phosphorylation (6). Evidence from several sources (21,25,27,29) suggests that cyanobacterial GS activity is regulated by allosteric processes. In contrast, Joseph and Meeks (9) suggested that GS activity in a symbiotic Nostoc strain might be regulated by a posttranslational mechanism. GS activity has also been shown to be regulated by environmental factors in two unicellular cyanobacteria. Marqués et al. (15) demonstrated a short-term regulation of activity in Synechoc...