Identification and nitrogen regulation of the cyanase gene from the cyanobacteria Synechocystis sp. strain PCC 6803 and Synechococcus sp. strain PCC 7942

Abstract

An open reading frame (slr0899) on the genome of Synechocystis sp. strain PCC 6803 encodes a polypeptide of 149 amino acid residues, the sequence of which is 40% identical to that of cyanase from Escherichia coli. Introduction into a cyanase-deficient E. coli strain of a plasmid-borne slr0899 resulted in expression of low but significant activity of cyanase. Targeted interruption of a homolog of slr0899 from Synechococcus sp. strain PCC 7942, encoding a protein 77% identical to that encoded by slr0899, resulted in loss of cellular cyanase activity. These results indicated that slr0899 and its homolog in the strain PCC 7942 represent the cyanobacterial cyanase gene (designated cynS). While cynS of strain PCC 6803 is tightly clustered with the four putative molybdenum cofactor biosynthesis genes located downstream, cynS of strain PCC 7942 was found to be tightly clustered with the two genes located upstream, which encode proteins similar to the subunits of the cyanobacterial nitrate-nitrite transporter. In both strains, cynS was transcribed as a part of a large transcription unit and the transcription was negatively regulated by ammonium. Cyanase activity was low in ammonium-grown cells and was induced 7-to 13-fold by inhibition of ammonium fixation or by transfer of the cells to ammoniumfree media. These findings indicated that cyanase is an ammonium-repressible enzyme in cyanobacteria, the expression of which is regulated at the level of transcription. Similar to other ammonium-repressible genes in cyanobacteria, expression of cynS required NtcA, a global nitrogen regulator of cyanobacteria.Cyanase (EC 4.3.99.1), which catalyzes the decomposition of cyanate (NCO Ϫ ) into CO 2 and NH 3 , has been shown to be present in plants, some heterotrophic bacteria, and the cyanobacterium Synechococcus sp. strain PCC 6301 (6, 22). In living organisms, cyanate is formed by the decomposition of carbamoylphosphate (CP) or urea (2, 14). Cyanate reacts with nucleophilic groups of proteins and hence is a toxic compound. With cyanase, however, Escherichia coli cells can utilize cyanate as the sole source of nitrogen (11). Cyanate has been also shown to reversibly inhibit CP synthetase of E. coli (4). The proposed biological functions of cyanase in E. coli therefore include detoxification of endogenously formed cyanate, utilization of cyanate as the nitrogen source (11), and regulation of enzyme activities through modulation of the intracellular cyanate level (12). For the cyanobacterium Synechococcus sp. strain PCC 6301, Miller and Espie found a high activity of cyanase and estimated the rate of cyanate decomposition per cell to be 20 times that in E. coli (22). The high cyanase activity implies the importance of cyanate metabolism in cyanobacterial cells, but the physiological role of the enzyme remains to be clarified.In the investigation of the mechanism of ammonium-promoted activation and repression of the carbon and nitrogen assimilation genes, respectively, in Synechococcus sp. strain PCC 7942, we found that exogenously adde...

Nitrogen Control in Cyanobacteria

“…AB005890). Although the tsp for cynABDS has not been reported, the expression of these genes, tested with a cynS probe, has been shown to be NtcA dependent (43).…”

“…strain PCC 7942, cynS is clustered together with three genes, cynABD, which might encode an ABCtype transporter. Cyanase activity and cynS and its accompanying genes are subject to repression by ammonium (43).…”

“…strain WH7805 (20) and are also present in the genomes of Synechocystis sp. strain PCC 6803 (57) (43,81), and the cynS gene encoding cyanase has been identified in both strains (43; F. Jalali and G. S. Espie, GenBank accession no. AF001333).…”

The cyanate utilization capacity of marine unicellular Cyanobacteria

“…Recently, Espie et al (2007) reported an abundance of cyanate transport genes among bacteria. An ABC-type transporter for cyanate was identified in the freshwater cyanobacteria Synechococcus elongatus strain PCC7942 (Harano et al 1997) and Synechococcus sp. strain PCC6301.…”

Distribution and expression of the cyanate acquisition potential among cyanobacterial populations in oligotrophic marine waters

“…A number of Synechococcus and Prochlorococcus strains grow on cyanate as the sole N source (Miller and Espie 1994;Kamennaya et al 2008;Kamennaya and Post 2010). Cyanase (Enzyme Commission [EC] 4.2.1.104), first characterized in Escherichia coli (Anderson 1980;Harano et al 1997), catalyzes a reaction of cyanate with bicarbonate producing ammonium and carbon dioxide. Growth on cyanate as the sole N source requires its transport over the cell membrane followed by its conversion to ammonium.…”