Polyamine transport into cyanobacterium Synechocystis sp. PCC 6803

Abstract

The transport of polyamines into a moderately salt tolerant cyanobacterium Synechocystis sp. PCC 6803 was characterized by measuring the uptake of radioactively-labeled polyamines namely putrescine and spermidine. The results showed that putrescine and spermidine transport exhibited saturation kinetics typical of Michaelis-Menten kinetics with an apparent Km of 98 and 67 µM, respectively, and Vmax of 0.33 and 0.45 nmol/min/mg protein, respectively. The transport of putrescine was pH-dependent with highest activity at pH 7.0, whilst spermidine transport was pH-dependent with pH optimum 8.0. The competition experiment showed strong inhibition of spermidine uptake by putrescine and spermine. Also strong inhibition of putrescine transport was caused by spermine and spermidine. Additionally, amino acids were hardly inhibitory to polyamine uptakes. The inhibition kinetics of spermidine transport by putrescine and spermine were found to be non-competitive with Ki values of 292 and 432 µM, respectively. These results suggest that the transport system in Synechocystis sp. PCC 6803 is highly specific for polyamines. The inhibition of putrescine and spermidine transport by various metabolic inhibitors and ionophores suggests that polyamine uptake is energy-dependent and proton motive force-dependent with the contribution of both ∆pH and ∆ψ. The diminution of cell growth was observed in cells grown at high concentration of NaCl. The addition of low concentration of putrescine and spermidine relieved growth inhibition by salt stress, suggesting that exogenous putrescine and spermidine could be transported into Synechocystis cells and could act as growth promoter in the presence of high salt concentration. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased uptake with an optimum 2-fold increase at 20 mosmol/kg and 1.5-fold increase at 10 mosmol/kg for putrescine and spermidine transport, respectively. At the post-transcriptional level, reverse transcription PCR demonstrated that the steady-state transcript amounts of the potD gene, encoding polyamine-binding protein were under regulation of a wide spectrum of long-term environmental stresses (3 days), namely light intensity, salt, osmotic, temperature and nutrient availability even if no short-term regulation (18 h) occurred under the same conditions. The decreased accumulation of the potD transcripts was not a result of a decreased stability of the transcript, but merely reflected an altered transcription activity by high temperature stress. Furthermore, the immunoblot analysis revealed that the recombinant Synechocystis His-PotD protein showed high specificity to monoclonal Anti-His antibody with a single protein band of 46 kDa. The sequence identity between Synechocystis PotD and Escherichia coli PotD was only 24 %, but the overall fold and the active site are well conserved. The putative polyamine-binding protein, PotD, has no amino terminal signal sequences.