Uptake and accumulation of putrescine and its lethality in Anacystis nidulans.

Abstract

The rate of uptake of putrescine by Anacystis nidulans has been shown to depend on the external pH and the extracellular concentration of putrescine. Accumulation of exogenous putrescine was also proportional to the concentration of putrescine in the medium, suggesting that putrescine uptake was not subject to cellular regulation. An equation was derived to test the hypothesis that putrescine accumulation was due to ion trapping. Comparison of the predicted and observed intracellular concentrations of putrescine under various conditions showed a close correlation in support of the hypothesis of ion trapping. Under conditions leading to cell death (e.g., 150 isM putrescine, pH 9.8), the correlation did not hold as a result of leakage of accumulated putrescine.It has been observed that exogenous putrescine is inhibitory to the photoautotrophic growth of Anacystis nidulans (1). The fact that exogenous putrescine was toxic at the concentration (0.15 mM) at which this normal metabolite is present within the cyanobacterium during exponential growth has led to additional studies of the mechanism of this effect. Uptake of putrescine and the other polyamines appears to be mediated by active transport in Escherichia coli (2) and in animal cells (3, 4), but nothing was known about polyamine transport in the cyanobacteria. A. nidulans is usually grown in a minimal medium that has a low buffering capacity, resulting in a large increase in the external pH during exponential growth (1). The cytoplasmic pH, however, remains relatively constant as the external pH is altered (5) and can be either acidic or basic relative to the external pH. The resultant pH gradient between the cytoplasm and the medium can cause the concentration of weak acids and bases (refs. 5 and 6; J. Gibson, unpublished results) which are permeable in the uncharged form but are impermeable when charged. This mechanism of accumulation (ion trapping or diffusion trapping) also accounts for the concentration of NH3 in green algae (7) and bicarbonate in chloroplasts (8). An analysis of the kinetics of putrescine uptake and of the equilibrium concentration of accumulated putrescine within the cell as a function of the pH of the medium and external concentration of putrescine is consistent with the theory that the concentration of putrescine is due to diffusion of the neutral molecule and trapping of the charged ion. MATERIALS AND METHODSGrowth and Viability of A. nidulans. A. nidulans 625 was obtained from the Indiana University Culture Collection (Bloomington, IN). Strain 625 was grown at 30'C with rotary shaking in Allen's medium containing 1% NaHCO3 with illumination from the top with white light at 7-8 W/cm2, as described (1). Cell number was determined in a Petroff-Hauser microscopic counting chamber. Viability was measured by the single cell plating technique (9).Uptake of Putrescine. Exponential phase cultures were harvested by centrifugation at 10,000 X g for 5 min, washed and resuspended in fresh growth medium containing 30 mM NaHCO3, and adjus...

Ustilago maydis spermidine synthase is encoded by a chimeric gene, required for morphogenesis, and indispensable for survival in the host

“…Apparently, this bifunctional gene is specific of basidiomycete fungi, because an in silico search for homologues gave similar results for all the basidiomycete species whose genomes have been sequenced, but not for any other eukaryotic organism belonging to different taxa. In other organisms, these same authors suggested that the polyamine was oxidized with the formation of a toxic aldehyde (Davis & Ristow, 1988), whereas in the cyanobacterium Anacystis nidulans toxicity was associated with its conjugation to ribosomes, affecting their protein-synthesizing activity (Guarino & Cohen, 1979). Kingsbury et al (2004) did not find evidence of any post-transcriptional regulation of the C. neoformans homologous bifunctional gene in response to exogenous addition or starvation of spermidine or lysine.…”

“…Accordingly, we considered the possibility that in spe mutants, putrescine would be accumulated, reaching toxic concentrations (Guarino & Cohen, 1979;Davis & Ristow, 1988;Tome et al, 1997). This result was rather peculiar because it is known that U. maydis homologous recombination is rather high (20-65%).…”

Inorganic Carbon Accumulation and Photosynthesis in a Blue-green Alga as a Function of External pH

“…The remaining basic solution (150 Ml) was acidified by the addition of 200 M1 of 2 M HC104, the acid-labile 14C removed in a CO2 stream and the remaining "C activity counted in ACS scintillation cocktail (10 ml, Amersham-Searle Corp. methylamine between the cell and the incubation medium as described by other workers (11,14,21). Cells used in these experiments were preincubated for 10 min at the appropriate pH as previously described.…”

Cellular systems for the study of the biosynthesis of polyamines and ethylene, as well as of virus multiplication

“…It was found early in our studies that exogenous putrescine is quite toxic to Cyanobacteria [13,14,18]. The bacteria concentrate putrescine to very high levels and the diamine is bound covalently to proteins of the ribosomal subunits as well as to cell wall polymers.…”