Requirement of the exopolyphosphatase gene for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803.

Polyphosphate, which is ubiquitous in cells in nature, is involved in a myriad of cellular functions, and has been recently focused on its metabolism related with microbial acclimation to phosphorus-source fluctuation. In view of the ecological importance of cyanobacteria as the primary producers, this study investigated the responsibility of polyphosphate metabolism for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803, with the use of a disruptant (Δppx) as to the gene of exopolyphosphatase that is responsible for polyphosphate degradation. Δppx was similar to the wild type in the cellular content of polyphosphate to show no defect in cell growth under phosphorus-replete conditions. However, under phosphorus-starved conditions, Δppx cells were defective in a phosphorus-starvation dependent decrease of polyphosphate to show deleterious phenotypes as to their survival and the stabilization of the photosystem complexes. These results demonstrated some crucial role of exopolyphosphatase to degrade polyP in the acclimation of cyanobacterial cells to phosphorus-starved conditions. Besides, it was found that ppx expression is induced in Synechocystis cells in response to phosphorus starvation through the action of the two-component system, SphS and SphR, in the phosphate regulon. The information will be a foundation for a fuller understanding of the process of cyanobacterial acclimation to phosphorus fluctuation.

High intracellular phosphorus contents exhibit a correlation with arsenate resistance in chlamydomonas mutants.

Pi in the medium relieved the toxicity of arsenate against cellular growth of Chlamydomonas reinhardtii. To investigate the relationship between intracellular P contents and arsenate resistance, we determined the intracellular P contents of arsenate-sensitive and arsenate-resistant mutants, which had been generated by random insertional mutagenesis. All 13 arsenate-resistant mutants showed higher P contents than the parent strain, while arsenate-sensitive mutants with high P contents were not found. In one of the arsenate-resistant mutants, AR3, the intracellular P content was about twice that in the wild type during growth in the absence of arsenate. Arsenate incorporation in AR3 was suppressed within 10 min after the addition of 1 mM arsenate, while Pi incorporation continued even after arsenate uptake ceased. Whereas the P content of the wild type decreased to half in the presence of 0.5 mM arsenate, almost the same degree (about 50%) of decrease was observed in AR3 cells grown in the presence of as much as 3 mM arsenate. AR3, in which PTB1, a homolog of a Pi transporter gene, had been disrupted, exhibited a higher activity of a high-affinity Pi transporter, suggesting that it may be due to a compensatory transport activity. These data suggest that the intracellular level of P is one of the important factors of arsenate resistance.

Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2.

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 microM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 microM; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 microM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE(2) (1 microM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). ACh and ionomycin increased PGE(2) release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca(2+)](i) activates Ca(2+)-regulated exocytotic events and PGE(2) release mediated by COX-1. The released PGE(2) induces the accumulation of cAMP, which enhances the Ca(2+)-regulated exocytosis. The autocrine mechanism mediated by PGE(2) maintains the high-level mucin release from antral mucous cells during ACh stimulation.

Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas.

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.