Study on a novel pathway for phosphate uptake in the cyanobacterium Synechocystis sp. PCC 6803

概要

Phosphorus (P), which has various roles in the cells, is an essential element for all living organisms. In this respect, improving the knowledge of the metabolism of P is directly connected to the understandings of lives.

　P is also an indispensable element for the present society. To support the increasing global population and their food demands, continuous fertilization containing P nutrition is required for maintaining agricultural production. However, most of P for fertilizers are obtained from phosphate (Pi) rock, a limited natural resource. On the other hand, ironically, P is considered a critical factor for eutrophication. It is a common understanding that the surplus of Pi to aquatic systems, which originated from human activity, induces eutrophication. Since the availability of Pi in the water environment is naturally very limited and excess emission of Pi promotes the growth of algae, a primary producer in the conditions, it causes severe water pollutions, defined as eutrophication. The spill out of P into the aquatic system must be restrained. Therefore, for the sustainable development of our society, the development of the recovery system for Pi is required.

　In the present study, I attempted to contribute to this requirement by improving the understanding of the metabolism of P in the freshwater cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis).

　Firstly, I attempted to develop an algal sustainable Pi recovery system using genetically engineered Synechocystis cells. In the previous studies, a mutant of the sphS gene for Pi- deficient sensor kinase, in which the PAS domain in the signal-input domain was deleted (∆PAS), constitutively expresses the pho-regulon including the high-affinity Pi transporters, Pst1 and Pst2. I, thus, expected this strain absorbs higher Pi than the wild-type strain. As the expectation, I found that the ∆PAS cells showed over four-fold higher Pi-uptake (461 ± 97 nM/d) than that of the wild-type cells (93 ± 15 nM/d), suggesting that the established phenotype of the ∆PAS cells, which expresses the Pi-uptake system constitutively, is appropriate for the Pi recovery system. Based on these results, I attempted to establish a transformant overexpressing the ppk gene for polyphosphate kinase, synthesizing polyphosphate (poly-P) in the wild-type and ∆PAS cells. In addition, the inactivation of the ppx gene for exopolyphosphatase, degrading poly-P, was accomplished for further accumulation of poly-P. Pi uptake in newly established strains was verified in the cultures containing 180 µM and 4 mM Pi. Regardless of whether the ppk gene was overexpressed, the uptake and storage of Pi in the cells were not altered. Moreover, the uptake of Pi in ΔPAS strains, which were showing consistent expression of the transporter for Pi, ceased for an unknown reason after 2 d of cultivation in 4 mM Pi conditions. Although this cease of Pi uptake in ∆PAS cells make difficult to develop the Pi-uptake system using ∆PAS cells, I found peculiar stimulation of the Pi uptake following severe chlorosis in the wild-type Synechocystis cells grown under the Pi-excess conditions was observed. Hence, I attempted to explore this phenomenon in detail.

　The Synechocystis cells were cultivated with the BG-11 medium containing an excess Pi concentration (4 mM) to explore the novel Pi uptake, while Pi concentration in the standard BG-11 medium is 0.18 mM. Pi uptake rates under both conditions were similar (94 ± 6 µM/d for 0.18 mM Pi condition and 80 ± 33 μM/d for 4 mM Pi condition) until 3 d, despite being more than 20-times different in the Pi concentration. Surprisingly, the Pi uptake rate was stimulated in the high Pi conditions after 3 d and continued until 5 d. Remarkably, the Synechocystis cells showed more than 14-folds higher Pi uptake (1,330 ± 45 µM/d) at that time compared to that in 0.18 mM conditions (94 ± 6 µM/d). To investigate the factor(s) causing this phenomenon, I cultivated the cells in the BG-11 medium in which the concentration of each component was altered and found that the shortage of MgSO4 under the high Pi conditions caused the phenomenon. More specifically, sulfate (SO42-) deficiency induced the rapid Pi uptake, and over-accumulation of Pi and magnesium (Mg2+) deficiency caused chlorosis and released the intracellularly accumulated Pi to the media. Moreover, reverse genetics revealed that the rapid Pi uptake occurred via an ABC transporter induced by sulfate deficiency, namely, a putative sulfate transporter, SbpA-CysTWA. Although it is thought that the transporters may have very high specificity to the substrate, my results contradicted this common sense.

　To sum up, I revealed a novel pathway for phosphate uptake in the cyanobacterium Synechocystis sp. PCC 6803 and my findings provide a new perspective; Pi uptake induced by the shortage of sulfate is originated from unspecific or undesirable uptake with sulfate transporter.