Highlights
The
adc1
disruption of
Synechocystis
sp. PCC 6803 did not inhibit cell growth, as evidently shown by its similar growth with wild type, as well as its intracellular pigments.
The
Δadc1
mutant has the highest capacity to produce PHB up to 36.1 %w/DCW under nitrogen and phosphorus-deprived medium containing acetate (BG
11
-N-P+A).
Abundant PHB granules stained by Nile-Red are markedly visualized in
Δadc1
mutant under BG
11
-N-P and BG
11
-N-P+A when compared to those of wild type.
Transcript levels of
phaA
and
phaB
are mainly responsible to PHB content whereas the changed proportion of
phaC
and
phaE
is noted when a certain
phaC
transcript level is decreased.
The transport of spermidine into a cyanobacterium, Synechocystis sp. PCC 6803, was characterized by measuring the uptake of 14 C-spermidine. Spermidine transport was shown to be saturable with an apparent affinity constant (K m ) value of 67 µM and a maximal velocity (V max ) value of 0.45 nmol/min/mg protein. Spermidine uptake was pHdependent with the pH optimum being 8.0. The competition experiment showed strong inhibition of spermidine uptake by putrescine and spermine, whereas amino acids were hardly inhibitory. The inhibition kinetics of spermidine transport by putrescine and spermine was found to be noncompetitive with K i values of 292 and 432 µM, respectively. The inhibition of spermidine transport by various metabolic inhibitors and ionophores suggests that spermidine uptake is energy-dependent. The diminution of cell growth was observed in cells grown at a high concentration of NaCl. Addition of a low concentration of spermidine at 0.5 mM relieved growth inhibition by salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased spermidine transport with about 30-40% increase at 10 mosmol/kg upshift.
The potD gene encodes the bacterial substrate-binding subunit of the polyamine transport system. The uptake system, which belongs to the ABC transporters, has been characterized in Escherichia coli, but it has not been previously studied in cyanobacteria. Although the overall sequence identity between Synechocystis sp. strain PCC 6803 (hereafter Synechocystis) PotD and Escherichia coli PotD is 24%, the ligand-binding site in the constructed homology model of Synechocystis PotD is well conserved. The conservation of the five polyamine-binding residues (Asp206, Glu209, Trp267, Trp293, and Asp295 in Synechocystis PotD) between these two species indicated polyamine-binding capacity for Synechocystis PotD. The Synechocystis potD gene is functional and its expression is under environmental regulation at transcriptional as well as post-transcriptional levels. Furthermore, an in vitro binding assay with the purified recombinant PotD protein demonstrated that the Synechocystis PotD protein is able to bind polyamines and favors spermidine over putrescine. Finally, we confirmed that Synechocystis PotD plays a physiological role in the uptake of polyamines in vivo using a constructed Synechocystis potD-disruption mutant.
The in vivo function of polyamine binding protein D (PotD) in Synechocystis sp. PCC 6803 for the transport of spermidine was investigated using Synechocystis mutant disrupted in potD gene. The growth rate of potD mutant was similar to that of wild-type when grown in BG11 medium. However, the mutant exhibited severely reduced growth compared to the wild-type when BG11 medium was supplemented with 0.5 mM spermidine. The mutant accumulated a higher spermidine level than the wild-type when grown in the medium with or without spermidine. Transport experiments revealed that the mutant had a reduction in both the uptake and the excretion of spermidine. Moreover, [(14)C]spermidine-loaded wild-type and mutant cells showed a decrease of [(14)C]spermidine excretion when the assay medium contained exogenous spermidine. These data suggest that PotD is involved in both the uptake and the excretion of spermidine in Synechocystis cells.
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