Na+-stimulated ATPase of alkaliphilic halotolerant cyanobacterium Aphanothece halophytica translocates Na+ into proteoliposomes via Na+ uniport mechanism

<p>Abstract</p> <p>Background</p> <p>When cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na⁺from cells to maintain the cytoplasmic Na⁺concentration. Until now, the ATPase involved in Na⁺transport in cyanobacteria has not been characterized. Here, the characterization of ATPase and its role in Na⁺transport of alkaliphilic halotolerant <it>Aphanothece halophytica </it>were investigated to understand the survival mechanism of <it>A. halophytica </it>under high salinity conditions.</p> <p>Results</p> <p>The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na⁺but not K⁺, Li⁺and Ca²⁺. The apparent K_{<it>m </it>}values for Na⁺and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F₁F₀-ATPase based on the usual subunit pattern and the protection against <it>N,N'</it>-dicyclohexylcarbodiimide inhibition of ATPase activity by Na⁺in a pH-dependent manner. Proteoliposomes reconstituted with the purified enzyme could take up Na⁺upon the addition of ATP. The apparent K_{<it>m </it>}values for this uptake were 3.3 and 0.5 mM for Na⁺and ATP, respectively. The mechanism of Na⁺transport mediated by Na⁺-stimulated ATPase in <it>A. halophytica </it>was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na⁺-stimulated ATPase was observed upon the addition of ATP with Na⁺but not with K⁺, Li⁺and Ca²⁺. The Na⁺- and ATP-dependent alkalization of the proteoliposome lumen was stimulated by carbonyl cyanide <it>m </it>- chlorophenylhydrazone (CCCP) but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na⁺uptake activity. The uptake of Na⁺was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na⁺-stimulated ATPase of the proteoliposomes.</p> <p>Conclusion</p> <p>The data demonstrate that Na⁺-stimulated ATPase from <it>A. halophytica</it>, a likely member of F-type ATPase, functions as an electrogenic Na⁺pump which transports only Na⁺upon hydrolysis of ATP. A secondary event, Na⁺- and ATP-dependent H⁺efflux from proteoliposomes, is driven by the electric potential generated by Na⁺-stimulated ATPase.</p

Dark fermentative hydrogen production and transcriptional analysis of genes involved in the unicellular halotolerant cyanobacterium Aphanothece halophytica under nitrogen and potassium deprivation

The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H2) producer. This study aimed to investigate the enhancement of H2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H2 production by A. halophytica, while no differences in H2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H2 production was investigated. The results showed that the highest H2 accumulation of 1,261.96 ± 96.99 µmol H2 g dry wt−1 and maximum hydrogenase activity of 179.39 ± 8.18 µmol H2 g dry wt−1 min−1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG110-K) for 48 h. An increase in hydrogenase activity was attributed to the decreased O2 concentration in the system, due to a reduction of photosynthetic O2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis (glgA, glgB, and glgP) were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells

Na+-stimulated phosphate uptake system in Synechocystis sp. PCC 6803 with Pst1 as a main transporter

<p>Abstract</p> <p>Background</p> <p>Most living cells uptake phosphate, an indispensable nutrient for growth from their natural environment. In <it>Synechocystis </it>sp. PCC 6803, the cells lack phosphate-inorganic transport (Pit) system but contain two phosphate-specific transport (Pst) systems, Pst1 and Pst2. We investigated the kinetics of Pi uptake of these two Pst systems by constructing the two mutants, ΔPst1 and ΔPst2, and comparing their kinetic properties with those of the wild-type cells under both Pi-sufficient and deficient conditions. The effects of pH and Na⁺on the uptake of phosphate in <it>Synechocystis </it>were also studied.</p> <p>Results</p> <p>Growth rates of the two mutants and wild type were similar either under phosphate-sufficient or deficient condition. The <it>K_m</it>for phosphate uptake was 6.09 μM in wild type and this was reduced to 0.13 μM in ΔPst1 cells and 5.16 μM in the ΔPst2 strain. The <it>V_max</it>values of 2.48, 0.22, and 2.17 μmol • (min • mg of chlorophyll <it>a</it>)^-1were obtained for wild type, the ΔPst1 and ΔPst2 strains, respectively. A monophasic phosphate uptake was observed in wild-type cells. The uptake of phosphate was energy and pH-dependent with a broad pH optimum between pH 7-10. Osmolality imposed by NaCl stimulated phosphate uptake whereas that imposed by sorbitol decreased uptake, suggesting stimulation of uptake was dependent upon ionic effects.</p> <p>Conclusion</p> <p>The data demonstrate that Pst2 system of <it>Synechocystis </it>has higher affinity toward phosphate with lower <it>V_max</it>than Pst1 system. The Pst1 system had similar <it>K_m</it>and <it>V_max</it>values to those of the wild type suggesting that Pst1 is the main phosphate transporter in <it>Synechocystis </it>sp. PCC 6803. The <it>K_m</it>of Pst1 of <it>Synechocystis </it>is closer to that of Pit system than to that of the Pst system of <it>E. coli</it>, suggesting that <it>Synechocystis </it>Pst1 is rather a medium/low affinity transporter whereas Pst2 is a high affinity transporter.</p

Synechocystis sp. PCC 6803 overexpressing genes involved in CBB cycle and free fatty acid cycling enhances the significant levels of intracellular lipids and secreted free fatty acids

The integrative aspect on carbon fixation and lipid production is firstly implemented in cyanobacterium Synechocystis sp. PCC 6803 using metabolic engineering approach. Genes related to Calvin-Benson-Bassham (CBB) cycle including rbcLXS and glpD and free fatty acid recycling including aas encoding acyl-ACP synthetase were practically manipulated in single, double and triple overexpressions via single homologous recombination. The significantly increased growth rate and intracellular pigment contents were evident in glpD-overexpressing (OG) strain among all strains studied under normal growth condition. The triple aas_glpD_rbcLXS-overexpressing (OAGR) strain notably gave the highest contents of both intracellular lipids and extracellular free fatty acids (FFAs) of about 35.9 and 9.6% w/DCW, respectively, when compared to other strains at day 5 of cultivation. However, the highest intracellular lipid titer and production rate were observed in OA strain at day 5 (228.7mg/L and 45.7mg/L/day, respectively) and OG strain at day 10 (358.3mg/L and 35.8mg/L/day, respectively) due to their higher growth. For fatty acid (FA) compositions, the main saturated fatty acid of palmitic acid (C16:0) was dominantly found in both intracellular lipid and secreted FFAs fractions. Notably, intracellular FA proportion of myristic acid (C14:0) was induced in all engineered strains whereas the increase of stearic acid (C18:0) composition was found in extracellular FFAs fraction. Altogether, these overexpressing strains efficiently produced higher lipid production via homeostasis balance on both its lipid synthesis and FFAs secretion

Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG11-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth