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Thermodynamics of copper and zinc distribution in the cyanobacterium Synechocystis PCC 6803.

ABSTRACT: Copper is supplied to plastocyanin for photosynthesis and cytochrome c oxidase for respiration in the thylakoids of Synechocystis PCC 6803 by the membrane-bound P-type ATPases CtaA and PacS, and the metallochaperone Atx1. We have determined the Cu(I) affinities of all of the soluble proteins and domains in this pathway. The Cu(I) affinities of the trafficking proteins range from 5 × 10(16) to 5 × 10(17) M(-1) at pH 7.0, consistent with values for homologues. Unusually, Atx1 binds Cu(I) significantly tighter than the metal-binding domains (MBDs) of CtaA and PacS (CtaA(N) and PacS(N)), and equilibrium copper exchange constants of approximately 0.2 are obtained for transfer to the MBDs. Dimerization of Atx1 increases the affinity for Cu(I), but the loop 5 His61 residue has little influence. The MBD of the zinc exporter ZiaA (ZiaA(N)) exhibits an almost identical Cu(I) affinity, and Cu(I) exchange with Atx1, as CtaA(N) and PacS(N), and the relative stabilities of the complexes must enable the metallochaperone to distinguish between the MBDs. The binding of potentially competing zinc to the trafficking proteins has been studied. ZiaA(N) has the highest Zn(II) affinity and thermodynamics could be important for zinc removal from the cell. Plastocyanin has a Cu(I) affinity of 2.6 × 10(17) M(-1), 15-fold tighter than that of the Cu(A) site of cytochrome c oxidase, highlighting the need for specific mechanisms to ensure copper delivery to both of these targets. The narrow range of Cu(I) affinities for the cytoplasmic copper proteins in Synechocystis will facilitate relocation when copper is limiting.

SUBMITTER: Badarau A

PROVIDER: S-EPMC3156197 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Thermodynamics of copper and zinc distribution in the cyanobacterium Synechocystis PCC 6803.

Badarau Adriana A Dennison Christopher C

Proceedings of the National Academy of Sciences of the United States of America 20110721 32

Copper is supplied to plastocyanin for photosynthesis and cytochrome c oxidase for respiration in the thylakoids of Synechocystis PCC 6803 by the membrane-bound P-type ATPases CtaA and PacS, and the metallochaperone Atx1. We have determined the Cu(I) affinities of all of the soluble proteins and domains in this pathway. The Cu(I) affinities of the trafficking proteins range from 5 × 10(16) to 5 × 10(17) M(-1) at pH 7.0, consistent with values for homologues. Unusually, Atx1 binds Cu(I) significa ...[more]

PMID: 21778408

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Project description:BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Dataset Information

Thermodynamics of copper and zinc distribution in the cyanobacterium Synechocystis PCC 6803.

Publications

Thermodynamics of copper and zinc distribution in the cyanobacterium Synechocystis PCC 6803.

Similar Datasets

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