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A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

ABSTRACT: Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily from (i) genes of two-component signal transducers of Synechocystis, (ii) Synechocystis genes whose homologues are conserved in the genome of Arabidopsis thaliana, and (iii) genes of unknown function on the Synechocystis chromosome. A total of 3236 independent two-hybrid interactions involving 1920 proteins (52% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure, as well as the general features of interacting partners. The interaction data obtained in this study should provide new insights and novel strategies for functional analyses of genes in Synechocystis, and, additionally, genes in other cyanobacteria and plant genes of cyanobacterial origin.

SUBMITTER: Sato S

PROVIDER: S-EPMC2779905 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

Sato Shusei S Shimoda Yoshikazu Y Muraki Akiko A Kohara Mitsuyo M Nakamura Yasukazu Y Tabata Satoshi S

DNA research : an international journal for rapid publication of reports on genes and genomes 20071001 5

Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily ...[more]

PMID: 18000013

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Project description:Background:Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p, in short: a 'mannitol cassette'), proved to be genetically unstable. In this study, we aim to overcome this genetic instability by conceiving a strategy to stabilize mannitol production using Synechocystis sp. PCC6803 as a model cyanobacterium. Results:Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild-type Synechocystis, complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions:Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.

Project description:BACKGROUND:The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. RESULTS:The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d-1 and 0.0303% (v/v) ethanol d-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. CONCLUSIONS:Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.

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A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

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A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

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