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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: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.

Project description:BackgroundThis study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product.ResultsIn total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level.ConclusionThe most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.

Dataset Information

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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