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ClpB1 overproduction in Synechocystis sp. strain PCC 6803 increases tolerance to rapid heat shock.

ABSTRACT: ClpB1 is a heat shock protein known to disaggregate large protein complexes. Constitutive, 16-fold ClpB1 overproduction in the cyanobacterium Synechocystis sp. strain PCC 6803 increased cell survival by 20-fold when cultures were heated quickly (1°C/s) to 50°C and delayed cell death by an average of 3 min during incubation at high temperatures (>46°C). Cooverexpression of ClpB1 and another heat shock protein, DnaK2, further increased cell survival. According to immunocytochemistry results, ClpB1 is dispersed throughout the cytoplasm but is concentrated in specific areas and is more prevalent near thylakoid membranes. However, ClpB1 overproduction does not lead to a change in the morphology, chlorophyll content, or photosystem ratio. Whereas electron microscopy demonstrated that apparent protein aggregation occurred after heat treatment in the control strain, protein aggregate size was maintained in the ClpB1 overexpresser. Constitutive ClpB1 overproduction allows an earlier response to heat shock and protects from rapid heating of cultures.

SUBMITTER: Gonzalez-Esquer CR

PROVIDER: S-EPMC3811215 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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ClpB1 overproduction in Synechocystis sp. strain PCC 6803 increases tolerance to rapid heat shock.

Gonzalez-Esquer C Raul CR Vermaas Wim F J WF

Applied and environmental microbiology 20130802 20

ClpB1 is a heat shock protein known to disaggregate large protein complexes. Constitutive, 16-fold ClpB1 overproduction in the cyanobacterium Synechocystis sp. strain PCC 6803 increased cell survival by 20-fold when cultures were heated quickly (1°C/s) to 50°C and delayed cell death by an average of 3 min during incubation at high temperatures (>46°C). Cooverexpression of ClpB1 and another heat shock protein, DnaK2, further increased cell survival. According to immunocytochemistry results, ClpB1 ...[more]

PMID: 23913426

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Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

Project description:Motile strains of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 readily aggregate into flocs, or floating multicellular assemblages, when grown in liquid culture. As described here, we used confocal imaging to probe the structure of these flocs, and we developed a quantitative assay for floc formation based on fluorescence imaging of 6-well plates. The flocs are formed from strands of linked cells, sometimes packed into dense clusters but also containing voids with very few cells. Cells within the dense clusters show signs of nutrient stress, as judged by the subcellular distribution of green fluorescent protein (GFP)-tagged Vipp1 protein. We analyzed the effects on flocculation of a series of mutations that alter piliation and motility, including ?hfq, ?pilB1, ?pilT1, and ?ushA mutations and deletion mutations affecting major and minor pilins. The extent of flocculation is increased in the hyperpiliated ?pilT1 mutant, but active cycles of pilus extension and retraction are not required for flocculation. Deletion of PilA1, the major subunit of type IV pili, has no effect on flocculation; however, flocculation is lost in mutants lacking an operon coding for the minor pilins PilA9 to -11. Therefore, minor pilins appear crucial for flocculation. We show that flocculation is a tightly regulated process that is promoted by blue light perception by the cyanobacteriochrome Cph2. Floc formation also seems to be a highly cooperative process. A proportion of nonflocculating ?hfq cells can be incorporated into wild-type flocs, but the presence of a high proportion of ?hfq cells disrupts the large-scale architecture of the floc.IMPORTANCE Some bacteria form flocs, which are multicellular floating assemblages of many thousands of cells. Flocs have been relatively little studied compared to surface-adherent biofilms, but flocculation could play many physiological roles, be a crucial factor in marine carbon burial, and enable more efficient biotechnological cell harvesting. We studied floc formation and architecture in the model cyanobacterium Synechocystis sp. strain PCC 6803, using mutants to identify specific cell surface structures required for floc formation. We show that floc formation is regulated by blue and green light perceived by the photoreceptor Cph2. The flocs have a characteristic structure based on strands of linked cells aggregating into dense clusters. Cells within the dense clusters show signs of nutrient stress, pointing to a disadvantage of floc formation.

Dataset Information

ClpB1 overproduction in Synechocystis sp. strain PCC 6803 increases tolerance to rapid heat shock.

Publications

ClpB1 overproduction in Synechocystis sp. strain PCC 6803 increases tolerance to rapid heat shock.

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