Dataset Information

A gene (ccmA) required for carboxysome formation in the cyanobacterium Synechocystis sp. strain PCC6803.

ABSTRACT: A high-CO2-requiring mutant, G7, of Synechocystis sp. strain PCC6803 capable of inorganic carbon transport but unable to utilize the intracellular inorganic carbon pool for photosynthesis was isolated. Transmission electron micrographs of the mutant indicated that the mutant does not have any carboxysomes. A clone (pHPG7) with a 7.5-kbp DNA insert that transforms the G7 mutant to the wild-type phenotype was isolated from a genomic library of wild-type Synechocystis sp. strain PCC6803. Complementation tests with subclones identified the mutation site in G7 within 208 bp. Sequencing of nucleotides in this region elucidated an open reading frame, designated ccmA, encoding a protein of 302 amino acids. Cloning and sequence analysis of the respective G7 gene revealed an A-to-G substitution that results in an Asp-to-Gly substitution in the deduced amino acid. The result indicated that the ccmA gene encodes a protein essential for the formation of carboxysomes. An open reading frame encoding a proline-rich protein of 271 amino acids was found downstream of the ccmA gene, but no ccm-like genes or rbc operon was found in this region.

SUBMITTER: Ogawa T

PROVIDER: S-EPMC205361 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

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A gene (ccmA) required for carboxysome formation in the cyanobacterium Synechocystis sp. strain PCC6803.

Ogawa T T Marco E E Orus M I MI

Journal of bacteriology 19940401 8

A high-CO2-requiring mutant, G7, of Synechocystis sp. strain PCC6803 capable of inorganic carbon transport but unable to utilize the intracellular inorganic carbon pool for photosynthesis was isolated. Transmission electron micrographs of the mutant indicated that the mutant does not have any carboxysomes. A clone (pHPG7) with a 7.5-kbp DNA insert that transforms the G7 mutant to the wild-type phenotype was isolated from a genomic library of wild-type Synechocystis sp. strain PCC6803. Complement ...[more]

PMID: 8157606

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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:BACKGROUND:The use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of product is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for maximising gene expression and subsequent product formation following genetic manipulation. RESULTS:A native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location. CONCLUSIONS:A significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing Synechocystis PCC6803 strains having high level, controlled expression may be more achievable.

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

A gene (ccmA) required for carboxysome formation in the cyanobacterium Synechocystis sp. strain PCC6803.

Publications

A gene (ccmA) required for carboxysome formation in the cyanobacterium Synechocystis sp. strain PCC6803.

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