A rapid and low-cost method for genomic DNA extraction from the cyanobacterium Synechocystis.
ABSTRACT: A two-step method is reported for preparation of genomic DNA from the model cyanobacterium Synechocystis that can be performed with minimal equipment and reagents in about an hour. High yields of genetic material can be obtained (200-450?ng/?l) with reasonable purity. A further ethanol precipitation step can be included but is not necessary if template is simply required for polymerase chain reaction (PCR) or digestion. This new protocol is helpful for amplification of genes of interest in early-stage research projects and for low throughput screening of transformants. It is more reliable than colony PCR of Synechocystis cultures, and less involved and cheaper than existing clean-DNA preparation methods. It represents an unusually simple and reliable extraction protocol for the growing body of research making use of this cyanobacterium.
Project description:Synechocystis sp. strain PCC 6714 is a unicellular cyanobacterium closely related to the popular model organism Synechocystis sp. strain PCC 6803. A combination of PacBio SMRT and Illumina GAIIx data results in a highly accurate finished genome sequence that provides a reliable resource for further comparative analyses.
Project description:The naturally transformable cyanobacterium Synechocystis sp. PCC 6803 is a widely used chassis strain for the photosynthetic production of chemicals. However, Synechocystis possesses multiple genome copies per cell which means that segregating mutations across all genome copies can be time-consuming. Here we use flow cytometry in combination with DNA staining to investigate the effect of phosphate deprivation on the genome copy number of the glucose-tolerant GT-P sub-strain of Synechocystis 6803. Like the PCC 6803 wild type strain, the ploidy of GT-P cells grown in BG-11 medium is growth phase dependent with an average genome copy number of 6.05 ± 0.27 in early growth (OD740 = 0.1) decreasing to 2.49 ± 0.11 in late stationary phase (OD740 = 7). We show that a 10-fold reduction in the initial phosphate concentration of the BG-11 growth medium reduces the average genome copy number of GT-P cells from 4.51 ± 0.20 to 2.94 ± 0.13 and increases the proportion of monoploid cells from 0 to 6% after 7 days of growth. In addition, we also show that the DnaA protein, which unusually for bacteria is not required for DNA replication in Synechocystis, plays a role in restoring polyploidy upon subsequent phosphate supplementation. Based on these observations, we have developed an alternative natural transformation protocol involving phosphate depletion that decreases the time required to obtain fully segregated mutants.
Project description:Cyanobacteria have evolved various strategies to sense and adapt to biotic and abiotic stresses including active movement. Motility in cyanobacteria utilizing the type IV pili (TFP) is useful to cope with changing environmental conditions. The model cyanobacterium Synechocystis sp. PCC 6803 (hereafter named Synechocystis) exhibits motility via TFP called thick pili, and uses it to seek out favorable light/nutrition or escape from unfavorable conditions. Recently, a number of studies on Synechocystis thick pili have been undertaken. Molecular approaches support the role of the pilin in motility, cell adhesion, metal utilization, and natural competence in Synechocystis. This review summarizes the most recent studies on the function of thick pili as well as their formation and regulation in this cyanobacterium.
Project description:The unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a photoautotrophic host for synthetic biology studies. However, as a green chassis to capture CO2 for biotechnological applications, the genetic toolbox for Synechocystis 6803 is still a limited factor.We systematically characterized endogenous genetic elements of Synechocystis 6803, including promoters, ribosome binding sites, transcription terminators, and plasmids. Expression from twelve native promoters was compared by measuring fluorescence from the reporter protein EYFP in an identical setup, exhibiting an 8000-fold range of promoter activities. Moreover, we measured the strength of twenty native ribosome binding sites and eight native terminators, indicating their influence on the expression of the reporter genes. In addition, two shuttle vectors, pCA-UC118 and pCB-SC101, capable of replication in both Synechocystis 6803 and E. coli were constructed. Expression of reporter proteins were significantly enhanced in cells containing these new plasmids, thus providing superior gene expression platforms in this cyanobacterium.The results of this study provide useful and well characterized native tools for bioengineering work in the model cyanobacterium Synechocystis 6803.
Project description:Cyanobacteria exhibit a great capacity to adapt to different environmental conditions through changes in gene expression. Although this plasticity has been extensively studied in the model cyanobacterium Synechocystis sp. PCC 6803, a detailed analysis of the coordinated transcriptional adaption across varying conditions is lacking. Here, we report a meta-analysis of 756 individual microarray measurements conducted in 37 independent studies-the most comprehensive study of the Synechocystis transcriptome to date. Using stringent statistical evaluation, we characterized the coordinated adaptation of Synechocystis' gene expression on systems level. Evaluation of the data revealed that the photosynthetic apparatus is subjected to greater changes in expression than other cellular components. Nevertheless, network analyses indicated a significant degree of transcriptional coordination of photosynthesis and various metabolic processes, and revealed the tight co-regulation of components of photosystems I, II and phycobilisomes. Detailed inspection of the integrated data led to the discovery a variety of regulatory patterns and novel putative photosynthetic genes. Intriguingly, global clustering analyses suggested contrasting transcriptional response of metabolic and regulatory genes stress to conditions. The integrated Synechocystis transcriptome can be accessed and interactively analyzed via the CyanoEXpress website (http://cyanoexpress.sysbiolab.eu).
Project description:Biophotovoltaic devices utilize photosynthetic organisms such as the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) to generate current for power or hydrogen production from light. These devices have been improved by both architecture engineering and genetic engineering of the phototrophic organism. However, genetic approaches are limited by lack of understanding of cellular mechanisms of electron transfer from internal metabolism to the cell exterior. Type IV pili have been implicated in extracellular electron transfer (EET) in some species of heterotrophic bacteria. Furthermore, conductive cell surface filaments have been reported for cyanobacteria, including Synechocystis. However, it remains unclear whether these filaments are type IV pili and whether they are involved in EET. Herein, a mediatorless electrochemical setup is used to compare the electrogenic output of wild-type Synechocystis to that of a ?pilD mutant that cannot produce type IV pili. No differences in photocurrent, i.e., current in response to illumination, are detectable. Furthermore, measurements of individual pili using conductive atomic force microscopy indicate these structures are not conductive. These results suggest that pili are not required for EET by Synechocystis, supporting a role for shuttling of electrons via soluble redox mediators or direct interactions between the cell surface and extracellular substrates.
Project description:A new sequence-specific endonuclease from the cyanobacterium Synechocystis species PCC 6701 has been purified and characterized. This enzyme, SecI, is unique in recognizing the nucleotide sequence: 5' -CCNNGG-3' 3' -GGNNCC-5' and cleaves it at the position indicated by the symbol. Two other restriction endonucleases, SecII and SecIII, found in this organism are isoschizomers of MspI and MstII, respectively.
Project description:Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) and Sycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in the loss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recovered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants. Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of the pilA9-pilA11 operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region of pilA9 Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.
Project description:Background:Cyanobacteria can be metabolically engineered to convert CO2 to fuels and chemicals such as ethylene. A major challenge in such efforts is to optimize carbon fixation and partition towards target molecules. Results:The efe gene encoding an ethylene-forming enzyme was introduced into a strain of the cyanobacterium Synechocystis PCC 6803 with increased phosphoenolpyruvate carboxylase (PEPc) levels. The resulting engineered strain (CD-P) showed significantly increased ethylene production (10.5?±?3.1 µg mL-1 OD-1 day-1) compared to the control strain (6.4?±?1.4 µg mL-1 OD-1 day-1). Interestingly, extra copies of the native pepc or the heterologous expression of PEPc from the cyanobacterium Synechococcus PCC 7002 (Synechococcus) in the CD-P, increased ethylene production (19.2?±?1.3 and 18.3?±?3.3 µg mL-1 OD-1 day-1, respectively) when the cells were treated with the acetyl-CoA carboxylase inhibitor, cycloxydim. A heterologous expression of phosphoenolpyruvate synthase (PPSA) from Synechococcus in the CD-P also increased ethylene production (16.77?±?4.48 µg mL-1 OD-1 day-1) showing differences in the regulation of the native and the PPSA from Synechococcus in Synechocystis. Conclusions:This work demonstrates that genetic rewiring of cyanobacterial central carbon metabolism can enhance carbon supply to the TCA cycle and thereby further increase ethylene production.