Overcoming Intrinsic Restriction Enzyme Barriers Enhances Transformation Efficiency in Arthrospira platensis C1.

The development of a reliable genetic transformation system for Arthrospira platensis has been a long-term goal, mainly for those trying either to improve its performance in large-scale cultivation systems or to enhance its value as food and feed additives. However, so far, most of the attempts to develop such a transformation system have had limited success. In this study, an efficient and stable transformation system for A. platensis C1 was successfully developed. Based on electroporation and transposon techniques, exogenous DNA could be transferred to and stably maintained in the A. platensis C1 genome. Most strains of Arthrospira possess strong restriction barriers, hampering the development of a gene transfer system for this group of cyanobacteria. By using a type I restriction inhibitor and liposomes to protect the DNA from nuclease digestion, the transformation efficiency was significantly improved. The transformants were able to grow on a selective medium for more than eight passages, and the transformed DNA could be detected from the stable transformants. We propose that the intrinsic endonuclease enzymes, particularly the type I restriction enzyme, in A. platensis C1 play an important role in the transformation efficiency of this industrial important cyanobacterium.

Physiological and transcriptional responses to high temperature in Arthrospira (Spirulina) platensis C1.

Arthrospira (Spirulina) platensis is a well-known commercial cyanobacterium that is used as a food and in feed supplements. In this study, we examined the physiological changes and whole-genome expression in A. platensis C1 exposed to high temperature. We found that photosynthetic activity was significantly decreased after the temperature was shifted from 35°C to 42°C for 2 h. A reduction in biomass production and protein content, concomitant with the accumulation of carbohydrate content, was observed after prolonged exposure to high temperatures for 24 h. Moreover, the results of the expression profiling in response to high temperature at the designated time points (8 h) revealed two distinct phases of the responses. The first was the immediate response phase, in which the transcript levels of genes involved in different mechanisms, including genes for heat shock proteins; genes involved in signal transduction and carbon and nitrogen metabolism; and genes encoding inorganic ion transporters for magnesium, nitrite and nitrate, were either transiently induced or repressed by the high temperature. In the second phase, the long-term response phase, both the induction and repression of the expression of genes with important roles in translation and photosynthesis were observed. Taken together, the results of our physiological and transcriptional studies suggest that dynamic changes in the transcriptional profiles of these thermal-responsive genes might play a role in maintaining cell homeostasis under high temperatures, as reflected in the growth and biochemical composition, particularly the protein and carbohydrate content, of A. platensis C1.

Draft genome sequence of Arthrospira platensis C1 (PCC9438).

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.

Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components.

The present study focused on comparative proteome analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed by using a bioinformatics approach, in response to both stress conditions.The data revealed two important points: first, the results indicate that low-temperature stress is tightly linked with oxidative stress as well as photosynthesis; however, no specific mechanism is revealed in the case of the high-temperature stress response. Second, temperature stress was revealed to be linked with nitrogen and ammonia assimilation. Moreover, the data also highlighted the cross-talk of signaling pathways. Some of the detected signaling proteins, e.g., Hik14, Hik26 and Hik28, have potential interactions with differentially expressed proteins identified in both temperature stress conditions. Some differentially expressed proteins found in the Spirulina protein-protein interaction network were also examined for their physical interactions by a yeast two hybrid system (Y2H). The Y2H results obtained in this study suggests that the potential PPI network gives quite reliable potential interactions for Spirulina. Therefore, the bioinformatics approach employed in this study helps in the analysis of phenomena where proteome analyses of knockout mutants have not been carried out to directly examine for specificity or cross-talk of signaling components.

A combined stress response analysis of Spirulina platensis in terms of global differentially expressed proteins, and mRNA levels and stability of fatty acid biosynthesis genes.

Changes in gene expression play a critical role in enhancing the ability of cyanobacteria to survive under cold conditions. In the present study, Spirulina platensis cultures were grown at the optimal growth temperature, in the light, before being transferred to dark conditions at 22 degrees C. Two dimensional-differential gel electrophoresis was then performed to separate differentially expressed proteins that were subsequently identified by MS. Among all differentiated proteins identified, a protein involved in fatty acid biosynthesis, (3R)-hydroxymyristoyl-[acyl-carrier-protein]-dehydratase encoded by fabZ, was the most up-regulated protein. However, the fatty-acid desaturation proteins were not significantly differentiated. This raised the question of how the unsaturated fatty acid, especially gamma-linolenic acid, content in the cells in the cold-dark shift remained stable compared with that of the cold shift. Thus, a study at the transcriptional level of these desaturase genes, desC, desA and desD, and also of the fabZ gene was conducted. The results indicated that in the dark, where energy is limited, mRNA stability was enhanced by exposure to low temperatures. The data demonstrate that when the cells encounter cold stress with energy limitation, they can maintain their homeoviscous adaptation ability via mRNA stability.

Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA.

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.