Biological nitrogen fixation in non-legume plants

Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO 2

/pdf/synechococcus-elongatus-utex-2973-a-fast-growing-3own9ta8eq.pdf

Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

Synthetic biology in cyanobacteria engineering and analyzing novel functions.

Porous silica and carbon derived materials from rice husk pyrolysis char

Cyanobacteria are a large group of photosynthetic prokaryotes of enormous environmental importance, being responsible for a large proportion of global CO 2 and N 2 fixation. They form symbiotic associations with a wide range of eukaryotic hosts including plants, fungi, sponges, and protists. The cyanobacterial symbionts are often filamentous and fix N 2 in specialized cells known as heterocysts, enabling them to provide the host with fixed nitrogen and, in the case of non-photosynthetic hosts, with fixed carbon. The best studied cyanobacterial symbioses are those with plants, in which the cyanobacteria can infect the roots, stems, leaves, and, in the case of the liverworts and hornworts, the subject of this review, the thallus. The symbionts are usually Nostoc spp. that gain entry to the host by means of specialized motile filaments known as hormogonia. The host plant releases chemical signals that stimulate hormogonia formation and, by chemoattraction, guide the hormogonia to the point of entry into the plant tissue. Inside the symbiotic cavity, host signals inhibit further hormogonia formation and stimulate heterocyst development and dinitrogen fixation. The cyanobionts undergo morphological and physiological changes, including reduced growth rate and CO 2 fixation, and enhanced N 2 fixation, and release to the plant much of the dinitrogen fixed. This short review summarizes knowledge of the cyanobacterial symbioses with liverworts and hornworts, with particular emphasis on the importance of pili and gliding motility for the symbiotic competence of hormogonia.

/pdf/cyanobacteria-bryophyte-symbioses-35cawn3woi.pdf

Cyanobacteria–bryophyte symbioses

Molecular mechanisms behind adaptations in the cyanobacterium (Nostoc sp.) to a life in endosymbiosis with plants are still not clarified, nor are the interactions between the partners. To get further insights, the proteome of a Nostoc strain, freshly isolated from the symbiotic gland tissue of the angiosperm Gunnera manicata Linden, was analyzed and compared with the proteome of the same strain when free-living. Extracted proteins were separated by two-dimensional gel electrophoresis and were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with tandem mass spectrometry. Even when the higher percentage of differentiated cells (heterocysts) in symbiosis was compensated for, the majority of the proteins detected in the symbiotic cyanobacteria were present in the free-living counterpart, indicating that most cellular processes were common for both stages. However, differential expression profiling revealed a significant number of proteins to be down-regulated or missing in the symbiotic stage, while others were more abundant or only expressed in symbiosis. The differential protein expression was primarily connected to i) cell envelope-associated processes, including proteins involved in exopolysaccharide synthesis and surface and membrane associated proteins, ii) to changes in growth and metabolic activities (C and N), including upregulation of nitrogenase and proteins involved in the oxidative pentose phosphate pathway and downregulation of Calvin cycle enzymes, and iii) to the dark, microaerobic conditions offered inside the Gunnera gland cells, including changes in relative phycobiliprotein concentrations. This is the first comprehensive analysis of proteins in the symbiotic state.

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-19-1251

Protein expression profiles in an endosymbiotic cyanobacterium revealed by a proteomic approach.

The filamentous cyanobacteria of the genus Nostoc are globally distributed, phenotypically complex organisms, capable of cellular differentiation and of forming symbiotic associations with a wide range of plants. To further our understanding of these processes and functions, the proteome of photoautotrophically and diazotrophically grown Nostoc sp. PCC 73102 (N. punctiforme) cells was examined. Extracted proteins were separated into membrane and soluble protein fractions and analysed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The analysis led to the identification of 82 proteins that could be divided into 12 functional categories. Significantly, 65 of these proteins have not been previously documented in the Nostoc proteome. Many of the proteins identified were readily recognized as housekeeping proteins involved in carbon, nitrogen and energy metabolism, but a number of proteins related to stress, motility, secretion and post-translational modifications were also identified. Ten unclassified proteins were also detected, representing potential novel functions. These proteins were highly expressed, suggesting that they play key roles during photoautotrophic and diazotrophic growth. Nineteen of the proteins expressed under the growth conditions examined contained putative thioredoxin (Trx) targets, a motif that functions in redox regulation via redox equivalent mediators and is known to be significant in a wide range of biological processes. These observations contribute to our understanding of the complex Nostoc life cycle.

Proteomic analyses of the photoauto- and diazotrophically grown cyanobacterium Nostoc sp. PCC 73102

Bacterial cell division is a highly co-ordinated and fine-tuned process. In the unicellular cyanobacterium Synechococcus sp. strain PCC 7942, inactivating mutations in the ftn2 and ftn6 genes block cell division and result in a phenotype with extensively elongated cells. In order to establish the pleiotropic responses induced and cellular processes affected by blocked cell division, the proteomes of wild-type and the cell division mutants Ftn2 and Ftn6 of Synechococcus sp. strain PCC 7942 were characterized and compared. By separating soluble extracted proteins on 2D gels, more than 800 protein spots were visualized on each SYPRO Ruby-stained gel. Quantitative differences in protein composition were detected by using the PDQuest software, and comparative analysis revealed that 76 protein spots changed significantly in the cell division mutants. These protein spots were selected for identification using peptide mass fingerprints generated by MALDI-TOF MS. Fifty-three protein spots were successfully identified, representing 44 different proteins. The upregulated proteins include proteins involved in cell division/cell morphogenesis, protein synthesis and processing, oxidative stress response, amino acid metabolism, nucleotide biosynthesis, and glycolysis, as well as unknown proteins. Among the downregulated proteins are those involved in chromosome segregation, protein processing, photosynthesis, redox regulation, carbon dioxide fixation, nucleotide biosynthesis, the biosynthetic pathway to fatty acids, and energy production. Besides eliciting common responses, inactivation of Ftn2 and Ftn6 in the mutants may result in different responses in protein levels between the mutants. Among 18 identified differentially affected protein spots, 75 % (9/12) of the protein spots affected in the Ftn2 mutant were upshifted, whereas in the Ftn6 mutant 70 % (7/10) of the affected protein spots were downshifted. Identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in cyanobacterial cell division.

Comparative proteomics of cell division mutants and wild-type of Synechococcus sp. strain PCC 7942

Cost-Effectiveness of 4CMenB Infant Vaccination in England: A Comprehensive Valuation Considering the Broad Impact of Serogroup B Invasive Meningococcal Disease.

Trypsin inhibitors have been suggested to constitute an inducible defense in the sedge Carex bigelowii, and some former studies suggest that this might be a cause for the cyclic population dynamics in many alpine and arctic small mammals, for example lemmings (Lemmus lemmus). We investigated this further by using a method of simulated grazing (clipping) at different intensities, in three different habitats with varying resource availability, with different harvest times (hours after clipping), and two different stages of ramets (reproductive/vegetative) in a study from the Swedish mountain range. Our results do not indicate that C. bigelowii has an inducible defense constituted by an increase in trypsin inhibitor activity (TIA), but rather that the amount of soluble plant proteins (SPP) is lowered in wounded plants. The responses were somewhat different in the three habitats, with ramets growing in the marsh showing the highest ratio of TIA to SPP, due to low amounts of SPP. We did not find any significant effects of harvest time, or of the stage of the ramet that could support the hypothesis of an inducible defense. To conclude, we could not find any evidence for an inducible defense consisting of trypsin inhibitors in Carex bigelowii ramets, but we did find variations in the amount of SPP that may have nutritional consequences for herbivores.

http://www.sysecol2.ethz.ch/Refs/EntClim/L/Li138.pdf

Defense mechanisms against grazing: a study of trypsin inhibitor responses to simulated grazing in the sedge Carex bigelowii

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