Slowdown of surface diffusion during early stages of bacterial colonization

We study the surface diffusion of the model cyanobacterium Synechocystis sp. PCC6803 during the incipient stages of cell contact with a glass surface in the dilute regime. We observe a twitching motility with alternating immobile tumble and mobile run periods, resulting in a normal diffusion described by a continuous-time random walk with a coefficient of diffusion D. Surprisingly, D is found to decrease with time down to a plateau. This is observed only when the cyanobacterial cells are able to produce released extracellular polysaccharides, as shown by a comparative study between the wild-type strain and various polysaccharides-depleted mutants. The analysis of the trajectories taken by the bacterial cells shows that the temporal characteristics of their intermittent motion depend on the instantaneous fraction of visited sites during diffusion. This describes quantitatively the time dependence of D, related to the progressive surface coverage by the polysaccharides. The observed slowdown of the surface diffusion may constitute a basic precursor mechanism for microcolony formation and provides clues for controlling biofilm formation

Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase ( PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of approximate to 86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions

Engineering the fatty acid synthesis pathway in Synechococcus elongatus PCC 7942 improves omega-3 fatty acid production

Background: The microbial production of fatty acids has received great attention in the last few years as feedstock for the production of renewable energy. The main advantage of using cyanobacteria over other organisms is their ability to capture energy from sunlight and to transform CO2 into products of interest by photosynthesis, such as fatty acids. Fatty acid synthesis is a ubiquitous and well-characterized pathway in most bacteria. However, the activity of the enzymes involved in this pathway in cyanobacteria remains poorly explored. Results: To characterize the function of some enzymes involved in the saturated fatty acid synthesis in cyanobacteria, we genetically engineered Synechococcus elongatus PCC 7942 by overexpressing or deleting genes encoding enzymes of the fatty acid synthase system and tested the lipid profile of the mutants. These modifications were in turn used to improve alpha-linolenic acid production in this cyanobacterium. The mutant resulting from fabF overexpression and fadD deletion, combined with the overexpression of desA and desB desaturase genes from Synechococcus sp. PCC 7002, produced the highest levels of this omega-3 fatty acid. Conclusions: The fatty acid composition of S. elongatus PCC 7942 can be significantly modified by genetically engineering the expression of genes coding for the enzymes involved in the first reactions of fatty acid synthesis pathway. Variations in fatty acid composition of S. elongatus PCC 7942 mutants did not follow the pattern observed in Escherichia coli derivatives. Some of these modifications can be used to improve omega-3 fatty acid production. This work provides new insights into the saturated fatty acid synthesis pathway and new strategies that might be used to manipulate the fatty acid content of cyanobacteria.Work in the FDLC laboratory was financed by the Spanish Ministry of Economy and Competitivity (MINECO) Grant BFU2014-55534-C2-1-P. MSM. was recipientof a Ph.D. fellowship (BES-2012-057387) from MINECO

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii: kinetics and specificity in deglutathionylation reactions

Protein deglutathionylation is mainly catalyzed by glutaredoxins (GRXs). We have analyzed the biochemical properties of four of the six different GRXs of Chlamydomonas reinhardtii. Kinetic parameters were determined for disulfide and dehydroascorbate reduction but also for deglutathionylation of artificial and protein substrates. The results indicate that GRXs exhibit striking differences in their catalytic properties, mainly linked to the class of GRX considered but also to the pK(a) of the N-terminal catalytic cysteine. Furthermore, glutathionylated proteins were found to exhibit distinct reactivities with GRXs. These results suggest that glutathionylation may allow a fine tuning of cell metabolism under stress conditions

Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey

Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in nonphotosynthetic organisms, whereas much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylated glutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways, among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase, and phosphoglycerate kinase was confirmed by Western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions