The Effect of Variable Light Source and Light Intensity on the Growth of Three Algal Species.

Light is the essential energy source for autotrophically growing organisms, including microalgae. Both light intensity and light quality affect cell growth and biomass composition. Here we used three green algae-Chlamydomonas reinhardtii, Desmodesmus quadricauda, and Parachlorella kessleri-to study the effects of different light intensities and light spectra on their growth. Cultures were grown at three different light intensities (100, 250, and 500 µmol m-2 s-1) and three different light sources: fluorescent lamps, RGB LEDs, and white LEDs. Cultures of Desmodesmus quadricauda and Parachlorella kessleri were saturated at 250 µmol m-2 s-1, and further increasing the light intensity did not improve their growth. Chlamydomonas reinhardtii cultures did not reach saturation under the conditions used. All species usually divide into more than two daughter cells by a mechanism called multiple fission. Increasing light intensity resulted in an increase in maximum cell size and division into more daughter cells. In Parachlorella kessleri cells, the concentration of photosynthetic pigments decreased with light intensity. Different light sources had no effect on algal growth or photosynthetic pigments. The results show a species-specific response of algae to light intensity and support the use of any white light source for their cultivation without negative effects on growth.

Improving microalgae for biotechnology - From genetics to synthetic biology - Moving forward but not there yet.

Microalgae are a diverse group of photosynthetic organisms that can be exploited for the production of different compounds, ranging from crude biomass and biofuels to high value-added biochemicals and synthetic proteins. Traditionally, algal biotechnology relies on bioprospecting to identify new highly productive strains and more recently, on forward genetics to further enhance productivity. However, it has become clear that further improvements in algal productivity for biotechnology is impossible without combining traditional tools with the arising molecular genetics toolkit. We review recent advantages in developing high throughput screening methods, preparing genome-wide mutant libraries, and establishing genome editing techniques. We discuss how algae can be improved in terms of photosynthetic efficiency, biofuel and high value-added compound production. Finally, we critically evaluate developments over recent years and explore future potential in the field.

Diclofenac Alters the Cell Cycle Progression of the Green Alga Chlamydomonas reinhardtii.

The aim of the study was to verify the hypothesis that a potential cause of the phytotoxicity of diclofenac (DCF, a non-steroidal anti-inflammatory drug) is an effect of cell cycle progression. This research was conducted using synchronous cultures of a model organism, green alga Chlamydomonas reinhardtii. The project examined DCF effects on selected parameters that characterize cell cycle progression, such as cell size, attainment of commitment points, DNA replication, number of nuclei formed during cells division and morphology of cells in consecutive stages of the cell cycle, together with the physiological and biochemical parameters of algae cells at different stages. We demonstrated that individual cell growth remained unaffected, whereas cell division was delayed in the DCF-treated groups grown in continuous light conditions, and the number of daughter cells from a single cell decreased. Thus, the cell cycle progression is a target affected by DCF, which has a similar anti-proliferative effect on mammalian cells.

Supra-Optimal Temperature: An Efficient Approach for Overaccumulation of Starch in the Green Alga Parachlorella kessleri.

Green algae are fast-growing microorganisms that are considered promising for the production of starch and neutral lipids, and the chlorococcal green alga Parachlorella kessleri is a favorable model, as it can produce both starch and neutral lipids. P. kessleri commonly divides into more than two daughter cells by a specific mechanism-multiple fission. Here, we used synchronized cultures of the alga to study the effects of supra-optimal temperature. Synchronized cultures were grown at optimal (30 °C) and supra-optimal (40 °C) temperatures and incident light intensities of 110 and 500 µmol photons m-2 s-1. The time course of cell reproduction (DNA replication, cellular division), growth (total RNA, protein, cell dry matter, cell size), and synthesis of energy reserves (net starch, neutral lipid) was studied. At 40 °C, cell reproduction was arrested, but growth and accumulation of energy reserves continued; this led to the production of giant cells enriched in protein, starch, and neutral lipids. Furthermore, we examined whether the increased temperature could alleviate the effects of deuterated water on Parachlorella kessleri growth and division; results show that supra-optimal temperature can be used in algal biotechnology for the production of protein, (deuterated) starch, and neutral lipids.

Characterization of Growth and Cell Cycle Events Affected by Light Intensity in the Green Alga Parachlorella kessleri: A New Model for Cell Cycle Research.

Multiple fission is a cell cycle variation leading to the production of more than two daughter cells. Here, we used synchronized cultures of the chlorococcal green alga Parachlorella kessleri to study its growth and pattern of cell division under varying light intensities. The time courses of DNA replication, nuclear and cellular division, cell size, total RNA, protein content, dry matter and accumulation of starch were observed at incident light intensities of 110, 250 and 500 µmol photons m-2s-1. Furthermore, we studied the effect of deuterated water on Parachlorella kessleri growth and division, to mimic the effect of stress. We describe a novel multiple fission cell cycle pattern characterized by multiple rounds of DNA replication leading to cell polyploidization. Once completed, multiple nuclear divisions were performed with each of them, immediately followed by protoplast fission, terminated by the formation of daughter cells. The multiple fission cell cycle was represented by several consecutive doublings of growth parameters, each leading to the start of a reproductive sequence. The number of growth doublings increased with increasing light intensity and led to division into more daughter cells. This study establishes the baseline for cell cycle research at the molecular level as well as for potential biotechnological applications, particularly directed synthesis of (deuterated) starch and/or neutral lipids as carbon and energy reserves.

A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I.

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation.

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme.

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.

Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I.

Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys-ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.