Mutants Of Cyanobacteria Research Articles

Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil-DNA glycosylase.

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4 to C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs.

Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria.

Here we describe a universal approach for plasmid-free genome engineering in cyanobacteria that exploits the polyploidy of their chromosomes as a natural counterselection system. Rather than being delivered via replicating plasmids, genes encoding for DNA modifying enzymes are instead integrated into essential genes on the chromosome by allelic exchange, as facilitated by antibiotic selection, a process that occurs readily and with only minor fitness defects. By virtue of the essentiality of these integration sites, full segregation is never achieved, with the strain instead remaining as a merodiploid so long as antibiotic selection is maintained. As a result, once the desired genome modification is complete, removal of antibiotic selection results in the gene encoding for the DNA modifying enzyme to then be promptly eliminated from the population. Proof of concept of this new and generalizable strategy is provided using two different site-specific recombination systems, CRE-lox and DRE-rox, in the fast-growing cyanobacterium Synechococcus sp. PCC 7002, as well as CRE-lox in the model cyanobacterium Synechocystis sp. PCC 6803. Reusability of the method, meanwhile, is demonstrated by constructing a high-CO2 requiring and markerless Δndh3 Δndh4 ΔbicA ΔsbtA mutant of Synechococcus sp. PCC 7002. Overall, this method enables the simple and efficient construction of stable and unmarked mutants in cyanobacteria without the need to develop additional shuttle vectors nor counterselection systems.

Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria.

The slow but temperature-insensitive adenosine triphosphate (ATP) hydrolysis reaction in KaiC is considered as one of the factors determining the temperature-compensated period length of the cyanobacterial circadian clock system. Structural units responsible for this low but temperature-compensated ATPase have remained unclear. Although whole-KaiC scanning mutagenesis can be a promising experimental strategy, producing KaiC mutants and assaying those ATPase activities consume considerable time and effort. To overcome these bottlenecks for in vitro screening, we optimized protocols for expressing and purifying the KaiC mutants and then designed a high-performance liquid chromatography system equipped with a multi-channel high-precision temperature controller to assay the ATPase activity of multiple KaiC mutants simultaneously at different temperatures. Through the present protocol, the time required for one KaiC mutant is reduced by approximately 80% (six-fold throughput) relative to the conventional protocol with reasonable reproducibility. For validation purposes, we picked up three representatives from 86 alanine-scanning KaiC mutants preliminarily investigated thus far and characterized those clock functions in detail.

Multiple Impacts of Loss of Plastidic Phosphatidylglycerol Biosynthesis on Photosynthesis during Seedling Growth of Arabidopsis.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane in cyanobacteria and plant chloroplasts. Although PG accounts only for ~10% of total thylakoid lipids, it plays indispensable roles in oxygenic photosynthesis. In contrast to the comprehensive analyses of PG-deprived mutants in cyanobacteria, in vivo roles of PG in photosynthesis during plant growth remain elusive. In this study, we characterized the photosynthesis of an Arabidopsis thaliana T-DNA insertional mutant (pgp1-2), which lacks plastidic PG biosynthesis. In the pgp1-2 mutant, energy transfer from antenna pigments to the photosystem II (PSII) reaction center was severely impaired, which resulted in low photochemical efficiency of PSII. Unlike in the wild type, in pgp1-2, the PSII complexes were susceptible to photodamage by red light irradiation. Manganese ions were mostly dissociated from protein systems in pgp1-2, with oxygen-evolving activity of PSII absent in the mutant thylakoids. The oxygen-evolving complex may be disrupted in pgp1-2, which may accelerate the photodamage to PSII by red light. On the acceptor side of the mutant PSII, decreased electron-accepting capacity was observed along with impaired electron transfer. Although the reaction center of PSI was relatively active in pgp1-2 compared to the severe impairment in PSII, the cyclic electron transport was dysfunctional. Chlorophyll fluorescence analysis at 77K revealed that PG may not be needed for the self-organization of the macromolecular protein network in grana thylakoids but is essential for the assembly of antenna-reaction center complexes. Our data clearly show that thylakoid glycolipids cannot substitute for the role of PG in photosynthesis during plant growth.

A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria.

Cyanobacteria can be utilized as a potential biocatalyst for the production of biofuels and biochemicals directly from CO2. Useful mutants of cyanobacteria, which can grow rapidly or are resistant to specific metabolic products, are essential to improve the productivity of biofuels. In this study, we developed a single cell culture system to effectively screen mutant cyanobacteria using magnetite nanoparticles and magnetic force. Lens culinaris Agglutinin (LCA) was selected as a lectin, which binds to the surface of Synechococcus elongatus PCC7942 cells and the LCA-conjugated magnetite cationic liposomes (MCLs) were developed for magnetic labeling of PCC7942 cells. The MCL-labeled PCC7942 cells were magnetically patterned at a single cell level by using 6,400 iron pillars of the pin-holder device. The device enabled 1,600 single cells to be arrayed in one square centimeter. We cultured the patterned cells in liquid medium and achieved higher colony-forming ratio (78.4%) than that obtained using conventional solid culture method (4.8%). Single cells with different properties could be distinguished in the single cell culture system depending on their growth. Furthermore, we could selectively pick up the target cells and subsequently perform efficient isolation culture. The ratio of successful isolation culture using the developed method was 13 times higher than that of the conventional methods. Thus, the developed system would serve as a powerful tool for screening mutant cyanobacteria.

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in Synechococcus sp. PCC 7002

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.

Chloroplast-encoded small subunits of the three multiprotein complexes in photosynthetic electron transport

The photosystem I, photosystem II, and cytochromeb 6 f complexes that are involved in electron transport of oxygenic photosynthesis consist of a number of subunits encoded by either the chloroplast or nuclear genomes. In addition to the major subunits that carry redox components or photosynthetic pigments, these complexes contain several to more than ten subunits with molecular masses of less than 10 kDa. Directed mutagenesis has served as a powerful tool for investigation of the roles of these small subunits in the organization or function of the complexes. Various chloroplast transformants of the green algaChlamydomonas reinhardtii and mutants of cyanobacteria in which a gene encoding a small subunit was deleted or altered have been constructed. Evidence has accumulated suggesting that these small subunits function in the assembly, stabilization, or protection from photoinhibition of the complexes or in the modulation or regulation of electron transport. This article presents an overview of the properties and functions of the chloroplast-encoded small subunits of the three multiprotein complexes of photosynthetic electron transport that have been mainly analyzed with chloroplast transformants ofC. reinhardtii and the corresponding cyanobacterial transformants.

Approach to Recognition of Regulatory Mutants of Cyanobacteria

Antimetabolite analogs of essential amino acids are useful as selective agents for isolation of regulatory mutants of cyanobacteria, although we observed striking microbiological differences from other widely used eubacterial systems. Regulatory mutants shown to overproduce and excrete tryptophan, phenylalanine, tyrosine, methionine, or arginine were isolated from four cyanobacteria: Anabaena sp. 29151, Synechococcus sp. 602, Synechococcus sp. AN Tx20, and Synechocystis sp. 29108. Surprisingly, regulatory-mutant colonies did not support a halo of cross-fed wild-type growth on selective medium. Since regulatory mutants were shown to excrete substantial levels of amino acids, it was deduced that poor cross-feeding must reflect a generally low nutritional responsiveness of the cyanobacterial background. This conclusion was confirmed by results which showed that regulatory-mutant cells of cyanobacteria dispersed among wild-type populations of Bacillus subtilis did produce halo colonies on solid analog-containing medium. Cross-feeding between one cyanobacterial pair (a phenylalanine excretor and a phenylalanine auxotroph) was successfully demonstrated in the absence of the analog under conditions in which relatively large masses of each cell population type were spread near one another on agar plates. These results suggest that amino acid excreted by regulatory mutants of cyanobacteria on analog-containing selective medium is transported into nearby wild-type cells too inefficiently to overcome the antimetabolite effects of the analog, thereby failing to generate halos of physiologically resistant background cells. Consistent with this interpretation was the finding that the pheA1 auxotroph from Synechococcus sp. 602 exhibited a linearly proportional dependence of growth rate upon exogenous concentration of l-phenylalanine (below 20 muM). Wild-type B. subtilis serves as a convenient and sensitive test lawn for screening obvious regulatory mutants from among collections of analog-resistant cyanobacterial mutants. Appropriate B. subtilis auxotrophs can be used as convenient indicator strains for the identification of regulatory mutants in cyanobacteria through the observation of syntrophic growth responses.

Metronidazole and the isolation of temperature-sensitive photosynthetic mutants in cyanobacteria.

A procedure has been developed for use of metronidazole (2-methyl-5-nitroimidazole-1-ethanol) as an enrichment agent during the isolation of temperature-sensitive, photosynthetic mutants in the cyanobacterium Synechococcus cedrorum. The protocol includes incubation with this drug following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Incubation of photosynthetically active S. cedrorum cells with 1 mM metronidazole causes a light-dependent reduction of cell viability. Maximum reduction in cell viability occurred following 6 h of incubation. Cessation of electron transport reduced the impact of the drug by five orders of magnitude. Yet during the time of incubation, metronidazole did not influence the electron transport capacities of the S. cedrorum cells, suggesting that the thylakoid membrane was not the target of the toxic effects of this drug. In addition, this drug was found to be an effective electron acceptor to photosystem I although high concentrations were required to observe maximum rates of electron transfer. Metronidazole interacted in a noncompetitive manner with methyl viologen, which suggested that those two acceptors to photosystem I have unique reduction sites on the S. cedrorum thylakoid membrane. The temperature-sensitive strains that were isolated using the procedure presented here were assessed for photosynthetic electron transport and chlorophyll fluorescence (induction kinetics and low-temperature emission spectra) characteristics. Approximately one-half of the temperature-sensitive mutants isolated possessed abnormal photosynthetic properties when shifted to the restrictive temperature (40 degrees C). A total of 31 throughout the photosynthetic electron-transport chain.