CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a “two-spacers” strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.
The coordination of carbon and nitrogen metabolism is essential for bacteria to adapt to nutritional variations in the environment, but the underlying mechanism remains poorly understood. In autotrophic cyanobacteria, high CO levels favor the carboxylase activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) to produce 3-phosphoglycerate, whereas low CO levels promote the oxygenase activity of RuBisCO, leading to 2-phosphoglycolate (2-PG) production. Thus, the 2-PG level is reversely correlated with that of 2-oxoglutarate (2-OG), which accumulates under a high carbon/nitrogen ratio and acts as a nitrogen-starvation signal. The LysR-type transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR) controls the expression of genes related to carbon metabolism. Based on genetic and biochemical studies, we report here that 2-PG is an inducer of NdhR, while 2-OG is a corepressor, as found previously. Furthermore, structural analyses indicate that binding of 2-OG at the interface between the two regulatory domains (RD) allows the NdhR tetramer to adopt a repressor conformation, whereas 2-PG binding to an intradomain cleft of each RD triggers drastic conformational changes leading to the dissociation of NdhR from its target DNA. We further confirmed the effect of 2-PG or 2-OG levels on the transcription of the NdhR regulon. Together with previous findings, we propose that NdhR can sense 2-OG from the Krebs cycle and 2-PG from photorespiration, two key metabolites that function together as indicators of intracellular carbon/nitrogen status, thus representing a fine sensor for the coordination of carbon and nitrogen metabolism in cyanobacteria.
Cyanobacteria were the first oxygenic photosynthetic organisms during evolution and were ancestors of plastids. Cyanobacterial cells exhibit an extraordinary diversity in their size and shape, and bacterial cell morphology largely depends on the synthesis and the dynamics of the peptidoglycan (PG) layer. Here, we used a fluorescence analog of the PG synthesis precursor D-Ala, 7-Hydroxycoumarin-amino-D-alanine (HADA), to probe the PG synthesis pattern in live cells of cyanobacteria with different morphology. They displayed diverse synthesis patterns, with some strains showing an intensive HADA incorporation at the septal region, whereas others gave an HADA signal distributed around the cells. Growth zones covering several cells at the tips of the filament were present in some filamentous strains such as in Arthrospira. In Anabaena PCC 7120, which is capable of differentiating heterocysts for N2 fixation, PG synthesis followed the cell division cycle. In addition, an HADA incorporation was strongly activated from 12 to 15 h following the initiation of heterocyst development, indicating a thickening of the PG layer in heterocysts. The PG synthesis pattern is diverse in cyanobacteria and responds to developmental regulation. The use of fluorescent analogs may serve as a useful tool for understanding the mechanisms of cell growth and morphogenesis operating in these organisms.
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