A CRISPR-Based Method for Constructing Conditional Mutations of Essential Genes in Cyanobacteria.

Cyanobacteria, a group of diverse bacteria capable of oxygenic photosynthesis, are excellent models for investigating many important cellular processes, such as photosynthesis, nitrogen fixation, and prokaryotic cell differentiation. They also have great potential to become the next-generation cell factories for sustainable biosynthesis of valuable products. However, genetic manipulation in cyanobacteria is not as convenient as in other model bacteria. Particularly, handling essential genes in cyanobacteria has been difficult due to the lack of appropriate tools, limiting our understanding of many important cellular functions encoded by them. We recently develop a CRISPR-based method for constructing the conditional mutants of cyanobacterial essential genes by engineering the ribosome binding site to a theophylline-responsive riboswitch. Here, we provide the details of this method. The principle of this method could be used to construct conditional mutants in a wide range of bacterial species.

The developmental regulator PatD modulates assembly of the cell-division protein FtsZ in the cyanobacterium Anabaena sp. PCC 7120.

FtsZ is a tubulin-like GTPase that polymerizes to initiate the process of cell division in bacteria. Heterocysts are terminally differentiated cells of filamentous cyanobacteria that have lost the capacity for cell division and in which the ftsZ gene is downregulated. However, mechanisms of FtsZ regulation during heterocyst differentiation have been scarcely investigated. The patD gene is NtcA dependent and involved in the optimization of heterocyst frequency in Anabaena sp. PCC 7120. Here, we report that the inactivation of patD caused the formation of multiple FtsZ-rings in vegetative cells, cell enlargement, and the retention of peptidoglycan synthesis activity in heterocysts, whereas its ectopic expression resulted in aberrant FtsZ polymerization and cell division. PatD interacted with FtsZ, increased FtsZ precipitation in sedimentation assays, and promoted the formation of thick straight FtsZ bundles that differ from the toroidal aggregates formed by FtsZ alone. These results suggest that in the differentiating heterocysts, PatD interferes with the assembly of FtsZ. We propose that in Anabaena FtsZ is a bifunctional protein involved in both vegetative cell division and regulation of heterocyst differentiation. In the differentiating cells PatD-FtsZ interactions appear to set an FtsZ activity that is insufficient for cell division but optimal to foster differentiation.

patD, a Gene Regulated by NtcA, Is Involved in the Optimization of Heterocyst Frequency in the Cyanobacterium Anabaena sp. Strain PCC 7120.

In the filamentous multicellular cyanobacterium Anabaena sp. strain PCC 7120, 5 to 10% of the cells differentiate into heterocysts, which are specialized in N2 fixation. Heterocysts and vegetative cells are mutually dependent for filament growth through nutrient exchange. Thus, the heterocyst frequency should be optimized to maintain the cellular carbon and nitrogen (C/N) balance for filament fitness in the environment. Here, we report the identification of patD, whose expression is directly activated in developing cells by the transcription factor NtcA. The inactivation of patD increases heterocyst frequency and promotes the upregulation of the positive regulator of heterocyst development hetR, whereas its overexpression decreases the heterocyst frequency. The change in heterocyst frequency resulting from the inactivation of patD leads to the reduction in competitiveness of the filaments under combined-nitrogen-depleted conditions. These results indicate that patD regulates heterocyst frequency in Anabaena sp. PCC 7120, ensuring its optimal filament growth.IMPORTANCE Microorganisms have evolved various strategies in order to adapt to the environment and compete with other organisms. Heterocyst differentiation is a prokaryotic model for studying complex cellular regulation. The NtcA-regulated gene patD controls the ratio of heterocysts relative to vegetative cells on the filaments of Anabaena sp. strain PCC 7120. Such a regulation provides a mechanism through which carbon fixation by vegetative cells and nitrogen fixation by heterocysts are properly balanced to ensure optimal growth and keep a competitive edge for long-term survival.

Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120.

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.