Single Crossover to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC 7120 (hereafter Anabaena 7120) is a model cyanobacterium for studying pathways such as photosynthesis and nitrogen fixation along with many other metabolic pathways common to plants. In addition, since Anabaena 7120 forms specialized N2-fixing cells, called heterocysts, to perform uniquely solar-powered, oxic nitrogen fixation under fixed-nitrogen depleted conditions, this cyanobacterium provides the unique opportunity to study cellular differentiation in bacteria. Since more than 155,810 sequenced prokaryotic genomes are currently available (Zhang et al., Microbiome 8(1):134, 2020), target gene inactivation, combined with analyses of the corresponding mutant's phenotype, has become a powerful tool to assess gene function through detecting a loss-of-function in the knockout mutant. In the method described here, a single crossover approach is used to knockout a target gene in Anabaena 7120. The method requires inserting an internal fragment of the target gene into the cyanobacterial integration vector pZR606 to create a knockout plasmid, and then is introduced to Anabaena 7120 via conjugative transformation. A single crossover, occurring via homologous recombination, disrupts the target gene, creating 3'- and 5'-deleted fragments (Fig. 1). The mutant containing the inactivated gene can then be studied to determine any loss of function, thereby defining the gene's function. This gene inactivation approach is based on an integrative vector pZR606 (Chen et al., Appl Microbiol Biotechnol 99:1779-1793, 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.