Enhanced Nitrogen Fixation in a glgX -Deficient Strain of Cyanothece sp. Strain ATCC 51142, a Unicellular Nitrogen-Fixing Cyanobacterium

Abstract

Cyanobacteria are oxygenic photosynthetic prokaryotes with important roles in the global carbon and nitrogen cycles. Unicellular nitrogen-fixing cyanobacteria are known to be ubiquitous, contributing to the nitrogen budget in diverse ecosystems. In the unicellular cyanobacterium Cyanothece sp. strain ATCC 51142, carbon assimilation and carbohydrate storage are crucial processes that occur as part of a robust diurnal cycle of photosynthesis and nitrogen fixation. During the light period, cells accumulate fixed carbon in glycogen granules to use as stored energy to power nitrogen fixation in the dark. These processes have not been thoroughly investigated, due to the lack of a genetic modification system in this organism. In bacterial glycogen metabolism, the glgX gene encodes a debranching enzyme that functions in storage polysaccharide catabolism. To probe the consequences of modifying the cycle of glycogen accumulation and subsequent mobilization, we engineered a strain of Cyanothece 51142 in which the glgX gene was genetically disrupted. We found that the ΔglgX strain exhibited a higher growth rate than the wild-type strain and displayed a higher rate of nitrogen fixation. Glycogen accumulated to higher levels at the end of the light period in the ΔglgX strain, compared to the wild-type strain. These data suggest that the larger glycogen pool maintained by the ΔglgX mutant is able to fuel greater growth and nitrogen fixation ability. IMPORTANCE Cyanobacteria are oxygenic photosynthetic bacteria that are found in a wide variety of ecological environments, where they are important contributors to global carbon and nitrogen cycles. Genetic manipulation systems have been developed in a number of cyanobacterial strains, allowing both the interruption of endogenous genes and the introduction of new genes and entire pathways. However, unicellular diazotrophic cyanobacteria have been generally recalcitrant to genetic transformation. These cyanobacteria are becoming important model systems to study diurnally regulated processes. Strains of the Cyanothece genus have been characterized as displaying robust growth and high rates of nitrogen fixation. The significance of our study is in the establishment of a genetic modification system in a unicellular diazotrophic cyanobacterium, the demonstration of the interruption of the glgX gene in Cyanothece sp. strain ATCC 51142, and the characterization of the increased nitrogen-fixing ability of this strain.

Natural competence inChlorogloeopsis fritschiiPCC 6912 and other ramified cyanobacteria

“…For example, the genome of Cyanothece sp. ATCC 51142 includes all NCGs, however, it is not naturally transformable (27). Similarly, although we could recover evidence for all NCGs in F. thermalis PCC 7521, no transformation could be established in that organism.…”

A Genetic Toolbox for the New Model Cyanobacterium Cyanothece PCC 7425: A Case Study for the Photosynthetic Production of Limonene

“…First, a single-stranded spectinomycin resistance DNA-cassette, which could be introduced (by electro-transformation) and integrated into the genome of Cyanothece PCC 7822 cells, yielded no spectinomycin resistant clones when tested with Cyanothece PCC 7425 ( Min and Sherman, 2010 ). More recently, Liberton et al (2019) using DNA methylases to protect the incoming DNA from the Cyanothece ATCC 51142 restriction enzymes, could insert a kanamycin resistance cassette into the glycogen-catabolism gene glgX, generating a glycogen-rich mutant. However, this technique was not tested with Cyanothece PCC 7425.…”

Modifying the Cyanobacterial Metabolism as a Key to Efficient Biopolymer Production in Photosynthetic Microorganisms

“…PCC 6803 strain, a mutant lacking GlgP was created that led to 420% of WT glycogen content in biomass [ 58 ]. Knock out of the glgX gene in the Cyanothece 51142 strain cyanobacterium resulted in faster growth and a higher rate of nitrogen fixation [ 67 ]. DNA methylation has recently been shown to influence the activity of genes associated with glycogen catabolism.…”