Isolation of Mutants of the Nitrogen-Fixing Actinomycete <i>Frankia</i>

Abstract

Frankia is a nitrogen (N)-fixing multicellular actinomycete which establishes root-nodule symbiosis with actinorhizal plants. Several aspects of Frankia N fixation and symbiosis are distinct, but genes involved in the specific features are largely unknown because of the lack of an efficient mutant screening method. In this study, we isolated mutants of Frankia sp. strain CcI3 using hyphae fragments mutagenized by chemical mutagens. Firstly, we isolated uracil auxotrophs as gain-of-function mutants resistant to 5-fluoroorotic acid (5-FOA). We obtained seven 5-FOA resistant mutants, all of which required uracil for growth. Five strains carried a frame shift mutation in orotidine-5′-phosphate decarboxylase gene and two carried an amino acid substitution in the orotate phosphoribosyltransferase gene. Secondly, we isolated mutants showing loss-of-function phenotypes. Mutagenized hyphae were fragmented by ultrasound and allowed to multiply at their tips. Hyphae were fragmented again and short fragments were enriched by filtration through 5 μm pores filters. Next-generation and Sanger sequencing revealed that colonies formed from the short hyphae fragments consisted of cells with an identical genotype. From the mutagenized colony population, we isolated three pigmentation mutants and a mutant with reduced N-fixation activity. These results indicate that our procedure is useful for the isolation of loss-of-function mutants using hyphae of Frankia.

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“…Several studies have enhanced our understanding on physiological methods associated with roots, chemical molecules produced by roots, signaling between microbes and root, and possible defense mechanisms. [101][102][103][104][105][106] Researchers have given special attention to microbes associated with root in soil among other symbiotic associations between many plants and microorganisms. Mycorrhiza is eminent by fungal colonization inside or outside the cell that helps in nutrient assumption.…”

Stable Transformation of the Actinobacteria Frankia spp

“…Initially, a few studies on spontaneous mutants in Frankia spp. have been reported (11)(12)(13), and chemical (N-methyl-N-nitro-N-nitrosoguanidine and ethyl methanesulfonate treatments) and physical (UV) mutagenesis approaches have also been investigated with some success but limited potential as tools (14)(15)(16)(17).…”

Nitrogen Fixation Mutants of the Actinobacterium <i>Frankia Casuarinae</i> CcI3

“…In an effort to identify these Frankia -specific genes with a forward genetic approach, we developed a method to isolate loss-of-function mutants using Frankia hyphae ( 13 ). In this “fragmentation and filtration” (FF) method, we grew mutagenized hyphae at their tips, fragmented them by ultrasonic waves, and purified short fragments by filtration with a 5-μm pore.…”

“…In this “fragmentation and filtration” (FF) method, we grew mutagenized hyphae at their tips, fragmented them by ultrasonic waves, and purified short fragments by filtration with a 5-μm pore. Most of the short hyphae fragments consisted of clonal mutant cells and we successfully obtained several loss-of-function mutants ( 13 ). In the present study, we applied the FF method to the isolation of N 2 -fixing mutants of Frankia .…”