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

Abstract

Frankia is a representative genus of nitrogen-fixing (N2-fixing) actinobacteria; however, the molecular mechanisms underlying various phenomena such as the differentiation of a N2 fixation-specific structure (vesicle) and the regulation of N2 fixation (nif) genes, have yet to be elucidated in detail. In the present study, we screened hyphal fragments of Frankia casuarinae that were mutagenized by 1-methyl-3-nitro-1-nitrosoguanidine or gamma rays, and isolated 49 candidate N2 fixation mutants. Twelve of these mutants were selected for further study, and their abilities to grow in NH3-deficient (N-) liquid media and their rates of acetylene reduction activities were evaluated. Eleven mutant strains were confirmed to lack the ability to fix N2. Five mutant strains formed significantly reduced numbers of vesicles, while some failed to form large mature vesicles. These vesicle mutants also exhibited an aberrant hyphal morphology, suggesting a relationship between vesicle differentiation and hyphal branching. Ten mutants showed significant reductions in the expression of nifE, nifH, and nifV genes under N- conditions. The genome sequencing of eight mutants identified 20 to 400 mutations. Although mutant strains N3H4 and N6F4 shared a large number of mutations (108), most were unique to each strain. Mutant strain N7C9 had 3 mutations in the nifD and nifH genes that may result in the inability to fix N2. The other mutant strains did not have any mutations in any known N2 fixation-related genes, indicating that they are novel N2 fixation mutants.

Characterization of Vesicle Differentiation Mutants of <i>Frankia casuarinae</i>

“…5 and Table S1). Genome analyses revealed that three of the mutants (G23C4, N7C9, and N10E6) did not carry mutations in these homologs (Kucho et al, 2017). Collectively, these results indicate that the genes responsible for the phenotypes of the three mutants are not related to global nitrogen regulation, but specifically function in the vesicle differentiation process (Fig.…”

“…We previously isolated five N 2 -fixation mutants of Frankia casuarinae (G21E10, G23C4, G23D3, N7C9, and N10E6), which had smaller numbers of vesicles (<ca. 15% of the wild type) (Kucho et al, 2017) (Table S1). These mutants are considered to have defects in the generation of vesicle primordia.…”

“…The cDNAs of the GSII (francci3_3143), ntrB (francci3_3178), and 16S rRNA (francci3_R0040, internal standard) genes were synthesized using PrimeScript reverse transcriptase (Takara Bio) in a 20-μL reaction mixture con- taining 1.5 μg of total RNA and 2 pmol of gene-specific reverse primers (GSII, Ghodhbane-Gtari et al, 2014; 16S rRNA, Kucho et al, 2017; ntrB, 5′-cccacatctcgggcagtt-3′) at 42°C for 30 min and then at 50°C for 15 min. Regarding GSII and 16S rRNA, real-time PCR was performed using the Probe qPCR mix (Takara Bio) in a 20-μL reaction mixture containing 4 pmol of forward and reverse primers (GSII, Ghodhbane-Gtari et al, 2014;16S rRNA, Kucho et al, 2017), 4 pmol of the TaqMan probe (GSII, 5′acgccatcgtcgcctgct-3′; 16S rRNA, Kucho et al, 2017), and cDNA derived from 100 ng (GSII) or 1 ng (16S rRNA) of 5. Semi-quantitative reverse transcription PCR of ntrB (francci3_3178) and 16S rRNA (16S) genes.…”

“…total RNA. Regarding ntrB and 16S rRNA, semiquantitative PCR was performed using EX Taq DNA polymerase (Takara Bio) in a 20-μL reaction mixture containing 4 pmol of a forward primer (ntrB, 5′-gccgctgaccagtgtgaa-3′; 16S rRNA, Kucho et al, 2017) and reverse primer (same primers used in reverse transcription), and cDNA derived from 100 ng (ntrB) or 1 ng (16S rRNA) of total RNA. A temperature regime (95°C for 30 s, 58°C for 30 s, and 72°C for 18 s) was repeated 28 times for ntrB or 23 times for 16S rRNA.…”

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).…”