Gamma Ray-induced Mutations in pyrEF Genes in Frankia casuarinae Strain CcI3

Abstract

Frankia spp. are multicellular actinobacteria with the ability to fix atmospheric dinitrogen (N₂). Frankia fixes N₂ not only in the free-living state, but also in root-nodule symbioses with more than 200 plant species called actinorhizal plants. In the present study, we isolated mutants of the pyrE (orotate phosphoribosyltransferase) and pyrF (orotidine-5′-phosphate decarboxylase) genes in Frankia casuarinae strain CcI3 using gamma rays as a mutagen and systematically identified the types of mutations that occurred in these genes. pyrEF mutants were isolated as uracil auxotrophs using the antimetabolite 5-fluoroorotic acid. We elucidated the nucleotide sequences of the pyrEF genes in 32 uracil auxotrophs, and detected eight substitutions, 17 single-nucleotide deletions, and seven large insertions. Large insertions were insertion sequences (IS elements); four belonged to the IS4 family, two to the IS66 family, and one to the IS110 family. This is the first study to demonstrate the mobilization of IS elements in the Frankia genome.

The genus Frankia belongs to the class Actinobacteria, order Frankiales, and family Frankiaceae. Frankia species possess the unique ability to fix atmospheric dinitrogen (N₂), a trait not observed in other actinobacterial genera. N₂ fixation occurs within vesicles that form at the tips of hyphae. These vesicles are encapsulated by multiple layers of hopanoid lipids, serving as a barrier against oxygen (Berry et al., 1993). Nitrogenase, an oxygen-sensitive enzyme, is exclusively synthesized within these vesicles (Meesters, 1987), enabling Frankia to effectively fix N₂ even under aerobic conditions.

Frankia establishes root nodule symbioses with more than 200 plant species across eight families, collectively known as actinorhizal plants (Benson and Dawson, 2007). Within these nodules, Frankia performs N₂ fixation and supplies the products to the host plants. This symbiotic relationship enables actinorhizal plants to grow rapidly even in nutrient-poor soils. Consequently, actinorhizal plants serve as vital pioneer species during the revegetation of disturbed lands. A phylogenetic ana­lysis classified Frankia strains into four lineages, each with distinct host preferences (Pozzi et al., 2018; Gtari et al., 2019). Lineage 1 comprises three subgroups (1a, 1b, and 1c), with 1a and 1b infecting plant species in Myricaceae and the genus Alnus of Betulaceae, while 1c infects Casuarinaceae. Lineage 2 infects plants from four families within the Rosales and Cucurbitales orders. Lineage 3 has a broader host range, infecting plant species from several families within the Fagales and Rosales orders. Lineage 4 comprises strains unable to fix N₂ or reinfect host plants.

To genetically identify novel Frankia genes involved in N₂ fixation, we isolated mutants that were unable to fix N₂ using 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and gamma rays (GR) as mutagens (Kucho et al., 2017). Mutants exhibited various phenotypes related to vesicle development (Kucho et al., 2017; Asukai and Kucho, 2020) and symbiosis (Kucho et al., 2023). In the NTG-induced mutant N3H4, we isolated cell lineages that restored N₂ fixation. We re-sequenced the genomes of the mutant and restored cell lineages using a short-read type next-generation sequencer and comprehensively identified nucleotide substitutions and short indels (Kucho et al., 2023). A comparative ana­lysis of these mutations detected a suppressor mutation in the NAD⁺ synthetase gene (Francci3_3146) of the restored cell lineages, and proved that this gene was responsible for aberrant N₂ fixation (Kucho et al., 2023). The same strategy was applied to GR-mutagenized mutants and their restored cell lineages. However, neither suppressor mutations nor reversions were detected (K. Kucho, unpublished). This result suggests that GR irradiation induced long insertions and deletions that were not detectable by our short-read mapping ana­lysis.

In the present study, we investigated mutation types (e.g., substitutions, short indels (a few bp), and long indels) induced by GR irradiation in Frankia. We previously established a method to selectively isolate mutants of the pyrE (orotate phosphoribosyltransferase) and pyrF (orotidine-5′-phosphate decarboxylase) genes in Frankia using the antimetabolite 5-fluoroorotic acid (5-FOA) with ethyl methanesulfonate as a mutagen (Kakoi et al., 2014). Briefly, 5-FOA is converted to 5-fluorouridine monophosphate (5-FUMP) by successive reactions catalyzed by the PyrE and PyrF proteins. Since 5-FUMP is toxic, wild-type (WT) cells cannot survive in the presence of 5-FOA. Mutants of the pyrE and pyrF genes are resistant to 5-FOA, but cannot synthesize uridine monophosphate (UMP), which is essential for growth. However, the growth of mutants may be supported by supplementing the medium with uracil. Therefore, pyrE and pyrF mutants (uracil auxotrophs) may be selectively isolated on minimal media containing 5-FOA and uracil. We utilized this method as a tool to easily identify mutations induced in GR-irradiated Frankia casuarinae strain CcI3 cells, which is a symbiont of Casuarina and Allocasuarina plant species (Zhang et al., 1984).

Materials and Methods

Bacterial strains and media

F. casuarinae strain CcI3 (Nouioui et al., 2016) was used as the parental WT strain. Buffered ammonium propionate medium used for transformation (BAP-T) (Kucho et al., 2009), a defined minimal medium, was employed to propagate WT Frankia cells.

Isolation of uracil auxotrophs with 5-FOA

Uracil auxotrophic mutants carrying mutations in the pyrE and pyrF genes were selectively isolated using 5-FOA and uracil according to the procedure described by Kakoi et al. (2014). Briefly, Frankia CcI3 cells grown in BAP-T medium were collected from a 14-mL culture by centrifugation and resuspended in 3 ml BAP-T. Hyphae were homogenized by forced passages through a 21G needle (TERUMO) five times and irradiated with GR from ⁶⁰Co (772 or 1,158 Gy). The two doses were tested because we used conditions for the isolation of N₂ fixation mutants (Kucho et al., 2017). Cells were cultivated in BAP-T supplemented with 100‍ ‍μg mL^–1 uracil and 100‍ ‍μg mL^–1 uridine at 28°C for 2 days. Cell suspensions were plated on solid CB media (Bassi and Benson, 2007) containing 0.5‍ ‍mg mL^–1 5-FOA, 100‍ ‍μg mL^–1 uracil, and 100‍ ‍μg mL^–1 uridine, and were incubated for 2 months.

Confirmation of uracil auxotrophy

Colonies that appeared on media containing 5-FOA were propagated in 0.5‍ ‍mL BAP-T with 100‍ ‍μg mL^–1 uracil. Cells were washed twice with sterilized distilled water to remove uracil, and half of them were inoculated into BAP-T and the remaining half into BAP-T with 100‍ ‍μg mL^–1 uracil. They were incubated at 28°C for one month, and cell density was assessed as OD₆₂₀. Strains that grew in BAP-T with uracil, but not in BAP-T without uracil were considered to be uracil auxotrophic mutants.

Identification of mutations in pyrEF genes

DNA segments containing the pyrE and pyrF genes were amplified by PCR using cell suspensions of the mutants as templates. Tks Gflex DNA polymerase (Takara) was used according to the manufacturer’s instructions. The primers 5′-GATGACATGAGGCAACGGT-3′ and 5′-ATCATGCTTCTACACGGGCT-3′ were used for pyrE, and 5′-ATCGTTGCAGGAATACCACG-3′ and 5′-AAGGCTTTGACGGACGAAAG-3′ for pyrF. Amplified products were electrophoresed on a 0.8% agarose gel containing 1×TAE buffer (40‍ ‍mM tris-acetate and 2‍ ‍mM EDTA, pH 8.0). To identify mutations in the amplified products, Sanger sequencing was performed by Eurofins Genomics. Detailed similarity searches for IS sequences were conducted using the ISfinder database (https://www-is.biotoul.fr/blast.php).

Results

Isolation of uracil auxotrophic mutants

Hyphae of Frankia CcI3 were irradiated with GR (772 and 1,158 Gy), and 17 and 85 5-FOA-resistant (5-FOA^R) colonies were obtained, respectively. The rate of 5-FOA^R colonies ranged between approximately 2×10^–3 and 4×10^–3. Forty 5-FOA^R colonies were isolated. They were cultivated in minimal media with or without uracil to test uracil auxotrophy (Fig. 1). While WT cells grew regardless of the presence of uracil, 36 of the 40 5-FOA^R strains only grew in the uracil-containing medium, indicating that they were uracil auxotrophic mutants. Mutant strains L1, L3 to L5, L10, and L12 to L15 were derived from 772-Gy GR irradiation. Mutant strains H1 to H9, H11, H13 to H18, and H20 to H30 were derived from 1,158-Gy GR irradiation. The remaining four strains (L8, H10, H19, and H31) also grew in the uracil-deficient medium, suggesting that they were false positives from screening.

Fig. 1.

Confirmation of uracil auxotrophy. Wild-type (WT) and 5-FOA-resistant strains were cultivated in minimal media with (gray box) or without (open box) uracil for one month. Strains obtained from 772-Gy (L1 to L15) and 1,158-Gy (H1 to H31) GR irradiation were tested. The average, along with the standard deviation, of three technical replicates is shown.

Identification of mutations in pyrEF genes

To identify mutations in the pyrEF genes, both genes were amplified by PCR from the uracil auxotrophs. Electrophoresis of the amplified products detected bands with larger sizes than WT in pyrE of strain H2 and pyrF of strains L10, H3, H11, H25, H26, and H30 (Fig. 2), which was indicative of the insertion of large DNA fragments within the genes. The other mutant strains showed bands of the same size as WT (data not shown).

Fig. 2.

PCR amplification of pyrEF genes in insertional mutants. Gel electrophoresis was performed on the PCR-amplified pyrE (A) and pyrF (B) genes. In the wild-type (WT) strain, the expected sizes of the amplified products were 0.73‍ ‍kbp for pyrE and 1.1‍ ‍kb for pyrF, respectively.

The nucleotide sequences of the amplified products were elucidated to identify mutations carried by 32 uracil auxotrophs. All the mutations detected are listed in Table S1. In summary, 10 mutations in pyrE and 22 mutations in pyrF were detected. The numbers of mutations roughly corresponded to the lengths of the genes (pyrE 537 bp and pyrF 825 bp). One-quarter of the mutations were nucleotide substitutions, consisting of seven transitions and one transversion (Table 1). More than half (17) of the mutations were single-nucleotide deletions that caused a frameshift in translation. Sixteen of the 17 deletions occurred at the 388th G in pyrF.

Table 1.

Summary of mutations detected in pyrEF genes.

Type of mutation	Number
Substitution (transition)	7
Substitution (transversion)	1
Frame shift (single-nucleotide deletion)	17
Large insertion	7
Total	32

Seven mutations were large insertions as described above. Nucleotide sequencing revealed that these large insertions were insertion sequences (IS), a type of mobile genetic element (Fig. 3). Six mutants had IS in the coding region of pyrF. Four of the pyrF mutants (H3, H11, H25, and H30) had IS4 elements with an identical sequence (Fig. 3A). The sequences of the IS4 elements were identical to those of the Francci3_2330 and Francci3_2941 genes (the two genes have identical sequences). The remaining two pyrF mutants (L10 and H26) had IS66 elements with an identical sequence (Fig. 3B), and the sequences of the IS66 elements were identical to those of the Francci3_0277 and Francci3_1922 genes (the two genes have identical sequences). One of the seven insertion mutants (H2) had an IS110 element in the promoter region of pyrE (Fig. 3C). The sequence of the IS110 element was identical to the Francci3_3436 gene.

Fig. 3.

Nucleotide sequences of insertion sites of IS4 (A), IS66 (B), and IS110 (C) elements. Lowercase letters represent the nucleotide sequences of the pyrE and pyrF genes, and uppercase letters represent those of the IS elements. Direct repeats (DR) are indicated by solid arrows and yellow highlights. Inverted repeats (IR) are indicated by dashed arrows and green highlights. Numbers represent positions relative to the translation initiation codon. An initiation codon (gtg) of the pyrE gene is underlined (C).

At the IS4 insertion sites, the partial sequences of the pyrF gene were duplicated, generating a 10-bp direct repeat (DR) (Fig. 3A). In addition, a 19-bp inverted repeat (IR) was found at both ends of the IS4 elements. DR and IR were also found at the IS66 insertion sites; DRs were 8 bp long and IRs were 23 bp long (Fig. 3B). Apparent DR and IR were not found at the IS110 insertion site (Fig. 3C).

Discussion

We previously isolated F. casuarinae mutants defective in nitrogen fixation using GR as a mutagen (Kucho et al., 2017). Using a next-generation sequencer, we re-sequenced the genomes of the mutants and identified nucleotide substitutions and short indels spanning several base pairs (Kucho et al., 2017). However, the short-read mapping ana­lysis conducted in that study overlooked large deletions and insertions. In the present study, we investigated mutation types (e.g., substitutions and short and long indels) induced by GR‍ ‍in the pyrEF mutants isolated by positive selection screening using the antimetabolite 5-FOA.

The most frequently observed mutations were single-nucleotide deletions (17 of 32 mutants, Table 1). Sixteen of these 17 mutations occurred at the same site in pyrF (G388_del, Table S1). The deletion of this base in the pyrF gene was also frequently found in mutants induced by ethyl methanesulfonate (Kakoi et al., 2014). Therefore, this single-nucleotide deletion did not appear to be a mutation specific to GR irradiation. The 388th G is one of seven consecutive Gs. In mono-nucleotide repeats, deletions frequently occur due to “replication slippage” (Viguera et al., 2001). During replication slippage, the newly synthesized strand by DNA polymerase and the template strand temporarily dissociate and reattach in a misaligned manner. This misalignment leads to indel mutations.

Eight of the 32 mutants carried nucleotide substitutions (Table 1). The induction of nucleotide substitutions by GR has been reported in various organisms (Glickman et al., 1980; Li et al., 2019; Yoshihara et al., 2013; Manaka et al., 2024). Ionizing radiation, such as GR, is known to induce the formation of hydroxyl radicals through the radiolysis of H₂O (Bjelland and Seeberg, 2003). These radicals may lead to the modification of DNA bases, resulting in substitutions.

We previously resequenced the genomes of GR-induced nitrogen fixation mutants (Kucho et al., 2017). In these genome ana­lyses, deletions were not observed as frequently as in the present study of pyrEF genes. Nucleotide substitutions, i.e., transitions (78% of all mutations) and transversions (15% of all mutations), were more common. Single-nucleotide deletions that were frequently observed in the pyrF gene (Table 1) occurred specifically in the consecu­tive‍ ‍G stretch, which is a hotspot for deletions by repli­cation‍ ‍slippage (Viguera et al., 2001). However, these mono-nucleotide repeats were not common across the entire‍ ‍genome. As a result, the relative number of single-nucleotide deletions to nucleotide substitutions (i.e., transitions and transversions) was lower than in the pyrF gene.

Seven of the 32 mutants had IS elements in the pyrEF genes (Table 1 and Fig. 3). ISs are the smallest mobile genetic elements, encoding only the enzymes necessary for their transposition, and are capable of repeated insertion into many different sites within a genome. IS elements are classified into major families based on the amino acid sequence of the transposase, terminal IR sequences, and flanking DR sequences generated upon insertion (Siguier et al., 2014; Siguier et al., 2015). Several environmental factors are known to induce the transposition of ISs in bacteria, such as UV irradiation, microaerobic conditions, oxidative stress, metals, antibiotics, and high temperatures (Vandecraen et al., 2017). In the radiation-resistant bacteria Deinococcus radiodurans and D. geothermalis, irradiation by GR induced the transposition of ISs. (Mennecier et al., 2006; Ye et al., 2022). In the present study, mutagenesis was performed using two doses of GR (772 and 1,158 Gy), and more IS transpositions were observed at the higher dose. Therefore, the transposition of these Frankia ISs may be activated by GR irradiation. The genome of F. casuarinae strain CcI3 was previously shown to be very rich in ISs (Normand et al., 2007), containing 148 ISs belonging to 12 families, which account for 3% of all coding sequences (Bickhart et al., 2009).

In the present study, the most numerous transposed IS element was IS4 (4 out of 7). IS4 is the most dominant IS family in F. casuarinae CcI3, with more than 50 homologs present in the genome (Bickhart et al., 2009). Sequence ana­lyses revealed that transposed IS4 homologs in the four mutants were the Francci3_2330 and Francci3_2941 genes. Since three of the four mutants had IS4 insertions at the same locus (Fig. 3A), this IS element may prefer a specific targeting site. More detailed similarity searches revealed that the Francci3_2330/2941 genes belong to the ISAs1 family. ISAs1 was identified in more than 50 bacterial species, including the fish pathogen Aeromonas salmonicida (Ross et al., 2021). ISAs1 contains a 14- to 22-bp terminal IR starting with “CAGGG”, and a similar IR was found in the Francci3_2330/2941 genes (Fig. 3A). Additionally, a 10-bp DR identified at the insertion site in other bacteria was present in our Frankia mutants (Fig. 3A).

The second most numerous transposed IS element was IS66 (2 out of 7). A previous study identified 16 IS66 homologs in the strain CcI3 genome (Bickhart et al., 2009). The transposed IS66 homologs in these two mutants were the Francci3_0277 or Francci3_1922 genes. IS66 was initially discovered in the Ti plasmid of Agrobacterium tumefaciens (Machida et al., 1984) and shortly after in a symbiotic plasmid of Rhizobium fredii (Ramakrishnan et al., 1986). Most IS66 members are found in Proteobacteria, with some also originating from Bacteroidetes/Chlorobi and Firmicutes. IS66 contains a 20- to 30-bp terminal IR starting with “GTAAGCG” (Ross et al., 2021), and similar IR was found in the Francci3_0227/1922 genes (Fig. 3B). Additionally, 8-bp DRs characteristic of the insertion site were present in our Frankia mutants (Fig. 3B).

The transposition of IS110 was detected in mutant strain H2. Nine IS110 homologs were previously shown to be pre­sent in the strain CcI3 genome (Bickhart et al., 2009). IS110 was initially discovered in the actinomycete Streptomyces coelicolor and has since been found in nearly 130 bacterial and archaeal species (Ross et al., 2021). The‍ ‍transposed IS110 homolog in the mutants was Francci3_3436. IS110 is notable in that it lacked DR and IR, and these sequences were also not found in our case (Fig. 3C).

This is the first study to demonstrate the mobilization of IS elements in the Frankia genome. Some N₂ fixation mutants (Kucho et al., 2017) may have been caused by insertional mutations by these IS elements. This result is expected to provide novel insights into the diversity and evolution of Frankia genomes. Additionally, the use of these IS elements as mutagens will be useful for genetic studies on the biology of Frankia.

Citation

Kucho, K., Han, O., and Yunoki, M. (2025) Gamma Ray-induced Mutations in pyrEF Genes in Frankia casuarinae Strain CcI3. Microbes Environ 40: ME24062.

https://doi.org/10.1264/jsme2.ME24062

Acknowledgements

We thank the Division of Isotope Science, Research Support Unit, Center for Advanced Science Research and Promotion (CASRaP), Kagoshima University for the use of the gamma ray radiation system. This work was supported by MEXT KAKENHI 19K05772.

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