Group-I Intron Containing a Putative Homing Endonuclease Gene in the Small Subunit Ribosomal DNA of Beauveria bassiana IFO 31676

Eiji Yokoyama*, Kenzo Yamagishi{dagger} and Akira Hara{ddagger}

*The Agricultural High-Tech Research Center,
{dagger}Laboratory of Entomology,
{ddagger}Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan

Group-I introns have been identified in the organellar and nuclear genomes of higher plants, fungi, amoebae, green algae, and archaea, and in the genomes of viruses and phages. This wide distribution and phylogenetically sporadic existence of group-I introns has led to the hypothesis of horizontal transfer. In the pyrenomycete fungi, Nikoh and Fukatsu (2001)Citation supposed that the common ancestor of Cordyceps prolifica and C. kanzashiana horizontally gained group-I introns of ribosomal DNA (rDNA) from distantly related fungi. An interkingdom transfer of group-I introns of rDNA between a plant, Youngia japonica, and a phytopathogenic fungus, Protomyces inouei, was also proposed by Nishida and Sugiyama (1995)Citation . Although the intron mobility of nuclear rDNA is widely accepted, few intron-encoded endonucleases are known. Because of the lack of the endonuclease gene, except for the cases of Nectria galligena (Johansen and Haugen 1999Citation ) and the ericoid fungal isolates (Perotto et al. 2000Citation ), most of the group-I introns in the fungal rDNA are principally thought to be immobile and vertically transmitted (Nikoh and Fukatsu 2001Citation ). The present study deals with the group-I intron in the small subunit (SSU) rDNA of Beauveria bassiana IFO 31676, which has a putative endonuclease gene.

During the course of the phylogenetic analysis of the clavicipitalean fungi, we determined the nucleotide sequences of SSU rDNA of B. bassiana and related fungi. A polymorphism with a nucleotide length similar to that of SSU rDNA was observed in the B. bassiana species. This polymorphism was due to the inserted group-I introns. Group-I introns are abundant in the rDNAs of the clavicipitalean fungi, their specific insertion positions are characterized at SSU516, SSU943, SSU989, SSU1199, LSU1921, LSU2066, LSU2449, and LSU2563, relative to Escherichia coli (Nikoh and Fukatsu 2001Citation ). Beauveria bassiana IFO 4848 (Nikoh and Fukatsu 2000Citation ) and IFO 31953 have no group-I introns in their SSU rDNAs. Beauveria bassiana IFO 31676 has a large group-I intron (1,866 bases) at the position SSU943 (fig. 1 ). Beauveria bassiana IFO 8554 and BCMU BB01 have two short group-I introns (about 380 bases) at positions SSU943 and SSU1199. All the SSU943 group-I introns have the conserved sequence elements P, Q, R, and S of the Cordyceps fungi (Cech 1988Citation ; Nikoh and Fukatsu 2001Citation ) (fig. 1 ). The introns at the same inserted position are closely related (Nikoh and Fukatsu 2001Citation ). The SSU943 introns of B. bassiana IFO 8554 and BCMU BB01 are identical. The nucleotide sequence (positions 1–300 in fig. 1 ) of the SSU943 intron of B. bassiana IFO 31676 has 92.0% identity with the sequences of the SSU943 introns of B. bassiana IFO 8554 and BCMU BB01. In fungi, except for N. galligena (Johansen and Haugen 1999Citation ) and the ericoid fungi (Perotto et al. 2000Citation ), the group-I introns reported so far were shorter by about 500 bases because of a lack of the endonuclease gene. The large SSU943 intron of B. bassiana IFO 31676 contains a putative homing endonuclease gene between the conserved elements R and S (fig. 1 ). The predicted amino acid sequence of the B. bassiana endonuclease has a His-Cys box (Johansen et al. 1993Citation ) conserved in the homing endonucleases (fig. 1 ). Comparisons of the amino acid sequence of the B. bassiana endonuclease with databases were done using BLAST (Altschul et al. 1990Citation ). High similarities were found at the His-Cys box, with the group-I intron–encoded homing endonucleases in rDNAs of fungi N. galligena NI3 and NI109 (Johansen and Haugen 1999Citation ), ericoid fungi isolate PSIV (Perotto et al. 2000Citation ), amoebae Naegleria sp. NG874 (De Jonckheere and Brown 2001Citation ), and Naegleria andersorni A2 (De Jonckheere 1994Citation ), and slime mold Physarum polycephalum Carolina (Muscarella et al. 1990Citation ). All homing endonucleases of the nuclear rDNA introns reported so far have the His-Cys box and belong to the ßß{alpha}-Me family (Gimble 2000Citation ), although most of the homing endonucleases belong to the LAGLI-DADG family or to the GIY-YIG family (Gimble 2000Citation ). His-Cys box endonucleases generally have two zinc-binding motifs (Cys–Xn–Cys–X4–Cys–X4–His and Arg–X2–Cys–X6–20–Cys–X–His–X3–Cys) (Elde et al. 2000Citation ). The homing endonuclease of B. bassiana IFO 31676 lacks the last Cys residue in the second zinc-binding motif (fig. 1 ). The homing endonuclease of Didymium iridis Pan2 (Johansen and Vogt 1994Citation ) also lacks the second zinc-binding motif and was proved to be functional (Johansen et al. 1997Citation ). The three residues (Arg61, His98, and Asn119) of the homing endonuclease of P. polycephalum are important for DNA cleavage reaction (Mannino et al. 1999Citation ). These three residues correspond to Arg192, His248, and Asn269 of the homing endonuclease of B. bassiana IFO 31676 (fig. 1 ). But the possibility that the homing endonuclease of B. bassiana IFO 31676 is not functional cannot be excluded. The endonuclease genes of B. bassiana IFO 31676 and the ericoid isolate PSIV locate on the sense strand of their introns, whereas those of N. galligena NI3 and NI109 locate on the antisense strand. The direction of transcription of the endonuclease gene may not be important.



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Fig. 1.—Nucleotide sequence of the SSU943 intron of B. bassiana IFO 31676. Partial alignment with the nucleotide sequence of SSU943 intron of B. bassiana IFO 8554 was prepared using ClustalX (Thompson et al. 1997Citation ). Dots and hyphens represent identical nucleotides and gaps in the alignment, respectively. The conserved elements P, Q, R, and S are boxed. The deduced amino acid sequence of a putative homing endonuclease is presented below the nucleotide sequence. The conserved His-Cys box is underlined. The amino acid residues required for the activity of the nuclease and binding of Zn are boldfaced. This sequence data was deposited in the DDBJ/EMBL/GenBank databases under the accession number AB079608

 
Phylogenetic trees based on the SSU rDNAs and on the SSU943 introns of B. bassiana and related fungi were prepared (fig. 2A ). Paecilomyces tenuipes IFO 30367 and IFO 31161 were used as an out-group in the SSU rDNA tree. The topology of the SSU rDNA tree agrees well with that of Nikoh and Fukatsu (2000)Citation . For investigating the possibility of horizontal transfer, an appropriate out-group could not be selected in the SSU943 intron tree. Except for B. bassiana IFO 31676, the topology of the SSU943 intron tree agrees well with that of the SSU rDNA tree. The monophyly of the SSU943 introns of Beauveria species, except for B. bassiana IFO 31676, is supported by a high bootstrap value, 86.5%. If the evolutionary rate of the endonuclease-carrying intron is equal to that of the truncated introns, the SSU943 intron of B. bassiana IFO 31676 is distantly related to other SSU943 introns of Beauveria species. This suggests that the SSU943 introns of B. bassiana IFO 8554 and BCMU BB01 are not the deletion derivatives of the SSU943 intron of B. bassiana IFO 31676. The SSU rDNA tree shows that B. bassiana IFO 31676 is closely related to other B. bassiana species. Discrepancy in the position of B. bassiana IFO 31676 in the SSU943 intron tree may be because of a horizontal transfer. The presence of the endonuclease gene in the SSU943 intron of B. bassiana IFO 31676 might support this hypothesis. Beauveria bassiana IFO 4848 and IFO 31953 have no intron. Intron loss and gain by a horizontal transfer could occur in the lineage of B. bassiana. Similar situations are predicted in the rDNAs of Naegleria species (De Jonckheere and Brown 1994Citation , 2001Citation ). In the group-I introns of yeast mitochondrial rDNA, a cyclical model of invasion, degeneration, loss, and reinvasion was proposed by Goddard and Burt (1999)Citation . Phylogenetic analysis based on the internal transcribed spacer 2 (ITS2) of Beauveria species reveals that the monophyly of B. bassiana IFO 31953 (no intron), IFO 4848 (no intron), and IFO 31676 (intron with a homing endonuclease gene) is supported by a high bootstrap value of 93.7% (fig. 2B ). Beauveria bassiana IFO 31676 once had lost a truncated intron corresponding to that of B. bassiana IFO 8554 and BCMU BB01 and then gained a mobile intron by a horizontal transfer.



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Fig. 2.—Phylogenetic trees based on SSU rDNA and SSU943 intron (A), and on ITS2 (B). Multiple alignments of the nucleotide sequences were prepared using ClustalX (Thompson et al. 1997Citation ). These alignments will be available upon request. Gap positions of alignments were excluded from a subsequent phylogenetic analysis. Ambiguously aligned positions were also excluded. The unambiguously aligned 1,701 (SSU rDNA), 342 (SSU943 intron), and 161 (ITS2) positions were analyzed by the neighbor-joining method (Saitou and Nei 1987Citation ). Distances were corrected using the Kimura's two-parameter method (Kimura 1980Citation ). The bootstrap tests were conducted with 1,000 resamplings. *The sequence data determined in this work were deposited in the DDBJ/EMBL/GenBank databases under the accession numbers AB079125 (B. bassiana IFO 8554), AB079608 (B. bassiana IFO 31676), AB079126 (B. bassiana IFO 31953), AB079609 (B. bassiana BCMU BB01), AB070374 (C. militaris IFO 9787), AB070375 (C. militaris IFO 30377), AB070371 (P. tenuipes IFO 30367), AB070372 (P. tenuipes IFO 31161). Fungi of the IFO collection were purchased from Institute for Fermentation, Osaka, Japan. Beauveria bassiana BCMU BB01 was obtained from a synnema formed on a wasp, Polistes jokahamae, at the Prefecture of Aichi, Japan. **These sequences are determined by Nikoh and Fukatsu (2000)Citation and are deposited under the accession numbers AB027336 and AB027382 (B. bassiana IFO 4848), AB027335 and AB027381 (B. brongniartii IFO 5299).

 
Recently, group-I twintrons (1.3–1.4 kb) containing a homing endonuclease gene were reported in the rDNAs of Didymium and Naegleria species (Einvik et al. 1998Citation ). The SSU943 intron of B. bassiana IFO 31676 (1.9 kb) is larger than these twintrons. We are analyzing the detailed secondary structure of the SSU943 intron of B. bassiana IFO 31676 and its in vitro self-cleavage reactions.

Acknowledgements

This work was supported by the Agricultural High-Tech Research Center, Meijo University, under the "Environmental Control through the Function of Microorganisms" project.

Footnotes

Pierre Capy, Reviewing Editor

Keywords: group-I intron Beauveria bassiana homing endonuclease mobile element small subunit ribosomal DNA Back

Address for correspondence and reprints: Akira Hara, Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan. hara{at}ccmfs.meijo-u.ac.jp Back

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Accepted for publication June 2, 2002.