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The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis

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The novel organization and complete sequence of the ribosomal

RNA gene of Nosema bombycis

Wei-Fone Huang,

a,1

Shu-Jen Tsai,

a,1

Chu-Fang Lo,

b

Yamane Soichi,

c

and Chung-Hsiung Wang

a,*

a

Department of Entomology, National Taiwan University, Taipei 106, Taiwan, ROC

bDepartment of Zoology, National Taiwan University, Taipei 106, Taiwan, ROC cBiological Laboratory, Faculty of Education, Ibaraki University, Mito 310-8512, Japan

Received 10 September 2003; accepted 15 December 2003

Abstract

We present here for the first time the complete DNA sequence data (4301 bp) of the ribosomal RNA (rRNA) gene of the mi-crosporidian type species, Nosema bombycis. Sequences for the large subunit gene (LSUrRNA: 2497 bp, GenBank Accession No. AY211393), the internal transcribed spacer (ITS: 179 bp, GenBank Accession No. AY211394), the small subunit gene (SSUrRNA: 1232 bp), intergenic spacer (IGS: 279 bp), and 5S region (114 bp) are also given, and the secondary structure of the large subunit is discussed. The organization of the N. bombycis rRNA gene is LSUrRNA-ITS-SSUrRNA-IGS-5S. This novel arrangement, in which the LSU is 50of the SSU, is the reverse of the organizational sequence (i.e., SSU-ITS-LSU) found in all previously reported

mi-crosporidian rRNAs, including Nosema apis. This unique character in the type species may have taxonomic implications for the members of the genus Nosema.

Ó 2003 Elsevier Inc. All rights reserved.

Index descriptors: Nosema; rRNA organization; microsporidia

1. Introduction

Microsporidia are tiny eukaryotic organisms (1– 10 lm) that infest all major animal groups, more than 1200 species from 143 genera of animals are reported (Wittner, 1999). These obligate, intracellular parasites are well adapted in pathogenicity, transmission, ecology, and resistance to the defense mechanisms of their hosts. In-sects in nearly all taxonomic orders are susceptible to this pathogen, but over half of the susceptible insect hosts occur in two orders, Lepidoptera and Diptera. Most of the entomopathogenic microsporidia occur in the genus Nosema, more than 150 described species found in 12 orders of insects (Becnel and Andreadis, 1999). Nosema bombycis, which is the type species of this genus (Sprague et al., 1992), has caused heavy losses in sericulture in

Europe, as well as in Asia and America, especially in the middle of 19th century (Steinhaus, 1949).

Since microsporidia lack mitochondria, for a long time they were considered to be extremely ancient eukaryotes (Vossbrinck et al., 1987). However, recent molecular data and phylogenetic analysis suggest that mitochondrial endosymbiosis occurred before the emergence of micro-sporidia (Germot et al., 1997; Hirt et al., 1997; Williams et al., 2002). The small genomic size (2.9–19.5 Mb) of these organisms indicates that may have developed strategies of packing genetic information tightly into the genome or they may have lost genetic information for a metabolic pathway and depend on host cell sources for these compounds (Weiss and Vossbrinck, 1999). Evidence from protein coding genes, especially a- and b-tubulins (Keeling, 2003; Keeling and Doolittle, 1996; Keeling and Fast, 2002; Keeling et al., 2000), and phylogenetic anal-ysis of microsporidia based on LSUrRNA sequences (Van de Peer et al., 2000) now suggest that in fact microsporidia share a common origin with fungi.

*

Corresponding author. Fax: +886-2-2736-4329. E-mail address:wangch@ccms.ntu.edu.tw(C.-H. Wang).

1

These two authors equally contributed to this work.

1087-1845/$ - see front matterÓ 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2003.12.005

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subunit rRNAs of microsporidia are highly conserved, but in contrast to the many microsporidian SSUrRNA sequences in GenBank, only 4 complete LSUrRNA gene sequences have been published. These are for Nosema apis (Gatehouse and Malone, 1998), Microsporidium 57864 (GenBank Accession No. U90885), Heterosporis anguil-larum (Tsai et al., 2002), and Encephalitozoon cuniculi (Peyretaillade et al., 1998). There are also several partial sequences for microsporidian LSUrRNAs in GenBank, including a portion (approximately 350 nucleotides) of the LSUrRNA from Vairimorpha and Nosema species (Baker et al., 1994). Baker et al. (1995) noted that N. apis bears a closer resemblance (in terms of its SSUrRNA se-quence) to some Vairimorpha species than it does to some other Nosema species. For N. bombycis, the full SSUrR-NA sequence (1232 bp) but only a partial LSUrRSSUrR-NA se-quence (292 bp) have been published (GenBank Accession Nos. D85503 and L28962) (Baker et al., 1994, 1995). Microsporidian rRNAs are hard to sequence completely, because it is difficult to design suitable primer sets when the microsporidan LSUrRNA sequences are highly diverse and, as we show here for N. bombycis, the rRNA gene has a novel organization.

the National Institute of Agrobiological Science, Ja-pan. The purification of spores was carried out as de-scribed previously (Huang et al., 1998; Tsai et al., 2002). Then, for DNA extraction, a suspension of purified spores (2 107 spores in 0.25 ml TE buffer) was mixed with an equal volume of zirconia/silica beads (0.1 mm diameter) in a 10 75 mm glass tube and shaken at maximum speed on a vortex mixer for 1 min (Undeen and Cockburn, 1989). The DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturerÕs in-structions. The DNA was eluted in TE buffer and stored at )20 °C. The DNA concentration was mea-sured by a GeneQuant II RNA/DNA Calculator (Pharmacia, Uppsala, Sweden).

2.2. Amplification and sequencing strategy of rRNA genes The primer sets used for rRNA gene amplification and the expected sizes of the amplicons are shown in Table 1 and Fig. 1. [Primer sets intended to amplify the ITS and adjoining 50 or 30 end of the LSUrRNA were also designed based on the published sequence for N.

Table 1

Primers used for amplification and sequencing of N. bombycis rRNA

Primer Sequence Amplicon size (bp)

Large subunit rRNA (LSU) 2108

LS228F 50-GGA GGA AAA GAA ACT AAC-30

ILSUR 50-ACC TGT CTC ACG ACG GTC TAA AC-30

50end of LSU

LSUF 50-ACT CTC CTC TTT GCC TCA ATC A-30

HG4R 50-CGC CGA ATT AAA CTG AGT TG-30

Internal transcribed spacer (ITS) 501

ILSUF 50-TGG GTT TAG ACC GTC GTG AG-30

S33R 50-ATA GCG TCT ACG TCA GGC AG-30

Small subunit rRNA (SSU) 1232

18f 50-CAC CAG GTT GAT TCT GCC-30

1537r 50-TTA TGA TCC TGC TAA TGG TTC-30

Intergenic spacer (IGS) and 5S rRNA 852

HG4F 50-GCG GCT TAA TTT GAC TCA AC-30

5SR 50-TAC AGC ACC CAA CGT TCC CAA G-30

LS228R 50-CCT CCT TTT CTT TGA TTG-30

Nosema bombycis putative pseudogene

KAI01N 50-GTA GTA GAG ACC CAA ATA TC-30

KAI02N 50-ACT GTT CAG ATA TGG TCC TTA TCG-30

(modified from KAI01 and KAI02 by removing the restriction enzyme site)

Primers LS228F, ILSUR, 18f, and 1537r are from Vossbrinck et al. (1993). HG4F and HG4R are from Gatehouse and Malone (1998). ILSUF (Tsai et al., 2002) is the complementary sequence to ILSUR. KAI01N and KAI02N (Tsai et al., 2003) are modified from Kawakami et al. (1994). 5SR was designed based on the conserved region of 5S, HG4R-c is the complementary sequence to HG4R, and S33R is the reverse sequence of the SSUrRNA located between nucleotides 14 and 33.

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apis (Gatehouse and Malone, 1998), but these primer sets failed; data not shown.]

For amplification, the genomic DNA (80 ng) of N. bombycis was mixed in a 100 ll PCR reaction mixture containing 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 100 mM of each dNTP, 100 pmol of each primer (Table 1), and 2.5 U Taq DNA polymerase (Promega). The amplification was performed in an AG-9600 Thermal Station (Biotronics) for 40 cycles, each with the following profile: 94°C for 30 s, 50 °C for 30 s, and 72°C for 2 min. A 10 ll aliquot from each reaction was run on a 1.0% agarose gel to visualize the PCR products. The gel was photographed using the Eagle-Eye II photo-documentation system (Stratagene). The PCR products were eluted by an E.Z.N.A. Gel Extrac-tion Kit (Omega Bio-tek). The eluted DNAs were then sequenced directly on an automated DNA Sequencer (DNA Sequencer 377, Applied Biosystems).

2.3. Confirmation of the rRNA gene organization of N. bombycis

The total length of N. bombycis rRNA was amplified by the primer set LSUF/5SR with Platinum Pfx DNA polymerase (Invitrogen). The amplicon was then eluted and used as a template to amplify fragments with the primer sets HG4R-c/HG4F-c and ILSUF/HG4F-c for partial LSUrRNA-ITS-partial SSUrRNA; ILSUF/ 1537R for partial LSUrRNA-ITS-SSUrRNA; and ILSUF/5SR for partial LSUrRNA-ITS-SSUrRNA-IGS-5S. The amplification protocol was as described above.

2.4. Secondary structure construction

The secondary structures of N. bombycis LSUrRNA were constructed by a combined manual and automatic method in which the N. bombycis LSUrRNA sequence was aligned to the rRNA database to generate DCSE alignment files (De Rijk and De Wachter, 1993). The helices in the LSUrRNA secondary structure elements were then located and labeled based on database on the LSUrRNA secondary structure (De Rijk et al., 1998a), while the hypervariable areas (V1-12) were numbered in accordance with N. apis and with all known eukaryotic

LSUrRNAs (De Rijk et al., 1998b; Wuyts et al., 2001). The final drawings were rendered by the RnaViz pro-gram (De Rijk and De Wachter, 1997; De Rijk et al., 2003). The secondary structures of N. bombycis SSUrRNA are already known and can be found in the European small subunit ribosomal RNA database (Van de Peer et al., 1998).

3. Results and discussion

3.1. The complete sequence and organization of N. bombycis rRNA

The complete DNA sequence of the N. bombycis rRNA gene contained 4301 bp (see Appendix A) and was submitted to the GenBank with Accession No. AY259631. The organization of the gene is shown in Fig. 1. Starting from the 50end, the N. bombycis rRNA gene consists of the large subunit gene (LSUrRNA: 2497 bp; submitted to GenBank with Accession No. AY211393), the internal transcribed spacer (ITS: 179 bp; GenBank with Accession No. AY211394), the small subunit gene (SSUrRNA: 1232 bp), the intergenic spacer (IGS: 279 bp), and the 5S region (114 bp). This organizational sequence, in which, the LSU gene is 50 of the SSU, is unique among microsporidia and is the reverse of the organizational sequence for all previ-ously reported microsporidian rRNAs (Gatehouse and Malone, 1998; Peyretaillade et al., 1998; Tsai et al., 2002).

PCR results with various combinations of the primers listed in Table 1 are shown in Figs. 2 and 3. The amplicon yielded by the primer set ILSUF/S33R (Fig. 2, lane 4) specifically confirms the novel orga-nization of this gene (i.e., LSU-ITS-SSU). Conversely, the primer set HG4F/LS228R would usually amplify the ITS region between the SSU and LSUrRNA, but the absence of any amplicon with these primers (Fig. 2, lane 7) provides further evidence that the LSU is 50 of the SSU (and also suggests that the N. bombycis rRNA gene is not a multiple gene). Further confirmation that the N. bombycis rRNA gene is organized as shown in Fig. 1 was provided by am-plifying the whole rRNA gene with the LSUF/5SR

Fig. 1. Schematic diagram of N. bombycis rRNA gene. Mature rRNA gene domains are boxed. Details of the primers are given in Table 1. The gray-shaded primer sets were used to amplify the SSUrRNA coding region and the main part of the LSUrRNA coding region. HG4R-c and HG4F-c are the complementary sequences of HG4R and HG4F, respectively.

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primer set (Fig. 3A). The resultant amplicon was of the predicted length (4442 bp), and when four internal fragments were amplified (Fig. 3B) and sequenced, results were completely consistent with the DNA se-quence listed in Appendix A.

3.2. LSUrRNA gene sequence

The main part of the LSUrRNA gene was amplified with the LS228F/ILSUR primer set, which produced an amplicon of 2108 bp (Fig. 2, lane 1). For sequencing the

Fig. 2. Agarose gel electrophoresis of PCR products. Lane 1, main part of LSUrRNA (primer set LS228F/ILSUR; 2108 bp amplicon). Lane 2, 50

region of LSUrRNA (primer set LSUF/LS228R). Lane 3, 50region and main part of LSUrRNA (primer set LSUF/ILSUR). Lane 4, 30end of

LSUrRNA- ITS-50end of SSUrRNA (arrow) (primer set ILSUF/S33R). Lane 5, SSUrRNA (primer set 18f /1537r). Lane 6, 30end of

SSUrRNA-IGS-5S (arrow) (primer set, HG4F/5SR). Lane 7, the result of amplification with HG4F/LS228R. Lane 8, (DNA quality control) the result of amplification with the N. bombycis specific primer set KAI01N/KAI02N (Tsai et al., 2003).

Fig. 3. Confirmation of the organization of the N. bombycis rRNA gene. (A) Amplification with the primer set LSUF/5SR yielded an amplicon of the predicted size (4401 bp). M, 1 kb DNA ladder (Promega). (B) Amplification of four internal rRNA fragments. Lane 1, 30end of LSUrRNA-

ITS-main part of SSUrRNA (1261 bp; primer set ILSUF/HG4F-c). Lane 2, 30end of LSUrRNA-ITS-SSUrRNA (1700 bp; primer set ILSUF/1537R).

Lane 3, 30 end of LSUrRNA-ITS-SSUrRNA-IGS-5S (2093 bp; primer set ILSUF/5SR). Lane 4, main part of LSUrRNA-ITS-main part of SSUrRNA (3031 bp; primer set HG4R-c/HG4F-c). M, 1 kb DNA ladder (Promega).

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50end of LSUrRNA, the primer set LSUF/LS228R was used (Fig. 2, lane 2). The putative start and terminal regions were determined by comparison to the N. apis LSUrRNA sequence (Gatehouse and Malone, 1998) and by the secondary structure construction of N. bombycis LSUrRNA gene (Fig. 4). The LSUrRNA gene

contains 2497 bp and its base composition is 31.9% G + C, which is the lowest G + C content of all known microsporidian LSUrRNA genes. The LSUrRNA se-quence identities between N. bombycis and N. apis (GenBank accession no. U97150; Gatehouse and Malone, 1998), Microsporidium 57864 (GenBank

Fig. 4. A model of the secondary structure of N. bombycis large subunit (LSU) rRNA. Helix numbering is according to De Rijk et al. (1998a). The boxed regions indicate parts of the structure where hypervariable areas are found in the eukaryotic rRNAs (De Rijk et al., 1998b; Wuyts et al., 2001).

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1994) had 100% identity with the sequence from 132 to 423 bp from the 50end.

Like all microsporidia, the internal transcribed spacer (ITS) region of N. bombycis lacks the 5.8S rRNA gene. However, as in Vairimorpha necatrix and N. apis, in which the 50 ends of the LSUrRNA genes include a se-quence that corresponds to 5.8S (Gatehouse and Ma-lone, 1998; Vossbrinck and Woese, 1986), the sequence of nucleotides in the N. bombycis LSUrRNA gene lo-cated at 1–160 from the 50end corresponds to the known fungal 5.8S rRNA sequences. Compared to Cystofi-lobasidium bisporidii (GenBank Accession No. M94511), Lactarius acerrimus (GenBank Accession No. AJ278139), Thanatephorus cucumeris (GenBank Acces-sion No. AB019008), Trichoderma reesei (GenBank Accession No. L27800), and Tuber cf. rapedorum (GenBank Accession No. AJ278140), the homologies are 34, 44, 44, 44, and 42%, respectively, by Clustal X and GeneDoc.

The secondary structure of the LSUrRNA of N. bombycis (Fig. 4) is basically similar to that of N. apis and H. anguillarum (De Rijk et al., 1998b; Tsai et al., 2002; Van de Peer et al., 2000). Based on the secondary structures of the eukaryotic LSUrRNA of Xenopus la-evis (De Rijk et al., 1998a) and the eukaryotic database (Wuyts et al., 2001), eight helical groups (B–I) can be distinguished clockwise from a core area. Eleven hy-pervariable areas (V1–5; V7–12) are also shown in Fig. 4. Nine helices (B6, B7, B8, B14, B21, D5, E9, E15, and G5) are missing. Six areas of the hypervariable areas are also almost entirely missing (V2, V3, V8, V10, V11, and V12), and two areas are extremely reduced (V5 and V9). V6 is almost absent. The secondary structure of N. bombycis LSUrRNA diverges most markedly from the LSUrRNAs of N. apis, Microsporidium 57864, H. an-guillarum, and E. cuniculi in the V4 area. The V3 areas of N. bombycis and N. apis LSUrRNAs are similar in conformation. By comparison, the LSUrRNA of E. cuniculi s has its own specific conformation in V3 (lack of the D3 helix), while the LSUrRNA of H. anguillarum has specific conformations in five other areas (V5, V6, V7, V9, and V10).

3.3. ITS sequence

In contrast to all known microsporidian rRNA genes, the ITS region of N. bombycis is 30of the LSU and 50of the SSUrRNA. It consists of 179 bp located between nucleotides 2498–2576 from the 50 end of rRNA gene (Fig. 1), and its G + C content is 19.6%. The N. bombycis

between nucleotides 2677–3908 relative to the 50end of the rRNA gene (Fig. 1). The G + C content of the SSUrRNA gene is 34.2%. The complete DNA sequence of the SSUrRNA gene of N. bombycis has 99% homol-ogy to the N. bombycis SSUrRNA sequence already held in GenBank (GenBank Accession No. D85503; different nucleotides at 3497 and 3874), to another mi-crosporidian isolate from Japan (GenBank Accession No. D85504; Hatakeyama et al., 1997), and also to five Nosema isolates from Taiwan (Tsai et al., 2003). 3.5. IGS

The IGS region consisted of a 279 bp sequence lo-cated between the SSU and 5S rRNA genes (nucleotides 3909–4187). Its G + C content is 30%. Homology with other known microsporidian ITS or IGS sequences is low; comparisons using standard nucleotide–nucleotide BLAST [blastn], Nucleotide Blast, and NCBI found only 20 matching nucleotides.

3.6. 5S rRNA gene

The 5S rRNA of N. bombycis consists of 114 bp (in-cluding the putative end), and is located between nu-cleotides 4188 and 4301 from the 50 end of the rRNA gene. Its G + C content is 47.3%. The sequence has a high homology, 91 and 92%, respectively, with the 5S rRNAs of two N. bombycis isolates from Japan (Gen-Bank Accession Nos. D14631 and AB097401; Kawa-kami et al., 1992), but a homology of only 77% with the 5S region of Microsporidium 57864 (GenBank Acces-sion No. U90885).

The members of the genus Nosema are often consid-ered the most important and widely distributed group of insect microsporidia (150 described species found in 12 orders of insects) (Becnel and Andreadis, 1999; Tanada and Kaya, 1993). As N. bombycis is the type species of this genus, its characteristics—not only as they relate to life cycle, development, and morphology, but also in terms of their biochemical and molecular characteris-tics—are critical. The unique organization of the N. bombycis rRNA therefore has implications for the taxonomy of the Nosema group.

Acknowledgments

This paper was supported by the National Science Council, ROC (Grant No. NSC 92-2313-B-002-030).

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We would like to thank Dr. R. Sugimoto of the MAFF GENE Bank of the National Institute of Agrobiological Science, Japan for donating spores of the type species,

N. bombycis. We would also like to thank Dr. J. Wuyts, University of Antwerp, Belgium, for his assistance with the secondary structure.

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Fig. 1. Schematic diagram of N. bombycis rRNA gene. Mature rRNA gene domains are boxed
Fig. 2. Agarose gel electrophoresis of PCR products. Lane 1, main part of LSUrRNA (primer set LS228F/ILSUR; 2108 bp amplicon)
Fig. 4. A model of the secondary structure of N. bombycis large subunit (LSU) rRNA. Helix numbering is according to De Rijk et al

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