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Journal of Bacteriology, March 2007, p. 1633-1640, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01210-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of a Group II Intron without an Open Reading Frame in the Alpha-Toxin Gene of Clostridium perfringens Isolated from a Broiler Chicken{triangledown}

Menglin Ma,1 Kaori Ohtani,2 Tohru Shimizu,2 and Naoaki Misawa1*

Department of Veterinary Public Health, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuenkibanadai-Nishi, Miyazaki, 889-2192, Japan,1 Department of Microbiology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-Machi, Kanazawa, Ishikawa 920-8640, Japan2

Received 3 August 2006/ Accepted 18 November 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Clostridium perfringens is a gram-positive anaerobic organism which is widely distributed in the environment and in the gastrointestinal tracts of animals and is associated with enteric and histotoxic diseases in humans and animals (12, 22). It has been reported that C. perfringens produces a variety of toxins, and these toxins are considered the most important virulence factors of this organism (22). C. perfringens strains are classified into five types (A, B, C, D, and E) according to the production of four major toxins (alpha, beta, epsilon, and iota) (1, 22). The alpha toxin is produced by all five types of C. perfringens and is the first bacterial protein from C. perfringens reported to show both enzymatic and toxic properties (12). The mature alpha toxin, produced by a single cpa gene and consisting of one single polypeptide with a molecular mass of 42.5 kDa, is a zinc-dependent phospholipase C (PLC) with 370 amino acid residues (30, 31, 41). The PLC produced by C. perfringens exhibits various biological activities, such as hemolysis, necrosis, and lethality to the intracellular metabolism of eukaryotic target cells (6, 22, 32).

Mobile genetic elements have important impacts on eukaryotic and prokaryotic genomes (5, 13, 18). In the prokaryotic world, transposable elements, such as insertion (IS) elements and transposons, which can disrupt the inserted genes, are involved in large-scale genome rearrangement and in the transfer of accessory genes and thus usually have important impacts on genome evolution (4). Introns, another type of interesting mobile element, are classified into four major classes, namely, self-splicing group I and group II introns, tRNA and rRNA archaeal introns, and spliceosomal introns in nuclear pre-mRNA (8, 14, 29, 34, 37). Bacterial introns belong to either group I or II, among which group I introns are mostly found in tRNAs or in protein-encoding genes associated with DNA metabolism of bacteriophages (8, 10, 13), while most group II introns are inserted in or associated with other mobile genetic elements, such as IS elements, transposons, or plasmids (10, 19, 21, 35, 45). After DNA insertion, they can remove themselves by protein-assisted autocatalytic RNA splicing (19). During the past several years, introns have been detected in surprising numbers in bacterial genomes as a result of genome sequencing projects (17, 20, 42).

In the genus Clostridium, introns have been reported for Clostridium difficile (2, 13, 28, 36). Although group I and group II introns have not been identified in C. perfringens, the sequence of a predicted group II intron reverse transcriptase (RT) gene, which was from plasmid pCPF4969 of strain F4969 (26), was recently deposited in GenBank with accession number AB236336.1. However, the group II intron present in the plasmid has not been characterized fully. In our previous experiments involving PCR screening of the cpa genes from 110 C. perfringens strains isolated from broiler chickens (unpublished data), we detected one isolate (designated CPBC16ML) with a larger amplicon for the cpa gene (Fig. 1A). Interestingly, strain CPBC16ML showed both PLC and hemolytic activities, as detected in reference strains (Fig. 1B and C). It was reported that the cpa gene is highly conserved, with an overall sequence difference of only about 1.5% (12, 16, 39). Therefore, we hypothesized that a mobile genetic element may be integrated into the cpa gene.


Figure 1
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  		FIG. 1. (A) PCR amplification of the cpa gene, using primers cpalphatox1-L and -1-R. Strain designations for the lanes are as follows: lane 1, CPBC16ML; lane 2, H4; and lane 3, CPBC102. The molecular size marker is indicated to the left of the gel. The amplicon of CPBC16ML represents a larger size than the others. (B) Colonies of strains H4, CPBC102, and CPBC16ML on egg yolk-CW agar plates. CPBC16ML showed typical C. perfringens colonies with a surrounding zone of precipitation, similar to those of the wild-type strain CPBC102 and the reference strain H4. (C) Colonies of strains H4, CPBC102, and CPBC16ML on horse blood agar plates. CPBC16ML also showed hemolytic activity, as seen in strains H4 and CPBC102.

 
Our goals in this study were to determine the nucleotide sequence of the cpa gene of CPBC16ML, to clone it for expression in Escherichia coli, to determine its molecular size and reactivity against an anti-Cpa antibody, and to examine the expression of the alpha toxin. As a result of these studies, the presence of a group II intron without an open reading frame (ORF) integrated into the cpa gene of C. perfringens was demonstrated.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and culture conditions. A total of 110 C. perfringens strains were isolated from the large intestinal contents of broiler chickens. Colonies indicating PLC activity on kanamycin-containing CW agar plates (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with egg yolk (EY-CW) were subjected to 16S rRNA gene sequence analysis for bacterial identification. PCR of the 16S rRNA gene was performed using bacterial universal primers F8 and R15 (Table 1), as described previously (25). The nucleotide sequences were determined directly from the PCR amplicons, using each universal primer. C. perfringens strains CPBC16ML and CPBC102 were selected from the 110 isolates and used for further studies. In addition to chicken isolates, C. perfringens strain H4 (type A, isolated from a human), which possesses a normally sized cpa gene, was used as a control. In addition to the PLC activity on EY-CW agar plates, the hemolytic activity of these strains was examined on GAM agar plates (Nissui) supplemented with 10% horse blood. C. perfringens strain 13, which has been sequenced completely (40), was used for Southern and Northern hybridization tests, as described below. Escherichia coli DH5{alpha} (TaKaRa, Tokyo, Japan) and M15 (QIAGEN Inc., Tokyo, Japan) were used for cloning and expression, respectively. E. coli DH5{alpha} was cultured on Luria-Bertani (LB) agar plates containing 100 µg/ml of ampicillin. Recombinant E. coli M15 was grown in LB broth or on LB agar plates containing 100 µg/ml of ampicillin and 25 µg/ml of kanamycin.


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  		TABLE 1. Primers used in this study

 
Genetic techniques. C. perfringens strains were grown aerobically in GAM broth (Nissui) at 37°C for 4 to 8 h, and DNAs were extracted using the hexadecyltrimethyl ammonium bromide (CTAB) method as described previously (11). Plasmids were isolated by using a QIAprep Spin plasmid kit (QIAGEN), and PCR products were purified with a QIAquick PCR purification kit (QIAGEN). RNAs of C. perfringens were extracted from GAM broth culture, using an RNeasy Mini kit (QIAGEN). All other standard molecular techniques were performed as described elsewhere (38). DNA sequencing was performed with a BigDye Terminator cycle sequencing kit, version 3.1 (Applied Biosystems Inc., Tokyo, Japan), and an ABI PRISM model 310 automated sequencer (Applied Biosystems) at the Genetic Center of the University of Miyazaki. Computer analysis of DNA and protein sequences was performed with GENETYX-WIN 5.4 software. RNA secondary structures were analyzed with GENETYX-WIN 5.4 and mfold (47), version 3.2, which is available at http://www.bioinfo.rpi.edu/applications/mfold/old/rna/. Database similarity searches were performed through the National Center for Biotechnology Information by use of the BLASTX and BLASTN algorithms.

PCR techniques. The primers used in this study are listed in Table 1. Primers cpasF1 and cpaR6 were designed from the sequence of C. perfringens strain 13, and the other primers for the cpa gene were designed based on nucleotide sequences from strain CPBC16ML.

(i) Standard PCR. To detect the cpa gene in C. perfringens, PCR was performed in a thermocycler (Applied Biosystems), using primer pair cpalphatox1-L/1-R (Table 1 and Fig. 2), as described elsewhere (3). PCR was performed in a final volume of 20 µl, which consisted of 20 pmol (each) of forward and reverse primers, a 200 µM concentration of each deoxynucleoside triphosphate, 0.5 U of Taq DNA polymerase (QIAGEN), 1x PCR buffer, and 50 ng of DNA template. Thermal cycle conditions consisted of denaturation at 94°C for 1 min, annealing at 45°C for 1 min, and extension at 72°C for 1 min. After 35 thermal cycles, the reaction was completed with a final extension step at 72°C for 10 min. Following amplification, the resulting amplicons were analyzed by electrophoresis on a 1% agarose gel.


Figure 2
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  		FIG. 2. Schematic structure of the cpa gene of C. perfringens strain CPBC16ML. Arrows indicate the orientations of the primers for the cpa gene. The CPF-G2Im sequence detected in cpa is shown as a gray box. Primers cpasF1 and cpaR6 were designed from the sequence of C. perfringens strain 13, and all other primers were designed based on the nucleotide sequence of strain CPBC16ML. Probes 1, 2, and 3 are PCR amplicons used as DNA probes for Southern and Northern hybridization.

 
(ii) RT-PCR. Prior to reverse transcription, RNAs purified from C. perfringens were treated with 10 U of DNase I (QIAGEN) at 37°C for 30 min. cDNA synthesis was carried out using a cDNA synthesis kit (TaKaRa). Reverse transcriptase was inactivated by heating at 70°C for 10 min prior to RT-PCR. RT-PCR was carried out with primer pair cpaF2/cpalphatox1-R, cpaF8/cpaR10, cpaF2/cpaR10, or cpaF8/cpalphatox1-R (Table 1 and Fig. 2). The reaction conditions and thermal cycles were the same as those for the standard PCR. Nucleotide sequencing of the RT-PCR products was performed as described above.

Cloning and protein expression of the C. perfringens cpa gene in E. coli. A DNA fragment of the intact cpa gene, including its promoter, was generated by PCR using primers cpasF1 and cpaR6 (Table 1 and Fig. 2) from C. perfringens strains CPBC16ML, CPBC102, and H4. A six-His-tagged protein expression vector, pQE30-UA (QIAGEN), was used to express the C. perfringens alpha toxin in E. coli M15 cells. Each cpa gene from the three strains was cloned into the pQE30-UA vector, yielding plasmids pTIC119, pTIC120, and pTIC121, respectively, and transformed into E. coli DH5{alpha} competent cells. Plasmids extracted from colonies were confirmed to include the intact cpa gene by PCR. Furthermore, each plasmid was transformed into E. coli M15 competent cells in accordance with the manufacturer's recommendations. Transformants were cultured in LB broth with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside; TaKaRa), and protein expressed in the cell lysate was purified with a Ni-nitrilotriacetic acid affinity chromatography kit (QIAGEN).

SDS-PAGE and Western blot analysis. To determine the molecular weight and reactivity to antibody of the alpha toxin protein expressed by E. coli M15 cells containing the cpa gene, the protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% resolving gel (Bio-Rad). After electrophoresis, one polyacrylamide gel was stained with 0.2% Coomassie brilliant blue R260 solution, and the other was electrotransferred onto a nitrocellulose membrane (Bio-Rad) for Western blotting analysis. The membrane was probed with a 1:10,000 dilution of a rabbit anti-alpha toxin polyclonal antibody (kindly provided by J. Sakurai, Tokushima Bunri University, Tokushima, Japan). In addition, supernatants of C. perfringens strains CPBC16ML, CPBC102, and H4 were also used for Western blotting analysis.

Hybridization. (i) Southern hybridization. Genomic DNAs from CPBC16ML and strain 13 were digested with EcoRI (TaKaRa), electrophoresed in a 1% agarose gel, and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The PCR products from CPBC16ML amplified with primer pairs cpaF2/cpalphatox1-R (probe 1), cpaF3/cpalphatox1-R (probe 2), and cpaF8/cpaR10 (probe 3) were used as probes (Fig. 2). Each probe was labeled with an AlkPhos-direct kit (Amersham Pharmacia Biotech), and signals were detected by CDPstar chemiluminescence. The sizes of detected bands were estimated using a {lambda}/StyI digestion marker (OneSTEP marker 6; Nippon Gene, Tokyo, Japan).

(ii) Northern hybridization. Northern hybridization was performed as previously described (41), except that the DNA fragments were labeled with an AlkPhos-direct kit (Amersham Pharmacia Biotech) and signals were detected by CDPstar chemiluminescence. The three probes (Fig. 2) examined for Southern hybridization, as described above, were also used in Northern hybridization. To estimate the sizes of the bands detected, 23S rRNA (2.9 kb) and 16S rRNA (1.6 kb) derived from C. perfringens strain 13 were used as markers.

Nucleotide sequence accession number. The 834-nucleotide sequence of CPF-G2Im in the cpa gene has been deposited in the GenBank data bank under accession number DQ787115.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
16S rRNA gene sequence analysis of strain CPBC16ML. Strain CPBC16ML has PLC activity on EY-CW agar plates containing kanamycin, which is a selective medium for the isolation of C. perfringens. To confirm whether this isolate is C. perfringens, its 16S rRNA gene sequence was determined. A 1,396-bp fragment of the gene showed 100% identity to C. perfringens (GenBank accession number Y12669.1) (data not shown). Thus, it was concluded that this isolate is C. perfringens.

PLC and hemolytic activities of strain CPBC16ML. To determine if the different size of the cpa gene affects the enzymatic activities of the alpha toxin of CPBC16ML, PLC and hemolytic activities were detected on EY-CW and GAM blood agar plates, respectively, and compared with those of reference strains CPBC102 and H4. CPBC16ML showed both activities, as did the reference strains (Fig. 1B and C). This suggested that the larger size of the cpa gene did not abolish the enzymatic activities of the alpha toxin produced by CPBC16ML.

Nucleotide sequencing of the cpa gene of strain CPBC16ML. Sequencing analysis of the cpa gene showed that a segment of 834 bp with no homology to cpa, designated CPF-G2Im, was inserted within the cpa ORF. The analysis revealed that CPF-G2Im was integrated into the cpa gene 340 nucleotides downstream of the initial codon. A BLASTn search revealed that a 661-bp region from the 5' end (positions 1 to 661) and a 120-bp region from the 3' end (positions 715 to 834) of CPF-G2Im have 95% and 97% identities, respectively, with two noncoding regions on plasmid pCPF4969 of C. perfringens (26) (GenBank accession number AB236336.1). These regions with high similarity to CPF-G2Im were located at positions flanking the ORF encoding a predicted group II intron RT. However, the remaining 53 bp of the intermediate region (positions 662 to 714) of CPF-G2Im had no similarity with any known nucleotide sequence. Furthermore, four short fragments, ranging from 42 to 86 bp, with similarity (90 to 96%) to the Tn554-related transposase A (GenBank accession number AE017195) carried on plasmid pBC10987 of Bacillus cereus ATCC 10987, were distributed in CPF-G2Im. The amino acid sequence deduced from the nucleotide sequence of CPF-G2Im revealed that an ORF encoding 44 amino acid residues could be predicted for the 3'-end region of CPF-G2Im. However, this short peptide had no similarity with any known proteins and is not likely to be a remnant of a known RT.

Southern hybridization. DNAs extracted from CPBC16ML and strain 13 were digested with EcoRI since the sequencing data showed that there is no restriction site for EcoRI within both the cpa gene and the CPF-G2Im fragment. When the DNAs were hybridized with probe 1, which consists of parts of cpa and the entire CPF-G2Im sequence (Fig. 2), three bands for strain CPBC16ML and a single band for strain 13 were detected. All of the bands detected for CPBC16ML were larger than that detected for strain 13 (Fig. 3). When probe 2, which does not contain CPF-G2Im, was used, both strains yielded single bands, but with different sizes (approximately 4.6 kb for strain 13 and 5.5 kb for CPBC16ML). In contrast, hybridization with probe 3, which contains a partial CPF-G2Im fragment, produced three bands for CPBC16ML, with the same sizes as those seen when probe 1 was used, but no band for strain 13. From these results, we confirmed that CPF-G2Im is present in the cpa gene of CPBC16ML. Moreover, it appears that the CPF-G2Im fragment is present not only in the cpa gene but also in other locations of the genomic DNA in CPBC16ML.


Figure 3
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  		FIG. 3. Southern hybridization analysis of strain CPBC16ML. Genomic DNAs from CPBC16ML and strain 13 were digested with EcoRI, electrophoresed in a 1% agarose gel, and transferred to a nitrocellulose membrane. Lanes 1, C. perfringens strain 13; lanes 2, CPBC16ML. Three probes (probe 1, probe 2, and probe 3), as shown in Fig. 2, were used.

 
Cloning and expression of the cpa gene in E. coli M15. To investigate the expression of the alpha toxin of CPBC16ML, an intact copy of cpa was cloned into an expression vector and transformed into E. coli M15. The attempt to transform E. coli M15 directly with the recombinant plasmid carrying the cpa gene initially failed. However, it was easy to transform DH5{alpha} cells directly. DH5{alpha} cells were then used as an intermediate host. E. coli M15 transformants harboring the intact cpa genes from strains CPBC16ML, CPBC102, and H4 were successfully isolated. All three transformants showed PLC activity, with the presence of a typical turbidity zone surrounding the colonies on LB agar supplemented with egg yolk (data not shown).

SDS-PAGE and Western blot analysis. The above results demonstrated that the CPBC16ML cpa gene was capable of producing functional alpha toxin protein in E. coli. Next, the molecular size of the expressed protein was determined. SDS-PAGE analysis revealed a single protein band with the same molecular mass (about 43 kDa) as the proteins of H4 and CPBC102 (Fig. 4A). Western blot analysis showed that single bands of about 43 kDa were detected from the three strains (Fig. 4B). However, the band from CPBC16ML was much weaker than those from the reference strains. Culture supernatants from the three strains of C. perfringens were also examined by Western blotting, and the reactivities against antibody were consistent among the strains examined (data not shown). These results confirmed that insertion of CPF-G2Im in CPBC16ML cpa neither affected alpha toxin expression nor the molecular weight of the alpha toxin produced.


Figure 4
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  		FIG. 4. (A) SDS-PAGE analysis of expressed proteins extracted from the periplasm of E. coli M15. Whole-cell lysates and purified His-tagged proteins from E. coli M15 carrying the cpa gene of the reference strain H4 (lanes 1), the wild-type strain CPBC102 (lanes 2), or the variant strain CPBC16ML (lanes 3) were separated in an SDS-PAGE gel (12%). Lane M contains molecular markers. The arrow indicates the position of the expressed protein purified by Ni-nitrilotriacetic acid affinity chromatography from a cell lysate. The positions (in kilodaltons) of molecular size markers are indicated on the left. (B) Western blot analysis of expressed proteins produced by strains H4 (lane 1), CPBC102 (lane 2), and CPBC16ML (lane 3). The alpha toxin protein was detected by a rabbit anti-alpha toxin polyclonal antibody.

 
Northern hybridization. The three probes used for Northern hybridization were the same as those used for Southern hybridization (Fig. 2). Hybridization of probe 1 to the RNAs of strain CPBC16ML yielded three bands (Fig. 5A). The largest band, with a size of about 3.0 kb, was faint (cannot be seen clearly on the photograph). This may be the primary transcript of mRNA for CPBC16ML cpa. The second band, with a size of about 1.5 kb, was stronger than the largest one and appeared to be present in C. perfringens strain 13 as well. The smallest band, with a size of about 0.9 kb, was detected in CPBC16ML but not in strain 13 (Fig. 5A). When probe 2 was used, CPBC16ML showed a single band of 1.5 kb, as detected in strain 13 (Fig. 5A). In contrast, using probe 3, a single band of approximately 0.9 kb was detected in CPBC16ML but not in strain 13 (Fig. 5A). Although the sizes of the bands detected were not precise because of estimation based on the migration of 23S and 16S rRNAs of C. perfringens strain 13 as size markers, these results suggested that the 1.5-kb and 0.9-kb bands corresponded to the cpa and CPF-G2Im mRNAs, respectively. From these results, it was assumed that CPF-G2Im may be excised from the flanking cpa gene at the mRNA level. The exons then ligate to form an intact cpa gene.


Figure 5
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  		FIG. 5. (A) Northern hybridization analysis of variant CPBC16ML cpa. Lanes 1, C. perfringens strain 13; lanes 2, strain CPBC16ML. Three bands for strain CPBC16ML, with sizes of 3.0 kb, 1.5 kb, and 0.9 kb, are indicated where probe 1 was used for hybridization. The arrow indicates a predicted primary transcript of CPBC16ML cpa mRNA. (B) RT-PCR of CPBC16ML and CPBC102 cpa genes. Primer set designations for the lanes are as follows: lanes 1, cpaF2/cpalphatox1-R; lanes 2, cpaF8/cpaR10; lane 3, cpaF2/cpaR10; and lane 4, cpaF8/cpalphatox1-R. Lane M contains molecular size markers. The positions of molecular size markers are indicated to the left of the gel.

 
RT-PCR analysis. The results of Northern hybridization indicated the possible deletion of CPF-G2Im from cpa during transcriptional regulation. To further understand CPF-G2Im regulation at the mRNA level and the possibility of deletion, RT-PCR amplification was performed. Using a combination of primers designed for CPF-G2Im and for the cpa ORF, there was no amplification of the cDNA (Fig. 5B). In contrast, primers designed from the sequences upstream and downstream of CPF-G2Im within cpa allowed the amplification of an ~0.9-kb fragment, which was of the expected size, similar to that of the wild-type strain, CPBC102. Using primers specific for the CPF-G2Im fragment, the expected PCR product size of about 0.6 kb was found for CPBC16ML but not for CPBC102 (Fig. 5B). Nucleotide sequencing of the RT-PCR products amplified with primers cpaF2 and cpalphatox1-R indicated that the 834-bp CPF-G2Im fragment was completely deleted from the transcript of its flanking cpa gene. Furthermore, using RNAs extracted from an E. coli M15 transformant harboring pTIC119 with the complete cpa gene of CPBC16ML, RT-PCR gave the same results as those described above (data not shown).

Secondary structure of CPF-G2Im. Although CPF-G2Im lacks a recognizable RT sequence, analysis of the predicted secondary structure of the CPF-G2Im sequence revealed the distinctive characteristics of group II introns, where six helical domains (D1 to D6) emerge around a central wheel (Fig. 6A). There is a consensus sequence of GUGCG at the 5' end. At the 3' end, there is a well-defined D5 domain with a 5' GAAA tetraloop (Fig. 6B.1) and a D6 domain with a short hairpin loop structure with a bulged A residue, the branching point which acts as the nucleophile during the first step in the self-splicing of group II introns, located 8 nucleotides upstream from the 3' end (Fig. 6B.2). With all of these important characteristics, CPF-G2Im matches well with the typical secondary structures of group II introns reported elsewhere (7, 44).


Figure 6
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  		FIG. 6. (A) Predicted secondary structure of CPF-G2Im. The six domains (D1 to D6) emanate from the central wheel. The exon binding sequences (EBS1, EBS2, and EBS3), intron binding sequences (IBS1, IBS2, and IBS3), and tertiary interaction sites ({alpha} and {alpha}') are marked. IBS1 and IBS2 are present in the 5'-flanking exon, and IBS3 is present in the 3'-flanking exon (IBS1, 5'-ATAGTT-3'; IBS2, 5'-GGTATT-3'; IBS3, T). (B) Sequence and predicted secondary structure of the 3' end of CPF-G2Im. The top panel shows the structure of D5, from residues 749 to 785, with a tetraloop (5'-GAAA), and the bottom panel shows D6, from residues 787 to 834, with a bulged A (A827) marked with an asterisk (*). Residues in domains 5 and 6 are numbered according to their positions in the intact CPF-G2Im sequence.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A unique C. perfringens strain, CPBC16ML, which showed a larger cpa gene by PCR, was isolated from a broiler chicken. Although nucleotide and amino acid sequence differences for the cpa gene of C. perfringens have been reported (12, 32, 39, 43), there is no previous report of a gene fragment inserted into the cpa gene. However, sequence analysis of the cpa gene demonstrated that it was not translated as an intact alpha toxin protein because several stop codons existed in the insertion fragment. Nevertheless, strain CPBC16ML showed both PLC and hemolytic activities. Two possibilities may help to explain these results. First, the truncated alpha toxin peptide from the 5' terminus of cpa possesses both PLC and hemolytic activities; second, CPF-G2Im may be deleted from the flanking cpa gene by some mechanism, resulting in the ligation of the flanking cpa fragments.

It is known that the C. perfringens alpha toxin protein comprises two domains: the N-terminal domain of 246 amino acid residues carries the active site for phospholipid hydrolysis, and the C-terminal domain, consisting of the remaining 124 residues, is essential for lethality to mice and for hemolytic and sphingomyelinase activities (16, 30, 31). It has been reported that the C terminus of alpha toxin is devoid of enzymatic and toxic activities; however, a mutant of the alpha toxin which lacked the 121 amino acids from the C terminus retained PLC activity but abolished hemolytic activity (30, 31), suggesting that the interaction of the two domains is required for hemolytic activity (31). Therefore, expression of both the N-terminal and C-terminal domains of the alpha toxin produced by CPBC16ML was deduced.

To verify that the CPF-G2Im fragment was integrated into the cpa gene of strain CPBC16ML, Southern hybridization analysis was also performed. The results showed that the fragment is present in the cpa gene of CPBC16ML. Moreover, it appeared that the insertion fragment (CPF-G2Im) is also inserted into other parts of the genomic DNA of CPBC16ML, suggesting the presence of a mobile element.

cpa gene expression at the transcriptional level was examined by Northern hybridization. C. perfringens strain 13 was used as a control since its whole genome sequence has been determined. The three probes shown in Fig. 2 were designed for the gene expression analysis. When probe 1, consisting of cpa and CPF-G2Im, was used, three bands were detected for strain CPBC16ML (Fig. 5A). These bands may suggest that the mRNA of cpa was first transcribed together with CPF-G2Im as a primary transcript (about 3.0 kb) and then separated into two fragments (about 1.5 kb and 0.9 kb). Since C. perfringens strain 13 has no sequence similarity with CPF-G2Im, the smallest band (about 0.9 kb) detected for CPBC16ML, but not for strain 13, might be derived from CPF-G2Im, which was spliced out of the flanking cpa exons after transcription.

Sequence analysis of the RT-PCR product from the cpa gene amplified from CPBC16ML confirmed that CPF-G2Im was excised completely from the precursor mRNA. However, RT-PCR using a combination of primers designed for the CPF-G2Im sequence and the cpa ORF (capF2/cpaR10 and cpaF8/cpalphatox1-R, respectively) could not amplify any of the cDNAs (Fig. 5B). These results, as well as a very faint 3.0-kb band detected by Northern hybridization, may imply that CPF-G2Im has a very efficient splicing activity.

These findings suggest that CPF-G2Im is likely a mobile element. Although group II introns are easily recognized through an RT encoded by an ORF within the introns (24, 44), the fragment in our study does not contain any known RT sequence. Although an ORF encoding 44 amino acid residues could be predicted for the 3'-end region of CPF-G2Im, the short peptide had no similarity with any known proteins and is not likely a remnant of a known RT. A predicted group II intron RT gene sequence in C. perfringens was recently deposited in GenBank under accession number AB236336.1. This was found during complete sequencing of the enterotoxin-encoding pCPF4969 plasmid of C. perfringens (26). A BLAST search of CPF-G2Im indicated that two regions in CPF-G2Im (bp 1 to 661 and 715 to 834) show high similarity (95% and 97%, respectively) to two noncoding regions flanking the predicted group II intron RT gene sequence. This suggests that CPF-G2Im may be a group II intron which lacks an ORF encoding an RT and that it is likely a remnant of the plasmid group II intron. The results of secondary structure prediction analysis showed that CPF-G2Im has the typical secondary structure of group II introns. It consists of six helical domains emerging from a central wheel (Fig. 6A) and seems to be a bacterial group II class B2-like intron (7, 44). Several ORF-less introns have been reported among prokaryotes. The first was found in Methanosarcina acetiborans, and some introns are inserted into the ORFs of other introns to form nested organizations called twintrons (9). Recently, ORF-less group II introns inserted in a conserved protein-encoding gene have been reported for a Cyanobacterium sp. (23). Furthermore, an ORF-less intron has also been found in a putative conjugative plasmid, pAW63, in the Bacillus cereus group (46), although functional analysis has not yet been carried out. To our knowledge, the CPF-G2Im sequence detected in the conserved alpha toxin-encoding gene is the first ORF-less group II intron which is inserted into an ORF encoding a functional toxin protein in C. perfringens.

It is known that the efficient splicing or reverse splicing (mobility) of group II introns requires proteins to help the intron RNA fold into the catalytically active structure (9, 18, 29). CPF-G2Im was capable of self-splicing, but nevertheless, it does not encode any RT. It is not clear whether the ORF-less CPF-G2Im sequence can move autonomously by using its intrinsic self-splicing activity or whether it requires additional proteins, such as possible RTs, carried by the organism that may act in trans. For a better understanding of the expression of the alpha toxin in CPBC16ML, and also in order to exclude the possibility that the PLC and hemolytic activities were produced by another gene(s), the cpa gene carrying CPF-G2Im was cloned into a pQE30 vector and transformed into E. coli M15 cells. The transformants harboring the intact cpa gene showed a PLC with the normal molecular weight, even though the reactivity of the expressed protein against anti-PLC antibody was relatively weak. This demonstrated that the phospholipase C was also produced in E. coli cells, despite the presence of CPF-G2Im. It should be noted that gene expression occurred in both C. perfringens and E. coli, implying that the splicing of CPF-G2Im may occur autonomously, using its intrinsic self-splicing activity alone, since E. coli M15 is derived from E. coli K-12, whose complete genome sequence has been determined (GenBank accession number NC_000913) and shows no evidence of group II introns being carried. However, it has been reported that group II introns can undergo splicing with the help of some proteins that function as RNA chaperones (15, 27) and/or other host-encoded protein splicing factors which are not related to intron-encoded proteins (33). Therefore, we cannot exclude the possibility that the splicing of CPF-G2Im may also be promoted by proteins with RNA chaperone activity or by other host-encoded splicing cofactors. It may be a rare occurrence, but the present study provides evidence for the existence of splicing-efficient bacterial group II introns. On the other hand, it remains unclear whether CPF-G2Im has the capacity for being mobile. The results of Southern hybridization with strain CPBC16ML demonstrated that CPF-G2Im is present in other parts of the genome (Fig. 3). Further studies are required to clarify the splicing mechanism and to determine the possibility of horizontal transfer of ORF-less group II introns.

BLAST analysis of CPF-G2Im showed another interesting finding. Four short fragments, ranging from 42 to 86 bp, with 90 to 96% sequence similarity to the Tn554-related transposase A (GenBank accession number AE017195) carried on plasmid pBC10987 of B. cereus ATCC 10987 were distributed in CPF-G2Im. The presence of transposase fragments within a catalytic intron RNA structure is very rare and unlikely. The possible significance of this finding remains unclear.

To date, the frequency of group II introns in C. perfringens remains unclear. Studies on the distribution and localization of group II introns, with or without RT gene sequences, in C. perfringens strains are ongoing in our laboratory. These studies may help to clarify not only the phylogenetic relationships among group II introns of bacteria but also the pathogenesis of C. perfringens.


   ACKNOWLEDGMENTS
 
We thank J. Sakurai, Tokushima Bunri University, Japan, for kindly supplying the rabbit anti-alpha toxin serum. We are grateful to Kingsley K. Amoako, Animal Disease Research Institute, Canadian Food Inspection Agency, Canada, for helpful discussions and assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Public Health, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuenkibanadai-Nishi, Miyazaki 889-2192, Japan. Phone: 81-985-58-7284. Fax: 81-985-58-7284. E-mail: a0d901u{at}cc.miyazaki-u.ac.jp. Back

{triangledown} Published ahead of print on 8 December 2006. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, March 2007, p. 1633-1640, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01210-06
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