development and application of a method for ...aem.asm.org/content/77/4/1375.full.pdftransformation...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2011, p. 1375–1382 Vol. 77, No. 40099-2240/11/$12.00 doi:10.1128/AEM.01572-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Development and Application of a Method for CounterselectableIn-Frame Deletion in Clostridium perfringens�†

Hirofumi Nariya,1* Shigeru Miyata,1 Motoo Suzuki,1 Eiji Tamai,2 and Akinobu Okabe1

Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan,1 andDepartment of Infectious Disease Science, Faculty of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho,

Matsuyama, Ehime 790-8578, Japan2

Received 2 July 2010/Accepted 13 December 2010

Many pathogenic clostridial species produce toxins and enzymes. To facilitate genome-wide identification ofvirulence factors and biotechnological application of their useful products, we have developed a markerlessin-frame deletion method for Clostridium perfringens which allows efficient counterselection and multiple-genedisruption. The system comprises a galKT gene disruptant and a suicide galK plasmid into which two fragmentsof a target gene for in-frame deletion are cloned. The system was shown to be accurate and simple by using itto disrupt the alpha-toxin gene of the organism. It was also used to construct of two different virulence-attenuated strains, ��1303 and HN1314: the former is a disruptant of the virRS operon, which regulates theexpression of virulence factors, and the latter is a disruptant of the six genes encoding the �, �, and � toxins;a clostripain-like protease; a 190-kDa secretory protein; and a putative cell wall lytic endopeptidase. Compar-ison of the two disruptants in terms of growth ability and the background levels of secreted proteins showedthat HN1314 is more useful than ��1303 as a host for the large-scale production of recombinant proteins.

Clostridium perfringens is a spore-forming anaerobic Gram-positive rod with a low DNA G�C content (28). It producespotent histolytic enzymes and toxins, causing various diseasesranging from mild food-borne diarrhea to fulminant histotoxicinfection (3, 29, 37). Other pathogenic clostridial species alsoproduce various toxins and enzymes (28). Some of such clos-tridial proteins are useful and commercially available, and oth-ers are potentially useful (15). Unfortunately, the expression ofclostridial proteins in a heterologous host is often inefficient,especially when high-molecular-weight proteins are expressed,mainly because of extremely biased codon usage as to the useof A and T at silent positions (39).

To circumvent such problems, we have developed an expres-sion system including C. perfringens using the following advan-tages of the organism: it grows very rapidly, its doubling timebeing less than 10 min under optimal conditions (19), and it isrelatively aerotolerant and can be simply batch cultured (28).Furthermore, strain 13, which is used in our system, is anextremely poorly sporulating strain with mutation of the mas-ter sporulation regulatory gene and hence is suitable in termsof environmental biohazard risk (20). Our system has so farbeen successfully applied to NanI sialidase (37), ColH colla-genase (39), and clostripain-like protease (11).

Since application of our system to the large-scale prepara-tion of clostridial proteins would cause a biosafety concern, wehave attempted to attenuate the virulence of C. perfringens bydeleting multiple virulence genes to extents comparable tothose in microorganisms generally regarded as safe (GRAS)

(16, 22, 30). In order to efficiently delete multiple genes, wechose the GalK/galactose (Gal)-based counterselection systemthat was developed for Myxococcus xanthus (21, 41) and ap-plied it to Streptococcus mutans (17). From C. perfringens strain13, we constructed a parental strain, HN13, that has an in-frame deletion (IF) in a galKT operon. As a suicide vector, weconstructed pGALK containing a drug resistance marker andthe galK gene to mediate the toxicity of galactose or a galactoseanalog (17, 41). The rationale of the markerless in-frame de-letion system is as follows. The first recombination occurs ateither of the two fragments of the target gene for the in-framedeletion. These two homologously recombined integrants arepositively selected by the drug resistance marker. However,when the second recombination event occurs in the integrants,the GalK-mediated toxicity allows selection of an in-framedeletion mutant and a revertant strain, which can be differen-tiated simply by colony PCR.

By using the HN13/pGALK system, we disrupted the virRSoperon encoding the VirR/VirS two-component signal trans-duction system involved in the regulation of virulence genes (5,25). In an attempt to reduce not only major virulence factorsbut also major secretory proteins, we disrupted the six genesencoding the � toxin (phospholipase C [PLC]), � toxin (per-fringolysin O [PFO]), � toxin (ColA collagenase), a clostripain-like protease (CLP), CPE1281 of a 190-kDa secretory protein(10), and CPE0278 of a CwlO homologue from Bacillus subti-lis, a cell wall lytic endopeptidase (43). The results presentedhere indicate that the six-gene mutation abolished the produc-tion of the corresponding proteins and also gave almost thesame growth rate and NanI productivity as those for the wild-type strain.

MATERIALS AND METHODS

Bacteria, growth conditions, and DNA manipulation. Escherichia coli Nova-Blue (Merck KGaA, Darmstadt, Germany) was used as the recipient strain for

* Corresponding author. Mailing address: Department of Microbi-ology, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Phone and fax: 81-87-891-2129. E-mail:[email protected].

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 23 December 2010.

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transformation and was grown in LB medium at 37°C. Clostridium acetobutylicumATCC 824 was obtained from the Riken BioResource Center and was used asthe source of the galK gene in the suicide vector. C. perfringens strain 13 (33) wasused as the host strain. It was grown on 2-fold-diluted Gifu anaerobic medium(GAM/2) agar plates (Nissui Pharmaceutical, Tokyo, Japan) under anaerobicconditions using an AnaeroPack system (Mitsubishi Gas Chemical, Tokyo,Japan), unless otherwise stated. Chloramphenicol was added to the agar plates at10 �g/ml (GAM/2-Cm10), when necessary. All recombinant strains constructedfrom C. perfringens strain 13 and used as IF mutants are listed in Table 1. Theywere grown in TY medium (3% tryptone, 2% yeast extract, 0.1% sodium thio-glycolate) or the same medium supplemented with sugar additive, as follows:TY-G1 and TY-G3 with 1 and 3% glucose, respectively; TY-GA1 and TY-GA3with 1 and 3% galactose, respectively; and TY-DGA1 with 1% 2-deoxygalactose(DGA). Cultures were performed under anaerobic conditions in tightly cappedtubes at 37°C. Twofold-diluted TY (TY/2) agar plates were also used for thein-frame deletion system.

DNA manipulation was performed using standard protocols (31). Transfor-mation of C. perfringens was performed by electroporation, as described previ-ously (12). The DNA sequences were determined with ABI Genetic Analyzers310 and 3130, using the methods recommended by the manufacturer and double-stranded plasmid DNA as the template. All the constructs, including PCR-amplified fragments were verified by DNA sequencing.

Construction of suicide vectors for the IF system. All the primers used in thisstudy are listed in Table S1 in the supplemental material. A suicide vector,pGALK (Fig. 1), expressing the galactokinase (galK) and chloramphenicol re-sistance (catP) genes was constructed from pJIR418 (36). The 3,548-bp FspI-NaeI fragment of pJIR418 containing oriCP and rep was removed and self-ligated. Furthermore, a 1,192-bp BspHI-BstZ17I fragment containing ermBP wasreplaced with a 119-bp fragment of Pfdx containing the ferredoxin (fdx) genepromoter region of pFN (39). Pfdx was PCR amplified by using FN5 and FN3 asprimers and pFN as the template. The resultant plasmid was designatedpJIR418-Pfdx. To express the galK gene of C. acetobutylicum ATCC 824 (galK-Ca)under Pfdx control, a 1,167-bp galK-Ca-coding region was PCR amplified by usingGalK-CaN and GalK-CaC as primers and was then cloned into pJIR418-Pfdx atthe NdeI-BstZ17I sites. The resultant plasmid was designated pGALK.

We also constructed another suicide vector for the IF system. A 113-bptranscriptional terminator region of the fdx gene (Tfdx) was PCR amplified byusing FTG5 and FTG3 as primers and was then introduced at the BstZ17I-ClaIsites in pGALK. The resultant plasmid was designated pGALK-Tfdx. A 1,384-bpAvrII-NruI fragment of pGALK-Tfdx was replaced with the AvrII-BsrBI frag-ment in pCM3 (GenBank/EMBL/DDBJ accession no. AB562893). The resultantsuicide vector was designated pCM-GALK (Fig. 1B). A 2,203-bp EcoRI-AvrIIfragment containing the plc gene was cloned from pJIR418� (23) into pCM3,and the resultant plasmid was designated pPLC.

Construction of a galactose metabolism-deficient C. perfringens strain. Usingthe sequence information for C. perfringens strain 13 (33) regarding galactosemetabolism (Fig. 1A), two homologous regions, the upstream region of galK(N fragment, 903 bp) and the downstream region of galT (C fragment, 926 bp),were PCR amplified using the following primers: primers GalK-N5 and GalK-N3and primers GalT-C5 and GalT-C3, respectively. Both the N and C fragmentswere cloned into pGALK at the BamHI-SalI sites, with pGalK-N and pGalT-C,respectively, thereby being obtained. The BglII-SalI fragment derived from

pGalT-C was inserted into pGalK-N at the BglII-SalI sites. The resultant plasmidwas designated pGalKT-IF and was used as the IF plasmid for disruption of thegalKT operon. The IF gene (GalKT-IF) in pGalKT-IF was fused in-frame at theBglII site between Arg27 (AGA) of GalK and Ser471 (TCT) of GalT, resultingin elimination of an internal 2,615-bp region (Fig. 1C; see Table S1 in thesupplemental material).

pGalKT-IF was electroporated into strain 13, and then chloramphenicol-resistant cells generated through single-crossover recombination were screenedby spreading cells on GAM/2-Cm10 plates and incubation for 8 h. Theoretically,the first homologous recombination occurs at two different crossover points, theupstream and downstream homologous recombination sites, i.e., the N and Csites, respectively, as shown in Fig. 1A and C. The crossovers at the two sites, theN and C crosses (Fig. 1D), were differentiated by colony PCR, using the follow-ing four sets of primers: for the N cross, primers FW and GalK-IN and primersGalT-IC and GalT-3, and for the C cross, primers GalK-5 and GalK-IN andprimers GalT-IC and RV.

Strains with the N and C crosses were individually cultured in TY medium for4 h. The cultures were then serially diluted 10-fold with TY medium and platedonto TY/2-DGA1 plates, followed by 8 h incubation for counterselection. The-oretically, the second homologous recombination causes looping out of theplasmid-derived region and includes two types of recombination, as shown in Fig.1D and E. One occurs at homologous regions different from those in the case ofthe first recombination. This generates an IF mutant. The other occurs at thesame sites as those in the case of the first recombination. This generates arevertant strain. However, the revertant wild-type strain expresses its own galKgene and hence cannot grow on a DGA-containing plate. Thus, DGA-resistantand chloramphenicol-sensitive clones were selected as IF mutant HN13 after thesecond recombination.

Strain HN13 was identified by colony PCR using three sets of primers: primersGalK-5 and GalK-IN, GalT-IC and GalT-3, and GalK-5 and GalT-3. ColonyPCR with primers FW and GalK-IN, GalT-IC and RV, GalK-CaN and GalK-CaC, and CatP-N and CatP-C (for detection of the catP gene) was also per-formed to confirm that HN13 was free of the plasmid. Furthermore, the IF muta-tions of the galKT operon were confirmed by DNA sequencing of the PCR productamplified by using GalK-5 and GalT-3 as primers and the isolated chromosomalDNA as the template. The phenotypes of HN13 obtained from the N- and C-crossstrains were confirmed to be identical, as described in the Results section, andtherefore, it was used as a parental strain for the IF system (Table 1).

Construction of IF mutants. IF mutation of the genes encoding PLC(CPE0036), VirS (CPE1500), VirR (CPE1501), CLP (CPE0846), ColA(CPE0173), PFO (CPE0163), CPE1281, and CwlO (CPE0278) was performed asdescribed above, except that 3% galactose instead of DGA was used for thecounterselection. As the suicide vector, pCM-GALK was used for the constructionof IF mutants other than HN1301, HN1302, and HN1303, which were constructedby using pGALK. IF genes such as PLC-IF, VirS-IF, VirRS-IF, CLP-IF, ColA-IF,PFO-IF, CPE1281-IF, and CwlO-IF were constructed by PCR amplification usingthe primers listed in Table S1 in the supplemental material. The N and C fragmentswere PCR amplified with primers N5 and N3 and primers C5 and C3, designed foreach construct, respectively. The N and C fragments were ligated adjacent to eachother in the suicide vector. In the case of CwlO-IF, they were fused by the overlapextension PCR method (8). The N and C crosses and the IF mutation were con-firmed by colony PCR and DNA sequencing using the sets of primers listed in TableS1 in the supplemental material.

TCA precipitation, SDS-PAGE, and N-terminal amino acid sequencing. Su-pernatant from an 8-h culture was obtained by centrifugation at 15,000 � g for2 min at 4°C, followed by passage through a 0.2-�m-pore-size membrane filterand was frozen at �80°C until use. One milliliter of the culture supernatant wassubjected to 20% trichloroacetic acid (TCA) precipitation. The precipitate waswashed with acetone, dissolved in 20 �l of 1 M Tris-HCl, pH 8.0, plus 100 �l ofSDS buffer (125 mM Tris-HCl, pH 6.8, 2.5% SDS, 5% 2-mercaptoethanol, 0.02%bromophenol blue, 20% glycerol), and then heated at 96°C for 3 min. SDS-PAGE was performed as described previously (40). Twenty-four microliters ofprotein sample, equivalent to 200 �l of the culture supernatant, was separated ona 14% gel and then stained with Coomassie brilliant blue R. The N-terminalamino acid sequences were determined with an automated protein sequencer(ABI 492; Perkin-Elmer, Waltham, MA), after transfer to polyvinylidene diflu-oride membranes as described previously (14).

Northern blotting. Preparation of total RNA from C. perfringens cells andNorthern blot analysis were carried out as described previously (6), with slightmodifications. In brief, the C. perfringens strain was grown at 37°C to the mid-exponential phase of growth (optical density at 600 nm [OD600] � 1.3), and thentotal RNA was prepared by the SDS/phenol method. For Northern blot analysis,10 �g of RNA was hybridized at 55°C with a DNA probe which had been PCR

TABLE 1. IF mutant strains derived from C. perfringens strain 13

Strain Relevant characteristic

HN13 ..........................................................................Strain 13 galKTHN1301 ......................................................................HN13 plcHN1302 ......................................................................HN13 virSHN1303 ......................................................................HN13 virRSHN1304 ......................................................................HN1302 plcHN1305 ......................................................................HN13 clpHN1306 ......................................................................HN13 colAHN1307 ......................................................................HN13 pfoHN1308 ......................................................................HN13 CPE1281HN1309 ......................................................................HN13 cwlOHN1310 ......................................................................HN1301 clpHN1311 ......................................................................HN1310 colAHN1312 ......................................................................HN1311 pfoHN1313 ......................................................................HN1312 CPE1281HN1314 ......................................................................HN1313 cwlO

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amplified with primers VirR-FW and VirR-RV using C. perfringens 13 chromo-some DNA as the template and labeled with digoxigenin-11-dUTP (RocheApplied Science, Mannheim, Germany). The hybridized signals were detected asLumi-Phos 530 (Lumigen, Southfield, MI) chemiluminescence on X-ray film.

Enzyme assaying of a culture supernatant. (i) PLC. The assaying of PLCactivity was performed using p-nitrophenylphosphorylcholine (Sigma-Aldrich, St.Louis, MO), as described previously (13). PLC activity was also examined byobserving the white halo formed around a colony on a GAM plate containing 5%(vol/vol) egg yolk (EY-GAM), after 1 �l of the culture was spotted and incuba-

tion under anaerobic conditions. Ten microliters of the culture supernatant wasalso applied to a well in an egg yolk agarose plate of EY-TBS (1% agarose, 5%egg yolk, 5 mM dithiothreitol [DTT], TBS buffer [20 mM Tris-HCl, pH 7.5, 150mM NaCl]), followed by incubation at 37°C.

(ii) CLP. CLP activity was assayed using benzoyl-L-arginine p-nitroanilide asdescribed previously (11).

(iii) ColA. Collagenase activity was measured by using 4-phenylazobenzyloxy-carbonyl–L-Pro–L-Leu–Gly–L-Pro–D-Arg (Sigma-Aldrich) as a synthetic sub-strate, as described previously (39).

FIG. 1. IF system for C. perfringens. (A) Gene organization in the region containing the gal operon in C. perfringens strain 13. The dark gray-and light gray-shaded regions, which are designated N and C, respectively, are the regions used to create the GalKT-IF gene. The region denotedby represents the deleted region in the galKT operon. The stem-loop structure shows a putative rho-independent transcriptional terminator.Genetic symbols are tagged with CPE numbers (33). (B) Schematic representation of the suicide vectors pGALK and pCM-GALK. Geneticsymbols: oriEC, the replication origin from pUC18; catP, the chloramphenicol acetyltransferase gene from C. perfringens; galK-Ca, the galactoki-nase gene from C. acetobutylicum ATCC 824; Pfdx, the fdx promoter region from pFN; Tfdx, the transcriptional terminator region of the fdx genefrom C. perfringens strain 13; and MCS, the multiple-cloning site. (C) Construction of the IF plasmid pGalKT-IF. (D and E) Schematic diagramsof the first (D) and second (E) recombinations with the IF system involving pGalKT-IF.

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(iv) PFO. Titration of hemolytic activity toward sheep red blood cells (SRBCs)in a culture supernatant was examined as described previously (23). The hemo-lytic activity of the culture supernatant was also examined on an SRBC agarose(1% agarose, 1.25% SRBC, 5 mM DTT, 5 mM CaCl2, TBS buffer) plate, withwhich the hemolytic activity of PLC was also measurable. Ten microliters of theculture supernatant was applied to a well in the SRBC agarose plate, followed byincubation at 37°C for 4 h. The hemolytic activity of the colony was detected onan SRBC-TY-G1 agarose plate (1% agarose, 1.25% SRBC, TY-G1 medium),after spotting of 1 �l of the culture and anaerobic incubation at 37°C for 8 h.

(v) Caseinase. Proteolytic activity in a culture supernatant was assayed usingazocasein (Sigma-Aldrich), as described previously (39).

(vi) NanI sialidase. Strains carrying pFFN (38) were cultured in TY-G1 me-dium containing 20 �g/ml of erythromycin. The sialidase activity in a culturesupernatant was determined using 2-(4-methylumbelliferyl)-�-D-N-acetylneura-minic acid, sodium salt hydrate (Sigma-Aldrich), as described previously (38).

RESULTS

Construction of an IF system for C. perfringens. Genomeanalysis of C. perfringens strain 13 (Fig. 1A) showed that theoperon (mglBAC) for the Gal ABC transporter system ispresent upstream of the galM-galKT cluster. Therefore, Galuptake seems to be mediated by the Gal ABC transportersystem. Furthermore, C. perfringens strain 13 possesses allgenes corresponding to the enzymes involved in the Leloirpathway for Gal utilization (9): GalM (Gal mutarotase), GalK(galactokinase), GalT (Gal-1-phosphate uridylyltransferase),GalE (UDP-Gal-4-epimerase), and GalU (UTP-glucose-1-phosphate uridylyltransferase). The ability of strain 13 to useGal as a sole carbon source was examined by growing it in TYmedium with and without Gal. Strain 13 could grow in TY-GA1 as well as in TY-G1. Moreover, strain 13 was vulnerableto the toxicity of DGA, a nonmetabolizable Gal analog, whichexhibits toxicity upon being taken up and phosphorylated.Thus, the development of a GalK/galactose-based IF systemfor C. perfringens requires the preparation of a parental mutantstrain (Fig. 1) in which the galKT operon is disrupted. It alsorequires the construction of a suicide vector that confers drugresistance on transformants and that simultaneously allowsconstitutive expression of GalK after being integrated into thechromosome. All data with respect to the growth of strain 13and galKT-disruptant strain HN13 in various media are shownin Fig. S1 in the supplemental material.

We constructed a suicide vector, pGALK (Fig. 1B), for theIF system which carries galK-Ca fused to the fdx promoter (39).galK-Ca was chosen on the basis of the following assumption:the galK genes of C. perfringens and C. acetobutylicum arephylogenetically close enough to be well expressed and func-tional but different enough in identity (61.2%) at the DNAlevel to prevent homologous recombination. The galK-Ca genein pGALK was suggested to be functional by the finding thatgalKT-disruptant strain HN13 (hereinafter named strainHN13) was obtained efficiently from the wild-type strain usingpGalKT-IF and DGA.

To confirm the phenotypic change in Gal utilization by strainHN13, we examined its growth in TY-GA1. It grew well inTY-G1 and similarly to the wild-type strain, but not in TY-GA1, unlike the wild-type strain. Furthermore, it was insensi-tive to the DGA toxicity, indicating that strain HN13 can beused as a parental strain for the IF system.

plc disruptant of strain HN13. PLC, one of the major viru-lence factors of C. perfringens, was disrupted using the IF

system to examine its accuracy and efficiency. The transforma-tion efficiency of strain HN13 was approximately 107 CFU per�g of pJIR418 DNA, with this being comparable to that ofstrain 13. Approximately 5,000 chloramphenicol-resistanttransformants were obtained per �g of pPLC-IF (see Table S1in the supplemental material) upon the first recombination. Ofthese transformants, 32 clones were randomly chosen and rep-lica plated onto GAM/2-Cm10 and TY/2-GA3 plates (Fig. 2A).Twenty-nine clones grew well on the GAM/2-Cm10 plates butnot on the TY/2-GA3 plates. Sixteen of these 29 clones werethen subjected to colony PCR to confirm the crossover point,with the clones with the N and C crosses being named strainsN and C, respectively. Eight and seven clones were confirmedto be strains N and C, respectively, whereas the rest remainedunidentified. Strains N and C were individually grown in TYmedium for 4 h and then plated onto TY/2-G3 and TY/2-GA3plates, respectively. As shown in Fig. 2B, Gal-resistant colonieswere formed on TY/2-GA3 plates at a level of approximately0.1% of the total number of CFU obtained on TY/2-G3 plates.The streaking of a colony of strain N or C on a TY/2-GA3 platealso allowed the isolation of a single Gal-resistant colony (Fig.2A). Sixteen Gal-resistant clones derived from each of strainsN and C were replica plated onto TY/2-GA3 and GAM/2-Cm10 plates. In both cases, 15 clones were Gal resistant andchloramphenicol sensitive, so they should be either an IF mu-tant or a revertant strain (strain R). Colony PCR revealed thatseven and five clones were the IF mutants derived from strainsN and C, respectively, and that the remaining clones werestrain R. The two IF clones derived from strains N and C wereconfirmed to be the plc IF mutant by DNA sequencing of the

FIG. 2. Gal counterselection with the IF system. (A) Selection bystreaking. Strain HN13 with pPLC-IF integrated was streaked on bothGAM/2-Cm10 and TY/2-GA3 plates. (B) Selection by spreading. Thestrain was also cultured in TY medium for 4 h, followed by 104-folddilution with the medium. One hundred microliters of the dilutedculture was spread onto both TY/2-G3 and TY/2-GA3 plates. Theplates were incubated anaerobically for 12 h at 37°C.



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PCR products and were used as HN1301. All data with respectto analysis of PCR products are shown in Fig. S2 in the sup-plemental material.

The PLC production of HN1301 was examined by SDS-PAGE. No band corresponding to PLC was detectable for theculture supernatant of HN1301, unlike for the culture super-natants of strains 13, HN13, and R (Fig. 3A). On the otherhand, there was no significant difference between these fourstrains in the levels of other major secretory proteins, such asCLP (heavy and light chains [11]), ColA, PFO, and 190-kDahypothetical protein CPE1281 (10), which was identified byN-terminal amino acid sequencing. This indicates that HN1301is disrupted properly and also suggests that the IF system doesnot affect the productivity of secretory proteins except for adisrupted one. Complete loss of the PLC production ofHN1301 was also shown by the egg yolk test involving a cultureand a culture supernatant (Fig. 3B and C) and also by assayingof PLC activity (Table 2). HN1301 transformed with pPLCcarrying the plc gene produced and secreted a large amount ofPLC (Fig. 3D), indicating that HN1301 retains the ability toproduce and secrete secretory proteins. Thus, HN1301 wasproved phenotypically to be the plc IF mutant.

virR/virS disruptant of strain HN13. Production of virulencefactors such as PLC, CLP, ColA, and PFO by C. perfringens isregulated at the transcriptional level by the VirR/VirS two-component signal transduction system (34). Furthermore, it

also regulates the expression of many virulence-related house-keeping genes through the function of VirR-regulated RNA(24, 35). Thus, the disruption of the virRS operon with the IFsystem can be expected to greatly attenuate the virulence.

By means of the IF system, we constructed virS IF mutantHN1302 and virRS IF mutant HN1303 (Table 1; see Table S1in the supplemental material). Met11 (ATG) and His399(CAT) were used as the IF fusion sites in the case of HN1302.Met1 (ATG) of VirR and His399 (CAT) of VirS were chosenas the IF fusion sites in the case of HN1303. As expected,Northern blot analysis showed that strains HN13 and HN1302expressed a single 2.1-kb virRS transcript (2) and a 0.9-kb virRtranscript, respectively, whereas no hybridized band was de-tected for HN1303 with the virR-specific probe (Fig. 4A). Thelevels of virRS transcripts in strain HN13 were almost the sameas those in strain 13 (data not shown). However, the level ofvirR transcripts in HN1302 seemed higher than that of thevirRS transcripts in strain HN13, probably due to the higherlevel of stability of the shorter transcript.

SDS-PAGE analysis of culture supernatants (Fig. 4B)showed that the levels of PLC, CLP, ColA, and PFO producedby HN1302 and HN1303 were far lower than those producedby strain HN13. The enzyme assay (Table 2) showed that noCLP or PFO activity and only marginal ColA and caseinaseactivities were detectable in the culture supernatant ofHN1303 and that the inhibitory effect on toxin production wasmore prominent in HN1303 than in HN1302. A likely expla-nation is that HN1302 expresses more virR than strain HN13,so the virS-defective phenotype could be partially comple-mented by the plasmid-encoded virR (2). In contrast to theseproteins, CPE1281 and several minor unidentified proteinsseemed to be produced at increased levels in culture superna-tants of HN1302 and HN1303. Large amounts of low-molecu-lar-weight substances, which correspond to those detectable ingrowth medium (data not shown), were detectable in the su-pernatants of the two disruptants. This seems to be due to lossof CLP protease regulated by the VirR/VirS system. The PLCproductivity of the two disruptants was completely abolishedwhen the plc gene was disrupted (Fig. 4B and C). The growthof the mutants in TY-G1 was slightly impaired, as shown bycomparison of the OD at 600 nm between the cultures of themutants and the wild-type strain (Fig. 4A).

TABLE 2. PLC, CLP, ColA, PFO, and caseinase activities inculture supernatants of the IF mutants

StrainActivitya

PLC CLP ColA PFO Caseinase

13 421 � 21 64.5 � 0.31 1719 � 6.5 923.9 � 4.7 7.08 � 0.15HN13 439 � 12 64.4 � 0.31 1722 � 4.6 930.9 � 6.1 7.11 � 0.08HN1301 — ND ND ND NDHN1302 39 � 1 — 37 � 0.0 �0.9 �0.09HN1303 29 � 4 — 24 � 5.1 — �0.09HN1305 ND — ND ND �0.09HN1306 ND ND — ND NDHN1307 ND ND ND — NDHN13014 — — — — �0.09

a The activities of PFO and the other enzymes are expressed in U and mU/mlof culture supernatant, respectively. Values are expressed as means � SDs fortriplicate determinations. — and ND, undetected and not determined, respec-tively.

FIG. 3. Expression of PLC in C. perfringens strains 13, HN13, andHN1301. (A) SDS-PAGE analysis of culture supernatants of C. per-fringens strains 13, HN13, and HN1301 and the revertant strain (R).The OD600s of the cultures are shown underneath the gel. Majorsecreted proteins are indicated by arrowheads on the left; CPE1281(187.0 kDa), ColA (121.4 kDa), PFO (52.7 kDa), PLC (42.6 kDa), andthe heavy chain (HC) and two light chains (LC and LC) of CLP(CLP-HC, 32.8 kDa; CLP-LC, 15.4 kDa; CLP-LC, 14.9 kDa [11]).(B) PLC activity exhibited by colonies on EY-GAM plates. (C) PLCactivity in the culture supernatants on EY-TBS agarose plates. Thesamples applied to the wells corresponded to those applied to the lanesin panel A. (D) Complementation test for HN1301. Strain HN1301transformed with pPLC or pCM3 was grown in TY-G1-Cm10 for 8 h,and then the culture supernatants were examined for PLC activity andsubjected to SDS-PAGE analysis. All assays were carried out as de-scribed under Materials and Methods.



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Sextuple disruptant of strain HN13. Another approach forconstructing GRAS strains from C. perfringens is to attenuatethe virulence and potential virulence determinants by deletingeach relevant gene. By means of the IF system, we disruptedthe genes encoding PLC, CLP, ColA, and PFO, which areregulated by the VirR/VirS system. Besides these virulencefactors, two major secretory proteins, CPE1281 and a putativecell wall lytic endopeptidase, CwlO, were also deleted to re-duce host extracellular proteins for purification of recombinantsecretory proteins. Our initial attempt to construct CLP-IF inpGALK involving E. coli was unsuccessful. However, pCM-GALK (Fig. 2B), which lacks a possible intrinsic strong pro-moter near the multiple-cloning site, allowed cloning of C.perfringens DNA with potential toxicity. pCM-GALK was usedto generate the following mutants (Table 1): single-IF mutantsHN1305, HN1306, HN1307 and HN1308; a double-IF one,HN1310; a triple-IF one, HN1311; a quadruple-IF one,HN1312; and a quintuple-IF one, HN1313.

SDS-PAGE analysis of the culture supernatants of these IFmutants (Fig. 5) showed that the expression of the relevant geneswas successfully abolished. In the culture supernatants of clp IFmutant HN1305 and its derivative mutants, low-molecular-weightsubstances were observed, as was the case for the virRS IF mutant,HN1303. Furthermore, we found that three proteins that mi-grated as 45-, 30-, and 15-kDa bands on SDS-PAGE had in-creased levels or were newly detected in the culture supernatantsof HN1305 and its derivatives. The N-terminal amino acid se-quences of the 45-, 30-, and 15-kDa proteins were determined tobe TPLTDDQK, TPLTDDQK, and GGDVNSG, respectively.Interestingly, these three proteins are derived from the same geneproduct, CPE0278, a 432-amino-acid hypothetical protein, whichexhibits homology to B. subtilis CwlO, a cell wall lytic endopepti-dase belonging to the NLPC/P60 family (43). Thus, we con-structed cwlO IF mutant HN1309 and sextuple-IF mutantHN1314, of which the phenotypic changes were confirmed by

FIG. 4. Northern blot analysis of virR mRNA and expression of PLC in the virRS IF mutants. (A) Northern blot analysis of the virR transcript.Ten micrograms of total RNA was subjected to Northern blot analysis using a digoxigenin-labeled virR-specific 520-bp DNA probe, as describedunder Materials and Methods. The calculated sizes of mRNA and the RNA size markers are given on the right and left, respectively (b, bases).(B) SDS-PAGE analysis of culture supernatants of strains HN13, HN1301, HN1302, HN1303, and HN1304. (C) PLC activity in the culturesupernatants on EY-TBS agarose plates. The samples applied to the wells corresponded to those applied to the lanes in panel B.

FIG. 5. SDS-PAGE analysis of culture supernatants of the IF mu-tants. (A) Culture supernatants of the single-IF mutants, HN1301,HN1305, HN1306, HN1307, HN1308, and HN1309, were subjected toSDS-PAGE analysis. (B) The culture supernatants of strains HN13,HN1301, HN1303, HN1305, HN1310, HN1311, HN1312, HN1313, andHN1314 were also subjected to SDS-PAGE analysis. Secreted proteinsderived from CwlO are indicated by arrowheads on the right.



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SDS-PAGE analysis (Fig. 5). Both strains exhibited normalgrowth, like the other mutant strains (Fig. 5).

Enzyme assaying of the culture supernatants showed thateach single-IF mutant produced no relevant protein and alsothat HN1314 produced no PLC, CLP, ColA, or PFO activity(Table 2). Furthermore, we examined the hemolytic activitiesof the culture supernatants using SRBC agarose plates (Fig.6A). virS IF mutant HN1302, virRS IF mutant HN1303, and pfoIF mutant HN1307 exhibited the hemolytic activity of PLC,and plc IF mutant HN1301 exhibited the hemolytic activity ofPFO, while HN1314 did not show the hemolytic activity ofeither one. This was also the case for hemolysis of coloniesformed on SRBC-TY-G1 agarose plates (Fig. 6B).

In order to assess the recombinant protein productivity ofstrain HN1314, 74-kDa NanI sialidase was expressed in strains13, HN13, and HN1314 transformed with pFFN (38). Theirgrowth was almost the same, and the NanI productivity dif-fered only slightly, i.e., only to such an extent that the NanIactivities of strains 13, HN13, and HN1314 grown for 8 h were120, 125, and 141 units/ml of culture supernatant, respectively,and those of strains grown for 12 h were 124, 128, and 142units/ml of culture supernatant, respectively. SDS-PAGE anal-ysis showed the presence of a large amount of NanI in theculture supernatant of HN1314 but the absence of other majorsecretory proteins (data not shown). Thus, sextuple-IF mutantHN1314 can be regarded as being more suitable than virRS IFmutant HN1303 in terms of the residual levels of toxins andhaving an inhibitory effect on cellular growth.

DISCUSSION

In this study we developed a precise in-frame allelic ex-change system for C. perfringens involving galK as a counters-electable marker. Genetic markers employed for IF systems sofar have been levansucrase (42), phosphoribosyltransferases

(18), and galactokinase (21, 41), which mediate the toxicity oftheir substrates or nonmetabolizable analogs in merodiploidsthat have not undergone the second recombination. Amongthem, we employed the GalK system because C. perfringenspossesses a chromosomally encoded galKT operon, utilizinggalactose as a sole carbon source, and also because the C.acetobutylicum galK homologue can be maintained stably on asuicide vector without recombination between the two homo-logues. We confirmed the excellent efficiency and reliability ofour IF system, on the basis of the results of deletion of the plcgenes, and applied it to the disruption of the virS and virRSgenes and also to the disruption of six genes encoding extra-cellularly secreted proteins.

In the case of the virRS IF mutant, the production of majortoxins decreased markedly, and thus it can be used as a viru-lence-attenuated host, depending on the purpose. However,the toxin productivity of the mutant was not completely abol-ished. Furthermore, it cannot be ruled out that the attenuatedvirulence may be restored under certain circumstances wherecross talk with other signaling pathways evokes the expressionof the virulence factors. In contrast, the sextuple-IF mutantwas completely deficient in the toxins, it thus being more suit-able for the large-scale preparation of recombinant proteins,especially when the products are to be used for medical pur-poses.

In the postgenomic era, comparative and functional genom-ics have been progressing (26). Aided by high-throughputmethods for proteomic (1, 34) and transcriptomic (32, 34, 44)analyses of C. perfringens, a genome-wide search for factorsinvolved in the networks of such biological processes as geneticexchange, development, virulence, and quorum sensing willreveal an increasing number of potentially key molecules ineach network.

Our method has several advantages over other methodsavailable for the mutagenesis of C. perfringens, including in-tron-mediated mutagenesis (4, 7): a selection marker can bereused for disruption of multiple genes, it does not involve apolar effect, and it allows allelic exchange of chromosomalregions with fragments of interest (27). The galK-based IFsystem will facilitate the construction of C. perfringens strainsthat are more useful for the biomedical and bioindustrial sci-ences.

ACKNOWLEDGMENTS

We thank N. J. Halewood for his assistance in preparing the manu-script.

This work was supported by a grant-in-aid from the Japan Societyfor the Promotion of Science (Grant for Scientific Research C21590482). It was also partly supported by the Kagawa UniversityCharacteristic Prior Research Funds 2010 and by Research for Pro-moting Technological Seeds Program of Japan Science and Technol-ogy Agency (no. 13-059).

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